Recombinant expression and characterization of GSTd3 from a resistant population of Anopheles arabiensis and comparison of DDTase activity with GSTe2.

The development of insecticide resistance in malaria vectors is a challenge for the global effort to control and eradicate malaria. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the detoxification of many classes of insecticides. For mosquitoes, it is known that overexpression of an epsilon GST, GSTe2, confers resistance towards DDT and pyrethroids. In addition to GSTe2, consistent overexpression of a delta class GST, GSTd3, has been observed in insecticide resistant populations of different malaria vector species. However, the functional role of GSTd3 towards DDT resistance has not yet been investigated. Here, we recombinantly expressed both GSTe2 and GSTd3 from Anopheles arabiensis and compared their metabolic activities against DDT. Both AaGSTd3 and AaGSTe2 exhibited CDNB-conjugating and glutathione peroxidase activity and DDT metabolism was observed for both GSTs. However, the DDT dehydrochlorinase activity exhibited by AaGSTe2 was much higher than for AaGSTd3, and AaGSTe2 was also able to eliminate DDE although the metabolite could not be identified. Molecular modeling revealed subtle differences in the binding pocket of both enzymes and a better fit of DDT within the H-site of AaGSTe2. The overexpression but much lower DDT metabolic activity of AaGSTd3, might suggest that AaGSTd3 sequesters DDT. These findings highlight the complexity of insecticide resistance in the major malaria vectors and the difficulties associated with control of the vectors using DDT, which is still used for indoor residual spraying.

Composition of mosquito fauna and insecticide resistance status of Anopheles gambiae sensu lato in Itang special district, Gambella, Southwestern Ethiopia.

BACKGROUND: Anopheles arabiensis, member species of the Anopheles gambiae complex, is the primary vector of malaria and is widely distributed in Ethiopia. Anopheles funestus, Anopheles pharoensis and Anopheles nili are secondary vectors occurring with limited distribution in the country. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are pillars for the interventions against malaria control and elimination efforts in Ethiopia. However, the emergence and widespread of insecticide resistance in An. gambiae sensu lato (s.l.), might compromise the control efforts of the country. The aim of this study was to investigate composition of mosquito fauna and insecticide resistance status of An. gambiae s.l. in Itang special district ( woreda), Gambella, southwestern Ethiopia. METHODS: Adult mosquitoes were sampled from September 2020 to February 2021 using the CDC light trap and pyrethrum spray catch (PSC). CDC light traps were placed in three selected houses for two consecutive days per month to collect mosquitoes indoor and outdoor from 6:00 P.M. to 06:00 A.M. and PSC was used to collect indoor resting mosquitoes from ten selected houses once in a month from October 2020 to February 2021. Moreover, mosquito larvae were also collected from different breeding sites and reared to adults to assess susceptibility status of populations of An. gambiae s.l. in the study area. Susceptibility tests were conducted on two to three days old non blood fed female An. gambiae s.l. using insecticide impregnated papers with deltamethrin (0.05%), alpha-cypermethrin (0.05%), propoxur (0.1%), pirimiphos-methyl (0.25%) and bendiocarb (0.1%) following World Health Organization (WHO) standard susceptibility test procedure. Molecular diagnostics were done for the identification of member species of An. gambiae s.l. and detection of knockdown resistance (kdr) allele using species specific polymerase chain reaction (PCR) and allele specific PCR. RESULTS: In total, 468 adult mosquitoes were collected from different houses. Culex mosquitoes were the most dominant (80.4%) followed by Anopheles mosquitoes. Three species of Anopheles (Anopheles coustani, An. pharoensis, and An. gambiae s.l.) were identified, of which An. coustani was the dominant (8.1%) species. Higher number of mosquitoes (231) were collected outdoor by CDC light traps. Out of 468 adult mosquitoes, 294 were blood fed, 46 were half-gravid and gravid whereas the remaining 128 were unfed. WHO bioassay tests revealed that the populations of An. gambiae s.l. in the study area are resistant against alpha-cypermethrin and deltamethrin, but susceptible to bendiocarb, pirimiphos-methyl and propoxur. Of the total 86 An. gambiae s.l. specimens assayed, 79 (92%) successfully amplified and identified as An. arabiensis. West African kdr (L1014F) mutation was detected with high kdr allele frequency ranging from 67 to 88%. CONCLUSION: The detection of target site mutation, kdr L1014F allele, coupled with the phenotypic resistance against alpha-cypermethrin and deltamethrin call for continuous resistance monitoring.

Residual efficacy of SumiShield™ 50WG for indoor residual spraying in Ethiopia.

BACKGROUND: The rate of decay of the biological efficacy of insecticides used for indoor residual spraying (IRS) is an important factor when making decisions on insecticide choice for national malaria control programmes. A key roadblock to IRS programme is insecticide resistance. If resistance is detected to most of the existing insecticides used for IRS (DDT, pyrethroids, organophosphates and carbamates), the logical next choice could be neonicotinoid insecticides, as pyrethroids are used to treat nets. SumiShield™ 50WG belongs to the neonicotinoid class of insecticides and has shown promising results in several phase I, II and III trials in different settings. The aim of this study was to assess the persistence of SumiShield™ 50WG by spraying on different wall surfaces and determine its decay rates over time in Ethiopia. METHODS: Five huts with different wall surface types (mud, dung, paint and cement) which represented the Ethiopian house wall surfaces were used to evaluate the residual efficacy of SumiShield™ 50WG. Actellic 300CS sprayed on similar wall surfaces of another five huts was used as a comparator insecticide and two huts sprayed with water were used as a control. All huts were sprayed uniformly by an experienced spray operator; non-stop starting from the door and moving clockwise to cover the entire wall surface of the hut. The treatments were assigned to huts randomly. The residual efficacy of the insecticide formulations was evaluated against a susceptible insectary-reared population of Anopheles arabiensis using WHO cone bioassays. RESULTS: SumiShield™ 50WG resulted in mortality rates of over 80% at 120 h post-exposure on all surface types for up to nine months post-spray, while Actellic 300CS yielded mortality rates of over 80% for eight months after spray. CONCLUSIONS: The results of this trial demonstrated that the residual efficacy of SumiShield™ 50WG extends up to nine months on all treated wall surface types. The long-lasting residual efficacy and unique mode of action of the SemiShield™ 50WG shows that it could be an ideal product to be considered as a potential candidate insecticide formulation for IRS in malaria endemic countries such as Ethiopia or other sub-Saharan countries where the transmission season lasts up to four months or longer.