Insecticide Resistance Testing in Malaria Vectors in Tanzania: Challenges in Mosquito Sampling and Rearing under Field Conditions

The National Institute for Medical Research, Amani centre, in collaboration with National Malaria Control Programme, has been conducting annual insecticide resistance surveillance since 1999, aimed at early detection of resistance to insecticides used for malaria control in Tanzania. The Standard WHO method for larvae collection and rearing were used but challenges and limitations were encountered. For example rearing the larvae and adult mosquitoes using the Standard WHO method experienced 100% mortality for larvae; and adults in three days. The researchers therefore made modifications to the Standard WHO method to create suitable tools for the field environment. A ladle was created from an empty water bottle in which an oval hole longitudinally cut halfway from the bottom. Instead of using TetraMin as mosquito larval food, green algae were collected from mosquito breeding sites and used as larval food. Improvised heater of charcoal stoves and humidifier of wet fabric such as “Kanga” and “Kitenge” were also used. There was 90% larval survival, adult mosquito survived much better and the scientists had a total of 467 mosquitoes to run the insecticide susceptibility tests. Innovative ways are necessary under field conditions for mosquito breeding in susceptibility studies

Genetic basis of pyrethroid resistance in a population of Anopheles arabiensis, the primary malaria vector in Lower Moshi, north-eastern Tanzania

Background Pyrethroid resistance has been slower to emerge in Anopheles arabiensis than in An. gambiae s.s and An. funestus and, consequently, studies are only just beginning to unravel the genes involved. Permethrin resistance in An. arabiensis in Lower Moshi, Tanzania has been linked to elevated levels of both P450 monooxygenases and β-esterases. We have conducted a gene expression study to identify specific genes linked with metabolic resistance in the Lower Moshi An. arabiensis population. Methods Microarray experiments employing an An. gambiae whole genome expression chip were performed on An. arabiensis, using interwoven loop designs. Permethrin-exposed survivors were compared to three separate unexposed mosquitoes from the same or a nearby population. A subsection of detoxification genes were chosen for subsequent quantitative real-time PCR (qRT-PCR). Results Microarray analysis revealed significant over expression of 87 probes and under expression of 85 probes (in pairwise comparisons between permethrin survivors and unexposed sympatric and allopatric samples from Dar es Salaam (controls). For qRT-PCR we targeted over expressed ABC transporter genes (ABC ‘2060’), a glutathione-S-transferase, P450s and esterases. Design of efficient, specific primers was successful for ABC ‘2060’and two P450s (CYP6P3, CYP6M2). For the CYP4G16 gene, we used the primers that were previously used in a microarray study of An. arabiensis from Zanzibar islands. Over expression of CYP4G16 and ABC ‘2060’ was detected though with contrasting patterns in pairwise comparisons between survivors and controls. CYP4G16 was only up regulated in survivors, whereas ABC ‘2060’ was similar in survivors and controls but over expressed in Lower Moshi samples compared to the Dar es Salaam samples. Increased transcription of CYP4G16 and ABC ‘2060’ are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Conclusions Increased transcription of a P450 (CYP4G16) and an ABC transporter (ABC 2060) are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Our study provides replication of CYP4G16 as a candidate gene for pyrethroid resistance in An. arabiensis, although its role may not be in detoxification, and requires further investigation

A significant association between deltamethrin resistance, Plasmodium falciparum infection and the Vgsc-1014S resistance mutation in Anopheles gambiae highlights the epidemiological importance of resistance markers.

BACKGROUND The success of malaria vector control is threatened by widespread pyrethroid insecticide resistance. However, the extent to which insecticide resistance impacts transmission is unclear. The objective of this study was to examine the association between the DDT/pyrethroid knockdown resistance mutation Vgsc-1014S, commonly termed kdr, and infection with Plasmodium falciparum sporozoites in Anopheles gambiae. METHODS WHO standard methods were used to characterize susceptibility of wild female mosquitoes to 0.05 % deltamethrin. PCR-based molecular diagnostics were used to identify mosquitoes to species and to genotype at the Vgsc-L1014S locus. ELISAs were used to detect the presence of P. falciparum sporozoites and for blood meal identification. RESULTS Anopheles mosquitoes were resistant to deltamethrin with mortality rates of 77.7 % [95 % CI 74.9-80.3 %]. Of 545 mosquitoes genotyped 96.5 % were A. gambiae s.s. and 3.5 % were Anopheles arabiensis. The Vgsc-1014S mutation was detected in both species. Both species were predominantly anthropophagic. In A. gambiae s.s., Vgsc-L1014S genotype was significantly associated with deltamethrin resistance (χ2 = 11.2; p < 0.001). The P. falciparum sporozoite infection rate was 4.2 %. There was a significant association between the presence of sporozoites and Vgsc-L1014S genotype in A. gambiae s.s. (χ2 = 4.94; p = 0.026). CONCLUSIONS One marker, Vgsc-1014S, was associated with insecticide resistance and P. falciparum infection in wild-caught mixed aged populations of A. gambiae s.s. thereby showing how resistance may directly impact transmission

The Dynamics of Pyrethroid Nesistance in Anopheles Arabiensis from Zanzibar and an Assessment of the Underlying Genetic Basis.

The emergence of pyrethroid resistance in the malaria vector, Anopheles arabiensis, threatens to undermine the considerable gains made towards eliminating malaria on Zanzibar. Previously, resistance was restricted to the island of Pemba while mosquitoes from Unguja, the larger of the two islands of Zanzibar, were susceptible. Here, we characterised the mechanism(s) responsible for resistance on Zanzibar using a combination of gene expression and target-site mutation assays. WHO resistance bioassays were conducted using 1-5d old adult Anopheles gambiae s.l. collected between 2011 and 2013 across the archipelago. Synergist assays with the P450 inhibitor piperonyl-butoxide were performed in 2013. Members of the An. gambiae complex were PCR-identified and screened for target-site mutations (kdr and Ace-1). Gene expression in pyrethroid resistant An. arabiensis from Pemba was analysed using whole-genome microarrays. Pyrethroid resistance is now present across the entire Zanzibar archipelago. Survival to the pyrethroid lambda-cyhalothrin in bioassays conducted in 2013 was 23.5-54.3% on Unguja and 32.9-81.7% on Pemba. We present evidence that resistance is mediated, in part at least, by elevated P450 monoxygenases. Whole-genome microarray scans showed that the most enriched gene terms in resistant An. arabiensis from Pemba were associated with P450 activity and synergist assays with PBO completely restored susceptibility to pyrethroids in both islands. CYP4G16 was the most consistently over-expressed gene in resistant mosquitoes compared with two susceptible strains from Unguja and Dar es Salaam. Expression of this P450 is enriched in the abdomen and it is thought to play a role in hydrocarbon synthesis. Microarray and qPCR detected several additional genes putatively involved in this pathway enriched in the Pemba pyrethroid resistant population and we hypothesise that resistance may be, in part, related to alterations in the structure of the mosquito cuticle. None of the kdr target-site mutations, associated with pyrethroid/DDT resistance in An. gambiae elsewhere in Africa, were found on the islands. The consequences of this resistance phenotype are discussed in relation to future vector control strategies on Zanzibar to support the ongoing malaria elimination efforts on the islands

Multiple insecticide resistance in Anopheles gambiae from Tanzania: a major concern for malaria vector control.

BACKGROUND: Malaria vector control in Tanzania is based on use of long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS), which both rely on the use of chemical insecticides. The effectiveness of these control tools is endangered by the development of insecticide resistance in the major malaria vectors. This study was carried out to monitor the susceptibility status of major malaria vectors to insecticides used for IRS and LLINs in mainland Tanzania. METHODS: Mosquito larvae were collected in 20 sites of Tanzania mainland in 2015. Phenotypic resistance was determined using standard WHO susceptibility tests. Molecular assay were used to determine distribution of Anopheles gambiae sub-species. A microplate assay approach was used for identifying enzyme levels on single mosquitoes from each sites compared with a susceptible reference strain, An. gambiae sensu stricto (s.s.) Kisumu strain. RESULTS: Anopheles arabiensis was the dominant malaria specie in the country, accounting for 52% of the sibling species identified, while An. gambiae s.s. represented 48%. In Arumeru site, the dominant species was An. arabiensis, which was resistant to both pyrethroids (permethrin and deltamethrin), and pirimiphos-methyl, and had significant elevated levels of GSTs, non-specific esterases, and oxidase enzymes. An. arabiensis was also a dominant species in Kilombero and Kondoa sites, both were resistant to permethrin and deltamethrin with significant activity levels of oxidase enzymes. Resistance to bendiocarb was recorded in Ngara site where specie composition is evenly distributed between An. gambiae s.s. and An.arabiensis. Also bendiocarb resistance was recorded in Mbozi site, where An. gambiae s.s. is the dominant species. CONCLUSIONS: Overall, this study confirmed resistance to all four insecticide classes in An. gambiae sensu lato in selected locations in Tanzania. Results are discussed in relation to resistance mechanisms and the optimization of resistance management strategies