Evidence of intensification of pyrethroid resistance in the major malaria vectors in Kinshasa, Democratic Republic of Congo

Assessing patterns and evolution of insecticide resistance in malaria vectors is a prerequisite to design suitable control strategies. Here, we characterised resistance profile in Anopheles gambiae and Anopheles funestus in Kinshasa and assess the level of aggravation by comparing to previous 2015 estimates. Both species collected in July 2021 were highly resistant to pyrethroids at 1×, 5× and 10× concentrations (mortality < 90%) and remain fully susceptible to bendiocarb and pirimiphos methyl. Compared to 2015, Partial recovery of susceptibility was observed in A. gambiae after PBO synergist assays for both permethrin and α-cypermethrin and total recovery of susceptibility was observed for deltamethrin in 2021. In addition, the efficacy of most bednets decreased significantly in 2021. Genotyping of resistance markers revealed a near fixation of the L1014-Kdr mutation (98.3%) in A. gambiae in 2021. The frequency of the 119F-GSTe2 resistant significantly increased between 2015 and 2021 (19.6% vs 33.3%; P = 0.02) in A. funestus. Transcriptomic analysis also revealed a significant increased expression (P < 0.001) of key cytochrome P450s in A. funestus notably CYP6P9a. The escalation of pyrethroid resistance observed in Anopheles populations from Kinshasa coupled with increased frequency/expression level of resistance genes highlights an urgent need to implement tools to improve malaria vector control.

Insecticide susceptibility assays.Bioassays with the discriminating concentration 1× (DC) in A. gambiae s.l. and A. funestus s.s.The F 1 progeny of A. 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 2.1 ± 1.2%.For deltamethrin and α-cypermethrin (Type II), mortality was 12.2 ± 4.9% and 23.6 ± 7.3% respectively.High resistance was also observed for the organochlorine (DDT) with 0% mortality.However, full susceptibility was observed with the organophosphate (pirimiphos-methyl) and carbamate (bendiocarb) with a 100% mortality rate (Fig. 1a).
PBO synergist assays with A. gambiae s.l.Because of the limited number of A. funestus, synergist assays were carried out only with A. 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 2.0 ± 1.2 to 33.8 ± 7.2% (χ 2 = 32.0;P < 0.0001) for permethrin and from 23.6 ± 7.3 to 88.4 ± 2.5% (χ 2 = 95.5;P < 0.0001) mortality for α-cypermethrin (Fig. 1a).However, PBO led to full recovery of susceptibility to deltamethrin from 12.2 ± 4.9 to 100% mortality (χ 2 = 77.5;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 A. gambiae population from NDjili.
Bioefficacy of LLINs using cone assays in A. funestus s.s. and A. gambiae s.l.Low efficacy was recorded against most of the nets tested in A. funestus except with Olyset plus and PermaNet 3.0 roof.The mortality rate was 0% for Olyset and PermaNet 2.0.However, Olyset Plus, and PermaNet 3.0 roof (PBO-based nets) did not show a difference of efficacy between 2015 and 2021 (Fig. 3b).

Discussion
The increase intensity in insecticide resistance in Anopheles vectors across Africa is threatening the effectiveness of the vector control tools.The extent of this resistance and the major molecular drivers were investigated in the Capital city of DRC revealing key findings in both major vectors A. funestus and A. gambiae.
Plasmodium infection rate.The two major malaria vectors were predominant (A.gambiae s.l., and A. funestus ss) in this location of Kinshasa.High Plasmodium infection rate was observed in A. gambiae (30.25%) and lower in A. funestus (9%) suggesting that malaria transmission is actually driven mainly by An gambiae in this area.This result is similar to that obtained by Riveron et al. 5 in DRC regarding the Plasmodium infection rate of A. gambiae during the rainy season but the Plasmodium infection rate observed in A. funestus was smaller than that observed in 2015 in the same location 5 .This greater infection rate in A. gambiae could be associated with the higher intensity of resistance in this species with for example low mortality rates observed even at 5× (46.92% ± 3.38%) and 10× (69.38 ± 3.34%) whereas higher mortalities were observed in A. funestus.Furthermore, with A. gambiae, the infection rate observed in this study was higher than those obtained in Cameroon  www.nature.com/scientificreports/(20%) 15 .Overall, the results reveal the high malaria transmission rate in Kinshasa, and corroborate that DRC is a highly endemic country with 12% of all malaria cases worldwide in 2021 1 .

Increasing resistance intensity in both A. gambiae and A. funestus. Overall, the bioassay results
showed a significantly increased in resistance to pyrethroids and DDT in A. funestus and A. gambiae in 2021 compared to 2015 profiles.This high resistance level against pyrethroids is likely driven by increasing insecticide pressures due to LLIN-based control interventions on roof of the local practice of agriculture.The same trend was observed in A. funestus with an increased resistance between 2015 and 2021 with the mortality reducing from 64.7 to 43.15% for permethrin and from 87.7 to 28.7% for deltamethrin.A similar increase resistance was seen against DDT with mortality reducing from 33.8 to 18.6% 5 .Interestingly no resistance was observed to organophosphates and carbamates as already reported back in 2015 suggesting that these insecticide classes could be alternatively used for IRS in this location.A similar level of increase resistance to pyrethroids and susceptibility to carbamates was also recently reported in Uganda (Tchouakui et al. 2020) and Cameroon 15 .This increase in level of insecticide resistance could be due to the massive distribution of LLINs in DRC (PermaNet 2.0, DawaPlus 2.0 in Kinshasa, 2016; Dawa + 2.0 and Yorkool in Kongo central, 2017) 9 .In addition, Ndjili-brasserie is also located in an area of intensive agriculture with massive use of pesticides, which could be another factor driving the increased level of resistance in this area.
A. funestus and A. gambiae populations were both resistant to type I and II pyrethroids at all diagnostic concentrations of 1×, 5×, and 10×.The high resistance to pyrethroids at all diagnostic doses in A. funestus is similar to the observations made in Uganda a neighbouring country of the DRC 10 and Malawi 2 .On the other hand, the resistance escalation observed in A. gambiae is similar to results made in DRC 9 and different from the results obtained by Tchouakui et al. 10 in Uganda where these mosquitoes were resistant to 1× and 5× and susceptible to 10×.This study showed a major contrast in the A. gambiae after pre-exposure to PBO, with a full (or nearly) recovery of susceptibility observed for type II (deltamethrin and alphacypermethrin) but only a moderate recovery with Type I (permethrin).This is similar to observation made in 2015 by Riveron et al. 5 .This variation in recovery rate suggests a difference in resistance mechanisms between these pyrethroids with P450 genes likely playing a greater role in type II (deltamethrin) than type I (permethrin).This suggests that LLINs combining deltamethrin and PBO would be more effective against this A. gambiae resistant population.

Drastic loss of bio-efficacy of LLINs between 2015 and 2021.
The results of the cone test showed a low efficacy of all pyrethroid-only LLINs tested against A. gambiae.The low efficacy of these bednets corroborates the high pyrethroid resistance results observed in this A. gambiae population using WHO bioassays.This increase in resistance intensity for most of the insecticide-treated bed nets (ITNs) tested is greater than those obtained across Africa by Riveron et   10 .This loss of bed net efficacy may be due to selection pressure induced by massive distribution of bed nets by the government 9 and observation of the massive use of pesticide in farming in this area.Even the net with synergist PBO combined with permethrin (Olyset Plus), induced a low mortality with A. gambiae, indicating that P450 genes may not be the main drivers of the permethrin resistance observed in DRC, but rather other mechanisms such the kdr mutation, which is nearly fixed in this population or cuticular resistance 5 .
The same pattern of low efficacy of bednet was observed in A. funestus except for Olyset Plus (containing PBO) which showed higher efficacy with 100% mortality.
Increasing allele and genotype frequencies between 2015 and 2021 contribute to increasing pyrethroid resistance aggravation.The L1014F (kdrw) mutation conferring insecticide resistance to permethrin and DDT in A. gambiae was closed to fixation in Kinshasa mosquito population.This result corroborates the extremely high resistance observed with permethrin and DDT in A. gambiae population.The same mutation has been previously detected in Kinshasa with the high frequency of the L1014F kdrw mutation (87.8%) by 5 .In addition, this mutation has been detected in other location in DRC 4,16 .
The 1014S resistance allele was recorded at the frequency of 10.1% in mosquito population that contributes to maintain the high resistance level to pyrethroids and DDT.Furthermore, the N1575Y mutation was still not detected in the Kinshasa samples as previously by Jones et al. 17 .
The G119S-Ace-1 mutation conferring bendiocarb resistance in A. gambiae s.l. was also not found in Kinshasa as it was already the case in 2015 5 .This absence of the 119S Ace-1 R supports the susceptibility to carbamates and organophosphates seen in this population.However, because this mutation was recently detected at a low frequency (0.11-0.19) in A. gambiae in eastern DRC 18 it is important to continue the monitoring.However, Metabolic mechanisms are more likely involved and a cost linked to this mutation.In A. funestus s.s the high resistance to DDT corroborates the high frequency of L119F-GSTe2 resistant allele in Ndjili mosquito population, confirming the results obtained by Riveron et al. 5 in this same mosquito population.This would further support the implication that the L119F-GSTe2 marker in the metabolic resistance to DDT/permethrin.On the other hand, the A296S-RDL GABA receptor mutation known to confer resistance to dieldrin 19 , was observed with high allelic frequency of 56.25% but lower than the 66.7% observed Riveron et al. 5 which confirm the past or reduced use of this insecticide in agricultural area.Such reduction could be explained by a fitness cost associated to this allele as commonly seen for resistance alleles 10,20 .On the other hand, the extremely low frequency of resistant CYP6P9a, CYP6P9b and CYP6P9a/b alleles observed in this study contrasts with the high frequency of this allele observed in the eastern DRC mosquito population 11,18 .This implies that migration and gene flow could be responsible for the presence of this mutation in the Kinshasa population, as the resistant allele is found in the south and part of East Africa and completely absent from other parts of Africa 21 .However, the first detection of Transcription profile of resistance genes in A. funestus.The transcription analyses show that, the CYP6P9a, CYP6P9b, and CYP6M7 genes, known to be involved in pyrethroid resistance in A. funestus genes 12,13,22 are significantly up-regulated in our field A. funestus s.s mosquito population (alive and unexposed to permethrin) compared to susceptible strain Fang.This expression result is higher than the one obtained by 5 and could explain the resistance escalation to pyrethroids observed in this location.Twelve (12) candidates genes were analysed in A. gambiae including, Cyp6Z2, Cyp6P4, CYP9K1 which had more than twofold-changes compared to the susceptible Kisumu strain; which is not surprizing and not different from the microarray results obtained by Nardini et al. in DRC 3 regarding CYP9K1 gene.

Methods
Study area and mosquito collection.Adult Anopheles female mosquitoes were collected inside households using electric aspirators at Ndjili-Brasserie, a suburb of Kinshasa (4° 19′ 39″ S, 15° 18′ 48″ E), in July 2021 in the same houses as done in May 2015 5 .Anopheles females mosquitoes collected were morphologically identified as belonging to A. funestus group or A. gambiae s.l complex according to morphological keys 23 .These mosquitoes were kept 4-5 days in paper cups and fed with sugar until they became fully gravid and forced to lay eggs individually in 1.5 mL Eppendorf tubes.After egg hatching, larvae were placed in trays and reared to adults mosquitoes as previously described 24 .
Identification of mosquito species.Whole mosquitoes (A.funestus s.l. and A. gambiae s.l.) collected in Ndjili-Brasserie, were used for genomic DNA extraction using the Livak protocol 25 .Members of the A. funestus s.l. group were identified using the cocktail polymerase chain reaction (PCR) assay 26 , whereas the SINE PCR protocol was used to identify those from the A. gambiae, A. coluzzii and A. arabiensis 27 .
Plasmodium infection rate determination.The Plasmodium infection rate was estimated using gDNA of the whole body of mosquito to detect the presence of P. falciparum (F+) and/or P. ovale, P. vivax and Plasmodium malariae (OVM+) in 60 A. gambiae s.l. and 60 A. funestus sensu stricto (s.s.) field-collected F 0 females individually using the TaqMan assay, as previously described 28,29 .We used nested PCR assay 30 to confirm the results of TaqMan assay and differentiate between the species belonging to the OVM+.
All the tests were performed at standard insectary conditions of 25 ± 2 °C temperature and 70-80% relative humidity.For each test, at least three replicates of 20-25 F 1 female mosquitoes of 2-5 day-old were exposed to insecticide-impregnated papers for 1 h and control mosquitoes were exposed to non-impregnated papers.After the exposure, mosquitoes were transferred to a holding tube provided with cotton soaked in a 10% sugar solution.The knockdown was recorded 60 min after exposure to insecticide and mortality was determined 24 h later.
Based on the results of resistance status with 1× (discriminating concentration (DC)) of pyrethroid (permethrin and deltamethrin), intensity bioassays were carried out with 5× DC and 10× DC of these insecticides.The intensity bioassays with 5× and 10× DC were performed following the WHO 2016 test procedure 31 .

PBO synergist assays.
In order to investigate the potential role of cytochrome P450s genes in the observed resistance, A. gambiae s.l.females were pre-exposed to 4% piperonyl butoxide (PBO) for 60 min and immediately exposed to permethrin (0.75%), deltamethrin (0.05%) and α-cypermethrin (0.05%).The mortality was recorded after 24 h and compared with the mortality obtained for mosquitoes not pre-exposed to PBO using unpaired Student t test.
Insecticide-treated bed nets efficacy assays.The efficacy of the LLINs was estimated by 3-min exposure cone bioassays following the WHO guidelines 32 .The nets tested for A. gambiae included Olyset, Olyset Plus, PermaNet 2.0, PermaNet 3.0-side and -roof, Royal guard, and an untreated net (as a control).Due to the low number of A. funestus, only Olyset, Olyset Plus, PermaNet 2.0 and PermaNet 3.0-roof were tested.As done in 2015, five replicates of 10 A. gambiae F 1 female (2-5 days old) were placed in plastic cones enclosed with the mosquito net for 3 min.But for A. funestus due to the very low number of mosquitoes, only three replicates with 5 mosquitoes/cone were tested.Mosquitoes were then transferred in small holding paper cups with cotton soaked in a 10% sugar solution.Mortality was determined 24 h after exposure.The assay was carried out at temperature of 25 °C ± 2 °C and 80% ± 10% relative humidity.

Figure 1 .
Figure 1.Susceptibility profile of Anopheles gambiae (a) and Anopheles funestus (b) population in Kinshasa in 2015 and 2021 using World Health Organization insecticide susceptibility tube assays.NM, no mortality.

Figure 3 .
Figure 3. Bioefficacy of Anopheles gambiae (a) and Anopheles funestus (b) from Kinshasa in 2015 and 2021 using different long-lasting insecticidal nets; NM, No Mortality; N/A, not applicable.

Figure 4 .
Figure 4. Genotype distribution for key resistance markers (a) and genotype comparison between Anopheles funestus F0 females collected in 2015 and 2021 (b).

Figure 5 .
Figure 5. Genotype distribution for key resistance markers (a) and genotype comparison between Anopheles gambiae F0 female collected 2015 and 2021 (b).

Figure 6 .
Figure 6.Differential gene expression of the P450 genes CYP6P9a, CYP6P9b, CYP9K1, CYP6M7 and CYP6P5 and the Gluthatione S-tranferase GSTe2 in Anopheles funestus from Ndjili brasserie (a) and Comparison of gene expression between mosquitoes collected in 2015 and 2021 (b).Error bars represent standard error of the mean.

Figure 7 .
Figure 7. Differential gene expression in Anopheles gambiae from Ndjili brasserie.Error bars represent standard error of the mean.
al. in DRC 5 , Menze et al. in Cameroon 2 , and Tchouakui et al. in Uganda