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1.  Insecticide resistance in Anopheles gambiae from south-western Chad, Central Africa 
Malaria Journal  2008;7:192.
Background
Indoor residual spraying and insecticide-treated nets (ITN) are essential components of malaria vector control in Africa. Pyrethroids are the only recommended compounds for nets treatment because they are fast-acting insecticides with low mammalian toxicity. However, there is growing concern that pyrethroid resistance may threaten the sustainability of ITN scaling-up programmes. Here, insecticide susceptibility was investigated in Anopheles gambiae sensu lato from an area of large scale ITN distribution programme in south-western Chad.
Methods
Susceptibility to 4% DDT, 0.05% deltamethrin, 0.75% permethrin, 0.1% bendiocarb and 5% malathion was assessed using the WHO standard procedures for adult mosquitoes. Tests were carried out with two to four days-old, non-engorged female mosquitoes. The An. gambiae Kisumu strain was used as a reference. Knockdown effect was recorded every 5 min and mortality scored 24 h after exposure. Mosquitoes were identified to species and molecular form by PCR-RFLP and genotypes at the kdr locus were determined in surviving specimens by Hot Oligonucleotide Ligation Assay (HOLA).
Results
During this survey, full susceptibility to malathion was recorded in all samples. Reduced susceptibility to bendiocarb (mortality rate of 96.1%) was found in one sample out of nine assayed. Increased tolerance to pyrethroids was detected in most samples (8/9) with mortality rates ranging from 70.2 to 96.6% for deltamethrin and from 26.7 to 96.3% for permethrin. Pyrethroid tolerance was not associated with a significant increase of knock-down times. Anopheles arabiensis was the predominant species of the An. gambiae complex in the study area, representing 75 to 100% of the samples. Screening for kdr mutations detected the L1014F mutation in 88.6% (N = 35) of surviving An. gambiae sensu stricto S form mosquitoes. All surviving An. arabiensis (N = 49) and M form An. gambiae s.s. (N = 1) carried the susceptible allele.
Conclusion
This first investigation of malaria vector susceptibility to insecticides in Chad revealed variable levels of resistance to pyrethroid insecticides (permethrin and deltamethrin) in most An. gambiae s.l. populations. Resistance was associated with the L1014F kdr mutation in the S form of An. gambiae s.s.. Alternative mechanisms, probably of metabolic origin are involved in An. arabiensis. These results emphasize the crucial need for insecticide resistance monitoring and in-depth investigation of resistance mechanisms in malaria vectors in Chad. The impact of reduced susceptibility to pyrethroids on ITN efficacy should be further assessed.
doi:10.1186/1475-2875-7-192
PMCID: PMC2566574  PMID: 18823537
2.  Evidence of carbamate resistance in urban populations of Anopheles gambiae s.s. mosquitoes resistant to DDT and deltamethrin insecticides in Lagos, South-Western Nigeria 
Parasites & Vectors  2012;5:116.
Background
Resistance monitoring is essential in ensuring the success of insecticide based vector control programmes. This study was carried out to assess the susceptibility status of urban populations of Anopheles gambiae to carbamate insecticide being considered for vector control in mosquito populations previously reported to be resistant to DDT and permethrin.
Methods
Two – three day old adult female Anopheles mosquitoes reared from larval collections in 11 study sites from Local Government Areas of Lagos were exposed to test papers impregnated with DDT 4%, deltamethrin 0.05% and propoxur 0.1% insecticides. Additional tests were carried out to determine the susceptibility status of the Anopheles gambiae population to bendiocarb insecticide. Members of the A. gambiae complex, the molecular forms, were identified by PCR assays. The involvement of metabolic enzymes in carbamate resistance was assessed using Piperonyl butoxide (PBO) synergist assays. The presence of kdr-w/e and ace-1R point mutations responsible for DDT-pyrethroid and carbamate resistance mechanisms was also investigated by PCR.
Results
Propoxur resistance was found in 10 out of the 11 study sites. Resistance to three classes of insecticides was observed in five urban localities. Mortality rates in mosquitoes exposed to deltamethrin and propoxur did not show any significant difference (P > 0.05) but was significantly higher (P < 0.05) in populations exposed to DDT. All mosquitoes tested were identified as A. gambiae s.s (M form). The kdr -w point mutation at allelic frequencies between 45%-77% was identified as one of the resistant mechanisms responsible for DDT and pyrethroid resistance. Ace-1R point mutation was absent in the carbamate resistant population. However, the possible involvement of metabolic resistance was confirmed by synergistic assays conducted.
Conclusion
Evidence of carbamate resistance in A. gambiae populations already harbouring resistance to DDT and permethrin is a clear indication that calls for the implementation of insecticide resistance management strategies to combat the multiple resistance identified.
doi:10.1186/1756-3305-5-116
PMCID: PMC3409038  PMID: 22686575
Carbamate; DDT; Pyrethroids; Insecticide resistance; Urban; Anopheles gambiae mosquitoes; Lagos; Nigeria
3.  Status of insecticide susceptibility in Anopheles gambiae s.l. from malaria surveillance sites in The Gambia 
Malaria Journal  2009;8:187.
Background
Vector control is an effective way of reducing malaria transmission. The main vector control methods include the use of insecticide-treated bed nets and indoor residual spraying (IRS). Both interventions rely on the continuing susceptibility of Anopheles to a limited number of insecticides. However, insecticide resistance, in particular pyrethroid-DDT cross-resistance, is a challenge facing malaria vector control in Africa because pyrethroids represent the only class of insecticides approved for treating bed nets and DDT is commonly used for IRS. Here baseline data are presented on the insecticide susceptibility levels of malaria vectors prior to The Gambian indoor residual spraying intervention programme.
Methods
Anopheles larvae were collected from six malaria surveillance sites (Brikama, Essau, Farafenni, Mansakonko, Kuntaur and Basse) established by the National Malaria Control Programme and the UK Medical Research Council Laboratories in The Gambia. The mosquitoes were reared to adulthood and identified using morphological keys and a species-specific polymerase chain reaction assay. Two- to three-day old adult female mosquitoes were tested for susceptibility to permethrin, deltamethrin and DDT using standard WHO protocols, insecticide susceptibility test kits and treated papers.
Results
All Anopheles mosquitoes tested belonged to the Anopheles gambiae complex. Anopheles arabiensis was predominant (54.1%), followed by An. gambiae s.s. (26.1%) and Anopheles melas (19.8%). Anopheles gambiae s.s. and An. arabiensis were found at all six sites. Anopheles melas was recorded only at Brikama. Mosquitoes from two of the six sites (Brikama and Basse) were fully susceptible to all three insecticides tested. However, DDT resistance was found in An. gambiae from Essau where the 24 hours post-exposure mortality was <80% but 88% for permethrin and 92% for deltamethrin.
Conclusion
This current survey of insecticide resistance in Anopheles provides baseline information for monitoring resistance in The Gambia and highlights the need for routine resistance surveillance as an integral part of the proposed nation wide IRS intervention using DDT.
doi:10.1186/1475-2875-8-187
PMCID: PMC3224992  PMID: 19656399
4.  Multiple insecticide resistance mechanisms in Anopheles gambiae s.l. populations from Cameroon, Central Africa 
Parasites & Vectors  2013;6:41.
Background
Increasing incidence of DDT and pyrethroid resistance in Anopheles mosquitoes is seen as a limiting factor for malaria vector control. The current study aimed at an in-depth characterization of An. gambiae s.l. resistance to insecticides in Cameroon, in order to guide malaria vector control interventions.
Methods
Anopheles gambiae s.l. mosquitoes were collected as larvae and pupae from six localities spread throughout the four main biogeographical domains of Cameroon and reared to adults in insectaries. Standard WHO insecticide susceptibility tests were carried out with 4% DDT, 0.75% permethrin and 0.05% deltamethrin. Mortality rates and knockdown times (kdt50 and kdt95) were determined and the effect of pre-exposure to the synergists DEF, DEM and PBO was assessed. Tested mosquitoes were identified to species and molecular forms (M or S) using PCR-RFLP. The hot ligation method was used to depict kdr mutations and biochemical assays were conducted to assess detoxifying enzyme activities.
Results
The An. arabiensis population from Pitoa was fully susceptible to DDT and permethrin (mortality rates > 98%) and showed reduced susceptibility to deltamethrin. Resistance to DDT was widespread in An. gambiae s.s. populations and heterogeneous levels of susceptibility to permethrin and deltamethrin were observed. In many cases, prior exposure to synergists partially restored insecticide knockdown effect and increased mortality rates, suggesting a role of detoxifying enzymes in increasing mosquito survival upon challenge by pyrethroids and, to a lower extent DDT. The distribution of kdr alleles suggested a major role of kdr-based resistance in the S form of An. gambiae. In biochemical tests, all but one mosquito population overexpressed P450 activity, whereas baseline GST activity was low and similar in all field mosquito populations and in the control.
Conclusion
In Cameroon, multiple resistance mechanisms segregate in the S form of An. gambiae resulting in heterogeneous resistance profiles, whereas in the M form and An. arabiensis insecticide tolerance seems to be essentially mediated by enzyme-based detoxification. Synergists partially restored susceptibility to pyrethroid insecticides, and might help mitigate the impact of vector resistance in the field. However, additional vector control tools are needed to further impact on malaria transmission in such settings.
doi:10.1186/1756-3305-6-41
PMCID: PMC3583743  PMID: 23433176
5.  Insecticide resistance profiles for malaria vectors in the Kassena-Nankana district of Ghana 
Malaria Journal  2009;8:81.
Background
Malaria is a major public health problem in Ghana. The current strategy of the National Malaria Control Programme is based on effective case management and the use of insecticide treated bed nets among vulnerable groups such as children under-five years of age and pregnant women. Resistance to pyrethroids by Anopheles gambiae s.l. and Anopheles funestus has been reported in several African countries including neighbouring Burkina Faso.
Methods
Indoor resting Anopheles mosquitoes were collected. Blood-fed and gravid females were allowed to oviposit, eggs hatched and larvae reared to 1–3 days old adults and tested against permethrin 0.75%, deltamethrin 0.05%, cyfluthrin 0.15%, lambdacyhalothrin 0.1% and DDT 4%, based on WHO methodology. PCR analyses were carried out on a sub-sample of 192 of the An. gambiae for sibling species complex determination. Resistance to pyrethroids and DDT was determined by genotyping the knock-down resistance kdr gene mutations in the study area.
Results
A total of 9,749 1–3 days-old F1 female Anopheles mosquitoes were exposed to the insecticides. Among the pyrethroids, permethrin, 0.75% had the least knockdown effect, whilst cyfluthrin 0.15%, had the highest knock-down effect. Overall, no difference in susceptibility between An. gambiae 93.3% (95% CI: 92.5–94.1) and An. funestus 94.5% (95% CI: 93.7–95.3) was observed when exposed to the pyrethroids. Similarly, there was no difference in susceptibility between the two vector species (An. gambiae = 79.1% (95% CI: 76.6–81.8) and An. funestus = 83.5% (95% CI: 80.2–86.4) when exposed to DDT. Overall susceptibility to the insecticides was between 80% and 98%, suggesting that there is some level of resistance, except for cyfluthrin 0.15%. The kdr PCR assay however, did not reveal any kdr mutations. The analysis also revealed only the molecular M (Mopti) form.
Conclusion
The findings in this study show that An. gambiae and An. funestus, the main malaria vector mosquitoes in the Kassena-Nankana district are susceptible to the insecticides being used in the treatment of bed nets in the malaria control programme. There is however, the need for continuous monitoring of the pyrethroids as the efficacy is not very high.
doi:10.1186/1475-2875-8-81
PMCID: PMC2685403  PMID: 19389257
6.  Multiple Insecticide Resistance: An Impediment to Insecticide-Based Malaria Vector Control Program 
PLoS ONE  2011;6(1):e16066.
Background
Indoor Residual Spraying (IRS), insecticide-treated nets (ITNs) and long-lasting insecticidal nets (LLINs) are key components in malaria prevention and control strategy. However, the development of resistance by mosquitoes to insecticides recommended for IRS and/or ITNs/LLINs would affect insecticide-based malaria vector control. We assessed the susceptibility levels of Anopheles arabiensis to insecticides used in malaria control, characterized basic mechanisms underlying resistance, and evaluated the role of public health use of insecticides in resistance selection.
Methodology/Principal findings
Susceptibility status of An. arabiensis was assessed using WHO bioassay tests to DDT, permethrin, deltamethrin, malathion and propoxur in Ethiopia from August to September 2009. Mosquito specimens were screened for knockdown resistance (kdr) and insensitive acetylcholinesterase (ace-1R) mutations using AS-PCR and PCR-RFLP, respectively. DDT residues level in soil from human dwellings and the surrounding environment were determined by Gas Chromatography with Electron Capture Detector. An. arabiensis was resistant to DDT, permethrin, deltamethrin and malathion, but susceptible to propoxur. The West African kdr allele was found in 280 specimens out of 284 with a frequency ranged from 95% to 100%. Ace-1R mutation was not detected in all specimens scored for the allele. Moreover, DDT residues were found in soil samples from human dwellings but not in the surrounding environment.
Conclusion
The observed multiple-resistance coupled with the occurrence of high kdr frequency in populations of An. arabiensis could profoundly affect the malaria vector control programme in Ethiopia. This needs an urgent call for implementing rational resistance management strategies and integrated vector control intervention.
doi:10.1371/journal.pone.0016066
PMCID: PMC3020220  PMID: 21264325
7.  Pyrethroid resistance in the major malaria vector Anopheles arabiensis from Gwave, a malaria-endemic area in Zimbabwe 
Malaria Journal  2008;7:247.
Background
Insecticide resistance can present a major obstacle to malaria control programmes. Following the recent detection of DDT resistance in Anopheles arabiensis in Gokwe, Zimbabwe, the underlying resistance mechanisms in this population were studied.
Methods
Standard WHO bioassays, using 0.75% permethrin, 4% DDT, 5% malathion, 0.1% bendiocarb and 4% dieldrin were performed on wild-collected adult anopheline mosquitoes and F1 progeny of An. arabiensis reared from wild-caught females. Molecular techniques were used for species identification as well as to identify knockdown resistance (kdr) and ace-1 mutations in individual mosquitoes. Biochemical assays were used to determine the relative levels of detoxifying enzyme systems including non-specific esterases, monooxygenases and glutathione-S-transferases as well as to detect the presence of an altered acetylcholine esterase (AChE).
Results
Anopheles arabiensis was the predominant member of the Anopheles gambiae complex. Of the 436 An. arabiensis females, 0.5% were positive for Plasmodium falciparum infection. WHO diagnostic tests on wild populations showed resistance to the pyrethroid insecticide permethrin at a mean mortality of 47% during February 2006 and a mean mortality of 68.2% in January 2008. DDT resistance (68.4% mean mortality) was present in February 2006; however, two years later the mean mortality was 96%. Insecticide susceptibility tests on F1 An. arabiensis families reared from material from two separate collections showed an average mean mortality of 87% (n = 758) after exposure to 4% DDT and 65% (n = 587) after exposure to 0.75% permethrin. Eight families were resistant to both DDT and permethrin. Biochemical analysis of F1 families reared from collections done in 2006 revealed high activity levels of monooxygenase (48.5% of families tested, n = 33, p < 0.05), glutathione S-transferase (25.8% of families tested, n = 31, p < 0.05) and general esterase activity compared to a reference susceptible An. arabiensis colony. Knockdown resistance (kdr) and ace-IR mutations were not detected.
Conclusion
This study confirmed the presence of permethrin resistance in An. arabiensis populations from Gwave and emphasizes the importance of periodic and ongoing insecticide susceptibility testing of malaria vector populations whose responses to insecticide exposure may undergo rapid change over time.
doi:10.1186/1475-2875-7-247
PMCID: PMC2613911  PMID: 19038063
8.  Pyrethroid-Resistance and Presence of Two Knockdown Resistance (kdr) Mutations, F1534C and a Novel Mutation T1520I, in Indian Aedes aegypti 
Background
Control of Aedes aegypti, the mosquito vector of dengue, chikungunya and yellow fever, is a challenging task. Pyrethroid insecticides have emerged as a preferred choice for vector control but are threatened by the emergence of resistance. The present study reports a focus of pyrethroid resistance and presence of two kdr mutations—F1534C and a novel mutation T1520I, in Ae. aegypti from Delhi, India.
Methodology/Principal Findings
Insecticide susceptibility status of adult-female Ae. aegypti against DDT (4%), deltamethrin (0.05%) and permethrin (0.75%) was determined using WHO's standard insecticide susceptibility kit, which revealed resistance to DDT, deltamethrin and permethrin with corrected mortalities of 35%, 72% and 76% respectively. Mosquitoes were screened for the presence of kdr mutations including those reported earlier (I1011V/M, V1016G/I, F1534C, D1794Y and S989P), which revealed the presence of F1534C and a novel mutation T1520I. Highly specific PCR-RFLP assays were developed for genotyping of these two mutations. Genotyping using allele specific PCR and new PCR-RFLP assays revealed a high frequency of F1534C (0.41–0.79) and low frequency of novel mutation T1520I (0.13). The latter was observed to be tightly linked with F1534C and possibly serve as a compensatory mutation. A positive association of F1534C mutation with DDT and deltamethrin resistance in Ae. aegypti was established. However, F1534C-kdr did not show significant protection against permethrin.
Conclusions/Significance
The Aedes aegypti population of Delhi is resistant to DDT, deltamethrin and permethrin. Two kdr mutations, F1534C and a novel mutation T1520I, were identified in this population. This is the first report of kdr mutations being present in the Indian Ae. aegypti population. Highly specific PCR-RFLP assays were developed for discrimination of alleles at both kdr loci. A positive association of F1534C mutation with DDT and deltamethrin resistance was confirmed.
Author Summary
Dengue and chikungunya are the two important human arboviral infections in India transmitted mainly by Aedes aegypti. In absence of any specific drug or vaccine for these infections, vector control and personal protection are the only control options available. The success of insecticide-based vector control heavily relies upon the knowledge of the status of insecticide resistance in vector populations and the underlying mechanisms of insecticide resistance, especially in the presence of cross-resistance. Knockdown resistance (kdr) is one of the mechanisms of resistance that confers cross-resistance to DDT and pyrethroids. Currently, pyrethroids are the only insecticide class recommended for use in long-lasting insecticide nets (LLIN) and have proven superior to all other insecticides used in vector control programme, due to low mammalian toxicity, low residual activity in nature and rapid knockdown action. The present study was undertaken to determine the susceptibility status of Ae. aegypti against DDT and pyrethroids, and identification of kdr mutations. Though the presence of kdr mutations in Ae. aegypti has been reported in many countries, such a report is not available from India. This study for the first time reports the presence of two kdr mutations, F1534C and a novel mutation T1520I, in an Indian Ae. aegypti population.
doi:10.1371/journal.pntd.0003332
PMCID: PMC4287524  PMID: 25569164
9.  Reduced susceptibility to pyrethroid insecticide treated nets by the malaria vector Anopheles gambiae s.l. in western Uganda 
Malaria Journal  2008;7:92.
Background
Pyrethroid insecticide-treated mosquito nets are massively being scaled-up for malaria prevention particularly in children under five years of age and pregnant mothers in sub-Saharan Africa. However, there is serious concern of the likely evolution of widespread pyrethroid resistance in the malaria vector Anopheles gambiae s.l. due to the extensive use of pyrethroid insecticide-treated mosquito nets. The purpose of this study was to ascertain the status of pyrethroid resistance in An. gambiae s.l. in western Uganda.
Methods
Wild mosquitoes (1–2 days old) were exposed in 10 replicates to new nets impregnated with K-othrine (Deltamethrin 25 mg/m2), Solfac EW50 (Cyfluthrin 50 mg/m2) and Fendona 6SC (Cypermethrin 50 mg/m2) and observed under normal room temperature and humidity (Temperature 24.8°C–27.4°C, Humidity 65.9–45.7). A similar set of mosquitoes collected from the control area 80 km away were exposed to a deltamethrin 25 mg/m2 impregnated net at the same time and under the same conditions. The 10-year mean KDT50 and mortality rates for each of the three pyrethroid insecticides were compared using the Student t-test.
Results
A significant increase in the mean knockdown time (KDT50) and mean mortality rate were observed in almost all cases an indication of reduced susceptibility. The overall results showed a four-fold increase in the mean knockdown time (KDT50) and 1.5-fold decrease in mortality rate across the three pyrethroid insecticides. There was a significant difference in the 10-year mean KDT50 between deltamethrin and cyfluthrin; deltamethrin and cypermethrin, but no significant difference between cyfluthrin and cypermethrin. The 10-year mean difference in KDT50 for mosquitoes exposed to deltamethrin from the control site was significantly different from that of mosquitoes from the intervention site (p<0.05, t=3.979, 9df). The 10-year mean difference in mortality rate between deltamethrin (84.64%); cyfluthrin (74.18%); cypermethrin (72.19%) and the control (90.45%) showed a significant decline in mortality across all the three insecticides.
Conclusion
Generally the results showed a trend of increase in mosquito resistance status with cross-resistance against all the three pyrethroid insecticides. This study reveals for the first time the development of pyrethroid resistance in An. gambiae s.l. in Western Uganda. It is therefore strongly recommended that the impact of this development on malaria control efforts be closely monitored and alternative fabric treatments be considered before this problem curtails community wide implementation of this malaria control strategy in Uganda.
doi:10.1186/1475-2875-7-92
PMCID: PMC2432068  PMID: 18503715
10.  CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae 
PLoS Genetics  2014;10(3):e1004236.
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.
Author Summary
Malaria control depends heavily on only four classes of insecticide to which Anopheles mosquitoes are increasingly resistant. It is important to manage insecticide application carefully to minimise increases in resistance, for example by using different compounds in combination or rotation. Recently, mosquitoes resistant to all available insecticides have been found in Tiassalé, West Africa, which could be problematic for resistance management, particularly if common genetic mechanisms are responsible (‘cross-resistance’). Tiassalé mosquitoes also exhibit extreme levels of resistance to the two most important classes, pyrethroids and carbamates. We investigated the genetic basis of extreme carbamate resistance and cross-resistance in Tiassalé, and the applicability of results in an additional population from Togo. We find that specific P450 enzymes are involved in both extreme and cross-resistance, including one, CYP6M2, which can cause resistance to three insecticide classes. However, amplification of a mutated version of the gene which codes for acetycholinesterase, the target site of both the carbamate and organophosphate insecticides, also plays an important role. Mechanisms involved in both extreme resistance and cross resistance are likely to be very resilient to insecticide management practices, and represent an alarming scenario for mosquito-targeted malaria control.
doi:10.1371/journal.pgen.1004236
PMCID: PMC3961184  PMID: 24651294
11.  Distribution of ace-1R and resistance to carbamates and organophosphates in Anopheles gambiae s.s. populations from Côte d'Ivoire 
Malaria Journal  2010;9:167.
Background
The spread of pyrethroid resistance in Anopheles gambiae s.s. is a critical issue for malaria vector control based on the use of insecticide-treated nets. Carbamates and organophosphates insecticides are regarded as alternatives or supplements to pyrethroids used in nets treatment. It is, therefore, essential to investigate on the susceptibility of pyrethroid resistant populations of An. gambiae s.s. to these alternative products.
Methods
In September 2004, a cross sectional survey was conducted in six localities in Côte d'Ivoire: Toumbokro, Yamoussoukro, Toumodi in the Southern Guinea savannah, Tiassalé in semi-deciduous forest, then Nieky and Abidjan in evergreen forest area. An. gambiae populations from these localities were previously reported to be highly resistant to pyrethroids insecticides. Anopheline larvae were collected from the field and reared to adults. Resistance/susceptibility to carbamates (0.4% carbosulfan, 0.1% propoxur) and organophosphates (0.4% chlorpyrifos-methyl, 1% fenitrothion) was assessed using WHO bioassay test kits for adult mosquitoes. Then, PCR assays were run to determine the molecular forms (M) and (S), as well as phenotypes for insensitive acetylcholinesterase (AChE1) due to G119S mutation.
Results
Bioassays showed carbamates (carbosulfan and propoxur) resistance in all tested populations of An. gambiae s.s. In addition, two out of the six tested populations (Toumodi and Tiassalé) were also resistant to organophosphates (mortality rates ranged from 29.5% to 93.3%). The M-form was predominant in tested samples (91.8%). M and S molecular forms were sympatric at two localities but no M/S hybrids were detected. The highest proportion of S-form (7.9% of An. gambiae identified) was in sample from Toumbokro, in the southern Guinea savannah. The G119S mutation was found in both M and S molecular forms with frequency from 30.9 to 35.2%.
Conclusion
This study revealed a wide distribution of insensitive acetylcholinesterase due to the G119S mutation in both M and S molecular forms of the populations of An. gambiae s.s. tested. The low cross-resistance between carbamates and organophosphates highly suggests involvement of other resistance mechanisms such as metabolic detoxification or F290V mutation.
doi:10.1186/1475-2875-9-167
PMCID: PMC2908637  PMID: 20553593
12.  Anopheles gambiae distribution and insecticide resistance in the cities of Douala and Yaoundé (Cameroon): influence of urban agriculture and pollution 
Malaria Journal  2011;10:154.
Background
Urban malaria is becoming a major health priority across Africa. A study was undertaken to assess the importance of urban pollution and agriculture practice on the distribution and susceptibility to insecticide of malaria vectors in the two main cities in Cameroon.
Methods
Anopheline larval breeding sites were surveyed and water samples analysed monthly from October 2009 to December 2010. Parameters analysed included turbidity, pH, temperature, conductivity, sulfates, phosphates, nitrates, nitrites, ammonia, aluminium, alkalinity, iron, potassium, manganese, magnesium, magnesium hardness and total hardness. Characteristics of water bodies in urban areas were compared to rural areas and between urban sites. The level of susceptibility of Anopheles gambiae to 4% DDT, 0.75% permethrin, 0.05% deltamethrin, 0.1% bendiocarb and 5% malathion were compared between mosquitoes collected from polluted, non polluted and cultivated areas.
Results
A total of 1,546 breeding sites, 690 in Yaoundé and 856 in Douala, were sampled in the course of the study. Almost all measured parameters had a concentration of 2- to 100-fold higher in urban compare to rural breeding sites. No resistance to malathion was detected, but bendiocarb resistance was present in Yaounde. Very low mortality rates were observed following DDT or permethrin exposure, associated with high kdr frequencies. Mosquitoes collected in cultivated areas, exhibited the highest resistant levels. There was little difference in insecticide resistance or kdr allele frequency in mosquitoes collected from polluted versus non-polluted sites.
Conclusion
The data confirm high selection pressure on mosquitoes originating from urban areas and suggest urban agriculture rather than pollution as the major factor driving resistance to insecticide.
doi:10.1186/1475-2875-10-154
PMCID: PMC3118161  PMID: 21651761
13.  Trends in DDT and pyrethroid resistance in Anopheles gambiae s.s. populations from urban and agro-industrial settings in southern Cameroon 
Background
Pyrethroid insecticides are widely used for insect pest control in Cameroon. In certain insect species, particularly the malaria vector Anopheles gambiae, resistance to this class of insecticides is a source of great concern and needs to be monitored in order to sustain the efficacy of vector control operations in the fields. This study highlights trends in DDT and pyrethroid resistance in wild An. gambiae populations from South Cameroon.
Methods
Mosquitoes were collected between 2001 and 2007 in four sites in South Cameroon, where insecticides are used for agricultural or personal protection purposes. Insecticide use was documented in each site by interviewing residents. Batches of 2-4 days old adult female mosquitoes reared from larval collections were tested for susceptibility to DDT, permethrin and deltamethrin using standard WHO procedures. Control, dead and survivors mosquitoes from bioassays were identified by PCR-RFLP and characterized for the kdr mutations using either the AS-PCR or the HOLA method.
Results
Four chemical insecticide groups were cited in the study sites: organochlorines, organophosphates, carbamates and pyrethroids. These chemicals were used for personal, crop or wood protection. In the four An. gambiae populations tested, significant variation in resistance levels, molecular forms composition and kdr frequencies were recorded in the time span of the study. Increases in DDT and pyrethroid resistance, as observed in most areas, were generally associated with an increase in the relative frequency of the S molecular form carrying the kdr mutations at higher frequencies. In Mangoum, however, where only the S form was present, a significant increase in the frequency of kdr alleles between 2003 to 2007 diverged with a decrease of the level of resistance to DDT and pyrethroids. Analyses of the kdr frequencies in dead and surviving mosquitoes showed partial correlation between the kdr genotypes and resistance phenotypes, suggesting that the kdr mechanism may act with certain co-factors to be identified.
Conclusion
These results demonstrate the ongoing spread of kdr alleles in An. gambiae in Central Africa. The rapid evolution of insecticide resistance in this highly dynamic and genetically polymorphic species remains a challenge for its control.
doi:10.1186/1471-2334-9-163
PMCID: PMC2764715  PMID: 19793389
14.  Insecticide resistance of Anopheles sinensis and An. vagus in Hainan Island, a malaria-endemic area of China 
Parasites & Vectors  2014;7:92.
Background
Malaria is one of the most important public health problems in Southeast Asia, including Hainan Island, China. Vector control is the main malaria control measure, and insecticide resistance is a major concern for the effectiveness of chemical insecticide control programs. The objective of this study is to determine the resistance status of the main malaria vector species to pyrethroids and other insecticides recommended by the World Health Organization (WHO) for indoor residual sprays.
Methods
The larvae and pupae of Anopheles mosquitoes were sampled from multiple sites in Hainan Island, and five sites yielded sufficient mosquitoes for insecticide susceptibility bioassays. Bioassays of female adult mosquitoes three days after emergence were conducted in the two most abundant species, Anopheles sinensis and An. vagus, using three insecticides (0.05% deltamethrin, 4% DDT, and 5% malathion) and following the WHO standard tube assay procedure. P450 monooxygenase, glutathione S-transferase and carboxylesterase activities were measured. Mutations at the knockdown resistance (kdr) gene and the ace-1gene were detected by DNA sequencing and PCR-RFLP analysis, respectively.
Results
An. sinensis and An. vagus were the predominant Anopheles mosquito species. An. sinensis was found to be resistant to DDT and deltamethrin. An. vagus was susceptible to deltamethrin but resistant to DDT and malathion. Low kdr mutation (L1014F) frequency (<10%) was detected in An. sinensis, but no kdr mutation was detected in An. vagus populations. Modest to high (45%-75%) ace-1 mutation frequency was found in An. sinensis populations, but no ace-1 mutation was detected in An. vagus populations. Significantly higher P450 monooxygenase and carboxylesterase activities were detected in deltamethrin-resistant An. sinensis, and significantly higher P450 monooxygenase, glutathione S-transferase and carboxylesterase activities were found in malathion-resistant An. vagus mosquitoes.
Conclusions
Multiple insecticide resistance was found in An. sinensis and An. vagus in Hainan Island, a malaria-endemic area of China. Cost-effective integrated vector control programs that go beyond synthetic insecticides are urgently needed.
doi:10.1186/1756-3305-7-92
PMCID: PMC3975898  PMID: 24589247
Anopheles sinensis; Anopheles vagus; Insecticide resistance; Kdr mutation; Ace-1 mutation; Metabolic detoxification enzymes
15.  Comparative susceptibility to permethrin of two Anopheles gambiae s.l. populations from Southern Benin, regarding mosquito sex, physiological status, and mosquito age 
Objective
To investigate what kind of mosquito sample is necessary for the determination of insecticide susceptibility in malaria vectors.
Methods
Larvae and pupae of Anopheles gambiae s.l. (An. gambiae) mosquitoes were collected from the breeding sites in Littoral and Oueme departments. The Centers for Disease Control and Prevention (CDC) susceptibility tests were conducted on unfed male and female mosquitoes aged 2-5 days old. CDC susceptibility tests were also conducted on unfed, blood fed and gravid female mosquitoes aged 2-5 days old. These susceptibility tests were also conducted on unfed and blood fed female mosquitoes aged 2-5 days old and 20 days old. CDC biochemical assay using synergist was also carried out to detect any increase in the activity of enzyme typically involved in insecticide metabolism.
Results
Female An. gambiae Ladji and Sekandji populations were more susceptible than the males when they were unfed and aged 2-5 days old. The mortality rates of blood fed female An. gambiae Ladji and Sekandji populations aged 2-5 days old were lower than those obtained when females were unfed. In addition, the mortality rates of gravid female An. gambiae Ladji and Sekandji populations aged 2-5 days old were lower than those obtained when they were unfed. The mortality rate obtained when female An. gambiae Sekandji populations were unfed and aged 20 days old was higher than the one obtained when these populations were unfed and aged 2-5 days old. The results obtained after effects of synergist penicillin in beeswax on F1 progeny of An. gambiae Ladji populations resistant to permethrin showed that mono-oxygenases were involved in permethrin resistant F1 progeny from Ladji.
Conclusions
The resistance is a hereditary and dynamic phenomenon which can be due to metabolic mechanisms like overproduction of detoxifying enzymes activity. Many factors influence vector susceptibility to insecticide. Among these factors, there are mosquito sex, mosquito age, its physiological status. Therefore, it is useful to respect the World Health Organization criteria in the assessment of insecticide susceptibility tests in malaria vectors. Otherwise, susceptibility testing is conducted using unfed female mosquitoes aged 3-5 days old. Tests should also be carried out at (25±2) °C and (80±10)% relative humidity.
doi:10.12980/APJTB.4.2014C1093
PMCID: PMC3929796  PMID: 25182557
Mosquito sex; Physiological status; Mosquito age; Susceptibility; Permethrin; Synergist
16.  Spatial and temporal variation in the kdr allele L1014S in Anopheles gambiae s.s. and phenotypic variability in susceptibility to insecticides in Western Kenya 
Malaria Journal  2011;10:10.
Background
Malaria vector control in Africa depends upon effective insecticides in bed nets and indoor residual sprays. This study investigated the extent of insecticide resistance in Anopheles gambiae s.l., Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya where ownership of insecticide-treated bed nets has risen steadily from the late 1990s to 2010. Temporal and spatial variation in the frequency of a knock down resistance (kdr) allele in A. gambiae s.s. was quantified, as was variation in phenotypic resistance among geographic populations of A. gambiae s.l.
Methods
To investigate temporal variation in kdr frequency, individual specimens of A. gambiae s.s. from two sentinel sites were genotyped using RT-PCR from 1996-2010. Spatial variation in kdr frequency, species composition, and resistance status were investigated in additional populations of A. gambiae s.l. sampled in western Kenya in 2009 and 2010. Specimens were genotyped for kdr as above and identified to species via conventional PCR. Field-collected larvae were reared to adulthood and tested for insecticide resistance using WHO bioassays.
Results
Anopheles gambiae s.s. showed a dramatic increase in kdr frequency from 1996 - 2010, coincident with the scale up of insecticide-treated nets. By 2009-2010, the kdr L1014S allele was nearly fixed in the A. gambiae s.s. population, but was absent in A. arabiensis. Near Lake Victoria, A. arabiensis was dominant in samples, while at sites north of the lake A. gambiae s.s was more common but declined relative to A. arabiensis from 2009 to 2010. Bioassays demonstrated that A. gambiae s.s. had moderate phenotypic levels of resistance to DDT, permethrin and deltamethrin while A. arabiensis was susceptible to all insecticides tested.
Conclusions
The kdr L1014S allele has approached fixation in A. gambiae s.s. populations of western Kenya, and these same populations exhibit varying degrees of phenotypic resistance to DDT and pyrethroid insecticides. The near absence of A. gambiae s.s. from populations along the lakeshore and the apparent decline in other populations suggest that insecticide-treated nets remain effective against this mosquito despite the increase in kdr allele frequency. The persistence of A. arabiensis, despite little or no detectable insecticide resistance, is likely due to behavioural traits such as outdoor feeding and/or feeding on non-human hosts by which this species avoids interaction with insecticide-treated nets.
doi:10.1186/1475-2875-10-10
PMCID: PMC3029224  PMID: 21235783
17.  Status of insecticide susceptibility in Anopheles arabiensis from Mwea rice irrigation scheme, Central Kenya 
Malaria Journal  2006;5:46.
Background
Control of the Anopheline mosquito vectors of malaria by use of insecticides has been shown to impact on both morbidity and mortality due to this disease. Evidence of insecticide resistance in different settings necessitates surveillance studies to allow prompt detection of resistance should it arise and thus enable its management. Possible resistance by Anopheles arabiensis mosquitoes from Mwea rice irrigation scheme in Central Kenya to insecticides in the four classes of insecticides approved by WHO for indoor residual spraying was investigated.
Methods
Susceptibility to DDT (an organochlorine), fenitrothion (an organophosphate), bendiocarb (a carbamate), lambdacyhalothrin and permethrin (both pyrethroids) was tested using standard WHO diagnostic bioassay kits. Bioassays were performed on non-blood fed mosquitoes one- to three-day old. Knockdown was recorded every 10 min and mortality 24 h post-exposure was noted.
Results
Mortality 24 h post-exposure was 100% for all insecticides except for lambdacyhalothrin, which averaged 99.46%. Knockdown rates at 10 min intervals were not significantly different between the Mwea population and the susceptible KISUMU strain of Anopheles gambiae sensu stricto control. The KDT50 and KDT95 values for the Mwea population were either lower than those for the control or higher by factors of no more than 2 for most comparisons and compared well with those of An. gambiae sensu lato categorized as susceptible in other studies.
Conclusion
These results suggest that the Mwea population of An. arabiensis is susceptible to all the insecticides tested. This implies that vector control measures employing any of these insecticides would not be hampered by resistance.
doi:10.1186/1475-2875-5-46
PMCID: PMC1501029  PMID: 16756645
18.  Insecticide resistance in Anopheles arabiensis (Diptera: Culicidae) from villages in central, northern and south west Ethiopia and detection of kdr mutation 
Parasites & Vectors  2010;3:40.
Background
Anopheles arabiensis is the major vector of malaria in Ethiopia. Malaria vector control in Ethiopia is based on selective indoor residual spraying using DDT, distribution of long lasting insecticide treated nets and environmental management of larval breeding habitats. DDT and pyrethroid insecticides are neurotoxins and have a similar mode of action on the sodium ion channel of insects. It was therefore necessary to verify the insecticide susceptibility status of An. arabiensis, to better understand the status of cross-resistance between DDT and the pyrethroids in this species as well as to detect a resistant gene.
Methods
Standard WHO insecticide susceptibility tests were conducted on adults reared from larval and pupal collections from breeding sites at three villages namely: Sodere in the Rift Valley, Gorgora in the north and Ghibe River Valley in the south west of Ethiopia. The occurrence of cross-resistance between pyrethroids and DDT was determined using a DDT selected laboratory colony originally collected from Gorgora. Phenotypically characterized mosquitoes were tested for the presence of knockdown resistance (kdr) alleles using the standard polymerase chain reaction assay.
Results
All An. gambiae s.l. specimens assayed by PCR were identified as An. arabiensis. The knockdown and mortality results showed An. arabiensis resistance to DDT in all villages, resistance to deltamethrin and permethrin in the Ghibe River Valley and permethrin resistance in Gorgora. Bioassay susceptibility tests also indicated the presence of cross-resistance between DDT and permethrin, but not between DDT and deltamethrin. The knockdown resistance (kdr) mutation of leucine to phenylalanine in the sodium ion channel gene was detected in populations from Gorgora and the Ghibe River Valley.
Conclusion
Since An. arabiensis shows high levels of resistance to DDT in all villages tested and varying pyrethroid resistance in Gorgora and the Ghibe River valley, precautionary measures should be taken in future vector control operations. Moreover, the status of resistance in other locations in Ethiopia and the spread of resistant gene (s) should be investigated.
doi:10.1186/1756-3305-3-40
PMCID: PMC2868498  PMID: 20416109
19.  Nationwide assessment of insecticide susceptibility in Anopheles gambiae populations from Zimbabwe 
Malaria Journal  2014;13(1):408.
Background
The scale-up of malaria interventions in sub-Saharan Africa has been accompanied by a dramatic increase in insecticide resistance in Anopheles spp. In Zimbabwe resistance to pyrethroid insecticides was reported in Gokwe District in 2008. This study reports results of the first nation-wide assessment of insecticide susceptibility in wild populations of Anopheles gambiae sensu lato (s.l.) in Zimbabwe, and provides a comprehensive review of the insecticide resistance status of An. gambiae s.l. in southern African countries.
Methods
World Health Organization (WHO) insecticide susceptibility tests were performed on 2,568 field collected mosquitoes originating from 13 sentinel sites covering all endemic regions in Zimbabwe in 2011–2012. At each site, 24-hour mortality and knock-down values for 50% and 90% of exposed mosquitoes (KD50 and KD90, respectively) were calculated for pools of 20–84 (mean, 54) mosquitoes exposed to 4% DDT, 0.1% bendiocarb, 0.05% λ-cyhalothrin or 5% malathion. Susceptibility results from Zimbabwe were compiled with results published during 2002–2012 for all southern African countries to investigate the resistance status of An. gambiae s.l. in the region.
Results
Using WHO criteria, insecticide resistance was not detected at any site sampled and for any of the insecticide formulations tested during the malaria transmission season in 2012. Knock-down within 1 hr post-insecticide exposure ranged from 95% to 100%; mortality 24 hours post-insecticide exposure ranged from 98% to 100%. Despite the lack of insecticide resistance, high variability was found across sites in KD50 and KD90 values. A total of 24 out of 64 (37.5%) sites in southern Africa with reported data had evidence of phenotypic insecticide resistance in An. gambiae s.l. to at least one insecticide.
Conclusion
Despite a long history of indoor residual spraying of households with insecticide, up to 2012 there was no evidence of phenotypic resistance to any of the four insecticide classes in An. gambiae s.l. collected across different eco-epidemiological areas in Zimbabwe. Results reinforce the need for careful monitoring over time in sentinel sites in order to detect the potential emergence and propagation of insecticide resistance as insecticidal vector control interventions in Zimbabwe continue to be implemented.
Electronic supplementary material
The online version of this article (doi:10.1186/1475-2875-13-408) contains supplementary material, which is available to authorized users.
doi:10.1186/1475-2875-13-408
PMCID: PMC4210579  PMID: 25322726
20.  Vectorial status and insecticide resistance of Anopheles funestus from a sugar estate in southern Mozambique 
Parasites & Vectors  2011;4:16.
Background
The dual problems of rising insecticide resistance in the malaria vectors and increasing human malaria cases since 2001 in southern Mozambique are cause for serious concern. The selection of insecticides for use in indoor residual spraying (IRS) programmes is highly dependent on the extent to which local mosquitoes are susceptible to the approved classes of insecticides. The insecticide resistance status and role in malaria transmission of Anopheles funestus was evaluated at the Maragra Sugar Estate in southern Mozambique where an IRS vector control programme has been in operation for seven years using the carbamate insecticide bendiocarb.
Results
No Anopheles species were captured inside the sugar estate control area. Anopheles funestus group captured outside of the estate represented 90% (n = 475) of the total collections. Of the specimens identified to species by PCR (n = 167), 95% were An. funestus s.s. One An. rivulorum was identified and seven specimens did not amplify. The Anopheles gambiae complex was less abundant (n = 53) and of those identified (n = 33) 76% were An. arabiensis and 24% An. merus. Insecticide susceptibility tests showed that wild-caught and F-1 family An. funestus were resistant to deltamethrin (32.5% mortality) and lambda-cyhalothrin (14.6% mortality), less so to bendiocarb (71.5% mortality) and fully susceptible to both malathion and DDT (100%). Bendiocarb and pyrethroid resistance was nullified using 4% piperonyl butoxide (Pbo), strongly suggesting that both are mediated by P450 monooxygenase detoxification. ELISA tests of An. funestus for Plasmodium falciparum, gave a sporozoite rate of 6.02% (n = 166). One unidentified member of the An. gambiae complex tested positive for P. falciparum sporozoites.
Conclusion
Anopheles funestus was found to be the most abundant and principle vector of malaria in this area, with members of the An. gambiae complex being secondary vectors. Despite the continual use of bendiocarb within the estate for seven years and the level of An. funestus resistance to this insecticide, the IVC programme is still effective against this and other Anopheles in that no vectors were found inside the control area. However, the Mozambique National Malaria Control Programme ceased the use of DDT and bendiocarb in this area of its operations in 2009, and replaced these insecticides with a pyrethroid which will increase insecticide resistance selection pressure and impact on control programmes such as the Maragra IVC.
doi:10.1186/1756-3305-4-16
PMCID: PMC3050845  PMID: 21306631
21.  Bioefficacy of long-lasting insecticidal nets against pyrethroid-resistant populations of Anopheles gambiae s.s. from different malaria transmission zones in Uganda 
Parasites & Vectors  2013;6:130.
Background
There are major concerns over sustaining the efficacy of current malaria vector control interventions given the rapid spread of resistance, particularly to pyrethroids. This study assessed the bioefficacy of five WHO-recommended long-lasting insecticidal nets (LLINs) against pyrethroid-resistant Anopheles gambiae field populations from Uganda.
Methods
Adult An. gambiae from Lira, Tororo, Wakiso and Kanungu districts were exposed to permethrin (0.75%) or deltamethrin (0.05%) in standard WHO susceptibility tests. Cone bioassays were used to measure the bioefficacy of four mono-treated LLINs (Olyset®, Interceptor®, Netprotect® and PermaNet® 2.0) and one combination LLIN (PermaNet® 3.0) against the four mosquito populations. Wireball assays were similarly conducted to determine knockdown rates. Species composition and kdr mutation frequency were determined for a sample of mosquitoes from each population. Chemical assays confirmed that test nets fell within target dose ranges.
Results
Anopheles gambiae s.s. predominated at all four sites (86 - 99% of Anopheles spp.) with moderate kdr L1014S allelic frequency (0.34 – 0.37). Confirmed or possible resistance to both permethrin and deltamethrin was identified for all four test populations. Reduced susceptibility to standard LLINs was observed for all four populations, with mortality rates as low as 45.8% even though the nets were unused. The combination LLIN PermaNet®3.0 showed the highest overall bioefficacy against all four An. gambiae s.l. populations (98.5 - 100% mortality). Wireball assays provided a more sensitive indicator of comparative bioefficacy, and PermaNet 3.0 was again associated with the highest bioefficacy against all four populations (76.5 – 91.7% mortality after 30 mins).
Conclusions
The bioefficacy of mono-treated LLINs against pyrethroid-resistant field populations of An. gambiae varied by LLIN type and mosquito population, indicating that certain LLINs may be more suitable than others at particular sites. In contrast, the combination LLIN PermaNet 3.0 performed optimally against the four An. gambiae populations tested. The observed reduced susceptibility of malaria vectors to mono-treated LLINs is of particular concern, especially considering all nets were unused. With ongoing scale-up of insecticidal tools in the advent of increasing resistance, it is essential that those interventions with proven enhanced efficacy are given preference particularly in areas with high resistance.
doi:10.1186/1756-3305-6-130
PMCID: PMC3656772  PMID: 23634798
Long-lasting insecticidal nets (LLIN); Pyrethroid-resistant An.gambiae s.s; Uganda
22.  Impacts of Agricultural Practices on Insecticide Resistance in the Malaria Vector Anopheles arabiensis in Khartoum State, Sudan 
PLoS ONE  2013;8(11):e80549.
Background
Agricultural pesticides may play a profound role in selection of resistance in field populations of mosquito vectors. The objective of this study is to investigate possible links between agricultural pesticide use and development of resistance to insecticides by the major malaria vector Anopheles arabiensis in northern Sudan.
Methodology/Principal Findings
Entomological surveys were conducted during two agricultural seasons in six urban and peri-urban sites in Khartoum state. Agro-sociological data were collected from 240 farmers subjected to semi-structured questionnaires based on knowledge attitude and practice (KAP) surveys. Susceptibility status of An. arabiensis (n=6000) was assessed in all sites and during each season using WHO bioassay tests to DDT, deltamethrin, permethrin, Malathion and bendiocarb. KAP analysis revealed that pesticide application was common practice among both urban and peri-urban farmers, with organophosphates and carbamates most commonly used. Selection for resistance is likely to be greater in peri-urban sites where farmers apply pesticide more frequently and are less likely to dispose of surpluses correctly. Though variable among insecticides and seasons, broad-spectrum mortality was slightly, but significantly higher in urban than peri-urban sites and most marked for bendiocarb, to which susceptibility was lowest. Anopheles arabiensis from all sites showed evidence of resistance or suspected resistance, especially pyrethroids. However, low-moderate frequencies of the L1014F kdr allele in all sites, which was very strongly associated with DDT, permethrin and deltamethrin survivorship (OR=6.14-14.67) suggests that resistance could increase rapidly.
Conclusions
Ubiquitous multiple-resistance coupled with presence of a clear mechanism for DDT and pyrethroids (kdr L1014F) in populations of An. arabiensis from Khartoum-Sudan suggests careful insecticide management is essential to prolong efficacy. Our findings are consistent with agricultural insecticide use as a source of selection for resistance and argue for coordination between the integrated vector control program and the Ministry of Agriculture to permit successful implementation of rational resistance management strategies.
doi:10.1371/journal.pone.0080549
PMCID: PMC3832379  PMID: 24260414
23.  Multiple Insecticide Resistance in Anopheles gambiae s.l. Populations from Burkina Faso, West Africa 
PLoS ONE  2012;7(11):e48412.
Malaria control programs are being jeopardized by the spread of insecticide resistance in mosquito vector populations. The situation in Burkina Faso is emblematic with Anopheles gambiae populations showing high levels of resistance to most available compounds. Although the frequency of insecticide target-site mutations including knockdown resistance (kdr) and insensitive acetylcholinesterase (Ace-1R) alleles has been regularly monitored in the area, it is not known whether detoxifying enzymes contribute to the diversity of resistance phenotypes observed in the field. Here, we propose an update on the phenotypic diversity of insecticide resistance in An. gambiae populations sampled from 10 sites in Burkina Faso in 2010. Susceptibility to deltamethrin, permethrin, DDT, bendiocarb and fenithrotion was assessed. Test specimens (N = 30 per locality) were identified to species and molecular form and their genotype at the kdr and Ace-1 loci was determined. Detoxifying enzymes activities including non-specific esterases (NSEs), oxydases (cytochrome P450) and Glutathione S-Transferases (GSTs) were measured on single mosquitoes (N = 50) from each test locality and compared with the An. gambiae Kisumu susceptible reference strain. In all sites, mosquitoes demonstrated multiple resistance phenotypes, showing reduced mortality to several insecticidal compounds at the same time, although with considerable site-to-site variation. Both the kdr 1014L and Ace-1R 119S resistant alleles were detected in the M and the S forms of An. gambiae, and were found together in specimens of the S form. Variation in detoxifying enzyme activities was observed within and between vector populations. Elevated levels of NSEs and GSTs were widespread, suggesting multiple resistance mechanisms segregate within An. gambiae populations from this country. By documenting the extent and diversity of insecticide resistance phenotypes and the putative combination of their underlying mechanisms in An. gambiae mosquitoes, our work prompts for new alternative strategies to be urgently developed for the control of major malaria vectors in Burkina Faso.
doi:10.1371/journal.pone.0048412
PMCID: PMC3506617  PMID: 23189131
24.  Synergy in Efficacy of Fungal Entomopathogens and Permethrin against West African Insecticide-Resistant Anopheles gambiae Mosquitoes 
PLoS ONE  2010;5(8):e12081.
Background
Increasing incidences of insecticide resistance in malaria vectors are threatening the sustainable use of contemporary chemical vector control measures. Fungal entomopathogens provide a possible additional tool for the control of insecticide-resistant malaria mosquitoes. This study investigated the compatibility of the pyrethroid insecticide permethrin and two mosquito-pathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, against a laboratory colony and field population of West African insecticide-resistant Anopheles gambiae s.s. mosquitoes.
Methodology/Findings
A range of fungus-insecticide combinations was used to test effects of timing and sequence of exposure. Both the laboratory-reared and field-collected mosquitoes were highly resistant to permethrin but susceptible to B. bassiana and M. anisopliae infection, inducing 100% mortality within nine days. Combinations of insecticide and fungus showed synergistic effects on mosquito survival. Fungal infection increased permethrin-induced mortality rates in wild An. gambiae s.s. mosquitoes and reciprocally, exposure to permethrin increased subsequent fungal-induced mortality rates in both colonies. Simultaneous co-exposure induced the highest mortality; up to 70.3±2% for a combined Beauveria and permethrin exposure within a time range of one gonotrophic cycle (4 days).
Conclusions/Significance
Combining fungi and permethrin induced a higher impact on mosquito survival than the use of these control agents alone. The observed synergism in efficacy shows the potential for integrated fungus-insecticide control measures to dramatically reduce malaria transmission and enable control at more moderate levels of coverage even in areas where insecticide resistance has rendered pyrethroids essentially ineffective.
doi:10.1371/journal.pone.0012081
PMCID: PMC2920335  PMID: 20711409
25.  Variations in susceptibility to common insecticides and resistance mechanisms among morphologically identified sibling species of the malaria vector Anopheles subpictus in Sri Lanka 
Parasites & Vectors  2012;5:34.
Background
Anopheles subpictus s.l., an important malaria vector in Sri Lanka, is a complex of four morphologically identified sibling species A-D. Species A-D reportedly differ in bio-ecological traits that are important for vector control. We investigated possible variations that had not been reported previously, in the susceptibility to common insecticides and resistance mechanisms among the An. subpictus sibling species.
Methods
Adult An. subpictus were collected from localities in four administrative districts in the dry zone of Sri Lanka. Single female isoprogeny lines were established and sibling species status determined according to reported egg morphology. World Health Organization's standard protocols were used for insecticide bioassays and biochemical assays to determine insecticide susceptibility and resistance mechanisms. Susceptibility of mosquitoes was tested against DDT (5%), malathion (4%), deltamethrin (0.05%) and λ-cyhalothrin (0.05%). Biochemical basis for resistance was determined through assaying for esterase, glutathione-S-transferase and monooxygenase activities and the insensitivity of acetycholinesterase (AChE) to propoxur inhibition.
Results
All sibling species were highly resistant to DDT. However there were significant differences among the sibling species in their susceptibility to the other tested insecticides. Few species A could be collected for testing, and where testing was possible, species A tended to behave more similarly to species C and D than to B. Species B was more susceptible to all the tested insecticides than the other sibling species. This difference may be attributed to the predominance of species B in coastal areas where selection pressure due to indoor residual spraying of insecticides (IRS) was lower. However there were significant differences between the more inland species C and D mainly towards pyrethroids. Higher GST activities in species C and D might have contributed to their greater DDT resistance than species B. Malathion resistance in both species C and D may be caused by elevated GST activity and an altered insensitive target site in AChE. In addition, a carboxylesterase based malathion resistance mechanisms was also detected in species C and D. Elevated esterase levels in species C and D might have contributed to the low levels of pyrethroid resistance. However an absence of elevated activity of monooxygenases in species B, C and D indicates that monooxygenases are unlikely to be the cause of this partial resistance to pyrethroids.
Conclusions
The differences in insecticide susceptibility and insecticide resistance mechanism shown by An. subpictus sibling species are important considerations for developing the malaria control and eradication program in Sri Lanka. Similar studies on species complexes of other anopheline vectors of malaria are necessary for effective malaria control worldwide. The differential susceptibility findings are also consistent with most, if not all, morphologically identified An. subpictus species B in Sri Lanka belonging to the An. sundaicus complex. There is a need therefore to develop molecular techniques that can be used to differentiate morphologically similar anopheline species in field conditions for more effective vector control.
doi:10.1186/1756-3305-5-34
PMCID: PMC3317438  PMID: 22325737
Anopheles subpictus s.l.; insecticide resistance; resistance mechanism; sibling species; Sri Lanka

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