Rousseau Djouaka

Expression of the cytochrome P450s, CYP6P3 and CYP6M2 are significantly elevated in multiple pyrethroid resistant populations of Anopheles gambiae s.s . from Southern Benin and Nigeria

BackgroundInsecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. This is particularly true in Benin where pyrethroid resistance has been linked to the failure of insecticide treated bed nets. The role of mutations in the insecticide target sites in conferring resistance has been clearly established. In this study, the contribution of other potential resistance mechanisms was investigated in Anopheles gambiae s.s. from a number of localities in Southern Benin and Nigeria. The mosquitoes were sampled from a variety of breeding sites in a preliminary attempt to investigate the role of contamination of mosquito breeding sites in selecting for resistance in adult mosquitoes.ResultsAll mosquitoes sampled belonged to the M form of An. gambiae s.s. There were high levels of permethrin resistance in an agricultural area (Akron) and an urban area (Gbedjromede), low levels of resistance in mosquito samples from an oil contaminated site (Ojoo) and complete susceptibility in the rural Orogun location. The target site mutation kdrW was detected at high levels in two of the populations (Akron f = 0.86 and Gbedjromede f = 0.84) but was not detected in Ojoo or Orogun. Microarray analysis using the Anopheles gambiae detox chip identified two P450s, CYP6P3 and CYP6M2 up regulated in all three populations, the former was expressed at particularly high levels in the Akron (12.4-fold) and Ojoo (7.4-fold) populations compared to the susceptible population. Additional detoxification and redox genes were also over expressed in one or more populations including two cuticular pre-cursor genes which were elevated in two of the three resistant populations.ConclusionMultiple resistance mechanisms incurred in the different breeding sites contribute to resistance to permethrin in Benin. The cytochrome P450 genes, CYP6P3 and CYP6M2 are upregulated in all three resistant populations analysed. Several additional potential resistance mechanisms were also identified that warrant further investigation. Metabolic genes were over expressed irrespective of the presence of kdr, the latter resistance mechanism being absent in one resistant population. The discovery that mosquitoes collected from different types of breeding sites display differing profiles of metabolic genes at the adult stage may reflect the influence of a range of xenobiotics on selecting for resistance in mosquitoes.

A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector

BackgroundMetabolic resistance to insecticides is the biggest threat to the continued effectiveness of malaria vector control. However, its underlying molecular basis, crucial for successful resistance management, remains poorly characterized.ResultsHere, we demonstrate that the single amino acid change L119F in an upregulated glutathione S-transferase gene, GSTe2, confers high levels of metabolic resistance to DDT in the malaria vector Anopheles funestus. Genome-wide transcription analysis revealed that GSTe2 was the most over-expressed detoxification gene in DDT and permethrin-resistant mosquitoes from Benin. Transgenic expression of GSTe2 in Drosophila melanogaster demonstrated that over-transcription of this gene alone confers DDT resistance and cross-resistance to pyrethroids. Analysis of GSTe2 polymorphism established that the point mutation is tightly associated with metabolic resistance to DDT and its geographical distribution strongly correlates with DDT resistance patterns across Africa. Functional characterization of recombinant GSTe2 further supports the role of the L119F mutation, with the resistant allele being more efficient at metabolizing DDT than the susceptible one. Importantly, we also show that GSTe2 directly metabolizes the pyrethroid permethrin. Structural analysis reveals that the mutation confers resistance by enlarging the GSTe2 DDT-binding cavity, leading to increased DDT access and metabolism. Furthermore, we show that GSTe2 is under strong directional selection in resistant populations, and a restriction of gene flow is observed between African regions, enabling the prediction of the future spread of this resistance.ConclusionsThis first DNA-based metabolic resistance marker in mosquitoes provides an essential tool to track the evolution of resistance and to design suitable resistance management strategies.

Screening of pesticide residues in soil and water samples from agricultural settings.

BackgroundThe role of agricultural practices in the selection of insecticide resistance in malaria vectors has so far been hypothesized without clear evidence. Many mosquito species, Anopheles gambiae in particular, lay their eggs in breeding sites located around agricultural settings. There is a probability that, as a result of farming activities, insecticide residues may be found in soil and water, where they exercise a selection pressure on the larval stage of various populations of mosquitoes. To confirm this hypothesis, a study was conducted in the Republic of Benin to assess the environmental hazards which can be generated from massive use of pesticides in agricultural settings.MethodsLacking an HPLC machine for direct quantification of insecticide residues in samples, this investigation was performed using indirect bioassays focussed on the study of factors inhibiting the normal growth of mosquito larvae in breeding sites. The speed of development was monitored as well as the yield of rearing An. gambiae larvae in breeding sites reconstituted with water and soil samples collected in agricultural areas known to be under pesticide pressure. Two strains of An. gambiae were used in this indirect bioassay: the pyrethroid-susceptible Kisumu strain and the resistant Ladji strain. The key approach in this methodology is based on comparison of the growth of larvae in test and in control breeding sites, the test samples having been collected from two vegetable farms.ResultsResults obtained clearly show the presence of inhibiting factors on test samples. A normal growth of larvae was observed in control samples. In breeding sites simulated by using a few grams of soil samples from the two vegetable farms under constant insecticide treatments (test samples), a poor hatching rate of Anopheles eggs coupled with a retarded growth of larvae and a low yield of adult mosquitoes from hatched eggs, was noticed.ConclusionToxic factors inhibiting the hatching of anopheles eggs and the growth of larvae are probably pesticide residues from agricultural practices. Samples used during this indirect assay have been stored in the laboratory and will be analysed with HPLC techniques to confirm hypothesis of this study and to identify the various end products found in soil and water samples from agricultural settings under pesticide pressure.

Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa.

Growing problems of pyrethroid resistance in Anopheles funestus have intensified efforts to identify alternative insecticides. Many agrochemicals target the GABA receptors, but cross-resistance from dieldrin resistance may preclude their introduction. Dieldrin resistance was detected in An. funestus populations from West (Burkina Faso) and central (Cameroon) Africa, but populations from East (Uganda) and Southern Africa (Mozambique and Malawi) were fully susceptible to this insecticide. Partial sequencing of the dieldrin target site, the γ-aminobutyric acid (GABA) receptor, identified two amino acid substitutions, A296S and V327I. The A296S mutation has been associated with dieldrin resistance in other species. The V327I mutations was detected in the resistant sample from Burkina Faso and Cameroon and consistently associated with the A296S substitution. The full-length of the An. funestus GABA-receptor gene, amplified by RT-PCR, generated a sequence of 1674 bp encoding 557 amino acid of the protein in An. funestus with 98% similarity to that of Anopheles gambiae. Two diagnostic assays were developed to genotype the A296S mutation (pyrosequencing and PCR-RFLP), and use of these assays revealed high frequency of the resistant allele in Burkina Faso (60%) and Cameroon (82%), moderate level in Benin (16%) while low frequency or absence of the mutation was observed respectively in Uganda (7.5%) or 0% in Malawi and Mozambique. The distribution of the RdlR mutation in An. funestus populations in Africa suggests extensive barriers to gene flow between populations from different regions.

Exploring Mechanisms of Multiple Insecticide Resistance in a Population of the Malaria Vector Anopheles funestus in Benin

BACKGROUND The insecticide resistance status of the malaria vector Anopheles funestus and the underlying resistance mechanisms remain uncharacterised in many parts of Africa, notably in Benin, West Africa. To fill this gap in our knowledge, we assessed the susceptibility status of a population of this species in Pahou, Southern Benin and investigated the potential resistance mechanisms. METHODOLOGY/PRINCIPAL FINDINGS WHO bioassays revealed a multiple resistance profile for An. funestus in Pahou. This population is highly resistant to DDT with no mortality in females after 1h exposure to 4%DDT. Resistance was observed against the Type I pyrethroid permethrin and the carbamate bendiocarb. A moderate resistance was detected against deltamethrin (type II pyrethroids). A total susceptibility was observed against malathion, an organophosphate. Pre-exposure to PBO did not change the mortality rates for DDT indicating that cytochrome P450s play no role in DDT resistance in Pahou. No L1014F kdr mutation was detected but a correlation between haplotypes of two fragments of the Voltage-Gated Sodium Channel gene and resistance was observed suggesting that mutations in other exons may confer the knockdown resistance in this species. Biochemical assays revealed elevated levels of GSTs and cytochrome mono-oxygenases in Pahou. No G119S mutation and no altered acetylcholinesterase gene were detected in the Pahou population. qPCR analysis of five detoxification genes revealed that the GSTe2 is associated to the DDT resistance in this population with a significantly higher expression in DDT resistant samples. A significant over-expression of CYP6P9a and CYP6P9b previously associated with pyrethroid resistance was also seen but at a lower fold change than in southern Africa. CONCLUSION The multiple insecticide resistance profile of this An. funestus population in Benin shows that more attention should be paid to this important malaria vector for the implementation and management of current and future malaria vector control programs in this country.

Does the spillage of petroleum products in Anopheles breeding sites have an impact on the pyrethroid resistance

BackgroundThe emergence of Anopheles populations capable of withstanding lethal doses of insecticides has weakened the efficacy of most insecticide based strategies of vector control and, has highlighted the need for further studies on the mechanisms of insecticide resistance and the various factors selecting resistant populations of mosquitoes. This research targeted the analysis of breeding sites and the oviposition behaviour of susceptible and resistant populations of Anopheles in localities of spilled petroleum products. The aim was to establish the possible contribution of oil spillage in the selection of pyrethroid resistance in malaria vectors.MethodsAnopheles breeding sites were identified and the insecticide susceptibility of the Anopheles gambiae populations mapped in 15 localities of South Western Nigeria. The presence of oil particles as well as the turbidity, the dissolved oxygen and the pH of each identified breeding site was recorded. Data were cross-analysed to correlate the habitat types and the insecticide susceptibility status of emerging mosquitoes. The second phase of this study was basically a laboratory model to provide more information on the implication of the spillage of petroleum on the selection of pyrethroid resistance in An. gambiae.ResultsModerate levels of resistance following exposure to permethrin-impregnated papers were recorded with the majority of An. gambiae samples collected in the South Western Nigeria. Data from this study established a link between the constituency of the breeding sites and the resistance status of the emerging Anopheles.ConclusionThis study has revealed the segregational occupation of breeding habitats by pyrethroid resistant and susceptible strains of An. gambiae in south-western Nigeria. Compiled results from field and laboratory research point out clear relationships between oil spillage and pyrethroid resistance in malaria vectors. The identification of this factor of resistance could serve as strong information in the management of insecticide resistance in some West African settings.

Zoom in at African country level: potential climate induced changes in areas of suitability for survival of malaria vectors

BackgroundPredicting anopheles vectors’ population densities and boundary shifts is crucial in preparing for malaria risks and unanticipated outbreaks. Although shifts in the distribution and boundaries of the major malaria vectors (Anopheles gambiae s.s. and An. arabiensis) across Africa have been predicted, quantified areas of absolute change in zone of suitability for their survival have not been defined. In this study, we have quantified areas of absolute change conducive for the establishment and survival of these vectors, per African country, under two climate change scenarios and based on our findings, highlight practical measures for effective malaria control in the face of changing climatic patterns.MethodsWe developed a model using CLIMEX simulation platform to estimate the potential geographical distribution and seasonal abundance of these malaria vectors in relation to climatic factors (temperature, rainfall and relative humidity). The model yielded an eco-climatic index (EI) describing the total favourable geographical locations for the species. The EI values were classified and exported to a GIS package. Using ArcGIS, the EI shape points were clipped to the extent of Africa and then converted to a raster layer using Inverse Distance Weighted (IDW) interpolation method. Generated maps were then transformed into polygon-based geo-referenced data set and their areas computed and expressed in square kilometers (km2).ResultsFive classes of EI were derived indicating the level of survivorship of these malaria vectors. The proportion of areas increasing or decreasing in level of survival of these malaria vectors will be more pronounced in eastern and southern African countries than those in western Africa. Angola, Ethiopia, Kenya, Mozambique, Tanzania, South Africa and Zambia appear most likely to be affected in terms of absolute change of malaria vectors suitability zones under the selected climate change scenarios.ConclusionThe potential shifts of these malaria vectors have implications for human exposure to malaria, as recrudescence of the disease is likely to be recorded in several new areas and regions. Therefore, the need to develop, compile and share malaria preventive measures, which can be adapted to different climatic scenarios, remains crucial.

Multiple insecticide resistance in an infected population of the malaria vector Anopheles funestus in Benin

BackgroundKnowledge on the spread and distribution of insecticide resistance in major malaria vectors such as Anopheles funestus is key to implement successful resistance management strategies across Africa. Here, by assessing the susceptibility status of an inland population of An. funestus Giles (Kpome) and investigating molecular basis of resistance, we show that multiple resistance and consistent plasmodium infection rate are present in Anopheles funestus populations from Kpome.MethodsThe insecticide susceptibility level of collected Anopheles funestus was assessed. Synergist (PBO) was used to screen resistance mechanisms. The TaqMan technique was used for genotyping of insecticide resistant alleles and detecting plasmodium infection levels. The nested PCR was used to further assess the plasmodium infection rate.ResultsThe TaqMan analysis of plasmodial infections revealed an infection rate (18.2 %) of An. funestus in this locality. The WHO bioassays revealed a multiple phenotypic resistance profile for An. funestus in Kpome. This population is highly resistant to pyrethroids (permethrin and deltamethrin), organochlorines (DDT), and carbamates (bendiocarb). A reduced susceptibility was observed with dieldrin. Mortalities did not vary after pre-exposure to PBO for DDT indicating that cytochrome P450s play little role in DDT resistance in Kpome. In contrast, we noticed, a significant increase in mortalities when PBO was combined to permethrin suggesting the direct involvement of P450s in pyrethroid resistance. A high frequency of the L119F-GSTe2 DDT resistance marker was observed in the wild DDT resistant population (9 %RS and 91 %RR) whereas the A296S mutation was detected at a low frequency (1 %RS and 99 %SS).ConclusionThe presence of multiple resistance in An. funestus populations in the inland locality of Kpome is established in this study as recently documented in the costal locality of Pahou. Data from both localities suggest that resistance could be widespread in Benin and this highlights the need for further studies to assess the geographical distribution of insecticide resistance across Benin and neighboring countries as well as a more comprehensive analysis of the resistance mechanisms involved.

Development of Reference Transcriptomes for the Major Field Insect Pests of Cowpea: A Toolbox for Insect Pest Management Approaches in West Africa

Cowpea is a widely cultivated and major nutritional source of protein for many people that live in West Africa. Annual yields and longevity of grain storage is greatly reduced by feeding damage caused by a complex of insect pests that include the pod sucking bugs, Anoplocnemis curvipes Fabricius (Hemiptera: Coreidae) and Clavigralla tomentosicollis Stål (Hemiptera: Coreidae); as well as phloem-feeding cowpea aphids, Aphis craccivora Koch (Hemiptera: Aphididae) and flower thrips, Megalurothrips sjostedti Trybom (Thysanoptera: Thripidae). Efforts to control these pests remain a challenge and there is a need to understand the structure and movement of these pest populations in order to facilitate the development of integrated pest management strategies (IPM). Molecular tools have the potential to help facilitate a better understanding of pest populations. Towards this goal, we used 454 pyrosequencing technology to generate 319,126, 176,262, 320,722 and 227,882 raw reads from A. curvipes, A. craccivora, C. tomentosicollis and M. sjostedti, respectively. The reads were de novo assembled into 11,687, 7,647, 10,652 and 7,348 transcripts for A. curvipes, A. craccivora, C. tomentosicollis and M. sjostedti, respectively. Functional annotation of the resulting transcripts identified genes putatively involved in insecticide resistance, pathogen defense and immunity. Additionally, sequences that matched the primary aphid endosymbiont, Buchnera aphidicola, were identified among A. craccivora transcripts. Furthermore, 742, 97, 607 and 180 single nucleotide polymorphisms (SNPs) were respectively predicted among A. curvipes, A. craccivora, C. tomentosicollis and M. sjostedti transcripts, and will likely be valuable tools for future molecular genetic marker development. These results demonstrate that Roche 454-based transcriptome sequencing could be useful for the development of genomic resources for cowpea pest insects in West Africa.

Quantification of the efficiency of treatment of Anopheles gambiae breeding sites with petroleum products by local communities in areas of insecticide resistance in the Republic of Benin

BackgroundThe emergence of Anopheles populations capable of withstanding lethal doses of insecticides has weakened the efficacy of most insecticide based strategies of vector control and, has highlighted the need for developing new insecticidal molecules or, improving the efficacy of existing insecticides or abandoning those to which resistance has emerged. The use of petroleum products (PP) against mosquito larvae had an immense success during early programmes of malaria control, but these compounds were abandoned and replaced in the 1950s by synthetic insecticides probably because of the high performances given by these new products. In the current context of vector resistance, it is important to elucidate the empirical use of PP by quantifying their efficiencies on resistant strains of Anopheles.MethodsLarvae of Anopheles Ladji a local resistant strain were exposed to increasing concentrations of various PP (kerosene, petrol and engine oils) for 24 hours and the lethal activities recorded. The highest concentration (HiC) having no lethal activity (also referred as the NOEL or no effect level) and the lowest concentration (LoC100) yielding 100% mortality were rated for each PP on the Ladji strain. Prior to laboratory analysis, KAP studies were conducted in three traditional communities were insecticide resistance is clearly established to confirm the use of PP against mosquitoes.ResultsLaboratory analysis of petrol, kerosene and engine oils, clearly established their lethal activities on resistant strains of Anopheles larvae. Contrary to existing references, this research revealed that exposed larvae of Anopheles were mostly killed by direct contact toxicity and not by suffocation as indicated in some earlier reports.ConclusionThis research could serve as scientific basis to backup the empirical utilisation of PP on mosquito larvae and to envisage possibilities of using PP in some traditional settings where Anopheles have developed resistance to currently used insecticides.