Taxinomie et variations spatio-temporelles de la résistance d'Anopheles gambiae s.l. Giles, 1902 (Diptera: Culicidae) à la deltaméthrine dans la Région du Nord-Cameroun

Abstract

The development and rapid expansion of Anopheles gambiae s.l. resistance to insecticides in Africa jeopardize the efforts to prevent and control malaria. The present study aimed to characterize the dynamics of deltamethrin resistance in A. gambiae s.l. from North Cameroon; more specifically the objectives are to identify morphologically and by molecular markers the species of the A. gambaie complex involved in insecticide resistance and to evaluate the spatiotemporal variations of this resistance while determining the different mechanisms involved. Mosquito larvae were collected every year between October and November, for five consecutive years (2011-2015) in the Garoua, Pitoa and Mayo Oulo Health Districts (HDs) from North Cameroon. Taxonomic studies and molecular biology analyzes were carried out between April 2013 and July 2017. Mosquito specimens used for the morphological studies were dissected; the wings were gently removed from the body and mounted between blade and lamina. Susceptibility of mosquitoes to deltamethrin was assessed using the WHO susceptibility test protocol for adult mosquitoes. The remaining parts of the dissected mosquito specimen and those tested for deltamethrin susceptibility were identified down to species using PCR-RFLP. The kdr 995 alleles were genotyped using the hot oligonucleotide ligation assay (HOLA). A total of 229 specimens were examined for taxonomic studies, including 142 females and 87 males. Among these specimens, three species of the A. gambiae complex were identified : mainly A. arabiensis (52%), followed by A. coluzzii (33%) and A. gambiae s.s. (15%). Morpho-taxonomic studies revealed the existence of a very high inter and intraspecific variability, especially in A. arabiensis and A. coluzzii, compared to A. gambiae s.s.; indeed, we observed 28 variants on the wing veins. In females, 13 pigment characters allowed to obtain 31 morphotypes. Among these morphotypes, three were only found in A. arabiensis (Mor1-Mor3), nine in A. coluzzii (Mor14-Mor22) and four in A. gambiae s.s. (Mor23-Mor26). Ten morphotypes were found in A. arabiensis and A. coluzzii (Mor4-Mor13) and five in the three species (Mor27- Mor31). On the other hand, in males, these different characters resulted in 28 morphotypes. Among these morphotypes, nine were observed only in A. arabiensis (Mor32-Mor37, Mor29, Mor30 and Mor23), eight in A. coluzzii (Mor43-Mor50) and one in A. gambaie s.s. (Mor52). Three morphotypes were found in A. arabiensis and A. coluzzii (Mor5, Mor38 and Mor14), three in A. arabiensis and A. gambiae s.s. (Mor39, Mor40 and Mor4) and two in A. coluzzii and A. gambiae s.s. (Mor51 and Mor15). Two morphotypes were found in the three species (Mor41 and Mor42). Moreover, in both males and females, three morphotypes were only found in A. arabiensis (Mor4, Mor29 and Mor30), two in A. coluzzii (Mor4, Mor29) and one (Mor5) was only common to A. arabiensis and A. coluzzii. Susceptibility tests of A. gambiae s.l. to 0.05% deltamethrin xxviii populations showed a significant increase in resistance between 2011 and 2015, with a decrease in mortality rates from 70-85% to 49-73% in the three HDs JT = 5638, p < 0.001). Resistance was higher in urban settings compared to peri-urban and rural settings (JT = 5282, p < 0.0001), with a positive spatial relationship during the whole study period from 2012 to 2015. Molecular analysis of 2687 mosquitoes tested with deltamethrin revealed three species : A. arabiensis (68%), A. coluzzii (25%) and A. gambiae s.s. (7%). Molecular analysis at the « kdr » 995 locus revealed the presence of the L995F kdr mutation responsible for resistance to pyrethroids and DDT, with 18% allelic frequencies in A. arabiensis, 65% in A. coluzzii and 58% in A. gambiae s.s.. In addition, allelic frequencies increased from 0-30% in 2011 to 18-61% between 2014 and 2015 in the three health districts (JT = 620, p < 0.001), especially in A. coluzzii. The L995S allele was only identified at very low frequencies in A. arabiensis (< 1%) and A. coluzzii (2%). The use of synergists (PBO 4%, DEF 0.25%) in combination with deltamethrin on the tested mosquito populations induced an increase of susceptibility i.e., decrease in knockdown times and a significant increase in mortality up to 100% (p < 0.05). The present study shows a diversity of pigmentary characters on mosquito wings and a rapid and widespread increase of deltamethrin resistance in A. arabiensis, A. coluzzii and A. gambiae s.s., associated with two main mechanisms: the kdr 995 mutations at and the activity of detoxification enzymes such as oxidases and esterases ; this multiple resistance could lead to a drastic decrease in the effectiveness of deltamethrin-based long-lasting insecticidal nets (LLINs). It is therefore essential to seek complementary or alternative tools such as mosquito nets impregnated with insecticides+PBO or impregnated with new classes of insecticides such as chlorphenapyr (Pyrrole class) which is a new generation insecticide, to strengthen the prevention of malaria in the North Region of Cameroon.