Studies on the bionomics and molecular insecticide resistance of anophelines in Gorgora, north-west Ethiopia

Abstract

Dembia district (wereda) in Gondar, north Ethiopia is one of the malarious areas in the country. An entomological study was carried out in one of the villages, Gorgora, during the malaria transmission seasons in 2006 and 2007 with the main objectives of documenting the species compositions of prevailing anophelines, study the feeding and resting habits, identify blood meal sources, detect Plasmodium infection, determine the susceptibility/resistance status of the main malaria vectors and detect the mechanisms of resistance. Anophelines were captured using Center for Disease Control (CDC) light trapnets, aspirators, space sprays and pit shelters. Morphological features were used to discriminate anophelines into species, and the polymerase chain reaction (PCR) was employed to screen members of the Anopheles gambiae complex. Sprozoite and blood meal ELISA analysis were carried out to detect infections and identify blood meal sources. Insecticide susceptibility status of An. arabiensis and An. pharoensis to DDT, permethrin, deltamethrin and malathion was evaluated using standard bioassay WHO test kits on adults reared from larvae and pupae. For comparative reasons, An. arabiensis from Sodere in central and Ghibe River Valley in south west Ethiopia were additionally included. Colonies were established from surviving individuals for assessing the occurrence of true as well as cross resistance. Genetic and metabolic resistance mechanisms on An. arabiensis were investigated by conducting PCR and micro plate biochemical assays, respectively. Overall, 5,664 in 2006 and 3,106 in 2007 adult female comprising 9 Anopheles species were captured predominantly from CDC light trap-nets. The dominant species was An. pharoensis Theobald comprising 70% of collections in 2006 and 54% in 2007 followed by An. arabiensis Patton accounting to 20% and 18% in the two consecutive years. The other species made up the remaining 10% and 28% of collections in those years. The density of An. arabiensis was highest from July to September and reduced in October and November, in contrast, An. pharoensis peaked from September to November. III CDC trap-nets collected all gonotrophic stages of An. arabiensis and An. pharoensis. The geometric mean of the blood unfed stages were considered relevant to determine the human biting rate (HBR) which corresponds to mosquitoes/trap/night, and hence, the HBR of An. arabiensis during the five months (July-November) in 2006 ranged from the lowest 0.4 to the highest 0.7 mosquitoes/man/night. The HBR of An. pharoensis for the same period was between 0.3 and 1.4 mosquitoes/man/night. There was no detectable difference in biting rates in 2007. From the CDC trap-net collections, blood meals of 255 An. arabiensis and 206 An. pharoensis were analyzed, out of which 64.7% and 36.9% were found to contain human blood, respectively. The proportion of cattle fed mosquitoes in that order was 27.5% and 44.7%. The rest of the blood meals contained both mixed human and cattle or were non-reactive. Only 4 An. arabiensis were captured from human habitations with aspirator in 2006 and 33 in 2007 using space sprays. Similarly, An. pharoensis was caught in small numbers. Both species were collected from animal shelters and the density of An. arabiesnsis varied from 0 to 7.5 mosquitoes/shelter/day while that of An. pharoensis was 0 to 14.8 mosquitoes/shelter/day at different months. However, the latter species density reached to 234 mosquitoes/shelter/day from animal shelters in Toka Agroindustrial Enterprise. It is, however, difficult to surmise animal shelters in the enterprise serve as resting sites since fresh fed mosquitoes were collected on window screens in the act of egression. From animal shelter collections, 59 An. arabiensis and 86 An. pharoensis blood meals were analyzed. The respective human blood index of the two species was 0.17 (n=1) and 0.58. (n=5). The proportion fed on cattle was 76.3% (n=45) and 50% (n=46), respectively while the rest of the blood meals were unidentified either human or bovine. More than 1000 An. arabiensis and 800 An. pharoensis were examined for parasite infection against circumsporozoite proteins of Plasmodium falciparum and P. vivax 210 and 247 polymorphs.but none was found with parasite infections. Insecticide bioassay test results of both mortality and knockdown time revealed very high DDT resistant An. arabiensis from Ghibe (96.2%) followed by Gorgora (50.5%). DDT IV failed to produce 100% knock-down in all test mosquitoes. An. arabiensis from Ghibe was also greatly resistant to permethrin (69.3%) and deltamethrin (69.7%), while the strain from Gorgora was less resistant (15-19%). Permethrin was less effective to cause complete knock-down in the vector from Ghibe while deltamethrin took an elevated KT50 of 37.6 minutes. The respective KT 50 values of the two pyrethroids on An. arabiensis from Gorgra were 19.6 and 25.3 minutes. The strain from Sodere can be considered moderately resistant (20%) to DDT and highly susceptible to the pyrethroids if mortality data are only taken into consideration, however, failure of DDT to knockdown all test mosquitoes and higher KT50 of 17.8 minutes for permethrin and 21.9 minutes for deltamethrin indicate presence of considerable resistance which needs regular follow up. The existence of cross resistance between DDT and permethrin was confirmed on DDT selected An. arabiensis from Gorgora, where as, it is absent between DDT and deltamethrin. The West African type kdr mutation was found in the vector population from Gorgora and Ghibe, and this molecular mechanism probably confers resistance to DDT and the pyrethroids in addition to metabolic enzymes. Biochemical assays on DDT selected An. arabiensis from Sodere revealed more glutathione-S-transferase and general estrases activities than the Debrezeit susceptible strain. DDT resistance in An. pharoensis from Gorgora was very high reaching up to 93.8%. The knock-down time for the two pyrethroids is longer than reference susceptible strains. Both species are susceptible to malathion