Resilience of transfluthrin to oxidative attack by duplicated CYP6P9 variants known to confer pyrethroid resistance in the major malaria mosquito Anopheles funestus.

Resistance to common pyrethroids, such as deltamethrin and permethrin is widespread in the malaria mosquito Anopheles funestus and mainly conferred by upregulated cytochrome P450 monooxygenases (P450s). In the pyrethroid resistant laboratory strain An. funestus FUMOZ-R the duplicated genes CYP6P9a and CYP6P9b are highly upregulated and have been shown to metabolize various pyrethroids, including deltamethrin and permethrin. Here, we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus using a baculovirus expression system and evaluated the interaction of the multifluorinated benzyl pyrethroid transfluthrin with these enzymes by different approaches. First, by Michaelis-Menten kinetics in a fluorescent probe assay with the model substrate 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC), we showed the inhibition of BOMFC metabolism by increasing concentrations of transfluthrin. Second, we tested the metabolic capacity of recombinantly expressed CYP6P9 variants to degrade transfluthrin utilizing UPLC-MS/MS analysis and detected low depletion rates, explaining the virtual lack of resistance of strain FUMOZ-R to transfluthrin observed in previous studies. However, as both approaches suggested an interaction of CYP6P9 variants with transfluthrin, we analyzed the oxidative metabolic fate and failed to detect hydroxylated transfluthrin, but low amounts of an M-2 transfluthrin metabolite. Based on the detected metabolite we hypothesize oxidative attack of the gem-dimethyl substituted cyclopropyl moiety, resulting in the formation of an allyl cation upon ring opening. In conclusion, these findings support the resilience of transfluthrin to P450-mediated pyrethroid resistance, and thus, reinforces its employment as an important resistance-breaking pyrethroid in resistance management strategies to control the major malaria vector An. funestus.

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment.

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

Biochemical profiling of functionally expressed CYP6P9 variants of the malaria vector Anopheles funestus with special reference to cytochrome b5 and its role in pyrethroid and coumarin substrate metabolism.

Cytochrome P450 monooxygenases (P450s) are well studied enzymes catalyzing the oxidative metabolism of xenobiotics in insects including mosquitoes. Their duplication and upregulation in agricultural and public health pests such as anopheline mosquitoes often leads to an enhanced metabolism of insecticides which confers resistance. In the laboratory strain Anopheles funestus FUMOZ-R the duplicated P450s CYP6P9a and CYP6P9b are highly upregulated and proven to confer pyrethroid resistance. Microsomal P450 activity is regulated by NADPH cytochrome P450 oxidoreductase (CPR) required for electron transfer, whereas the modulatory role of cytochrome b5 (CYB5) on insect P450 activity is less clear. In previous studies CYP6P9a and CYP6P9b were recombinantly expressed in tandem with An. gambiae CPR using E. coli-expression systems and CYB5 added to the reaction mix to enhance activity. However, the precise role of CYB5 on substrate turn-over when combined with CYP6P9a and CYP6P9b remains poorly investigated, thus one objective of our study was to address this knowledge gap. In contrast to the CYP6P9 variants, the expression levels of both CYB5 and CPR were not upregulated in the pyrethroid resistant FUMOZ-R strain when compared to the susceptible FANG strain, suggesting no immediate regulatory role of these genes in pyrethroid resistance in FUMOZ-R. Here, for the first time we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus in a baculovirus expression system using High-5 insect cells. Co-expression of each enzyme with CPR from either An. gambiae or An. funestus did not reveal noteworthy differences in catalytic capacity. Whereas the co-expression of An. funestus CYB5 - tested at different multiplicity of infection (MOI) ratios - resulted in a significantly higher metabolization of coumarin substrates as measured by fluorescence assays. This was confirmed by Michaelis-Menten kinetics using the most active substrate, 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC). We observed a similar increase in coumarin substrate turnover by adding human CYB5 to the reaction mix. Finally, we compared by UPLC-MS/MS analysis the depletion rate of deltamethrin and the formation of 4'OH-deltamethrin by recombinantly expressed CYP6P9a and CYP6P9b with and without CYB5 and detected no difference in the extent of deltamethrin metabolism. Our results suggest that co-expression (or addition) of CYB5 with CYP6P9 variants, recombinantly expressed in insect cells, can significantly enhance their metabolic capacity to oxidize coumarins, but not deltamethrin.

Improving houses in the Bolivian Chaco increases effectiveness of residual insecticide spraying against infestation with Triatoma infestans, vector of Chagas disease.

OBJECTIVE: Failure to control domestic Triatoma infestans in the Chaco is attributed to vulnerable adobe construction, which provides vector refuges and diminishes insecticide contact. We conducted a pilot to test the impact of housing improvement plus indoor residual spraying (IRS) on house infestation and vector abundance in a rural community in the Bolivian Chaco. METHODS: The intervention included three arms: housing improvement + IRS [HI], assisted IRS [AS] in which the team helped to clear the house pre-IRS and routine IRS [RS]. HI used locally available materials, traditional construction techniques and community participation. Vector parameters were assessed by Timed Manual Capture for 2 person-hours per house at baseline and medians of 114, 173, 314, 389 and 445 days post-IRS-1. A second IRS round was applied at a median of 314 days post-IRS-1. RESULTS: Post-intervention infestation indices and abundance fell in all three arms. The mean odds of infestation was 0.29 (95% CL 0.124, 0.684) in the HI relative to the RS arm. No difference was observed between AS and RS. Vector abundance was reduced by a mean 44% (24.8, 58.0) in HI compared to RS, with no difference between AS and RS. Median delivered insecticide concentrations per house were lower than the target of 50 mg/m2 in >90% of houses in all arms. CONCLUSION: Housing improvement using local materials and community participation is a promising strategy to improve IRS effectiveness in the Bolivian Chaco. A larger trial is needed to quantify the impact on reinfestation over time.

Improving the performance of spray operators through monitoring and evaluation of insecticide concentrations of pirimiphos-methyl during indoor residual spraying for malaria control on Bioko Island.

BACKGROUND: Quality control of indoor residual spraying (IRS) is necessary to ensure that spray operators (SOs) deposit the correct concentration of insecticide on sprayed structures, while also confirming that spray records are not being falsified. METHODS: Using high-performance liquid chromatography (HPLC), this study conducted quality control of the organophosphate insecticide pirimiphos-methyl (Actellic 300CS), during the 2018 IRS round on Bioko Island, Equatorial Guinea. Approximately 60 SOs sprayed a total of 67,721 structures in 16,653 houses during the round. Houses that were reportedly sprayed were randomly selected for quality control testing. The SOs were monitored twice in 2018, an initial screening in March followed by sharing of results with the IRS management team and identification of SOs to be re-trained, and a second screening in June to monitor the effectiveness of training. Insecticide samples were adhesive-lifted from wooden and cement structures and analysed using HPLC. RESULTS: The study suggests that with adequate quality control measures and refresher training, suboptimal spraying was curtailed, with a significant increased concentration delivered to the bedroom (difference = 0.36, P < 0.001) and wooden surfaces (difference 0.41, P = 0.001). Additionally, an increase in effective coverage by SOs was observed, improving from 80.7% in March to 94.7% in June after re-training (McNemar's test; P = 0.03). CONCLUSIONS: The ability to randomly select, locate, and test houses reportedly sprayed within a week via HPLC has led to improvements in the performance of SOs on Bioko Island, enabling the project to better evaluate its own performance.

Cross-resistance profiles of malaria mosquito P450s associated with pyrethroid resistance against WHO insecticides.

Extensive use of pyrethroids for malaria control in Africa has led to widespread pyrethroid resistance in the two major African vectors of malaria An. gambiae and An. funestus. This is often associated with constitutively elevated levels of cytochrome P450s involved with pyrethroid metabolism and detoxification. P450s have the capacity to metabolise diverse substrates, which raises concerns about their potential to cause cross-resistance. A bank of seven recombinant P450s from An. gambiae (CYPs 6M2, 6P2, 6P3, 6P4, 6P5, 9J5) and An. funestus (CYP6P9a) commonly associated with pyrethroid resistance were screened against twelve insecticides representing the five major classes of insecticides recommended by WHO for malaria control; permethrin, etofenprox and bifenthrin (type I pyrethroids), deltamethrin, lambda cyhalothrin and cypermethrin (type II pyrethroids), DDT (organochlorine), bendiocarb (carbamate), malathion, pirimiphos methyl and fenitrothion (organophosphates) and pyriproxyfen (juvenile hormone analogue). DDT was not metabolised by the P450 panel, while bendiocarb was only metabolised by CYP6P3. Pyrethroids and pyriproxyfen were largely susceptible to metabolism by the P450 panel, as were organophosphates, which are activated by P450s. Primiphos-methyl is increasingly used for malaria control. Examination of the pirimiphos-methyl metabolites generated by CYP6P3 revealed both the active pirimiphos-methyl-oxon form and the inactive oxidative cleavage product 2-diethylamino-6-hydroxy-4-methylpyrimidine. The inhibition profile of CYPs 6M2, 6P2, 6P3, 6P9a and 9J5 was also examined using diethoxyfluorescein (DEF) as the probe substrate. Bendiocarb was the weakest inhibitor with IC50 > 100 µM across the P450 panel, while CYP6M2 showed strongest inhibition by malathion (IC50 0.7 µM). The results suggest that P450s present at elevated levels in two major Anopheline vectors of malaria in Africa have the capacity to metabolise a diverse range of pyrethroid and organophosphate insecticides as well as pyriproxyfen that could impact vector control.

Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector.

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val109Ile, Asp335Glu and Asn384Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.

DDT-based indoor residual spraying suboptimal for visceral leishmaniasis elimination in India.

Indoor residual spraying (IRS) is used to control visceral leishmaniasis (VL) in India, but it is poorly quality assured. Quality assurance was performed in eight VL endemic districts in Bihar State, India, in 2014. Residual dichlorodiphenyltrichloroethane (DDT) was sampled from walls using Bostik tape discs, and DDT concentrations [grams of active ingredient per square meter (g ai/m(2))] were determined using HPLC. Pre-IRS surveys were performed in three districts, and post-IRS surveys were performed in eight districts. A 20% threshold above and below the target spray of 1.0 g ai/m(2) was defined as "in range." The entomological assessments were made in four districts in IRS and non-IRS villages. Vector densities were measured: pre-IRS and 1 and 3 mo post-IRS. Insecticide susceptibility to 4% DDT and 0.05% deltamethrin WHO-impregnated papers was determined with wild-caught sand flies. The majority (329 of 360, 91.3%) of pre-IRS samples had residual DDT concentrations of <0.1 g ai/m(2). The mean residual concentration of DDT post-IRS was 0.37 g ai/m(2); 84.9% of walls were undersprayed, 7.4% were sprayed in range, and 7.6% were oversprayed. The abundance of sand flies in IRS and non-IRS villages was significantly different at 1 mo post-IRS only. Sand flies were highly resistant to DDT but susceptible to deltamethrin. The Stockholm Convention, ratified by India in 2006, calls for the complete phasing out of DDT as soon as practical, with limited use in the interim where no viable IRS alternatives exist. Given the poor quality of the DDT-based IRS, ready availability of pyrethroids, and susceptibility profile of Indian sand flies, the continued use of DDT in this IRS program is questionable.

CYP6 P450 enzymes and ACE-1 duplication produce extreme and multiple insecticide resistance in the malaria mosquito Anopheles gambiae.

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.

Directionally selected cytochrome P450 alleles are driving the spread of pyrethroid resistance in the major malaria vector Anopheles funestus.

Pyrethroid insecticides are critical for malaria control in Africa. However, resistance to this insecticide class in the malaria vector Anopheles funestus is spreading rapidly across Africa, threatening the success of ongoing and future malaria control programs. The underlying resistance mechanisms driving the spread of this resistance in wild populations remain largely unknown. Here, we show that increased expression of two tandemly duplicated P450 genes, CYP6P9a and CYP6P9b, is the main mechanism driving pyrethroid resistance in Malawi and Mozambique, two southern African countries where this insecticide class forms the mainstay of malaria control. Genome-wide transcription analysis using microarray and quantitative RT-PCR consistently revealed that CYP6P9a and CYP6P9b are the two genes most highly overexpressed (>50-fold; q < 0.01) in permethrin-resistant mosquitoes. Transgenic expression of CYP6P9a and CYP6P9b in Drosophila melanogaster demonstrated that elevated expression of either of these genes confers resistance to both type I (permethrin) and type II (deltamethrin) pyrethroids. Functional characterization of recombinant CYP6P9b confirmed that this protein metabolized both type I (permethrin and bifenthrin) and type II (deltamethrin and Lambda-cyhalothrin) pyrethroids but not DDT. Variability analysis identified that a single allele of each of these genes is predominantly associated with pyrethroid resistance in field populations from both countries, which is suggestive of a single origin of this resistance that has since spread across the region. Urgent resistance management strategies should be implemented in this region to limit a further spread of this resistance and minimize its impact on the success of ongoing malaria control programs.

Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions.

Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or "pyrethrome." Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of unique tools for disease control.

Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana.

In the last decade there have been marked reductions in malaria incidence in sub-Saharan Africa. Sustaining these reductions will rely upon insecticides to control the mosquito malaria vectors. We report that in the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide. This is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of insecticide routinely used for malaria control. The gene-expression profile of a highly DDT-resistant population of A. gambiae s.s. from Ghana was characterized using a unique whole-genome microarray. A number of genes were significantly overexpressed compared with two susceptible West African colonies, including genes from metabolic families previously linked to insecticide resistance. One of the most significantly overexpressed probe groups (false-discovery rate-adjusted P < 0.0001) belonged to the cytochrome P450 gene CYP6M2. This gene is associated with pyrethroid resistance in wild A. gambiae s.s. populations) and can metabolize both type I and type II pyrethroids in recombinant protein assays. Using in vitro assays we show that recombinant CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in the presence of solubilizing factor sodium cholate.

Evaluating the feasibility of using insecticide quantification kits (IQK) for estimating cyanopyrethroid levels for indoor residual spraying in Vanuatu.

BACKGROUND: The quality of routine indoor residual spraying (IRS) operations is rarely assessed because of the limited choice of methods available for quantifying insecticide content in the field. This study, therefore, evaluated a user-friendly, rapid colorimetric assay for detecting insecticide content after routine IRS operations were conducted. METHODS: This study was conducted in Tafea Province, Vanuatu. Routine IRS was conducted with lambda cyhalothrin. Two methods were used to quantify the IRS activities: 1) pre-spray application of small felt pads and 2) post-spray removal of insecticide with adhesive. The insecticide content was quantified using a colorimetric assay (Insecticide Quantification Kit [IQK]), which involved exposing each sample to the test reagents for 15 mins. The concentration of insecticide was indicated by the depth of red colour. RESULTS: The IQK proved simple to perform in the field and results could be immediately interpreted by the programme staff. The insecticide content was successfully sampled by attaching felt pads to the house walls prior to spraying. The IRS operation was well conducted, with 83% of houses being sprayed at the target dose (20 - 30 mg AI/m2). The average reading across all houses was 24.4 ± 1.5 mg AI/m2. The results from the felt pads applied pre-spray were used as a base to compare methods for sampling insecticide from walls post-spray. The adhesive of Sellotape did not collect adequate samples. However, the adhesive of the felt pads provided accurate samples of the insecticide content on walls. CONCLUSION: The IQK colorimetric assay proved to be a useful tool that was simple to use under realistic field conditions. The assay provided rapid information on IRS spray dynamics and spray team performance, facilitating timely decision making and reporting for programme managers. The IQK colorimetric assay will have direct applications for routine quality control in malaria control programmes globally and has the potential to improve the efficacy of vector control operations.

Chlorfenapyr metabolism by mosquito P450s associated with pyrethroid resistance identifies potential activation markers.

Chlorfenapyr is a pro-insecticide increasingly used in combination with pyrethroids such as a-cypermethrin or deltamethrin in insecticide treated bednets (ITNs) to control malaria transmitted by pyrethroid-resistant mosquito populations. Chlorfenapyr requires P450 activation to produce tralopyril and other bioactive metabolites. Pyrethroid resistance is often associated with elevated levels of chemoprotective P450s with broad substrate specificity, which could influence chlorfenapyr activity. Here, we have investigated chlorfenapyr metabolism by a panel of eight P450s commonly associated with pyrethroid resistance in An. gambiae and Ae. aegypti, the major vectors of malaria and arboviruses. Chlorfenapyr was activated to tralopyril by An. gambiae CYP6P3, CYP9J5, CYP9K1 and Ae. aegypti, CYP9J32. The Kcat/KM value of 0.66 µM-1 min-1 for CYP9K1 was, 6.7 fold higher than CYP6P3 and CYP9J32 (both 0.1 µM-1 min-1) and 22-fold higher than CYP9J5 (0.03 µM-1 min-1). Further investigation of the effect of -cypermethrin equivalent to the ratios used with chlorfenapyr in bed nets (~ 1:2 molar ratio) resulted in a reduction in chlorfenapyr metabolism by CYP6P3 and CYP6K1 of 76.8% and 56.8% respectively. This research provides valuable insights into the metabolism of chlorfenapyr by mosquito P450s and highlights the need for continued investigation into effective vector control strategies.

Role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase: implications for increasing the activity against sensitive and multidrug-resistant leukaemia HL60 cells.

The aim of this study was to examine the role of structural factors of antitumour anthraquinone derivatives and analogues in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase (CPR) and determine the impact of this activation on increasing the activity especially with regard to multidrug resistant (MDR) tumour cells. It was found that at a high NADPH concentration (500 µmol/l), the anthracenedione agent ametantrone, with an unmodified quinone structure, was susceptible to CPR-dependent reductive activation. In contrast, it was shown that compounds with modified quinone grouping (benzoperimidine BP1, anthrapyridone CO1 and pyrazolopyrimidoacridine PPAC2) did not undergo reductive activation by CPR. This suggests that the presence of a modified quinone function is the structural factor excluding reductive activation of antitumour anthraquinone derivatives and analogues by CPR. In the second part of the work, the ability of antitumour anthraquinone derivatives and analogues to inhibit the growth of the human promyelocytic, sensitive leukaemia HL60 cell line as well as its MDR sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX) was studied in the presence of exogenously added CPR. A significant increase in the activity of ametantrone with an unmodified quinone structure after its reductive conversion by CPR was observed against HL60 as well as HL60/VINC and HL60/DOX cells, whereas in the case of quinone-modified compounds (BP1, CO1 and PPAC2), the presence of the activation system had no effect on their activity against the sensitive and MDR tumour cells examined.

Sequential phase I metabolism of pyrethroids by duplicated CYP6P9 variants results in the loss of the terminal benzene moiety and determines resistance in the malaria mosquito Anopheles funestus.

Pyrethroid resistance in Anopheles funestus is threatening the eradication of malaria. One of the major drivers of pyrethroid resistance in An. funestus are cytochrome P450 monooxygenases CYP6P9a and CYP6P9b, which are found upregulated in resistant An. funestus populations from Sub-Saharan Africa and are known to metabolise pyrethroids. Here, we have functionally expressed CYP6P9a and CYP6P9b variants and investigated their interactions with azole-fungicides and pyrethroids. Some azole fungicides such as prochloraz inhibited CYP6P9a and CYP6P9b at nanomolar concentrations, whereas pyrethroids were weak inhibitors (>100 µM). Amino acid sequence comparisons suggested that a valine to isoleucine substitution at position 310 in the active site cavity of CYP6P9a and CYP6P9b, respectively, might affect substrate binding and metabolism. We therefore swapped the residues by site directed mutagenesis to produce CYP6P9aI310V and CYP6P9bV310I. CYP6P9bV310I produced stronger metabolic activity towards coumarin substrates and pyrethroids, particularly permethrin. The V310I mutation was previously also detected in a pyrethroid resistant field population of An. funestus in Benin. Additionally, we found the first metabolite of permethrin and deltamethrin after hydroxylation, 4'OH permethrin and 4'OH deltamethrin, were also suitable substrates for CYP6P9-variants, and were depleted by both enzymes to a higher extent than as their respective parent compounds (approximately 20% more active). Further, we found that both metabolites were toxic against An. funestus FANG (pyrethroid susceptible) but not towards FUMOZ-R (pyrethroid resistant) mosquitoes, the latter suggesting detoxification by overexpressed CYP6P9a and CYP6P9b. We confirmed by mass-spectrometric analysis that CYP6P9a and CYP6P9b are capable of cleaving phenoxybenzyl-ethers in type I pyrethroid permethrin and type II pyrethroid deltamethrin and that both enzymes preferentially metabolise trans-permethrin. This provides new insight into the metabolism of pyrethroids and a greater understanding of the molecular mechanisms of pyrethroid resistance in An. funestus.

A high-throughput HPLC method for simultaneous quantification of pyrethroid and pyriproxyfen in long-lasting insecticide-treated nets.

Long-lasting insecticide-treated nets (LLINs) play a crucial role in preventing malaria transmission. LLINs should remain effective for at least three years, even after repeated washings. Currently, monitoring insecticides in LLINs is cumbersome, costly, and requires specialized equipment and hazardous solvents. Our aim was to develop a simple, high-throughput and low-resource method for measuring insecticides in LLINs. To extract insecticides, polyethylene-LLIN samples were heated at 85 °C for 45 min in a non-hazardous solvent mix containing dicyclohexylphthalate as an internal standard. The extraction solvent was reduced from 50 to 5 ml using a 0.2 g sample, 90% smaller than the recommended sample size. By optimizing HPLC chromatography, we simultaneously detected pyrethroid and pyriproxyfen insecticides with high sensitivity in LLIN's extract. The method can quantify levels ≥ 0.0015% permethrin, 0.00045% alpha-cypermethrin and 0.00025% pyriproxyfen (w/w) in polyethylene, allowing for insecticide tracking before and after the use of LLINs. This method can be used to assess LLINs with 1% pyriproxyfen (pyriproxyfen-LLIN) or 2% permethrin (Olyset® Net), 1% pyriproxyfen and 2% permethrin (Olyset® Duo), or 0.55% pyriproxyfen and 0.55% alpha-cypermethrin (Royal Gaurd®). One can run 120 samples (40 nets) simultaneously with high precision and accuracy, improving throughput and reducing labour, costs, and environmental impact.

Tandem duplication of a genomic region encoding glutathione S-transferase epsilon-2 and -4 genes in DDT-resistant Anopheles stephensi strain from India.

The glutathione S-transferases (GST) genes are a multigene family of enzymes involved in the metabolism of endogenous and xenobiotic compounds by catalysing the conjugation of the reduced form of glutathione to the substrate. The epsilon class of GST (GSTe), unique to arthropods, is known to be involved in the detoxification process of several classes of insecticides, and GSTe2 in particular is known to have DDT dehydrochlorinase activity. This communication reports a tandem duplication of a genomic region encoding GSTe2 and GSTe4 genes in a laboratory-colonized DDT-resistant Anopheles stephensi. We identified duplication breakpoints and the organization of gene duplication through Sanger sequencing performed on long-PCR products. Manual annotation of sequences revealed a tandemly-arrayed duplication of a 3.62 kb segment of GST epsilon gene clusters comprised of five genes: a partial GSTe1, GSTe2, GSTe2-pseudogene, GSTe4 and partial GSTe5, interconnected by a conserved 2.42 kb DNA insert segment major part of which is homologous to a genomic region located on a different chromosome. The tandemly duplicated array contained a total of two GSTe2 and three GSTe4 functional paralog genes. Read-depth coverage and split-read analysis of Illumina-based whole-genome sequence reads confirmed the presence of duplication in the corresponding region of the genome. The increased gene dose in mosquitoes as a result of the GSTe gene-duplication may be an adaptive process to increase levels of detoxifying enzymes to counter insecticide pressure.

Field-caught permethrin-resistant Anopheles gambiae overexpress CYP6P3, a P450 that metabolises pyrethroids.

Insects exposed to pesticides undergo strong natural selection and have developed various adaptive mechanisms to survive. Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is receiving increasing attention because it threatens the sustainability of malaria vector control programs in sub-Saharan Africa. An understanding of the molecular mechanisms conferring pyrethroid resistance gives insight into the processes of evolution of adaptive traits and facilitates the development of simple monitoring tools and novel strategies to restore the efficacy of insecticides. For this purpose, it is essential to understand which mechanisms are important in wild mosquitoes. Here, our aim was to identify enzymes that may be important in metabolic resistance to pyrethroids by measuring gene expression for over 250 genes potentially involved in metabolic resistance in phenotyped individuals from a highly resistant, wild A. gambiae population from Ghana. A cytochrome P450, CYP6P3, was significantly overexpressed in the survivors, and we show that the translated enzyme metabolises both alpha-cyano and non-alpha-cyano pyrethroids. This is the first study to demonstrate the capacity of a P450 identified in wild A. gambiae to metabolise insecticides. The findings add to the understanding of the genetic basis of insecticide resistance in wild mosquito populations.

High concentrations of membrane-fed ivermectin are required for substantial lethal and sublethal impacts on Aedes aegypti.

BACKGROUND: With widespread insecticide resistance in mosquito vectors, there is a pressing need to evaluate alternatives with different modes of action. Blood containing the antihelminthic drug ivermectin has been shown to have lethal and sub-lethal effects on mosquitoes. Almost all work to date has been on Anopheles spp., but impacts on other anthropophagic vectors could provide new options for their control, or additional value to anti-malarial ivermectin programmes. METHODS: Using dose-response assays, we evaluated the effects of ivermectin delivered by membrane feeding on daily mortality (up to 14 days post-blood feed) and fecundity of an Indian strain of Aedes aegypti. RESULTS: The 7-day lethal concentration of ivermectin required to kill 50% of adult mosquitoes was calculated to be 178.6 ng/ml (95% confidence intervals 142.3-218.4) for Ae. aegypti, which is much higher than that recorded for Anopheles spp. in any previous study. In addition, significant effects on fecundity and egg hatch rates were only recorded at high ivermectin concentrations (≥ 250 ng/ul). CONCLUSION: Our results suggest that levels of ivermectin present in human blood at current dosing regimes in mass drug administration campaigns, or even those in a recent higher-dose anti-malaria trial, are unlikely to have a substantial impact on Ae. aegypti. Moreover, owing to the strong anthropophagy of Ae. aegypti, delivery of higher levels of ivermectin in livestock blood is also unlikely to be an effective option for its control. However, other potential toxic impacts of ivermectin metabolites, accumulation in tissues, sublethal effects on behaviour, or antiviral action might increase the efficacy of ivermectin against Ae. aegypti and the arboviral diseases it transmits, and require further investigation.