This year, the Pan-African Mosquito Control Association (PAMCA)-Cameroon annual conference gave its stakeholders the opportunity to think critically on the quality of research they are doing. CRID researchers contributed to the brainstorming process.
Binyang Jerome Achille, PhD student
“L1014F allele is associated with low plasmodium sporozoites prevalence and intensity in anopheles coluzzii mosquito salivary glands”.
Pyrethroid resistance in African vector mosquitoes is a threat to malaria control. However, in the context of escalation of insecticide resistance, very little information is available on the impact of this phenomenon on the development of malaria parasite in anopheles mosquito. This study therefore explores the impact of L1014F (knockdown resistance “kdr”) mutation on the outcome of Plasmodium falciparum sporozoite in the salivary glands of Anopheles coluzzii mosquito species.
It was observed that, infection rate among live mosquitoes was high (74.47%). Also, infection rate according to kdr genotype showed that, prevalence of infections was high in homozygote susceptible (SS) compared to homozygote resistant (RR). The Intensity of infection was higher in SS than in RR mosquitoes. The expression of salivary glands receptor trio was associated with the intensity of infection in SS and RS compared to RR. For this study, resistant mosquitoes were collected at immature stages in breeding sites from the field and further cross with the full susceptible lab strain (Ngousso strain). Females from F3 (Third filial generation of offspring) generation aged from 3 to 5 days were experimentally infected with natural isolates of parasites. The infection prevalence (proportion of infected mosquitoes) and intensity (number of Plasmodium falciparum sporozoites in the salivary gland) in female mosquitoes were measured using qPCR (Quantitative Polymerase Chain Reaction) assay and were compared with mosquitoes bearing Kdr resistant and susceptible genotypes. We also assess the expression level of some salivary gland receptor of plasmodium: Trio, Saglin, Cspbp, Sgs4 and Sgs5 in different gene type. We can therefore conclude that, prevalence of infection is low in RR and RS than SS. The expression of genes encoding sporozoite protein-receptors in salivary gland is under express in RR and RS in comparison to SS.
Yvan Gaétan FOTSO TOGUEM, PhD student
“Polymorphism of Cyp6m2, a main metabolic resistance gene in Anopheles gambiae from Yaoundé, Cameroon”.
The rapid spread of insecticide resistance, mainly caused by a recent selection of resistance in vectors population linked to LLINs (Long lasting insecticidal nets) usage, is a major threat to the effectiveness of malaria control tools. In Africa, increased expression of Cyp6m2 (Cytochrome P450 annotated gene), a metabolic detoxification enzyme in resistant mosquitoes has been associated to Permethrin resistance in many studies. However, mutations that may modulate the expression of Cyp6m2 in anopheles gambiae remains unknown.
High levels of Permethrin resistance was observed in anopheles gambiae mosquitoes caught in the field with a partial recovery to susceptibility after pre-exposure to PBO compared to susceptible lab strain. Analysis of 930 bp fragments of 5’UTR region revealed 70 polymorphic sites and low haplotype diversity, with an InDel of 8bp, while analysis of 1.5kb (Kilo base pairs) of gene showed 92 polymorphic sites and high haplotype diversity with one non-synonymous mutation A392S. However, no strong association was observed between the polymorphism of these genes and their overexpression. In this study, anopheles gambiae mosquitoes were collected in Nkolondom in the Central Cameroon and bioassays were performed to determine the level of resistance. Real-time quantitative polymerase chain reaction (qPCR) was used to measure the expression level of Cyp6m2, the gene and 5’UTR region were sequenced for polymorphism analysis. This work provides important preliminary data that will be useful for any study on the genetic polymorphism of Cyp6m2 and the identification of markers for this gene.
Amen N. Fadel, CRID post-doc researcher
“Exploring the molecular mechanisms underlying potential escalation of insecticide resistance in a major malaria vector anopheles coluzzii from Northern Cameroon”.
High pyrethroid resistance and profound loss of efficacy of Piperonyl Butoxide (PBO ), an inhibitor of cytochrome P450s contained in long-lasting insecticidal nets was recently observed in anopheles coluzzii from Gounougou. The intensity of resistance and its underlying molecular mechanisms for its escalation was investigated in anopheles Coluzzii, a major malaria vector from Northern Cameroon.
Blood fed anopheles gambiae sensu lato collected at Gounougou in August, 2019 were forced to lay eggs and Plasmodium infection in 77 F0 (Offspring field collected) females was determined successfully using TaqMan assay. Resistance profiles and intensity of resistance were established. Synergist bioassays were conducted with Diethyl maleate for Dichlorodiphenyltrichloroethane (DDT) and Piperonyl Butoxide for Permethrin. Anopheles coluzzii was the only malaria vector collected in Gounougou with Plasmodium falciparum infection rate of 3.89 %. High Permethrin and Deltamethrin resistance were observed in F1 females. Intense Pyrethroid resistance was obtained with exposure to 5x and 10x discriminating concentrations of Permethrin. High DDT resistance and moderate Bendiocarb resistance were observed, while full susceptibility to Malathion was obtained. Synergist bioassays with DEM (Diethyl maleate, an inhibitor of Glutathion S-transferases) significantly recovered some susceptibility to Permethrin and DDT, implicating Glutathion S-transferases. Synergist bioassays with PBO recovered significant susceptibility with increased mortality with 5x and 10x Permethrin. RNA-seq identified GSTe2 and CYP6Z2 as the most commonly overexpressed metabolic genes in Pyrethroid-resistant mosquitoes compared with Ngousso colony. High frequency of 1014F kdr was observed in F0 females, in contrast to a low frequency of 1014S and 1575Y. No ace-1 mutation was detected in the bendiocarb-resistant populations suggesting metabolic resistance mechanisms.The finding of metabolic resistance genes GSTe2 and CYP6Z2 (Glutathion S-transferase epsilon2 and Cytochrome P450s) highly overexpressed in field populations could explain the escalated resistance, calling for caution in implementation of resistance management.