Species Composition
297 adult female Anopheles mosquitoes (55.5% An. gambiae s.l and 44.5% An. funestus s.s) were collected indoor in Ndjili-Brasserie. All samples (60) from the An. funestus group were molecularly confirmed as being An. funestus s.s. while 59 out of the 60 F0 (98.33%) An. gambiae s.l, belonged to An. gambiae s.s. species and one (1.67%) was An. coluzzii.
Plasmodium Infection Rate
A total of 101 An. funestus was tested for Plasmodium infection using TaqMan. The analysis revealed 8.9% (9/101) of mosquitoes infected with P. falciparum only with 7 out of these 9 confirmed by nested PCR as P. falciparum (Pf+). Infection rate was higher in An. gambiae s.l. with 25.21% (29/115) of mosquitoes infected with P. falciparum, 0.8% (1/115) infected with P. ovale, P. vivax, and/or P. malariae (OVM+) and 4% (5/115) were co-infected (Pf+/OVM+). The nested PCR failed to confirm 2 Pf + infections and 2 mix infections (Pf+/OVM+). Overall, the nested PCR confirmed 29 mosquitoes infected with Pf+, one (1) infected with P. malariae, one (1) infected with P. falciparum + P.ovale and 3 infected with P. falciparum + P. malariae.
Compared to the Plasmodium infection rate results obtained in 2015, we broadly observed a reduction in the Plasmodium infection rate in the An. funestus population (30% in 2015 vs. 8.9% in 2021; P = 0.0003), and not difference in An. gambiae population (41.28% in 2015 vs. 30.4% in 2021; P = 0.09) .
Insecticide Susceptibility Assays
Bioassays with the Discriminating Concentration 1X (DC) in An. gambiae s.l. and An. funestus s.s.
F1 progeny of An. gambiae s.l. from this field population showed an extremely high resistance to type I and type II pyrethroids. For permethrin (Type I), mortality was 2.05 ± 1.19%. For deltamethrin and α-cypermethrin (Type II), mortality was 12.18 ± 4.88% and 23.64 ± 7.27% respectively. High resistance was also observed for the organochlorine (DDT) with 0% mortality. However, full susceptibility was observed with the organophosphate (pyrimiphos-methyl) and carbamate (bendiocarb) with a 100% mortality rate (Fig. 1a).
The An. funestus s.s. population was resistant to permethrin (43.15% ± 4.7%), deltamethrin (28.7% ± 5.92%), α-cypermethrin (45.47% ± 17.5%) and DDT (18.6% ± 1.58%), but only moderately resistant to bendiocarb (94.44% ± 1.2%) (Fig. 1b). There were not enough of mosquitoes to perform pyrimiphos-methyl bioassay test with An. funestus.
Bioassays with Pyrethroid 5X and 10X DC in An. funestus s.s. and An. gambiae s.l.
Bioassays were carried out with 5X DC and 10X DC of permethrin (3.75% and 7.5%) and deltamethrin (0.25% and 0.5%) to assess the resistance intensity. According to WHO criteria this population is highly resistant because we have mortality < 98% at 10X. Hence, An. funestus s.s exhibited a mortality rate of 87.48% ± 1.96% and 92.62 ± 2.76% to permethrin 5X and 10X respectively (Fig. 2b). An. gambiae s.l showed a mortality rate of 82.68% ± 1.91% and 92.7% ± 1.05% respectively with permethrin 5X and 10X (Fig. 2a). However, Higher intensity resistance was observed with deltamethrin 5X and 10X with respective prevalence of 46.92% ± 3.38% and 69.38% ± 3.34% (Fig. 2a).
PBO synergist assays with An. gambiae s.l.
Because of the limited number of An. funestus, synergist assays were carried out only with An. gambiae s.l. The synergist assay results showed a slight recovery of susceptibility to deltamethrin and α-cypermethrin. An increased mortality was observed after PBO exposure from 2.05 ± 1.19% to 33.82 ± 7.21% (ꭓ2 = 32.05; p < 0.0001) for permethrin and from 23.64 ± 7.27% to 88.44 ± 2.54% (ꭓ2 = 95.47; p < 0.0001) mortality for α-cypermethrin (Fig. 1a). However, PBO led to full recovery of susceptibility to deltamethrin from 12.18 ± 4.88–100% mortality (ꭓ2 = 77.48; p < 0.0001). These results show that cytochrome P450s are playing a greater role in the escalation of resistance to type II pyrethroids (deltamethrin and alphacypermethrin) than to type I (permethrin) in An. gambiae population from NDjili.
Aggravation of Pyrethroid resistance in An. gambiae s.l. and An. funestus s.s. between 2015 and 2021
Bioassay results showed a significantly overall increase resistance intensity to pyrethroids and carbamates in 2021 compared to 2015. The mortality rate of An. gambiae after exposure to deltamethrin reduces from 67% in 2015 to 12% in 2021 (ꭓ2 = 58, P < 0.0001). (Fig. 1a). In contrast, Increase mortality rate was observed for the Bendiocarb (79% vs. 100%; ꭓ2 = 16, P < 0.0001). However, no difference in mortality was observed when we compared mosquitoes exposed to permethrin 1X in 2015 and 2021 (0 vs 2%; P < 0.05) (Fig. 1a).
In An funestus, a significant reduction in mortality rate was noticed between 2021 and 2015 for DDT (34% vs. 19%; ꭓ2 = 18.7, P < 0.001), deltamethrin (64.6% vs. 29.6%; ꭓ2 = 18.7, P < 0.0001), and Permethrin (64.6% vs. 43%; ꭓ2 = 6.9, P = 0.008) (Fig. 1b) while no statistical difference was observed in An. funestus population exposed to bendiocarb.
Bioefficacy of LLINs using cone assays in An. funestus s.s. and An. gambiae s.l
Low efficacy was recorded against most of the nets tested in An. funestus except with Olyset plus. The mortality rate was 0% for Olyset Net and PermaNet 2.0. However, Olyset Plus, and PermaNet 3.0 Top (PBO-based nets) showed a significant increased efficacy with 100% and 84.52 ± 1.19% of mortality respectively (Fig. 3b).
In An. gambiae s.l., a lower efficacy was observed for all the nets tested with mortality rate of 0 ± 0%, 6.11 ± 3.09%, 3.64 ± 3.64% and 5.45 ± 3.64% respectively for Olyset net, PermaNet 2.0, PermaNet 3.0 side and Interceptor (α-cypermethrin-based net) (Fig. 3a). The PBO-based nets showed a significant increased efficacy with 43.64 ± 6.03% for Olyset Plus and 26.36 ± 9.11% for PermaNet 3.0 Top. Royal guard (α-cypermethrin + pyriproxyfen), a new generation net, showed mortality rate of 28.69 ± 12.67 (Fig. 3a).
As observed with WHO tube tests, cone assays for bed net efficacy revealed also a significant reduction in mortality rate between 2021 and 2015 in An. gambiae mosquitoes after exposure to PermaNet 2.0 (33% vs. 6%; ꭓ2 = 7, P = 0.007), PermaNet 3.0 side (82% vs. 4%; ꭓ2 = 35.8, P < 0.0001) and PermaNet 3.0 top (86% vs. 35%; ꭓ2 = 16; P < 0.0001) and an increase in mortality with Olyset+ (43.6% vs 32.7%; ꭓ2 = 1.2, P = 0.25) (Fig. 3a). In An funestus, we recorded the same decrease in mortality with PermaNet 2.0 (24% vs. 0%; ꭓ2 = 5 P = 0.02), PermaNet 3.0 top (86% vs. 35%; ꭓ2 = 9, P < 0.0001), and Olyset (68% vs. 0%; ꭓ2 = 24.8, P < 0.0001) (Fig. 3b).
Genotyping of insecticide resistance markers in An. funestus
In An. funestus s.s., the A296S-RDL mutation which confers resistance to dieldrin, was detected. The 296S-resistant allele (R) frequency was low (16.7%) with genotype frequency of 26.4% RS and 71.6% SS and 2% RR (Fig. 4a). This frequency was not different (P = 0.13) compared to 2015 (10%). In contrast, the high allelic frequency of the 119F-GSTe2 significantly increased in 2021 (80.4%) compared to 2015 (66.6%) (P = 0.02) with 70.5% of the individuals homozygote resistant (RR), 9.8% of homozygous susceptible (SS) and 19.6% heterozygous (RS) (Fig. 4b). The, Cyp6P9a_R and Cyp6P9b_R resistance markers conferring pyrethroid resistance were for the first time detected although at low frequency: 1.7% and 1.8% respectively in contrast to 2015 where they were completely absent (Fig. 4a). The 6.5kb insertion conferring the resistance to pyrethroid in the malaria vector remains absent in this population (100% SS).
Changes in the frequency of Insecticide Resistance Markers in An. gambiae between 2021 and 2015
The L1014F-KdrW mutation was close to fixation in NDjili population with 98.3% of homozygous resistant individuals (RR) and 1.7% homozygous susceptible ones (SS). No difference was obtain with samples collected in 2015 (94% RR and 6% SS). However, the L1014S-KdrE resistant (R) allele was low (6%) with 89.9% SS, 8.4% RS and 1.7% RR (Fig. 5a). The Kdr East susceptible allele frequency has significantly increased in 2021 (94% (2021) Vs 77.5% (2015); P = 0.004) (Fig. 5b). The N1575Y-kdr mutation associated with pyrethroid resistance and the G119S-Ace1 mutation conferring carbamate and organophosphate resistance were completely absent (Fig. 5a).
Transcriptional profiling of metabolic resistance genes in An. funestus s.s.
The cytochrome P450 genes CYP6P9a, CYP6P9b, CYP6M7, CYP9K1 and the glutathione s-transferase GSTe2 previously shown to be conferring pyrethroid resistance in An funestus 25–27 were significantly over-expressed in DDT and permethrin resistant mosquitoes of Kinshasa compared to the susceptible FANG strain. A respective fold change of 19.69, 24.81, 7.69, 2.72, and 2.202 was obtained for these genes whereas the other P450, CYP6P4b, was rather downregulated with a fold change of 0.3 (Fig. 6a). Furthermore, expression level of CYP6M7, GSTe2 and P450 duplicated genes CYP6P9a and CYP6P9b was significantly higher in An. funestus collected 2021 compared to in 2015 especially for CYP6P9a and CYP6P9b (P < 0.001). (Fig. 6b).
For An. gambiae, the transcription pattern of CYP4G16 and CYP4G17, (associated with cuticular resistance) SAP1, SAP2, SAP3, GSTe2, CYP6P1, CYP6P3, CYP6P4, CYP6Z1, CYP6Z2, CYP9K1 and CYP6M2, evolving in P450 resistance were assessed and most of them were down- regulated (Fig. 7).