Insecticide Resistance Profile and Mechanisms in An. gambiae s.l. from Ebolowa, South Cameroon.

Monitoring the trend of insecticide resistance and understanding associated genetic mechanisms is important for designing efficient malaria vector control strategies. This study was conducted to provide temporal data on insecticide resistance status and mechanisms in the major malaria vector Anopheles gambiae s.l. from Ebolowa, Southern Cameroon. Methods: Larvae of An. gambiae s.l. were collected from typical breeding sites throughout the city and reared to adulthood. Emerging adults were morphologically identified and WHO tube assays were performed to determine their susceptibility to carbamate, organophosphate and pyrethroid insecticides at diagnostic doses. When resistance was observed, its intensity was determined by performing WHO tube tests using 5 and 10 times the concentration of the diagnostic dose. Metabolic resistance mechanisms were investigated using insecticide-synergist assays. Sibling species of the An. gambiae complex were identified using SINE-PCR protocol. TaqMan assay was used to genotype the L1014F and L1014S kdr mutations, and the N1575Y mutation, an amplifier of the resistance conferred by the L1014F mutation. Results: Anopheles coluzzii was by far the dominant (99%) member of the An. gambiae s.l. complex in Ebolowa. The species was fully susceptible to carbamates and organophosphates, but resistant to all pyrethroid insecticides tested. Resistance was of moderate intensity for deltamethrin (mortality: 37%, 70% and 99% for 1×, 5× and 10× insecticide concentration, respectively) but rather of high intensity for permethrin (5% for 1×; 62% for 5× and 75% for 10×) and for alphacypermethrin (4.4% for 1×; 57% for 5× and 80% for 10×). Pre-exposure to the synergist PBO resulted in a full recovery of the susceptibility to delthametrin, but this was not observed for the other two pyrethroids tested. L1014S (kdr-East) and the N1575Y mutations were absent, whereas the L1014F (kdr-West) mutation was present at a high frequency (75%), showing a significant association with resistance to permethrin (OR = 3.8; 95%; CI [1.9−7.4]; p < 0.0001) and alphacypermethrin (OR = 3; 95%; CI [1.6−5.4]; p = 0.0002). Conclusion: The increased resistance of An. gambiae s.l. to pyrethroid insecticides as observed in Ebolowa poses a threat to the efficacy of LLINs used to protect populations from the bites of Anopheles mosquitoes that transmit malaria parasites. The present study further highlights the urgent need to implement resistance management strategies in order to maintain the effectiveness of insecticide-based vector control interventions and prevent a rebound in malaria-related mortality.

Isolation and characterization of a temperature-sensitive lethal strain of Anopheles arabiensis for SIT-based application.

BACKGROUND: Malaria is still a global health problem and vector control is the cornerstone of disease control strategies using indoor residual insecticide spraying (IRS) and insecticide-treated nets. The situation is becoming acute with widespread resistance to the limited arsenal of available insecticide classes. Therefore, new and innovative tools to reduce Plasmodium transmission are in need and this situation raised considerable interest in using sterile insect technique (SIT) against human pest insects, particularly Anopheles malaria vectors. When considering a mosquito release programme, one of the first issues to be addressed is how to eliminate/separate the hematophagous vector females. In this paper, we report the development and evaluation of an Anopheles arabiensis temperature-sensitive lethal (tsl) strain towards its potential use for the development of a genetic sexing strain (GSS). Anopheles arabiensis male mosquitoes originated from North Cameroon were treated with 0.05% ethyl methanesulfonate (EMS). The mutagen was added to 10% sucrose solution and mosquitoes fed ad-libitum for 24h and 48h on a cotton wool soaked with sucrose-mutagen solution placed at the top of the cup. Treated males were then allowed to mate with wild virgin females and their progenies were screened for heat sensitivity from F3 to F8, until the isolation and establishment of a tsl strain which was further characterized by assessing its productivity (fecundity and fertility), larval development, adult longevity as well as nature and inheritance pattern of the tsl. RESULTS: Observations showed that the number of eggs laid and their hatch rate were similar between females that mated with mutagenized males and those from the control suggesting that mutagenesis didn't affect An. arabiensis male fecundity and fertility. At F3, fourteen isofemale families out of 88 tested, which showed mortalities ranged between 50 and 80 %, were selected as lines potentially containing a tsl mutation. From F4 to F8, a tsl strain was isolated and established by screening L1 larvae at 41°C for 3 hours. This strain showed similar life history traits compared to the wild type strain in terms of fertility, larval development time and adult's emergence. Crossing experiments to further assess the nature and inheritance pattern of the tsl phenotype showed that it is due to a recessive allele located on an autosome. CONCLUSION: The successful establishment of the An. arabiensis tsl strain is a valuable tool towards the development of a GSS for SIT applications against this species. This will be done by induction of a Y-autosome translocation to link the wild-type allele to the Y chromosome in such manner that males are wild-type (temperature resistant) and females are mutant (temperature sensitive).

A review on the progress of sex-separation techniques for sterile insect technique applications against Anopheles arabiensis.

The feasibility of the sterile insect technique (SIT) as a malaria vector control strategy against Anopheles arabiensis has been under investigation over the past decade. One of the critical steps required for the application of this technique to mosquito control is the availability of an efficient and effective sex-separation system. Sex-separation systems eliminate female mosquitoes from the production line prior to irradiation and field release of sterile males. This is necessary because female mosquitoes can transmit pathogens such as malaria and, therefore, their release must be prevented. Sex separation also increases the efficiency of an SIT programme. Various sex-separation strategies have been explored including the exploitation of developmental and behavioural differences between male and female mosquitoes, and genetic approaches. Most of these are however species-specific and are not indicated for the major African malaria vectors such as An. arabiensis. As there is currently no reliable sex-separation method for An. arabiensis, various strategies were explored in an attempt to develop a robust system that can be applied on a mass-rearing scale. The progress and challenges faced during the development of a sexing system for future pilot and/or large-scale SIT release programmes against An. arabiensis are reviewed here. Three methods of sex separation were examined. The first is the use of pupal size for gender prediction. The second is the elimination of blood-feeding adult females through the addition of an endectocide to a blood meal source. The third is the establishment of a genetic sexing strain (GSS) carrying an insecticide resistance selectable marker (dieldrin-resistance rdl gene and/or other GABA receptor antagonists that can be used as alternative insecticides to dieldrin) or a temperature-sensitive lethal marker.

Rapid evolution of pyrethroid resistance prevalence in Anopheles gambiae populations from the cities of Douala and Yaoundé (Cameroon).

BACKGROUND: The adaptation of malaria vectors to urban areas is becoming a serious challenge for malaria control. The study presents the evolution of pyrethroid resistance in mosquito populations from the cities of Douala and Yaoundé between 2010 and 2013. METHODS: Susceptibility tests to permethrin and deltamethrin were carried out with two- to four-day old unfed Anopheles gambiae sensu lato adults raised from larvae collected from the field. Mosquitoes resistant to permethrin and deltamethrin and control were screened to detect the presence of the kdr alleles using the TaqMan assays. Mosquitoes belonging to the An. gambiae complex were subjected to PCR assays designed for species and molecular forms identifications. The genomic region containing the upstream of intron-1 of the voltage-gated sodium channel was sequenced and compared between mosquitoes originating from different breeding habitats. RESULTS: Anopheles gambiae s.l. specimens collected from the city of Douala were all Anopheles coluzzii. In Yaoundé, both An. gambiae and An. coluzzii were recorded. A rapid decrease of mosquito mortality to permethrin and deltamethrin was recorded between 2010 and 2013 in the two cities. The mortality rate varied from 80.3 to 22.3% and 94.4 to 59.7% for permethrin and deltamethrin, respectively. Both kdr alleles L1014F and L1014S were recorded. The frequency of kdr alleles increased rapidly over the study period, varying from 44 to 88.9% in Yaoundé and from 68 to 81% in Douala. The sequencing of a 1,228 bp region of intro-1 of the voltage-gated sodium channel revealed the presence of five different haplotypes. A high number of these haplotypes were recorded in An. coluzzii samples. No evidence for a recent selective sweep on intron-1 sequence within samples originating from different breeding habitat was detected using Fu's and Tajima Fs statistics. CONCLUSION: The present study supports rapid evolution of pyrethroid resistance in vector populations from the cities of Douala and Yaoundé and calls for immediate action to fight against the increasing prevalence of pyrethroid-resistant mosquitoes.