Investigating the molecular basis of multiple insecticide resistance in a major malaria vector Anopheles funestus (sensu stricto) from Akaka-Remo, Ogun State, Nigeria.

BACKGROUND: Understanding the mechanisms used by Anopheles mosquitoes to survive insecticide exposure is key to manage existing insecticide resistance and develop more suitable insecticide-based malaria vector control interventions as well as other alternative integrated tools. To this regard, the molecular basis of permethrin, DDT and dieldrin resistance in Anopheles funestus (sensu stricto) at Akaka-Remo was investigated. METHODS: Bioassays were conducted on 3-5-day-old adult An. funestus (s.s.) mosquitoes for permethrin, DDT and dieldrin susceptibility test. The molecular mechanisms of mosquito resistance to these insecticides were investigated using microarray and reverse transcriptase PCR techniques. The voltage-gated sodium channel region of mosquitoes was also screened for the presence of knockdown resistance mutations (kdr west and east) by sequencing method. RESULTS: Anopheles funestus (s.s.) population was resistant to permethrin (mortality rate of 68%), DDT (mortality rate of 10%) and dieldrin (mortality rate of 8%) insecticides. Microarray and RT-PCR analyses revealed the overexpression of glutathione S-transferase genes, cytochrome P450s, esterase, trypsin and cuticle proteins in resistant mosquitoes compared to control. The GSTe2 was the most upregulated detoxification gene in permethrin-resistant (FC = 44.89), DDT-resistant (FC = 57.39) and dieldrin-resistant (FC = 41.10) mosquitoes compared to control population (FC = 22.34). The cytochrome P450 gene, CYP6P9b was also upregulated in both permethrin- and DDT-resistant mosquitoes. The digestive enzyme, trypsin (hydrolytic processes) and the cuticle proteins (inducing cuticle thickening leading to reduced insecticides penetration) also showed high involvement in insecticide resistance, through their overexpression in resistant mosquitoes compared to control. The kdr east and west were absent in all mosquitoes analysed, suggesting their non-involvement in the observed mosquito resistance. CONCLUSIONS: The upregulation of metabolic genes, especially the GSTe2 and trypsin, as well as the cuticle proteins is driving insecticide resistance of An. funestus (s.s.) population. However, additional molecular analyses, including functional metabolic assays of these genes as well as screening for a possible higher cuticular hydrocarbon and lipid contents, and increased procuticle thickness in resistant mosquitoes are needed to further describe their distinct roles in mosquito resistance.

Impact of PermaNet 3.0 on entomological indices in an area of pyrethroid resistant Anopheles gambiae in south-western Nigeria.

BACKGROUND: PermaNet® 3.0 is an insecticide synergist-combination long-lasting insecticidal net designed to have increased efficacy against malaria vectors with metabolic resistance, even when combined with kdr. The current study reports on the impact of this improved tool on entomological indices in an area with pyrethroid-resistant malaria vectors in Nigeria. METHODS: Baseline entomological indices across eight villages in Remo North LGA of Ogun State provided the basis for selection of three villages (Ilara, Irolu and Ijesa) for comparing the efficacy of PermaNet® 3.0 (PN3.0), PermaNet® 2.0 (PN2.0) and untreated polyester nets as a control (UTC). In each case, nets were distributed to cover all sleeping spaces and were evaluated for insecticidal activity on a 3-monthly basis. Collection of mosquitoes was conducted monthly via window traps and indoor resting catches. The arithmetic means of mosquito catches per house, entomological inoculation rates before and during the intervention were compared as well as three other outcome parameters: the mean mosquito blood feeding rate, mean mortality and mean parity rates. RESULTS: Anopheles gambiae s.l. was the main malaria vector in the three villages, accounting for >98% of the Anopheles population and found in appreciable numbers for 6-7 months. Deltamethrin, permethrin and lambdacyhalothrin resistance were confirmed at Ilara, Irolu and Ijesa. The kdr mutation was the sole resistance mechanism at Ilara, whereas kdr plus P450-based metabolic mechanisms were detected at Irolu and Ijesa. Bioassays repeated on domestically used PN 2.0 and PN 3.0 showed persistent optimal (100%) bio-efficacy for both net types after the 3rd, 6th, 9th and 12th month following net distribution. The use of PN 3.0 significantly reduced mosquito densities with a 'mass killing' effect inside houses. Households with PN 3.0 also showed reduced blood feeding as well as lower mosquito parity and sporozoite rates compared to the PN 2.0 and the UTC villages. A significant reduction in the entomological inoculation rate was detected in both the PN 2.0 village (75%) and PN 3.0 village (97%) post LLIN-distribution and not in the UTC village. CONCLUSION: The study confirms the efficacy of PN 3.0 in reducing malaria transmission compared to pyrethroid-only LLINs in the presence of malaria vectors with P450-based metabolic- resistance mechanisms.

Abundance and diversity of Anopheles species (Diptera: Culicidae) associated with malaria transmission in human dwellings in rural and urban communities in Oyo State, Southwestern Nigeria.

Mosquito samples were collected from rural and urban communities in three selected major towns in Southwestern Nigeria to determine the impact of urbanization on the diversity and abundance of Anopheles species associated with malaria transmission in human habitations. A total of ten Anopheles species were identified in the rural communities, while eight Anopheles species were identified in the urban communities. Out of the ten Anopheles species identified, only four species, Anopheles gambiae (Giles), Anopheles funestus (Giles), Anopheles moucheti (Evans), and Anopheles nili (Theobald), were established to be vectors of malaria occurring in greater than 50% of the rural communities. Only A. gambiae occurred in all the urban communities, while the other three major vectors occurred in not more than 20% of the urban communities. Margalef's and Shannon-Wiener indices showed that diversity and species richness were higher in the rural compared to the urban. Comprehensive information on malaria vector abundance and diversity in rapidly changing communities is an important tool in planning and implementing successful vector control programs.