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Background: Insecticides are a crucial component of vector control. However, resistance constitute a threat on their efficacy and the gains obtained over the years through malaria vector control. In Gabon, little data on phenotypic insecticide resistance in Anopheles vectors are published, compromising the rational implementation of resistance management strategies. We assessed the susceptibility to pyrethroids, carbamates and organophosphates of Anopheles gambiae sensu lato (s.l.) and discuss the mechanisms involved in the pyrethroid resistance-phenotype., Methods: A. gambiae s.l. larvae were collected from breeding sites in Lambaréné. Emerging adults were used in WHO tube assays at an insecticide concentration that defines resistance (diagnostic concentration). Subsequently, deltamethrin and permethrin were used at 5x and 10x diagnostic concentrations and after preexposure with the cytochrome p450 (and glutathione S-transferase) inhibitor piperonyl butoxide (PBO). A subset of mosquitoes was typed by molecular methods and screened using Taqman assays for mutations conferring target site resistance at the Voltage-gated sodium channel 1014 (Vgsc-1014) locus and the acetylcholinesterase (Ace-1) gene., Results: All mosquitoes were A. gambiae sensu stricto (s.s.) and resistant to permethrin, deltamethrin and alphacypermethrin (mortality less than 98%). However, mosquitoes were susceptible to malathion but resistant to bendiocarb. The level of resistance was high for permethrin and at least moderate for deltamethrin. Pre-exposure to PBO significantly increased the mortality of resistant mosquitoes (P < 0.0001). They became fully susceptible to deltamethrin and permethrin-induced mortality increased 4-fold. The G119S Ace-1 resistance allele, which confers resistance to both organophosphates and carbamates, was not present. All sampled mosquitoes were either homozygous for the Vgsc-L1014F or heterozygous for Vgsc-L1014F/L1014S, a marker for resistance to pyrethroids and organochlorides., Conclusion: These findings demonstrate a role of cytochrome P450 monooxygenases in the pyrethroid-resistance of A. gambiae s.s. from Lambaréné. Combining PBO with pyrethroids, as done in second generation bednets, may be used to revert resistance. In addition, malathion could also be used in combination with pyrethroids-based methods for resistance management., (© 2024. The Author(s).)

Background: Brown 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., Methods: Adult 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., Results: RaTexT ® 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., Conclusions: RaTexT ® is a valuable tool for monitoring the development of resistance to synthetic pyrethroids or phenylpyrazoles in tick-infested dogs., (© 2024. The Author(s).)

Background: Aedes aegypti (L.) is the main vector of dengue, yellow fever, Zika, and chikungunya viruses in many parts of the world, impacting millions of people worldwide each year. Insecticide-based interventions have been effective in controlling Aedes mosquito populations for several years, but in recent times, resistance to these compounds has developed, posing a global threat to the control of this mosquito., Methods: Ovitraps were used to collect A. aegypti eggs in the cities of Tartagal and San Ramón de la Nueva Orán (Salta), Puerto Iguazú (Misiones), and Clorinda (Formosa). World Health Organization (WHO)-impregnated papers with the discriminating concentration (DC) of permethrin, 5X, 10X and pirimiphos methyl were used for the toxicological bioassays. We also genotyped each sample for the three kdr single nucleotide polymorphisms (SNP): V410L, V1016I, and F1534C in individual TaqMan quantitative PCR (qPCR) reactions., Results: All investigated A. aegypti populations were highly resistant to permethrin, as the mortality percentage with the permethrin 10×DC remained below 98%. However, all populations were 100% susceptible to pirimiphos-methyl. Kdr genotyping demonstrated the presence of the V410L mutation for the first time in Argentina in all the populations studied. A prevalence of the triple mutant genotype (LL + II + CC) was observed in the northeastern cities of Clorinda (83.3%) and Puerto Iguazú (55.6%)., Conclusions: This study demonstrates for the first time the presence and intensity of resistance to permethrin in different populations from Argentina, and correlates the observed phenotype with the presence of kdr mutations (genotype)., (© 2024. The Author(s).)

Background: Anopheles stephensi is recognized as the main malaria vector in Iran. In recent years, resistance to several insecticide classes, including organochlorine, pyrethroids, and carbamate compounds, has been reported for this medically important malaria vector. The main objective of the present study was to evaluate the insecticide susceptibility status of An. stephensi collected from the southern part of Iran, and to clarify the mechanism of resistance, using bioassay tests and molecular methods comparing the sequence of susceptible and resistant mosquitoes., Methods: Mosquito larvae were collected from various larval habitats across six different districts (Gabrik, Sardasht, Tidar, Dehbarez, Kishi and Bandar Abbas) in Hormozgan Provine, located in the southern part of Iran. From each district standing water areas with the highest densities of Anopheles larvae were selected for sampling, and adult mosquitoes were reared from them. Finally, the collected mosquito species were identified using valid keys. Insecticide susceptibility of An. stephensi was tested using permethrin 0.75%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, and DDT 4%, following the World Health Organization (WHO) test procedures for insecticide resistance monitoring. Additionally, knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene was sequenced and analysed among resistant populations to detect possible molecular mechanisms of observed resistance phenotypes., Results: The susceptibility status of An. stephensi revealed that resistance to DDT and permethrin was found in all districts. Furthermore, resistance to all tested insecticides in An. stephensi was detected in Gabrik, Sardasht, Tidar, and Dehbarez. Analysis of knockdown resistance (kdr) mutations at the vgsc did not show evidence for the presence of this mutation in An. stephensi., Conclusion: Based on the results of the current study, it appears that in An. stephensi from Hormozgan Province (Iran), other resistance mechanisms such as biochemical resistance due to detoxification enzymes may be involved due to the absence of the kdr mutation or non-target site resistance. Further investigation is warranted in the future to identify the exact resistance mechanisms in this main malaria vector across the country., (© 2024. The Author(s).)

Background: The Dual-Active Ingredient long-lasting insecticidal nets (Dual-AI LLIN) have been developed to counteract the reduced efficacy of pyrethroid (PY)-only nets due to widespread pyrethroid insecticide resistance in malaria vector mosquitoes. They constitute half of the nets distributed in sub-Saharan Africa between 2022 and 2024. However, their effectiveness once they develop holes is unclear, particularly in pyrethroid-resistant settings. This study evaluates the textile integrity of three dual- AI LLINs compared to standard PY LLN, over 3 years of use in a community in Tanzania and the associated impact on malaria prevalence and incidence., Methods: A secondary analysis of data from a randomized controlled trial (RCT) in North-western Tanzania was conducted to evaluate the effectiveness of α-cypermethrin only; pyriproxyfen and α-cypermethrin (PPF-PY); chlorfenapyr and α-cypermethrin (chlorfenapyr-PY); and the synergist piperonyl butoxide and permethrin (PBO-PY) LLINs on malaria infection prevalence and case incidence. The association between the net textile condition and 1/malaria prevalence over 3 years of use between 2019 and 2022, and 2/malaria case incidence in a cohort of children over 2 years of follow-up was assessed between 2019 and 2021., Results: There was no significant association between damaged (OR 0.98, 95% CI 0.71-1.37, p-value  = 0.655) and too-torn (OR 1.07, 95% CI 0.77-1.47, p-value = 0.694) compared to intact nets on malaria prevalence for all net types. However, there were reduced rates of malaria case incidence in children sleeping under a net in good condition compared to too-torn nets (incidence rate ratio (IRR) 0.76 [95% CI 0.63-0.92], p = 0.005). Malaria incidence was also consistently lower in too-torn PBO-PY LLIN (IRR = 0.37 [95% CI 0.19-0.72], p = 0.003) and chlorfenapyr-PY LLIN (IRR = 0.45 [95% CI 0.33-0.97], p = 0.053) compared to an intact PY-only LLIN during the first year of follow up. In year 2, the incidence was only significantly lower in intact chlorfenapyr-PY LLIN (IRR = 0.49 [95% CI 0.29-0.81], p = 0.006) compared to intact PY LLIN., Conclusion: The study confirmed that sleeping under a chlorfenapyr-PY LLIN or PBO-PY LLIN offered superior protection to pyrethroid-only nets even when torn. Preventing the development of holes is essential as they impact the level of protection offered against malaria infection., Trial Registration: ClinicalTrials.gov, number (NCT03554616)., (© 2024. The Author(s).)

Background: "Regeneration time" (RT) denotes the time required to obtain a stable mortality rate for mosquitoes exposed to insecticide-treated nets (ITNs) after three consecutive washes of a net in a day. The RT informs the wash interval used to artificially age ITNs to simulate their lifetime performance under user conditions (20 washes). RT was estimated following World Health Organization (WHO) longitudinal method (LM) procedures. Longitudinal evaluation may introduce heterogeneity due to mosquito batch variability, complicating RT determination. To overcome this, nets at each stage of regeneration (i.e., 1, 2, 3, 5 and 7 days post wash) were prepared in advance and refrigerated; then, a complete regeneration series was tested with a single mosquito batch on 1 testing day, completing four series over 4 days. This study compared the complete series method (CSM) against the LM., Methods: The overall heterogeneity in the methods for estimating RT of one incorporated alpha-cypermethrin and piperonyl butoxide (PBO) and one incorporated permethrin with PBO ITNs was determined using laboratory-reared resistant Anopheles arabiensis under standard laboratory conditions. LM methods and CSM were compared in two experiments with refrigerated nets acclimated for (i) 2 h (test 1) and (ii) 3 h (test 2). Four regeneration replicates per day were tested per ITN product with 50 mosquitoes exposed per replicate (equivalent sample size to LM). The heterogeneity from these methods was compared descriptively., Results: The intra-method variability for unwashed pieces was minimal, with variance of 1.26 for CSM and 1.18 for LM. For unwashed nets, LM had substantially greater variance and ratio of LM:CSM was 2.66 in test 1 and 2.49 in test 2. The magnitude of mortality measured in bioassays depended on sample acclimation after refrigeration., Conclusions: The CSM is a convenient method for determining the regeneration times. ITNs are prepared in advance, reducing pressure to prepare all samples to start on a single day. A complete regeneration series of samples is removed from the refrigerator, defrosted and evaluated on a single day with one mosquito batch reducing the influence of mosquito batch heterogeneity on results. Replicates can be conducted over several days but do not have to be conducted on consecutive days, allowing easy facility scheduling., (© 2024. The Author(s).)

Background: Outbreaks of Aedes-borne arboviral diseases are becoming rampant in Africa. In Ghana, there is no organized arboviral control programme with interventions restricted to mitigate outbreaks. Insecticide application is a crucial part of outbreak responses and future preventative control measures. Thus, knowledge of the resistance status and underlying mechanisms of Aedes populations is required to ensure optimal insecticide choices. The present study assessed the insecticide resistance status of Aedes aegypti populations from southern Ghana (Accra, Tema and Ada Foah) and northern Ghana (Navrongo) respectively., Methods: Phenotypic resistance was determined with WHO susceptibility tests using Ae. aegypti collected as larvae and reared into adults. Knockdown resistance (kdr) mutations were detected using allele-specific PCR. Synergist assays were performed with piperonyl butoxide (PBO) to investigate the possible involvement of metabolic mechanisms in resistance phenotypes., Results: Resistance to DDT was moderate to high across sites (11.3 to 75.8%) and, for the pyrethroids deltamethrin and permethrin, moderate resistance was detected (62.5 to 88.8%). The 1534C kdr and 1016I kdr alleles were common in all sites (0.65 to 1) and may be on a trajectory toward fixation. In addition, a third kdr mutant, V410L, was detected at lower frequencies (0.03 to 0.31). Pre-exposure to PBO significantly increased the susceptibility of Ae. aegypti to deltamethrin and permethrin (P < 0.001). This indicates that in addition to kdr mutants, metabolic enzymes (monooxygenases) may be involved in the resistance phenotypes observed in the Ae. aegypti populations in these sites., Conclusion: Insecticide resistance underpinned by multiple mechanisms in Ae. aegypti indicates the need for surveillance to assist in developing appropriate vector control strategies for arboviral disease control in Ghana., (© 2023. The Author(s).)

Background: Head louse infestation is an important public health problem, and expanding resistance to permethrin is a major challenge to its control. The mapping and detection of pyrethroid resistance are essential to the development of appropriate treatments and ensure the effectiveness of current measures. The aim of this study was to present the phenotypic and genotypic basis of permethrin resistance and identify knockdown resistance (kdr) mutations in head louse populations in northwestern Iran., Methods: Adult head lice were collected from 1059 infested girls in Ardebil, East Azerbaijan, West Azerbaijan and Zanjan Provinces, northwestern Iran. The toxicity of permethrin and the possible synergistic effect of piperonyl butoxide (PBO) on this toxicity were assessed using bioassays. Fragments of voltage-sensitive sodium channels (vssc) and cytochrome b (cytb) genes were amplified and analyzed for the detection of knockdown resistance (kdr) mutations and mitochondrial groups. Moreover, genotypes of the two hot spot regions of the vssc gene were determined by melting curve analysis of amplicons., Results: A total of 1450 adult head lice were collected during 2016-2021. Live lice were exposed to a dose of 1% permethrin for 12 h, and the median lethal time (LT 50 ) and time to achieve 90% mortality (LT 90 ) were determined to be 6 and 14.8 h, respectively. Topical application of 2 and 16 ng permethrin per louse resulted in 25% and 42.11% mortality, respectively. Pre-exposure of samples to 3% piperonyl butoxide had no synergistic effect on the effects of permethrin. Analysis of the 774-bp vssc gene fragment showed the presence of the M815I, T917I and L920F mutations, wild-type and T917I mutation, in 91.6%, 4.2% and 4.2% of samples, respectively. Investigation of the mitochondrial cytb gene demonstrated the predominance of clade B. The frequency of domain II segment 4 (S4)-S5 kdr genotypes in mitochondrial groups was identical, and heterozygotes were present in 93.5% of samples. A significant difference was detected in the frequency of domain IIS1-S3 kdr genotypes, and the frequency of resistant alleles and heterozygotes was higher in clade B than in clade A., Conclusions: The presence of kdr mutations in the vssc gene and the non-synergist effect of PBO indicate that pyrethroid target site insensitivity is the main resistance mechanism. This phenomenon and the high frequency of resistant alleles necessitate that new pediculosis management programs be developed. Further studies need to be conducted to identify all factors contributing this resistance and to develop alternative pediculicides., (© 2023. The Author(s).)

Background: Mass distribution of insecticide-treated nets (ITNs) is the principal malaria vector control strategy adopted by Niger. To better inform on the most appropriate ITN to distribute, the National Malaria Control Programme (NMCP) of Niger and its partners, conducted insecticide resistance monitoring in selected sites across the country., Methods: The susceptibility of Anopheles gambiae sensu lato (s.l.) to chlorfenapyr and pyrethroid insecticides was investigated in a total of sixteen sites in 2019 and 2020, using 2-5-day-old adults reared from wild collected larvae per site. The susceptibility status, pyrethroid resistance intensity at 5 and 10 times the diagnostic concentrations, and piperonyl butoxide (PBO) synergism with diagnostic concentrations of deltamethrin, permethrin and alpha-cypermethrin were assessed using WHO bioassays. Two doses (100 and 200 µg/bottle) of chlorfenapyr were tested using the CDC bottle assay method. Species composition and allele frequencies for knock-down resistance (kdr-L1014F and L1014S) and acetylcholinesterase (ace-1 G119S) mutations were further characterized using polymerase chain reaction (PCR)., Results: High resistance intensity to all pyrethroids tested was observed in all sites except for alpha-cypermethrin in Gaya and Tessaoua and permethrin in Gaya in 2019 recording moderate resistance intensity. Similarly, Balleyara, Keita and Tillabery yielded moderate resistance intensity for alpha-cypermethrin and deltamethrin, and Niamey V low resistance intensity against deltamethrin and permethrin in 2020. Pre-exposure to PBO substantially increased susceptibility with average increases in mortality between 0 and 70% for tested pyrethroids. Susceptibility to chlorfenapyr (100 µg/bottle) was recorded in all sites except in Tessaoua and Magaria where susceptibility was recorded at the dose of 200 µg/bottle. Anopheles coluzzii was the predominant malaria vector species in most of the sites followed by An. gambiae sensu stricto (s.s.) and Anopheles arabiensis. The kdr-L1014S allele, investigated for the first time, was detected in the country. Both kdr-L1014F (frequencies [0.46-0.81]) and L1014S (frequencies [0.41-0.87]) were present in all sites while the ace-1 G119S was between 0.08 and 0.20., Conclusion: The data collected will guide the NMCP in making evidence-based decisions to better adapt vector control strategies and insecticide resistance management in Niger, starting with mass distribution of new generation ITNs such as interceptor G2 and PBO ITNs., (© 2022. The Author(s).)

Background: Malaria transmission can be highly heterogeneous between and within localities, and is influenced by factors such as survival and biting frequencies of Anopheles mosquitoes. This study investigated the relationships between the biological age, distance from aquatic habitats and pyrethroid resistance status of Anopheles funestus mosquitoes, which currently dominate malaria transmission in south-east Tanzania. The study also examined how such relationships may influence malaria transmission and control., Methods: Female An. funestus were collected in houses located 50-100 m, 150-200 m or over 200 m from the nearest known aquatic habitats. The mosquitoes were exposed to 1×, 5× and 10× the diagnostic doses of deltamethrin or permethrin, or to the synergist, piperonyl butoxide (PBO) followed by the pyrethroids, then monitored for 24 h-mortality. Ovaries of exposed and non-exposed mosquitoes were dissected to assess parity as a proxy for biological age. Adults emerging from larval collections in the same villages were tested against the same insecticides at 3-5, 8-11 or 17-20 days old., Findings: Mosquitoes collected nearest to the aquatic habitats (50-100 m) had the lowest mortalities compared to other distances, with a maximum of 51% mortality at 10× permethrin. For the age-synchronized mosquitoes collected as larvae, the insecticide-induced mortality assessed at both the diagnostic and multiplicative doses (1×, 5× and 10×) increased with mosquito age. The highest mortalities at 1× doses were observed among the oldest mosquitoes (17-20 days). At 10× doses, mortalities were 99% (permethrin) and 76% (deltamethrin) among 8-11 day-olds compared to 80% (permethrin) and 58% (deltamethrin) among 3-5 day-olds. Pre-exposure to PBO increased the potency of both pyrethroids. The proportion of parous females was highest among mosquitoes collected farthest from the habitats., Conclusion: In this specific setting, older An. funestus and those collected farthest from the aquatic habitats (near the centre of the village) were more susceptible to pyrethroids than the younger ones and those caught nearest to the habitats. These findings suggest that pyrethroid-based interventions may remain at least moderately effective despite widespread pyrethroid-resistance, by killing the older, less-resistant and potentially-infective mosquitoes. Further studies should investigate how and whether these observations could be exploited to optimize malaria control in different settings., (© 2022. The Author(s).)

Background: Dengue (DENV), chikungunya (CHIKV) and Zika virus (ZIKV), are mosquito-borne viruses of medical importance in most tropical and subtropical regions. Vector control, primarily through insecticides, remains the primary method to prevent their transmission. Here, we evaluated insecticide resistance profiles and identified important underlying resistance mechanisms in populations of Aedes aegypti and Ae. albopictus from six different regions in Cameroon to pesticides commonly used during military and civilian public health vector control operations., Methods: Aedes mosquitoes were sampled as larvae or pupae between August 2020 and July 2021 in six locations across Cameroon and reared until the next generation, G1. Ae. aegypti and Ae. albopictus adults from G1 were tested following World Health Organization (WHO) recommendations and Ae. aegypti G0 adults screened with real time melting curve qPCR analyses to genotype the F1534C, V1016I and V410L Aedes kdr mutations. Piperonyl butoxide (PBO) assays and real time qPCR were carried out from some cytochrome p450 genes known to be involved in metabolic resistance. Statistical analyses were performed using Chi-square test and generalized linear models., Results: Loss of susceptibility was observed to all insecticides tested. Mortality rates from tests with 0.25% permethrin varied from 24.27 to 85.89% in Ae. aegypti and from 17.35% to 68.08% in Ae. albopictus. Mortality rates for 0.03% deltamethrin were between 23.30% and 88.20% in Ae. aegypti and between 69.47 and 84.11% in Ae. albopictus. We found a moderate level of resistance against bendiocarb, with mortality rates ranging from 69.31% to 90.26% in Ae. aegypti and from 86.75 to 98.95% in Ae. albopictus. With PBO pre-exposure, we found partial or fully restored susceptibility to pyrethroids and bendiocarb. The genes Cyp9M6F88/87 and Cyp9J10 were overexpressed in Ae. aegypti populations from Douala sites resistant to permethrin and deltamethrin. Cyp6P12 was highly expressed in alphacypermethrin and permethrin resistant Ae. albopictus samples. F1534C and V1016I mutations were detected in A. aegypti mosquitoes and for the first time V410L was reported in Cameroon., Conclusions: This study revealed that Ae. aegypti and Ae. albopictus are resistant to multiple insecticide classes with multiple resistance mechanisms implicated. These findings could guide insecticide use to control arbovirus vectors in Cameroon., (© 2022. The Author(s).)

Background: More than 90% of malaria cases occur in Africa where the disease is transmitted by Anopheles gambiae and Anopheles arabiensis. This study evaluated the anti-mosquito properties of Juniperus virginiana (JVO) and Pelargonium roseum (PRO) essential oils (EOs) against larvae and adults of An. gambiae sensu lato (s.l.) from East Africa in laboratory and semi-field conditions., Methods: EOs was extracted from the aerial green parts of Asian herbs by hydrodistillation. Their constituents were characterized by gas chromatography-mass spectrometry (GC-MS). Larvicidal activities of JVO, PRO, and PRO components [citronellol (CO), linalool (LO), and geraniol (GO)] were investigated against An. gambiae sensu stricto (s.s.). The percentage of knockdown effects and mortality rates of all oils were also evaluated in the adults of susceptible An. gambiae s.s. and permethrin-resistant An. arabiensis., Results: GC-MS analyses identified major constituents of JVO (sabinene, dl-limonene, β-myrcene, bornyl acetate, and terpinen-4-ol) and PRO (citronellol, citronellyl formate, L-menthone, linalool, and geraniol). Oils showed higher larvicidal activity in the laboratory than semi-field trials. The LC 50 values for JVO/PRO were computed as 10.82-2.89/7.13-0.9 ppm and 10.75-9.06/13.63-8.98 ppm in laboratory and semi-field environments, respectively at exposure time of 24-72 h. The percentage of knockdown effects of the oils were also greater in An. gambiae s.s. than in An. arabiensis. Filter papers impregnated with JVO (100 ppm) and PRO (25 ppm) displayed 100% mortality rates for An. gambiae s.s. and 3.75% and 90% mortality rates, for An. arabiensis populations, respectively. Each component of CO, LO, and GO exhibited 98.13%, 97.81%, and 87.5%, respectively, and a mixture of the PRO components indicated 94.69% adult mortality to permethrin-resistant An. arabiensis., Conclusions: The findings of this study show that PRO and its main constituents, compared to JVO, have higher anti-mosquito properties in terms of larvicidal, knockdown, and mortality when applied against susceptible laboratory and resistant wild populations of An. gambiae s.l. Consequently, these oils have the potential for the development of new, efficient, safe, and affordable agents for mosquito control., (© 2022. The Author(s).)

Background: Malaria vector control using insecticide-based approaches has proven to be an effective strategy. However, widespread insecticide resistance among malaria vector populations across sub-Saharan Africa threatens to derail control efforts. This study was conducted in Chikwawa district, an area in rural southern Malawi characterised by persistent malaria transmission and reports of insecticide resistance in the local mosquito population. The aim of the was to characterise the intensity of insecticide resistance within a population of Anopheles funestus sensu lato (s.l.), a major vector of malaria in this district., Methods: Live adult females belonging to the An. funestus group were collected from households by indoor aspiration. The CDC bottle assay was used for phenotypic quantification of resistance to deltamethrin, permethrin and alpha-cypermethrin at 1×, 2.5×, 5× and 10× the recommended diagnostic dose for each of these insecticides. WHO tube assays were used to determine susceptibility to bendiocarb, dichlorodiphenyltrichloroethane (DDT) and pirimiphos-methyl insecticides at diagnostic concentrations., Results: Anopheles funestus s.l. exposed to 10× the recommended diagnostic dose was highly resistant to alpha-cypermethrin (mortality 95.4%); in contrast, mortality was 100% when exposed to both deltamethrin and permethrin at the same dose. Despite showing susceptibility to deltamethrin and permethrin at the 10× concentration, mortality at the 5× concentration was 96.7% and 97.1%, respectively, indicating moderate resistance to these two insecticides. WHO susceptibility assays indicated strong resistance against bendiocarb (mortality 33.8%, n = 93), whereas there was full susceptibility to DDT (mortality 98.9%, n = 103) and pirimiphos-methyl (mortality 100%, n = 103)., Conclusions: Strategies for managing resistance to insecticides, particularly against pyrethroids, must be urgently implemented to maintain the effectiveness of insecticide-based vector control interventions in the area. Such strategies include the wide-scale introduction of third-generation synergist insecticide-treated bed nets (ITNs) and next-generation dual active ingredient ITNs. The use of effective non-pyrethroids, such as pirimiphos-methyl, clothianidin and potentially DDT, could provide a window of opportunity for indoor residual spraying across the district. This strategy would support the current Malawi Insecticide Resistance Management Plan which aims at rotating insecticides to minimise selection pressure and slow down the evolution of resistance to approved insecticides. These actions will help to prevent malaria vector control failure and improve progress towards malaria elimination., (© 2022. The Author(s).)

Background: The practice of agriculture in urban settings contributes to the rapid expansion of insecticide resistance in malaria vectors. However, there is still not enough information on pesticide usage in most urban settings. The present study aims to assess the evolution of Anopheles gambiae (s.l.) population susceptibility to insecticides and patterns of pesticide usage in agriculture in the city of Yaoundé, Cameroon., Methods: WHO susceptibility tests and synergist PBO bioassays were conducted on adult An. gambiae (s.l.) mosquitoes aged 3 to 5 days emerging from larvae collected from the field. Seven insecticides (deltamethrin, permethrin, DDT, bendiocarb, propoxur, fenitrothion and malathion) were evaluated. The presence of target site mutation conferring knockdown (kdr) resistance was investigated using TaqMan assay, and mosquito species were identified using SINE-PCR. Surveys on 81 retailers and 232 farmers were conducted to assess general knowledge and practices regarding agricultural pesticide usage., Results: High resistance intensity to pyrethroids was observed with a high frequency of the kdr allele 1014F and low frequency of the kdr 1014S allele. The level of susceptibility of An. gambiae (s.l.) to pyrethroids and carbamates was found to decrease with time (from > 34% in 2017 to < 23% in 2019 for deltamethrin and permethrin and from 97% in 2017 to < 86% in 2019 for bendiocarb). Both An. gambiae (s.s.) and An. coluzzii were recorded. Over 150 pesticides and fertilizers were sold by retailers for agricultural purposes in the city of Yaoundé. Most farmers do not respect safety practices. Poor practices including extensive and inappropriate application of pesticides as well as poor management of perished pesticides and empty pesticide containers were also documented., Conclusions: The study indicated rapid evolution of insecticide resistance and uncontrolled usage of pesticides by farmers in agriculture. There is an urgent need to address these gaps to improve the management of insecticide resistance., (© 2022. The Author(s).)

Background: Malaria remains one of the most devastating diseases globally, and the control of mosquitoes as the vector is mainly dependent on chemical insecticides. Elevated temperatures associated with future warmer climates could affect mosquitoes' metabolic enzyme expression and increase insecticide resistance, making vector control difficult. Understanding how mosquito rearing temperatures influence their susceptibility to insecticide and expression of metabolic enzymes could aid in the development of novel tools and strategies to control mosquitoes in a future warmer climate. This study evaluated the effects of temperature on the susceptibility of Anopheles gambiae sensu lato (s.l.) mosquitoes to pyrethroids and their expression of metabolic enzymes., Methods: Anopheles gambiae s.l. eggs obtained from laboratory-established colonies were reared under eight temperature regimes (25, 28, 30, 32, 34, 36, 38, and 40 °C). Upon adult emergence, 3- to 5-day-old female non-blood-fed mosquitoes were used for susceptibility tests following the World Health Organization (WHO) bioassay protocol. Batches of 20-25 mosquitoes from each temperature regime (25-34 °C) were exposed to two pyrethroid insecticides (0.75% permethrin and 0.05% deltamethrin). In addition, the levels of four metabolic enzymes (α-esterase, β-esterase, glutathione S-transferase [GST], and mixed-function oxidase [MFO]) were examined in mosquitoes that were not exposed and those that were exposed to pyrethroids., Results: Mortality in An. gambiae s.l. mosquitoes exposed to deltamethrin and permethrin decreased at temperatures above 28 °C. In addition, mosquitoes reared at higher temperatures were more resistant and had more elevated enzyme levels than those raised at low temperatures. Overall, mosquitoes that survived after being exposed to pyrethroids had higher levels of metabolic enzymes than those that were not exposed to pyrethroids., Conclusions: This study provides evidence that elevated temperatures decreased An. gambiae s.l. mosquitoes' susceptibility to pyrethroids and increased the expression of metabolic enzymes. This evidence suggests that elevated temperatures projected in a future warmer climate could increase mosquitoes' resistance to insecticides and complicate malaria vector control measures. This study therefore provides vital information, and suggests useful areas of future research, on the effects of temperature variability on mosquitoes that could guide vector control measures in a future warmer climate., (© 2022. The Author(s).)

Background: Improved understanding of the molecular basis of insecticide resistance may yield new opportunities for control of relevant disease vectors. In this current study, we investigated the quantification responses for the phenotypic and genotypic resistance of Aedes aegypti populations from different states in Malaysia., Methods: We tested the insecticide susceptibility status of adult Ae. aegypti from populations of three states, Penang, Selangor and Kelantan (Peninsular Malaysia), against 0.25% permethrin and 0.25% pirimiphos-methyl using the World Health Organisation (WHO) adult bioassay method. Permethrin-resistant and -susceptible samples were then genotyped for domains II and III in the voltage-gated sodium channel (vgsc) gene using allele-specific polymerase chain reaction (AS-PCR) for the presence of any diagnostic single-nucleotide mutations. To validate AS-PCR results and to identify any possible additional point mutations, these two domains were sequenced., Results: The bioassays revealed that populations of Ae. aegypti from these three states were highly resistant towards 0.25% permethrin and 0.25% pirimiphos-methyl. Genotyping results showed that three knockdown (kdr) mutations (S989P, V1016G and F1534C) were associated with pyrethroid resistance within these populations. The presence of a novel mutation, the A1007G mutation, was also detected., Conclusions: This study revealed the high resistance level of Malaysian populations of Ae. aegypti to currently used insecticides. The resistance could be due to the widespread presence of four kdr mutations in the field and this could potentially impact the vector control programmes in Malaysia and alternative solutions should be sought., (© 2022. The Author(s).)

Background: The tropical bed bug, Cimex hemipterus, is an important ectoparasite causing various health problems. This species is mainly confined to tropical regions; however, insecticide resistance, global warming, and globalization have changed its distribution map. Molecular information on pyrethroid resistance, which is essential for the development of control programs, is unknown for C. hemipterus in expanded areas. The present study was designed to determine the permethrin resistance status, characterize the pyrethroid receptor sites in voltage-gated sodium channel (vgsc) gene, and identify the resistance-related mutations in the populations of tropical bed bug in Iran., Methods: Live bed bugs were collected, and adults of C. hemipterus were selected for bioassay and molecular surveys. Bioassay was performed by tarsal contact with permethrin 0.75% in mixed-sex of samples. Conventional and quantitative TaqMan and SYBR Green real-time PCR assays were conducted to characterize the vgsc gene and genotypes of studied populations., Results: In the bioassay tests, the mortality rates were in the range of 30.7-38.7% and 56.2-77.4% in 24 and 48 h, respectively. The knockdown rates of studied populations were in the range of 32.2-46.6% and 61.5-83.8% in the first and second days, respectively. The KT 50 and KT 90 values in the Cimex lectularius Kh1 strain were presented as 5.39 and 15.55 h, respectively. These values in the selected populations of C. hemipterus varied from 27.9 to 29.5 and from 82.8 to 104.4 h, respectively. Knockdown time ratios (KR 50 and KR 90 ) in these populations varied from 5.17 to 6.17-fold compared with those of the C. lectularius Kh1 strain. Fragments of vgsc gene with 355 bp and 812 bp were amplified. Analysis of sequences revealed the A468T substitution, kdr-associated D953G, and super-kdr M918I and L1014F mutations in all populations., Conclusions: The specific/sensitive, safe, and rapid diagnostic assays developed in this study are recommended for detection of kdr/super-kdr mutations and frequency of mutant alleles. The presence of super-kdr mutations and high resistance to permethrin in all the populations necessitate the reconsideration of control approaches against C. hemipterus., (© 2021. The Author(s).)

Background: The selection and the spread of insecticide resistance in malaria vectors to the main classes of insecticides used in vector control tools are a major and ongoing challenge to malaria vector control programmes. This study aimed to determine the intensity of vector resistance to insecticides in three regions of Benin with different agro-ecological characteristics., Methods: Larvae of Anopheles gambiae sensu lato (s.l.) were collected from September to November 2017 in different larval sites in three northern Benin communes: Parakou, Kandi and Malanville. Two to five-day-old, non-blood-fed, female mosquitoes were exposed to papers impregnated with deltamethrin, permethrin and bendiocarb at dosages of 1 × the diagnostic dose, 5 × and 10 × to determine the intensity of resistance in these vectors. Molecular frequencies of the kdr L1014F and ace-1R G119S insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled at each study site., Results: Resistance to pyrethroids (permethrin and deltamethrin) was recorded in all three communes with mortality rates below 60% using the diagnostic dose (1x). The results obtained after exposure of An. gambiae to permethrin 10 × were 99% in Kandi, 98% in Malanville and 99% in Parakou. With deltamethrin 10x, mortality rates were 100% in Kandi, 96% in Malanville and 73% in Parakou. For the diagnostic dose of bendiocarb, suspected resistance was recorded in the communes of Malanville (97%) and Kandi (94%) while sensitivity was observed in Parakou (98%).Using the 10 × dose, mortality was 98% in Kandi, 100% in Malanville and 99% in Parakou. The frequencies of the kdr L1014F allele varied between 59 and 83% depending on the sites and species of the An. gambiae complex, while the frequency of the ace-1R G119S gene varied between 0 and 5%. Biochemical tests showed high levels of oxidase and esterase activity compared to the susceptible colony strain of An. gambiae sensu stricto (Kisumu strain)., Conclusion: Anopheles gambiae showed a generalized loss of susceptibility to permethrin and deltamethrin but also showed moderate to high intensity of resistance in different regions of Benin. This high intensity of resistance is a potential threat to the effectiveness of vector control., (© 2021. The Author(s).)

Background: Malaria control in Kenya is based on case management and vector control using long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). However, the development of insecticide resistance compromises the effectiveness of insecticide-based vector control programs. The use of pesticides for agricultural purposes has been implicated as one of the sources driving the selection of resistance. The current study was undertaken to assess the status and mechanism of insecticide resistance in malaria vectors in irrigated and non-irrigated areas with varying agrochemical use in western Kenya., Methods: The study was carried out in 2018-2019 in Homa Bay County, western Kenya. The bioassay was performed on adults reared from larvae collected from irrigated and non-irrigated fields in order to assess the susceptibility of malaria vectors to different classes of insecticides following the standard WHO guidelines. Characterization of knockdown resistance (kdr) and acetylcholinesterase-inhibiting enzyme/angiotensin-converting enzyme (Ace-1) mutations within Anopheles gambiae s.l. species was performed using the polymerase chain reaction (PCR) method. To determine the agricultural and public health insecticide usage pattern, a questionnaire was administered to farmers, households, and veterinary officers in the study area., Results: Anopheles arabiensis was the predominant species in the irrigated (100%, n = 154) area and the dominant species in the non-irrigated areas (97.5%, n = 162), the rest being An. gambiae sensu stricto. In 2018, Anopheles arabiensis in the irrigated region were susceptible to all insecticides tested, while in the non-irrigated region reduced mortality was observed (84%) against deltamethrin. In 2019, phenotypic mortality was decreased (97.8-84% to 83.3-78.2%). In contrast, high mortality from malathion (100%), DDT (98.98%), and piperonyl butoxide (PBO)-deltamethrin (100%) was observed. Molecular analysis of the vectors from the irrigated and non-irrigated areas revealed low levels of leucine-serine/phenylalanine substitution at position 1014 (L1014S/L1014F), with mutation frequencies of 1-16%, and low-frequency mutation in the Ace-1R gene (0.7%). In addition to very high coverage of LLINs impregnated with pyrethroids and IRS with organophosphate insecticides, pyrethroids were the predominant chemical class of pesticides used for crop and animal protection., Conclusion: Anopheles arabiensis from irrigated areas showed increased phenotypic resistance, and the intensive use of pesticides for crop protection in this region may have contributed to the selection of resistance genes observed. The susceptibility of these malaria vectors to organophosphates and PBO synergists in pyrethroids offers a promising future for IRS and insecticide-treated net-based vector control interventions. These findings emphasize the need for integrated vector control strategies, with particular attention to agricultural practices to mitigate mosquito resistance to insecticides.

Background: The rapid and widespread evolution of insecticide resistance has emerged as one of the major challenges facing malaria control programs in sub-Saharan Africa. Understanding the insecticide resistance status of mosquito populations and the underlying mechanisms of insecticide resistance can inform the development of effective and site-specific strategies for resistance prevention and management. The aim of this study was to investigate the insecticide resistance status of Anopheles gambiae (s.l.) mosquitoes from coastal Kenya., Methods: Anopheles gambiae (s.l.) larvae sampled from eight study sites were reared to adulthood in the insectary, and 3- to 5-day-old non-blood-fed females were tested for susceptibility to permethrin, deltamethrin, dichlorodiphenyltrichloroethane (DDT), fenitrothion and bendiocarb using the standard World Health Organization protocol. PCR amplification of rDNA intergenic spacers was used to identify sibling species of the An. gambiae complex. The An. gambiae (s.l.) females were further genotyped for the presence of the L1014S and L1014F knockdown resistance (kdr) mutations by real-time PCR., Results: Anopheles arabiensis was the dominant species, accounting for 95.2% of the total collection, followed by An. gambiae (s.s.), accounting for 4.8%. Anopheles gambiae (s.l.) mosquitoes were resistant to deltamethrin, permethrin and fenitrothion but not to bendiocarb and DDT. The L1014S kdr point mutation was detected only in An. gambiae (s.s.), at a low allelic frequency of 3.33%, and the 1014F kdr mutation was not detected in either An. gambiae (s.s.) or An. arabiensis., Conclusion: The findings of this study demonstrate phenotypic resistance to pyrethroids and organophosphates and a low level of the L1014S kdr point mutation that may partly be responsible for resistance to pyrethroids. This knowledge may inform the development of insecticide resistance management strategies along the Kenyan Coast.

Background: Insecticide resistance is threatening the effectiveness of efforts to control malaria vectors in Benin. This study explores the levels and mechanisms of insecticide resistance in An. gambiae s.l. to pyrethroids., Methods: Larvae were collected from August 2017 to July 2018 in five communes in southern Benin (Adjohoun, Allada, Bohicon, Cotonou, and Porto-Novo) representing diverse ecological regions, and were reared in Benin's insectary. Two- to five-day-old female mosquitoes from each district were exposed to multiple doses of deltamethrin and permethrin (1×, 2×, 5×, and 10×) using the WHO insecticide resistance intensity bioassay. The effect of pre-exposure to the synergist, piperonyl butoxide (PBO), was also tested at different pyrethroid doses. Molecular allele frequencies of kdr (1014F) and ace-1R (119S) insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled from each study area., Results: An. gambiae s.l. were resistant to pyrethroid-only exposure up to 10× the diagnostic doses in all the study sites for both deltamethrin and permethrin. Mortality was significantly higher in An. gambiae s.l. pre-exposed to PBO followed by exposure to deltamethrin or permethrin compared to mosquitoes exposed to deltamethrin or permethrin only (p < 0.001). The difference in mortality between deltamethrin only and PBO plus deltamethrin was the smallest in Cotonou (16-64%) and the greatest in Bohicon (12-93%). The mortality difference between permethrin only and PBO plus permethrin was the smallest in Cotonou (44-75%) and the greatest in Bohicon (22-72%). In all the study sites, the kdr resistance allele (1014F) frequency was high (75-100%), while the ace-1 resistance allele (G119S) frequency was low (0-3%). Analysis of the metabolic enzymatic activity of An. gambiae s.l. showed overexpression of nonspecific esterases and glutathione S-transferases (GST) in all study sites. In contrast to the PBO results, oxidase expression was low and was similar to the susceptible An. gambiae s.s. Kisumu strain in all sites., Conclusion: There is high-intensity resistance to pyrethroids in southern Benin. However, pre-exposure to PBO significantly increased susceptibility to the pyrethroids in the different An. gambiae s.l. populations sampled. The use of PBO insecticide-treated bed nets may help maintain the gains in An. gambiae (s.l.) control in southern Benin.

Background: Vector control for malaria prevention relies most often on the use of insecticide-treated bed net (ITNs) and indoor residual spraying. Little is known about the longevity of long-lasting insecticidal nets (LLINs) in the Americas. The physical integrity and insecticide retention of LLINs over time were monitored after a bed net distribution campaign to assess community practices around LLIN care and use in Waspam, northeastern Nicaragua., Methods: At least 30 nets were collected at 6, 12, 24, and 36 months post distribution. Physical integrity was measured by counting holes and classifying nets into categories (good, damaged, and too torn) depending on a proportionate hole index (pHI). Insecticide bioefficacy was assessed using cone bioassays, and insecticide content measured using a cyanopyrethroid field test (CFT)., Results: At 6 months, 87.3 % of LLINs were in good physical condition, while by 36 months this decreased to 20.6 %, with 38.2 % considered 'too torn.' The median pHI increased from 7 at the 6-month time point to 480.5 by 36 months. After 36 months of use, median mortality in cone bioassays was 2 % (range: 0-6 %) compared to 16 % (range: 2-70 %) at 6 months. There was a decrease in the level of deltamethrin detected on the surface of the LLINs with 100 % of tested LLINs tested at 12 months and 24 months crossing the threshold for being considered a failed net by CFT., Conclusions: This first comprehensive analysis of LLIN durability in Central America revealed rapid loss of chemical bioefficacy and progressive physical damage over a 36-month period. Use of these findings to guide future LLIN interventions in malaria elimination settings in Nicaragua, and potentially elsewhere in the Americas, could help optimize the successful implementation of vector control strategies.

Background: Insecticide resistance poses a growing challenge to malaria vector control in Kenya and around the world. Following evidence of associations between the mosquito microbiota and insecticide resistance, the microbiota of Anopheles gambiae sensu stricto (s.s.) from Tulukuyi village, Bungoma, Kenya, with differing permethrin resistance profiles were comparatively characterized., Methods: Using the CDC bottle bioassay, 133 2-3 day-old, virgin, non-blood fed female F 1 progeny of field-caught An. gambiae s.s. were exposed to five times (107.5 µg/ml) the discriminating dose of permethrin. Post bioassay, 50 resistant and 50 susceptible mosquitoes were subsequently screened for kdr East and West mutations, and individually processed for microbial analysis using high throughput sequencing targeting the universal bacterial and archaeal 16S rRNA gene., Results: 47 % of the samples tested (n = 133) were resistant, and of the 100 selected for further processing, 99 % were positive for kdr East and 1 % for kdr West. Overall, 84 bacterial taxa were detected across all mosquito samples, with 36 of these shared between resistant and susceptible mosquitoes. A total of 20 bacterial taxa were unique to the resistant mosquitoes and 28 were unique to the susceptible mosquitoes. There were significant differences in bacterial composition between resistant and susceptible individuals (PERMANOVA, pseudo-F = 2.33, P = 0.001), with presence of Sphingobacterium, Lysinibacillus and Streptococcus (all known pyrethroid-degrading taxa), and the radiotolerant Rubrobacter, being significantly associated with resistant mosquitoes. On the other hand, the presence of Myxococcus, was significantly associated with susceptible mosquitoes., Conclusions: This is the first report of distinct microbiota in An. gambiae s.s. associated with intense pyrethroid resistance. The findings highlight differentially abundant bacterial taxa between resistant and susceptible mosquitoes, and further suggest a microbe-mediated mechanism of insecticide resistance in mosquitoes. These results also indicate fixation of the kdr East mutation in this mosquito population, precluding further analysis of its associations with the mosquito microbiota, but presenting the hypothesis that any microbe-mediated mechanism of insecticide resistance would be likely of a metabolic nature. Overall, this study lays initial groundwork for understanding microbe-mediated mechanisms of insecticide resistance in African mosquito vectors of malaria, and potentially identifying novel microbial markers of insecticide resistance that could supplement existing vector surveillance tools.

Background: Long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) have greatly reduced malaria transmission in sub-Saharan Africa, but are threatened by insecticide resistance. In south-eastern Tanzania, pyrethroid-resistant Anopheles funestus are now implicated in > 80% of malaria infections, even in villages where the species occurs at lower densities than the other vector, Anopheles arabiensis. This study compared the insecticide resistance phenotypes between the two malaria vectors in an area where pyrethroid-LLINs are widely used., Methods: The study used the World Health Organization (WHO) assays with 1×, 5× and 10× insecticide doses to assess levels of resistance, followed by synergist bioassays to understand possible mechanisms of the observed resistance phenotypes. The tests involved adult mosquitoes collected from three villages across two districts in south-eastern Tanzania and included four insecticide classes., Findings: At baseline doses (1×), both species were resistant to the two candidate pyrethroids (permethrin and deltamethrin), but susceptible to the organophosphate (pirimiphos-methyl). Anopheles funestus, but not An. arabiensis was also resistant to the carbamate (bendiocarb). Both species were resistant to DDT in all villages except in one village where An. arabiensis was susceptible. Anopheles funestus showed strong resistance to pyrethroids, surviving the 5× and 10× doses, while An. arabiensis reverted to susceptibility at the 5× dose. Pre-exposure to the synergist, piperonyl butoxide (PBO), enhanced the potency of the pyrethroids against both species and resulted in full susceptibility of An. arabiensis (> 98% mortality). However, for An. funestus from two villages, permethrin-associated mortalities after pre-exposure to PBO only exceeded 90% but not 98%., Conclusions: In south-eastern Tanzania, where An. funestus dominates malaria transmission, the species also has much stronger resistance to pyrethroids than its counterpart, An. arabiensis, and can survive more classes of insecticides. The pyrethroid resistance in both species appears to be mostly metabolic and may be partially addressed using synergists, e.g. PBO. These findings may explain the continued persistence and dominance of An. funestus despite widespread use of pyrethroid-treated LLINs, and inform new intervention choices for such settings. In short and medium-term, these may include PBO-based LLINs or improved IRS with compounds to which the vectors are still susceptible.

Background: Understanding the mechanisms used by Anopheles mosquitoes to survive insecticide exposure is key to manage existing insecticide resistance and develop more suitable insecticide-based malaria vector control interventions as well as other alternative integrated tools. To this regard, the molecular basis of permethrin, DDT and dieldrin resistance in Anopheles funestus (sensu stricto) at Akaka-Remo was investigated., Methods: Bioassays were conducted on 3-5-day-old adult An. funestus (s.s.) mosquitoes for permethrin, DDT and dieldrin susceptibility test. The molecular mechanisms of mosquito resistance to these insecticides were investigated using microarray and reverse transcriptase PCR techniques. The voltage-gated sodium channel region of mosquitoes was also screened for the presence of knockdown resistance mutations (kdr west and east) by sequencing method., Results: Anopheles funestus (s.s.) population was resistant to permethrin (mortality rate of 68%), DDT (mortality rate of 10%) and dieldrin (mortality rate of 8%) insecticides. Microarray and RT-PCR analyses revealed the overexpression of glutathione S-transferase genes, cytochrome P450s, esterase, trypsin and cuticle proteins in resistant mosquitoes compared to control. The GSTe2 was the most upregulated detoxification gene in permethrin-resistant (FC = 44.89), DDT-resistant (FC = 57.39) and dieldrin-resistant (FC = 41.10) mosquitoes compared to control population (FC = 22.34). The cytochrome P450 gene, CYP6P9b was also upregulated in both permethrin- and DDT-resistant mosquitoes. The digestive enzyme, trypsin (hydrolytic processes) and the cuticle proteins (inducing cuticle thickening leading to reduced insecticides penetration) also showed high involvement in insecticide resistance, through their overexpression in resistant mosquitoes compared to control. The kdr east and west were absent in all mosquitoes analysed, suggesting their non-involvement in the observed mosquito resistance., Conclusions: The upregulation of metabolic genes, especially the GSTe2 and trypsin, as well as the cuticle proteins is driving insecticide resistance of An. funestus (s.s.) population. However, additional molecular analyses, including functional metabolic assays of these genes as well as screening for a possible higher cuticular hydrocarbon and lipid contents, and increased procuticle thickness in resistant mosquitoes are needed to further describe their distinct roles in mosquito resistance.

Background: Between 2011 and 2018, an estimated 134.8 million pyrethroid-treated long-lasting insecticidal nets (LLINs) were distributed nationwide in the Democratic Republic of Congo (DRC) for malaria control. Pyrethroid resistance has developed in DRC in recent years, but the intensity of resistance and impact on LLIN efficacy was not known. Therefore, the intensity of resistance of Anopheles gambiae sensu lato (s.l.) to permethrin and deltamethrin was monitored before and after a mass distribution of LLINs in Kinshasa in December 2016, and in 6 other sites across the country in 2017 and 11 sites in 2018., Methods: In Kinshasa, CDC bottle bioassays using 1, 2, 5, and 10 times the diagnostic dose of permethrin and deltamethrin were conducted using An. gambiae s.l. collected as larvae and reared to adults. Bioassays were conducted in four sites in Kinshasa province 6 months before a mass distribution of deltamethrin-treated LLINs and then two, six, and 10 months after the distribution. One site in neighbouring Kongo Central province was used as a control (no mass campaign of LLIN distribution during the study). Nationwide intensity assays were conducted in six sites in 2017 using CDC bottle bioassays and in 11 sites in 2018 using WHO intensity assays. A sub-sample of An. gambiae s.l. was tested by PCR to determine species composition and frequency of kdr-1014F and 1014S alleles., Results: In June 2016, before LLIN distribution, permethrin resistance intensity was high in Kinshasa; the mean mortality rate was 43% at the 5× concentration and 73% at the 10× concentration. Bioassays at 3 time points after LLIN distribution showed considerable variation by site and time and there was no consistent evidence for an increase in pyrethroid resistance intensity compared to the neighbouring control site. Tests of An. gambiae s.l. in 6 sites across the country in 2017 and 11 sites in 2018 showed all populations were resistant to the diagnostic doses of 3 pyrethroids. In 2018, the intensity of resistance varied by site, but was generally moderate for all three pyrethroids, with survivors at ×5 the diagnostic dose. Anopheles gambiae sensu stricto (s.s.) was the most common species identified across 11 sites in DRC, but in Kinshasa, An. gambiae s.s. (91%) and Anopheles coluzzii (8%) were sympatric., Conclusions: Moderate or high intensity pyrethroid resistance was detected nationwide in DRC and is a serious threat to sustained malaria control with pyrethroid LLINs. Next generation nets (PBO nets or bi-treated nets) should be considered for mass distribution.

Background: Insecticide resistance is a growing concern for malaria control and vector control effectiveness relies on assessing it distribution and understanding its evolution., Methods: We assessed resistance levels and the frequencies of two major target-site mutations, L1014F-VGSC and G119S-ace-1, conferring resistance to pyrethroids (PYRs) and carbamates/organophosphates (CXs/OPs) insecticides. These data were compared to those acquired between 2006 and 2010 to follow resistance evolutionary trends over ten years., Results: We report the results of a 3-year survey (2013-2015) of insecticide resistance in 13 localities across the whole country of Benin. Permethrin (PYR) resistance was found in all populations tested, L1014F-VGSC being almost fixed everywhere, while bendiocarb resistance was limited to a few localities, G119S-ace-1 remaining rare, with very limited variations during surveyed period. Interestingly, we found no effect of the type of insecticide pressure on the dynamics of these mutations., Conclusions: These results confirm both the high prevalence of PYR resistance and the potential of CXs/OPs as short- to medium-term alternatives in Benin. They also underline the need for regular resistance monitoring and informed management in their usage, as the G119S-ace-1 mutation is already present in Benin and surrounding countries. Their unwise usage would rapidly lead to its spread, which would jeopardize PYR-resistant Anopheles control.

Background: Mosquitoes and other vectors are often exposed to sublethal doses of insecticides. Larvae can be exposed to the run-off of agricultural use, and adults can be irritated by insecticides used against them and move away before they have picked up a lethal dose. This sublethal exposure may affect the success of control of insect-borne diseases, for it may affect the competence of insects to transmit parasites, in particular if the insects are undernourished., Methods: We assessed how exposure of larvae and adults to a sublethal dose of permethrin (a pyrethroid) and how larval competition for food affect several aspects of the vector competence of the mosquito Anopheles gambiae for the malaria parasite Plasmodium berghei. We infected mosquitoes with P. berghei and measured the longevity and the prevalence and intensity of infection to test for an effect of our treatments., Results: Our general result was that the exposure to the insecticide helped mosquitoes deal with infection by malaria. Exposure of either larvae or adults decreased the likelihood that mosquitoes were infected by about 20%, but did not effect the parasite load. Exposure also increased the lifespan of infected mosquitoes, but only if they had been reared in competition. Larval competition had no effect on the prevalence of infection, but increased parasite load. These effects may be a consequence of the machinery governing oxidative stress, which underlies the responses of mosquitoes to insecticides, to food stress and to parasites., Conclusions: We conclude that insecticide residues are likely to affect the ability of mosquitoes to carry and transmit pathogens such as malaria, irrespective of the stage at which they are exposed to the insecticide. Our results stress the need for further studies to consider sublethal doses in the context of vector ecology and vector-borne disease epidemiology.

Background: Increasing resistance of head lice against neurotoxic agents and safety concerns have led to the search for treatment alternatives. Dimeticones with a physical mode of action are safe, and bear a reduced risk for the development of resistance., Methods: We performed in vitro bioassays to assess pediculicidal and ovicidal activities of a new dimeticone-based product, and a randomized controlled clinical trial to assess efficacy, following 10 min application. Of 153 individuals screened, 100 participants with active head louse infestations were randomly assigned to treatment with either a dimeticone-based test product, or a 0.5% permethrin-based reference product (50 participants per group). Participants received two topical applications of either the test (10 min) or reference products (45 min) at days 0 and 7 or 8. Outcome measures included the efficacies of treatment and their safety, as well as global and local tolerability at baseline, and days 1, 7, and 10., Results: After 10 min exposure, all lice treated with the dimeticone test product were classified as non-viable in the in vitro assay. Ovicidal activity after treatment of eggs with the dimeticone test product was 96.8%. In the clinical trial, 96 patients completed all study visits. In the full analysis set (FAS) population, on day 1 after one application, 98% of patients were cured in the test group, as compared to 84% cured in the reference group. All participants in both groups were free of head lice on day 10, following two applications (100% cure rate). In total, 42 adverse events (AEs) in 23 patients of both treatment groups were recorded, with the majority of AEs classified as mild., Conclusions: We have shown a high level of pediculicidal and ovicidal activity, and clinical efficacy and safety, of a brief application of a new dimeticone-based product. The short application time and reduced risk for the development of resistance are key drivers for improved patients' compliance., Trial Registration: EU Clinical Trials Register EudraCT  2016-004635-20 . Registered 14 November 2016.

Background: Aedes aegypti were found developing in the water in open public drains (drain-water, DW) in Jaffna city in northern Sri Lanka, a location where the arboviral diseases dengue and chikungunya are endemic., Methods: Susceptibilities to the common insecticides dichlorodiphenyltrichloroethane (DDT), malathion, propoxur, permethrin and deltamethrin and activities of the insecticide-detoxifying enzymes carboxylesterase (EST), glutathione S-transferase (GST) and monooxygenase (MO) were compared in adult Ae. aegypti developing in DW and fresh water (FW)., Results: DW Ae. aegypti were resistant to the pyrethroids deltamethrin and permethrin, while FW Ae. aegypti were susceptible to deltamethrin but possibly resistant to permethrin. Both DW and FW Ae. aegypti were resistant to DDT, malathion and propoxur. Greater pyrethroid resistance in DW Ae. aegypti was consistent with higher GST and MO activities., Conclusions: The results demonstrate the potential for insecticide resistance developing in Ae. aegypti adapted to DW. Urbanization in arboviral disease-endemic countries is characterized by a proliferation of open water drains and therefore the findings identify a potential new challenge to global health.

Background: PermaNet ® 3.0 is a deltamethrin-treated combination long-lasting insecticidal net with the addition of synergist piperonylbutoxide (PBO) on its roof section. It is designed to overcome the challenge posed by pyrethroid resistant vector populations against mainstream long-lasting insecticidal nets impregnated with pyrethroids only. The objective of this study was to determine insecticide resistance status of Anopheline and Culicine mosquitoes, to evaluate the bio-efficacy of PermaNet ® 3.0 nets and to assess household factors affecting the physical integrity of PermaNet ® 3.0 after 3 years of use., Methods: Insecticide susceptibility test was conducted using the WHO tube test. Bio-activity of PermaNet ® 3.0 samples was evaluated using the WHO cone bioassay. Cross-sectional survey was conducted on 150 randomly selected households from two districts to determine household factors affecting net utilization. One hundred fifty PermaNet ® 3.0 nets were randomly collected from the community with replacement after 3 years of deployment and physical integrity of each net was assessed., Results: Both Anopheles gambiae sensu lato and Culex quinquefasciatus developed resistance against permethrin and deltamethrin. However, following pre-exposure to synergist PBO the susceptibility of mosquito population increased to both permethrin (from 39% without to 92% with PBO against An. gambiae and from 28% without to 94% with PBO against Culex quinquefasciatus) and deltamethrin (from 52% without to 99% with PBO against An. gambiae and from 43% without to 98% with PBO against Culex quinquefasciatus). Eighty percent (80%) mortality was recorded in wild population of An. gambiae s.l. exposed to unused PermaNet ® 3.0, but its bioactivity subsequently declined as washing frequency increased from 0 to 20. The PBO coated roof section of unused PermaNet ® 3.0 resulted in higher mosquito mortality (100%) compared to the side panels without PBO (85%). House structure, cooking and washing habits, and damage due to household pests were cited as determinants associated with bed net deterioration. Bed net proportionate hole index (pHI) was ranged from 0 to 6064. Of the 150 PermaNet ® 3.0 nets assessed 80, 29 and 41 were considered as 'good', 'acceptable' and 'too torn', respectively., Conclusions: The bio-efficacy evaluation of PermaNet ® 3.0 from Jimma area, southwestern Ethiopia showed moderate efficacy against pyrethroid resistant population of An. gambiae and Culex quinquefasciatus. Thus, NMCPs in parallel to deployment of LLINs, should implement timely insecticide resistance management and integrated vector management strategies to slowdown the evolution and further spread of insecticide resistance. Household factors such as, housing conditions, open flame fire used for cooking and rodent attack were identified as factors contributing to the observed reduced bed net physical integrity in the study area. Universal coverage of bed nets should be accompanied with community awareness creation and training on net utilization and handling.

Background: Insecticide-treated bed nets (ITNs) help to control malaria by mechanically impeding the biting of mosquitoes, by repelling and irritating them and by killing them. In contrast to spatial repellency, irritancy implies that mosquitoes contact the ITN and are exposed to at least a sub-lethal dose of insecticide, which impedes their further blood-seeking. This would weaken the transmission of malaria, if mosquitoes are infectious., Methods: It was therefore tested whether sub-lethal exposure to permethrin impedes blood-feeding differently in uninfected mosquitoes and in mosquitoes carrying the non-transmissible stage (oocysts) or the infectious stage (sporozoites) of the malaria parasite Plasmodium berghei. In addition, as the degree of irritancy determines the dose of insecticide the mosquitoes may receive, the irritancy to permethrin of infected and uninfected mosquitoes was compared., Results: In this laboratory setting, sub-lethal exposure to permethrin inhibited the blood-seeking behaviour of Anopheles gambiae mosquitoes for almost 48 h. Although infection by malaria did not affect the irritancy of the mosquitoes to permethrin at either the developmental stage or the infectious stage, both stages of infection shortened the duration of inhibition of blood-seeking., Conclusions: The results suggest that the impact of ITNs may be weaker for malaria-infected than for uninfected mosquitoes. This will help to understand the global impact of ITNs on the transmission of malaria and gives a more complete picture of the effectiveness of that vector control measure.

Background: Pyrethroid and organophosphate resistance in the malaria vector Anopheles gambiae has led to the search for not only alternative insecticides, but also repellent chemical compounds. However, little is known about the potential actions of repellents and the cross-resistance risk between insecticide and repellent compounds., Methods: Here we show the action of permethrin, DEET, geraniol, carvacrol, culminaldehyde and cinnamaldehyde against three A. gambiae strains: 'Kis' (Kisumu susceptible strain), 'KdrKis' (pyrethroid resistant strain) and 'AcerKis' (organophosphate resistant strain), the last two differing from the first by a mutation on the kdr and ace1 genes, respectively., Conclusions: Results from the DEET assays show it induced repellency for the resistant KdrKis and AcerKis strains but maintained irritancy for the susceptible strain. More generally, we show resistance genes modify the behavior of An. gambiae, increasing or decreasing the effectiveness of DEET and natural compounds, depending on the mutation. These findings offer a new avenue for research on the target and mechanism of repellent compounds. We discuss these findings in the context of vector control strategies.

Background: Protecting people outdoors against mosquito-borne diseases is a major challenge. Here we compared commercially available personal protection methods to identify the most effective method for outdoor use in northern Lao PDR., Methods: From June to August 2016 the protective efficacy of treatments were compared in a secondary forest during the afternoon and a village during the evening. Comparisons were made using a replicated Latin square design between: (i) short permethrin-treated overalls; (ii) long permethrin-treated overalls; (iii) short untreated overalls with para-menthane-3,8-diol (PMD) applied topically; (iv) short permethrin-treated overalls plus PMD applied topically; (v) short untreated overalls with metofluthrin coils in a metal casing worn on a belt; and (vi) long untreated overalls. Short untreated overalls served as the control. Cone tests were conducted on the treated and untreated fabric before and after field experiments. A questionnaire survey was used to measure social acceptability., Results: Mosquito coils in a metal casing worn on a belt resulted in 92.3% (95% confidence interval, CI: 88.9-94.6%). landing protection from female mosquitoes in the afternoon and 68.8% (95% CI: 41.7-83.3%) protection in the evening compared to short untreated clothing. PMD was protective both when combined with short permethrin-treated overalls (afternoon, 68.2%, 95% CI: 52.6-78.7%; evening, 52.3%, 95% CI: 33.8-65.7%) and when used in combination with short untreated overalls (afternoon, 55.0%, 95% CI: 41.7-65.2%; evening, 25.2%, 95% CI: 9.4-38.2%). Whilst long permethrin-treated overalls were protective (afternoon, 61.1%, 95% CI: 51.4-68.8%; evening, 43.0%, 95% CI: 25.5-56.4%), short permethrin-treated overalls and long untreated overalls were not. Exposure to new permethrin-treated fabric in cone tests resulted in 25.0% (95% CI, 17.8-32.2%) and 26.2% (95% CI 16.7-35.8%) mortality for susceptible Ae. albopictus and susceptible Ae. aegypti, respectively. There was a loss of efficacy of permethrin-treated clothing after use in the field, with 3 min knockdown rates of Ae. albopictus and 1 h knockdown of Ae. aegypti decreasing over time. Participants considered all treatments acceptable., Conclusions: The portable mosquito coils were highly protective against outdoor biting mosquitoes, although there are safety concerns related to its use. The combination of permethrin-treated clothing and PMD repellent represent an alternative treatment for protection against outdoor-biting mosquitoes.

Background: Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. In order to implement suitable insecticide resistance management strategies, it is necessary to understand the underlying mechanisms involved. To achieve this, the molecular basis of permethrin and DDT resistance in the principal malaria vector, Anopheles funestus from inland Benin (Kpome), was investigated., Results: Here, using a microarray-based genome-wide transcription and qRT-PCR analysis, we showed that metabolic resistance mechanisms through over-expression of cytochrome P450 and glutathione S-transferase genes (GSTs) are a major contributor to DDT and permethrin resistance in Anopheles funestus from Kpome. The GSTe2 gene was the most upregulated detoxification gene in both DDT- [fold-change (FC: 16.0)] and permethrin-resistant (FC: 18.1) mosquitoes suggesting that upregulation of this gene could contribute to DDT resistance and cross-resistance to permethrin. CYP6P9a and CYP6P9b genes that have been previously associated with pyrethroid resistance were also significantly overexpressed with FC 5.4 and 4.8, respectively, in a permethrin resistant population. Noticeably, the GSTs, GSTd1-5 and GSTd3, were more upregulated in DDT-resistant than in permethrin-resistant Anopheles funestus suggesting these genes are more implicated in DDT resistance. The absence of the L1014F or L1014S kdr mutations in the voltage-gated sodium channel gene coupled with the lack of directional selection at the gene further supported that knockdown resistance plays little role in this resistance., Conclusions: The major role played by metabolic resistance to pyrethroids in this An. funestus population in Benin suggests that using novel control tools combining the P450 synergist piperonyl butoxide (PBO), such as PBO-based bednets, could help manage the growing pyrethroid resistance in this malaria vector in Benin.

Background: Eugenol is a botanical monoterpene that hyperactivates the blood-sucking bug Triatoma infestans, and permethrin is a pyrethroid with a strong triatomicide effect. In the present work, we tested the hypothesis that eugenol-hyperactivated nymphs of T. infestans pick up more insecticide, and then become intoxicated faster, than non-hyperactivated nymphs when exposed to a permethrin-treated surface., Results: Values of knockdown time 50% (KT50) for third-instar T. infestans exposed to a paper impregnated with permethrin were obtained under the following situations: (a.i.) immediately after topical application of eugenol (KT50: 66.75 min for acetone pre-treated controls, and 46.27 min for eugenol pre-treated nymphs); (a.ii.) 30 min after topical application of eugenol (KT50: 66.79 min for controls, and 66.79 min for eugenol pre-treated nymphs); (b) simultaneously with exposure to eugenol vapors (KT50: 51.90 min for controls, and 39.5 min for nymphs exposed to an eugenol-treated filter paper); and (c) immediately after an injection of eugenol (on average, controls were knocked down after 63.00 min, whereas nymphs injected with eugenol were knocked down after 65.30 min). In other experimental series, the distance traveled (DT) by nymphs exposed to eugenol was quantified in the same situations previously described, but without exposure to permethrin. In (a.i.), the DT in interval 0-30 min after topical application of eugenol was 487.00 (control) and 1127.50 (eugenol) cm; in (a.ii.), the DT in the interval 31-60 min after topical application was 336.75 (control) and 256.75 (eugenol) cm; in (b), DT was 939.08 (control) and 1048.53 (eugenol) cm; and in (c), it was 589.20 (control) and 700.00 (eugenol) cm. The KT50 values for permethrin decreased significantly in situations (a.i.) and (b), and eugenol only produced a significant hyperactivity in the same situations. Finally, the amount of permethrin picked up by non-hyperactivated and hyperactivated nymphs exposed to a film of permethrin was quantified by gas chromatography. Non-hyperactivated nymphs picked up 0.34 μg/insect of permethrin, while hyperactivated nymphs picked up 0.65 μg/insect., Conclusion: Results support the hypothesis that eugenol-hyperactivated nymphs of T. infestans pick up more insecticide, and then become intoxicated faster, than non-hyperactivated nymphs when exposed to a permethrin-treated surface.

Background: Increased use of long-lasting insecticidal nets (LLINs) over the last decade has considerably improved the control of malaria in sub-Saharan Africa. However, there is still a paucity of data on the influence of LLIN use and other factors on mosquito bionomics in different epidemiological foci. The objective of this study was to provide updated data on the evolution of vector bionomics and malaria transmission patterns in the equatorial forest region of Cameroon over the period 2000-2017, during which LLIN coverage has increased substantially., Methods: The study was conducted in Olama and Nyabessan, two villages situated in the equatorial forest region. Mosquito collections from 2016-2017 were compared to those of 2000-2001. Mosquitoes were sampled using both human landing catches and indoor sprays, and were identified using morphological taxonomic keys. Specimens belonging to the An. gambiae complex were further identified using molecular tools. Insecticide resistance bioassays were undertaken on An. gambiae to assess the susceptibility levels to both permethrin and deltamethrin. Mosquitoes were screened for Plasmodium falciparum infection and blood-feeding preference using the ELISA technique. Parasitological surveys in the population were conducted to determine the prevalence of Plasmodium infection using rapid diagnostic tests., Results: A change in the species composition of sampled mosquitoes was recorded between the 2000-2001 collections and those of 2016-2017. A drop in the density of the local primary vectors An. nili and An. moucheti in the forest region was recorded, whereas there was an increase in the density of An. gambiae (s.l.), An. marshallii, An. ziemannii and An. paludis. A change in the biting behaviour from indoor to outdoor was recorded in Olama. Very few indoor resting mosquitoes were collected. A change in the night biting cycle was recorded with mosquitoes displaying a shift from night biting to late evening/early in the night. Several mosquitoes were found positive for Plasmodium infection, thus sustaining continuous transmission of malaria in both sites. Reduction of malaria transmission in Nyabessan was lower than that seen in Olama and associated with deforestation and the construction of a dam that may have enabled a more efficient vector, An. gambiae (s.l.), to invade the area. A high level of resistance to pyrethroids (permethrin and deltamethrin) was detected for An. gambiae in both sites. High parasite prevalence was recorded in both sites, with children of 0-16 years being the most affected. In both Olama and Nyabessan, bed net usage appeared to correlate to protection against malaria infection., Conclusions: The study shows important changes in the bionomics of vector populations and malaria transmission patterns in the equatorial forest region. The changes call for more concerted efforts to address challenges such as insecticide resistance, environmental modifications or behavioural changes affecting the performance of current control measures.

Background: Safety and efficacy of the combined monthly use of spot-on fipronil 6.76% w/v / permethrin 50.48% w/v (Frontline Tri-Act®) and chewable tablets of afoxolaner 1.9% w/w / milbemycin oxime 0.4% w/w (NexGard Spectra®) in dogs was evaluated in a field study over a period of 6 months., Methods: Forty-one healthy dogs living in highly endemic area for canine leishmaniosis and other canine vector borne diseases (VBD) were included in the study at the beginning of the Leishmania transmission season. Sixteen dogs were pet dogs living each in a single household; twenty-five dogs were hunting dogs living in three kennels. At inclusion, the dogs were ELISA (rapid test) negative for antibodies to Anaplasma, Borrelia, Ehrlichia, and for antigens of Dirofilaria. The dogs were also negative for blood microfilariae at the Knott's test, and no clinical or haematological abnormalities were observed. Of the included dogs, six hunting, apparently healthy, dogs were ELISA (rapid test) positive to Leishmania, and some were naturally infected by gastrointestinal nematodes (58.5%) and/or infested by fleas (58.5%) and ticks (9.8%). All the included dogs were treated at Days 0, 28, 56, 84, 112 and 140, and followed-up for efficacy until the study end (Day 168)., Results: No adverse events related to the two products, nor skin reactions, general signs, or changes in the haematological profile, were observed during the study. At Day 14, anthelminthic efficacy was 100% for Toxocara canis, Toxascaris leonina and Capillaria aerophila, while few hunting dogs were still shedding eggs of Trichuris vulpis (1/25 hunting dog) and Ancylostomatidae (9/25 hunting dogs). All pet dogs were nematode free at the end of the study. Hunting dogs were free of roundworms and whipworms. Twenty-four hours after the first treatment, 95.8% of the ectoparasite infested dogs were free from fleas and ticks. Ectoparasites were significantly controlled during the 6-month study period, with 100% efficacy on both fleas and ticks from Day 56 to Day 168. Blood and serum samples collected on Day 168 were tested for vector-borne pathogens using same methods of the inclusion and no new seroconversions or circulating blood microfilariae were observed., Conclusions: Concomitant use of Frontline Tri-Act® and NexGard Spectra® in dogs for six months was well tolerated. The combination was effective in controlling fleas, ticks, gastro-intestinal nematodes, and neither new seroconversion to the tested vector-borne pathogens nor blood microfilariae were detected in treated dogs at the end of the study.

Background: In a previous screening program for mosquitocides from local edible plants in Thailand, essential oils (EOs) of Cyperus rotundus, Alpinia galanga and Cinnamomum verum, were found to possess promising adulticidal activity against Aedes aegypti. With the aim of reducing usage of conventional insecticides and improving the management of resistant mosquito populations, this study was designed to determine the potential synergism in the adulticidal efficacy of EOs on permethrin toxicity against Ae. aegypti, both pyrethroid-resistant and -susceptible strains., Methods: EOs extracted from rhizomes of C. rotundus and A. galanga as well as C. verum barks were evaluated for chemical compositions and adulticidal activity against Muang Chiang Mai-susceptible (MCM-S) and Pang Mai Dang-resistant (PMD-R) strains of Ae. aegypti. Adulticidal bioassays of EO-permethrin mixtures for synergistic activity were also performed on these Ae. aegypti strains., Results: Chemical characterization by the GC-MS analytical technique demonstrated that 48 compounds were identified from the EOs of C. rotundus, A. galanga and C. verum, representing 80.22%, 86.75% and 97.24%, respectively, of all compositions. Cyperene (14.04%), β-bisabolene (18.27%) and cinnamaldehyde (64.66%) were the main constituents of C. rotundus, A. galanga and C. verum oils, respectively. In adulticidal bioassays, EOs of C. rotundus, A. galanga and C. verum were effective in killing Ae. aegypti, both MCM-S and PMD-R strains, with LD 50 values of 10.05 and 9.57 μg/mg female, 7.97 and 7.94 μg/mg female, and 3.30 and 3.22 μg/mg female, respectively. The adulticidal efficacy against MCM-S and PMD-R Ae. aegypti of these EOs was close to that of piperonyl butoxide (PBO, LD 50 values = 6.30 and 4.79 μg/mg female, respectively) but less pronounced than that of permethrin (LD 50 values = 0.44 and 3.70 ng/mg female, respectively). Nevertheless, combination-based bioassays discovered the accomplished synergism of EOs together with permethrin. Significant synergistic effects with permethrin against both the strains of Ae. aegypti were recorded in the EOs of C. rotundus and A. galanga. Addition of C. rotundus and A. galanga oils decreased the LD 50 values of permethrin against MCM-S dramatically from 0.44 to 0.07 and 0.11 ng/mg female, respectively, with synergism ratio (SR) values of 6.28 and 4.00, respectively. Furthermore, EOs of C. rotundus and A. galanga also reduced the LD 50 values of permethrin against PMD-R drastically from 3.70 to 0.42 and 0.003 ng/mg female, respectively, with SR values of 8.81 and 1233.33, respectively., Conclusions: The synergy of enhanced adulticidal toxicity recorded from EO-permethrin combinations against both strains of Ae. aegypti presents a promising role of EOs as a synergist for improving mosquitocidal efficacy, particularly in situations where conventional compounds are ineffective or inappropriate.

Background: Members of the Anopheles gambiae (s.l.) complex are one of the major vectors of malaria in Africa. LLINs and IRS are the most effective tools used in vector control of malaria. However, their effectiveness may be hampered by the development and spread of insecticide resistance in the target vectors species. The objective of this study was to assess the susceptibility of Anopheles gambiae (s.l.) mosquitoes from South-West Cameroon to deltamethrin, permethrin and to malathion, four years after the mass deployment of LLINs., Methods: Anopheles larvae were collected from Limbe, Tiko and Buea, three cities of the Fako division and reared until adult emergence. Adult mosquitoes from field larvae were identified as belonging to the Anopheles gambiae (s.l.) complex using standard identification keys. Susceptibility of mosquito samples to deltamethrin, permethrin and malathion was assessed using WHO susceptibility tests protocol for adult mosquitoes. Molecular identification of tested samples was performed using the PCR SINE200 protocol and by PCR-RFLP. The kdr alleles were genotyped using the hot ligation oligonucleotide assay (HOLA)., Results: Two species of the An. gambiae (s.l.) complex, An. coluzzii and An. gambiae (s.s.) were identified in all three study locations with high proportions of An. coluzzii in Limbe (84.06%) and Tiko (92.2%), while in Buea, An. coluzzii (55.6%) and An. gambiae (s.s.) (44.4%) occurred almost in the same proportions. Tested samples were found resistant to pyrethroids (deltamethrin and permethrin) in all locations (< 90% mortality), with > 3-fold increase of KDT 50 values compared with the Kisumu susceptible reference strain of An. gambiae (s.s.). However, the mosquito populations from Limbe and Buea were fully susceptible to malathion. The L1014F kdr was found in both An. coluzzii and An. gambiae (s.s.) with the highest frequencies found in An. gambiae (s.l.) populations from Tiko (94%) and Buea (90%) compared with the Limbe population (66%) (P = 0.00063, df = 2). No kdr L1014S was observed in analyzed samples., Conclusions: These findings reemphasize the ongoing development of An. gambiae (s.l.) resistance to pyrethroids used in impregnating LLINs and suggest the use of malathion as an alternative insecticide for IRS in complementarity with LLINs.

Background: Insecticides targeting adult mosquitoes are the main way of controlling malaria. They work not only by killing mosquitoes, but also by repelling and irritating them. Indeed their repellent action gives valuable personal protection against biting mosquitoes. In the context of malaria control this personal protection is especially relevant when mosquitoes are infectious, whereas to protect the community we would prefer that the mosquitoes that are not yet infectious are killed (so, not repelled) by the insecticide. As the infectious stage of malaria parasites increases the motivation of mosquitoes to bite, we predicted that it would also change their behavioural response to insecticides., Results: With two systems, a laboratory isolate of the rodent malaria Plasmodium berghei infecting Anopheles gambiae and several isolates of P. falciparum obtained from schoolchildren in Tanzania that infected Anopheles arabiensis, we found that mosquitoes harbouring the infectious stage (the sporozoites) of the parasite were less repelled by permethrin-treated nets than uninfected ones., Conclusions: Our results suggest that, at least in the laboratory, malaria infection decreases the personal protection offered by insecticide-treated nets at the stage where the personal protection is most valuable. Further studies must investigate whether these results hold true in the field and whether the less effective personal protection can be balanced by increased community protection.

Background: Lutzomyia longipalpis is the main vector of Leishmania infantum, the agent of canine and human visceral leishmaniosis in the Americas. Considering that the dog is the main domestic host of the parasite, repellent treatment is a measure that might contribute to the prevention of canine visceral leishmaniosis. The repellent efficacy of a single treatment of a new spot-on topical combination of fipronil and permethrin (Frontline Tri-Act®, Merial, now part of Boehringer-Ingelheim) to repel Lu. longipalpis sand flies was evaluated., Methods: Sixteen healthy Beagle dogs, eight females and eight males, weighing 8.4-14.4 kg, and 2 to 4 years-old were included in the study. Animals were blocked on decreasing body weight and randomly allocated within the blocks to one of two treatment groups of eight animals each. Dogs in Group 1 were untreated and Dogs in Group 2 were treated with a combination containing 67.6 mg/ml fipronil + 504.8 mg/ml permethrin (Frontline Tri-Act®) once on Day 0. Sand fly exposures were performed on Days 1, 14, 21 and 30 with Lu. longipalpis female sand flies. After 65 (± 15 min), sand flies were assessed for engorgement status., Results: The percent repellency of the treated group compared to the untreated control group was 95.7, 94.3, 81.7 and 72.2% for exposure days 1, 14, 21 and 30, respectively. The two treatment groups were significantly different for all exposure days (P ≤ 0.016 for days 1, 14, 21 and 30). No adverse reactions were observed during the study., Conclusion: A single topical administration of a new combination of fipronil and permethrin demonstrated a significant repellent effect against Lu. longipalpis bites as soon as it was applied on the dogs and its repellent efficacy lasted for 4 weeks with results greater than 80% for 3 weeks. The results suggest that in endemic areas the regular application of the new combination could contribute to protect dogs from Leishmania infection and therefore serve as an additional tool for the prevention of canine visceral leishmaniosis.

Background: Uganda's malaria burden includes the sixth highest number of annual deaths in Africa (10,500) with approximately 16 million cases (2013) and the entire population at risk. The President's Malaria Initiative has been supporting the malaria control interventions of indoor residual spraying (IRS) and distribution of long-lasting insecticidal nets (LLIN) in Uganda since 2007. These interventions are threatened by emerging and spreading insecticide resistance, known to exist in Ugandan malaria vectors. Pyrethroid insecticides have been used in agriculture since the early 1990s and in IRS programmes from the mid-2000s until 2010. A universal LLIN coverage campaign was executed in 2013-2014, distributing pyrethroid-treated LLINs throughout the country. This study investigated insecticide susceptibility, intensity, and oxidase detoxification in Anopheles gambiae sensu lato and Anopheles funestus to permethrin and deltamethrin in four eastern Ugandan sites., Methods: The susceptibility status of An. gambiae and An. funestus to bendiocarb, permethrin and deltamethrin was determined using the CDC (Centers for Disease Control and Prevention) bottle bioassay. Presence of oxidative enzyme detoxification mechanisms were determined by pre-exposing mosquitoes to piperonyl butoxide followed with exposure to discriminating doses of deltamethrin- and permethrin-coated CDC bottles. Resistance intensity was investigated using serial dosages of 1×, 2×, 5× and 10× the diagnostic dose and scored at 30 min to determine the magnitude of resistance to both of these LLIN pyrethroids. Testing occurred in the Northern and Eastern Regions of Uganda., Results: Anopheles gambiae and An. funestus were fully susceptible to bendiocarb where tested. Anopheles gambiae resistance to deltamethrin and permethrin was observed in all four study sites. Anopheles funestus was resistant to deltamethrin and permethrin in Soroti. Oxidative resistance mechanisms were found in An. gambiae conferring pyrethroid resistance in Lira and Apac. 14.3% of An. gambiae from Tororo survived exposure of 10× concentrations of permethrin., Conclusions: Both An. gambiae and An. funestus are resistant to pyrethroids but fully susceptible to bendiocarb at all sites. Susceptibility monitoring guided the Ministry of Health's decision to rotate between IRS insecticide classes. Intensity bioassay results may indicate encroaching control failure of pyrethroid-treated LLINs and should inform decision-makers when choosing LLINs for the country.

Background: Globally, the Democratic Republic of Congo (DRC) accounted for 9% of malaria cases and 10% of malaria deaths in 2015. As part of control efforts, more than 40 million long-lasting insecticidal nets (LLINs) were distributed between 2008 and 2013, resulting in 70% of households owning one or more LLINs in 2014. To optimize vector control efforts, it is critical to monitor vector behaviour and insecticide resistance trends. Entomological data was collected from eight sentinel sites throughout DRC between 2013 and 2016 in Kingasani, Mikalayi, Lodja, Kabondo, Katana, Kapolowe, Tshikaji and Kalemie. Mosquito species present, relative densities and biting times were monitored using human landing catches (HLC) conducted in eight houses, three times per year. HLC was conducted monthly in Lodja and Kapolowe during 2016 to assess seasonal dynamics. Laboratory data included resistance mechanism frequency and sporozoite rates. Insecticide susceptibility testing was conducted with commonly used insecticides including deltamethrin and permethrin. Synergist bioassays were conducted with PBO to determine the role of oxidases in permethrin resistance., Results: In Lodja, monthly Anopheles gambiae s.l. biting rates were consistently high at > 10 bites/person/night indoors and outdoors. In Kapolowe, An. gambiae s.l. dominated during the rainy season, and Anopheles funestus s.l. during the dry season. In all sites, An. gambiae and An. funestus biting occurred mostly late at night. In Kapolowe, significant biting of both species started around 19:00, typically before householders use nets. Sporozoite rates were high, with a mean of 4.3% (95% CI 3.4-5.2) for An. gambiae and 3.3% (95% CI 1.3-5.3) for An. funestus. Anopheles gambiae were resistant to permethrin in six out of seven sites in 2016. In three sites, susceptibility to deltamethrin was observed despite high frequency permethrin resistance, indicating the presence of pyrethroid-specific resistance mechanisms. Pre-exposure to PBO increased absolute permethrin-associated mortality by 24%, indicating that resistance was partly due to metabolic mechanisms. The kdr-1014F mutation in An. gambiae was present at high frequency (> 70%) in three sites (Kabondo, Kingasani and Tshikaji), and lower frequency (< 20%) in two sites (Lodja and Kapolowe)., Conclusion: The finding of widespread resistance to permethrin in DRC is concerning and alternative insecticides should be evaluated.

Background: Understanding the contribution of community-level long-lasting, insecticidal net (LLIN) coverage to malaria control is critical to planning and assessing intervention campaigns. The Democratic Republic of Congo (DRC), which has one of the highest burdens of malaria cases and deaths and has dramatically scaled up LLIN ownership in recent years thus it is an ideal setting to evaluate the effect of individual versus community-level use to prevent malaria among children under the age of 5., Results: Data were derived from the 2013-2014 DRC Demographic and Health Survey. Community-level LLIN usage was significantly associated with protection against malaria, even when individual-level LLIN usage was included in the model. In stratified analysis, higher levels of community LLIN coverage enhanced the protective effect of individual LLIN usage, resulting in lower malaria prevalence among individuals who used a LLIN. A sub-analysis of individual LLIN usage by insecticide type revealed deltamethrin-treated nets were more protective than permethrin-treated nets, suggesting that mosquitoes in the DRC are more susceptible to deltamethrin., Conclusions: This study examines the effects of individual and community-level LLIN usage in young children in an area of high ITN usage. Individual and community LLIN usage were significantly associated with protection against malaria in children under 5 in the DRC. Importantly, the protective effect of individual LLIN usage against malaria is enhanced when community LLIN coverage is higher, demonstrating the importance of increasing community-level LLIN usage. LLINs treated with deltamethrin were shown to be more protective against malaria than LLINs treated with permethrin. Demographic and Health Surveys are thus a novel and important means of surveillance for insecticide resistance.

Background: The strategy for malaria vector control in the context of reducing malaria morbidity and mortality has been the scale-up of long-lasting insecticidal nets to universal coverage and indoor residual spraying. This has led to significant decline in malaria transmission. However, these vector control strategies rely on insecticides which are threatened by insecticide resistance. In this study the status of pyrethroid resistance in malaria vectors and it's implication in malaria transmission at the Kenyan Coast was investigated., Results: Using World Health Organization diagnostic bioassay, levels of phenotypic resistance to permethrin and deltamethrin was determined. Anopheles arabiensis showed high resistance to pyrethroids while Anopheles gambiae sensu stricto (s.s.) and Anopheles funestus showed low resistance and susceptibility, respectively. Anopheles gambiae sensu lato (s.l.) mosquitoes were further genotyped for L1014S and L1014F kdr mutation by real time PCR. An allele frequency of 1.33% for L1014S with no L1014F was detected. To evaluate the implication of pyrethroid resistance on malaria transmission, Plasmodium falciparum infection rates in field collected adult mosquitoes was determined using enzyme linked immunosorbent assay and further, the behaviour of the vectors was assessed by comparing indoor and outdoor proportions of mosquitoes collected. Sporozoite infection rate was observed at 4.94 and 2.60% in An. funestus s.l. and An. gambiae s.l., respectively. A higher density of malaria vectors was collected outdoor and this also corresponded with high Plasmodium infection rates outdoor., Conclusions: This study showed phenotypic resistance to pyrethroids and low frequency of L1014S kdr mutation in An. gambiae s.l. The occurrence of phenotypic resistance with low levels of kdr frequencies highlights the need to investigate other mechanisms of resistance. Despite being susceptible to pyrethroids An. funestus s.l. could be driving malaria infections in the area.

Background: The development and spread of resistance among local vectors to the major classes of insecticides used in Long-Lasting Insecticidal Nets (LLINs) and Indoor Residual Spraying (IRS) poses a major challenge to malaria vector control programs worldwide. The main methods of evaluating insecticide resistance in malaria vectors are the WHO tube bioassay and CDC bottle assays, with their weakness being determination of resistance at a fixed dose for variable populations. The CDC bottle assay using different insecticide dosages has proved applicable in ascertaining the intensity of resistance., Methods: We determined the status and intensity of permethrin resistance and investigated the efficacy of commonly used LLINs (PermaNet® 2.0, PermaNet® 3.0 and Olyset®) against 3-5 day-old adult female Anopheles mosquitoes from four sub-counties; Teso, Bondo, Rachuonyo and Nyando in western Kenya. Knockdown was assessed to 4 doses of permethrin; 1× (21.5 μg/ml), 2× (43 μg/ml), 5× (107.5 μg/ml) and 10× (215 μg/ml) using CDC bottle assays., Results: Mortality for 0.75% permethrin ranged from 23.5% to 96.1% in the WHO tube assay. Intensity of permethrin resistance was highest in Barkanyango Bondo, with 84% knockdown at the 30 min diagnostic time when exposed to the 10× dose. When exposed to the LLINs, mortality ranged between- 0-39% for Olyset®, 12-88% for PermaNet® 2.0 and 26-89% for PermaNet® 3.0. The efficacy of nets was reduced in Bondo and Teso. Results from this study show that there was confirmed resistance in all the sites; however, intensity assays were able to differentiate Bondo and Teso as the sites with the highest levels of resistance, which coincidentally were the two sub-counties with reduced net efficacy., Conclusions: There was a reduced efficacy of nets in areas with high resistance portraying that at certain intensities of resistance, vector control using LLINs may be compromised. It is necessary to incorporate intensity assays in order to determine the extent of threat that resistance poses to malaria control.

Background: Aedes aegypti and Ae. albopictus are two highly invasive mosquito species, both vectors of several viruses, including dengue, chikungunya and Zika. While Ae. aegypti is the primary vector in the tropics and sub-tropics, Ae. albopictus is increasingly under the public health watch as it has been implicated in arbovirus-transmission in more temperate regions, including continental Europe. Vector control using insecticides is the pillar of most control programmes; hence development of insecticide resistance is of great concern. As part of a Brazilian-Swiss Joint Research Programme we set out to assess whether there are any signs of existing or incipient insecticide resistance primarily against the larvicide Bacillus thuringiensis svar. israelensis (Bti), but also against currently applied and potentially alternative insecticides in our areas, Recife (Brazil) and the Swiss-Italian border region., Methods: Following World Health Organization guidelines, dose-response curves for a range of insecticides were established for both colonized and field caught Ae. aegypti and Ae. albopictus. The larvicides included Bti, two of its toxins, Cry11Aa and Cry4Ba, Lysinibacillus sphaericus, Vectomax CG®, a formulated combination of Bti and L. sphaericus, and diflubenzuron. In addition to the larvicides, the Swiss-Italian Ae. albopictus populations were also tested against five adulticides (bendiocarb, dichlorodiphenyltrichloroethane, malathion, permethrin and λ-cyhalothrin)., Results: Showing a similar dose-response, all mosquito populations were fully susceptible to the larvicides tested and, in particular, to Bti which is currently used both in Brazil and Switzerland. In addition, there were no signs of incipient resistance against Bti as larvae were equally susceptible to the individual toxins, Cry11Aa and Cry4Ba. The field-caught Swiss-Italian populations were susceptible to the adulticides tested but DDT mortality rates showed signs of reduced susceptibility., Conclusions: The insecticides currently used for mosquito control in Switzerland and Brazil are still effective against the target populations. The present study provides an important reference as relatively few insecticide susceptibility surveys have been carried out with Ae. albopictus.

Background: Insecticide-based vector control, which comprises use of insecticide-treated bed nets (ITNs) and indoor residual spraying (IRS), is the key method to malaria control in Madagascar. However, its effectiveness is threatened as vectors become resistant to insecticides. This study investigated the resistance status of malaria vectors in Madagascar to various insecticides recommended for use in ITNs and/or IRS., Methods: WHO tube and CDC bottle bioassays were performed on populations of Anopheles gambiae (s.l.), An. funestus and An. mascarensis. Adult female An. gambiae (s.l.) mosquitoes reared from field-collected larvae and pupae were tested for their resistance to DDT, permethrin, deltamethrin, alpha-cypermethrin, lambda-cyhalothrin, bendiocarb and pirimiphos-methyl. Resting An. funestus and An. mascarensis female mosquitoes collected from unsprayed surfaces were tested against permethrin, deltamethrin and pirimiphos-methyl. The effect on insecticide resistance of pre-exposure to the synergists piperonyl-butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) also was assessed. Molecular analyses were done to identify species and determine the presence of knock-down resistance (kdr) and acetylcholinesterase resistance (ace-1 R ) gene mutations., Results: Anopheles funestus and An. mascarensis were fully susceptible to permethrin, deltamethrin and pirimiphos-methyl. Anopheles gambiae (s.l.) was fully susceptible to bendiocarb and pirimiphos-methyl. Among the 17 An. gambiae (s.l.) populations tested for deltamethrin, no confirmed resistance was recorded, but suspected resistance was observed in two sites. Anopheles gambiae (s.l.) was resistant to permethrin in four out of 18 sites (mortality 68-89%) and to alpha-cypermethrin (89% mortality) and lambda-cyhalothrin (80% and 85%) in one of 17 sites, using one or both assay methods. Pre-exposure to PBO restored full susceptibility to all pyrethroids tested except in one site where only partial restoration to permethrin was observed. DEF fully suppressed resistance to deltamethrin and alpha-cypermethrin, while it partially restored susceptibility to permethrin in two of the three sites. Molecular analysis data suggest absence of kdr and ace-1 R gene mutations., Conclusion: This study suggests involvement of detoxifying enzymes in the phenotypic resistance of An. gambiae (s.l.) to pyrethroids. The absence of resistance in An. funestus and An. mascarensis to pirimiphos-methyl and pyrethroids and in An. gambiae (s.l.) to carbamates and organophosphates presents greater opportunity for managing resistance in Madagascar.

Background: Mosquito nets containing synergists designed to overcome metabolic resistance mechanisms in vectors have been developed. These may enhance excitability in the mosquitoes and affect how they respond to CDC light-traps. Investigating the behaviour of vectors of disease in relation to novel mosquito nets is, therefore, essential for the design of sampling and surveillance systems., Methods: In an initial experiment in Muleba, Tanzania, nine bedrooms from three housing clusters were sampled. CDC light-traps were operated indoors next to occupied untreated nets (UTN), Olyset ® long lasting insecticidal net (LLIN) and Olyset Plus ® LLIN containing piperonyl butoxide (PBO) synergist. Nets were rotated daily between the nine rooms over nine nights. A further series of experiments using the nets on alternate nights in a single room was undertaken during the short rains. Anopheles gambiae s.l. were collected in CDC light-traps, a window-trap and Furvela tent-trap. Anopheles gambiae s.l. were identified to species by polymerase chain reaction (PCR)., Results: In the initial experiment 97.7% of the 310 An. gambiae s.l. were An. gambiae s.s., the remainder being Anopheles arabiensis. The number of mosquitoes collected from 81 light-trap collections was greater in the presence of an Olyset [density rate ratio 1.81, 95% CI (1.22-2.67), p = 0.003] relative to an UTN. In a second experiment, in the wet season 84% of the 180 An. gambiae s.l. identified were An. arabiensis. The number of An. gambiae s.l. collected from a light-trap compared to a tent-trap was significantly higher when an Olyset Plus net was used compared to an UTN. Survival of the mosquitoes in the window trap was not reduced by the use of an Olyset Plus net in the bedroom relative to an Olyset net., Conclusion: Mosquitoes entering bedrooms, even those susceptible to pyrethroids, were not killed by contact with an Olyset Plus LLIN. The enhanced numbers of An. gambiae or An. arabiensis collected in light-traps when a treated net is used requires further experimentation and may be because of a heightened escape reaction on the part of the mosquito.