Comparative Study of the Effect of Solvents on the Efficacy of Neonicotinoid Insecticides Against Malaria Vector Populations Across Africa

Background: New insecticides with novel modes of action such as neonicotinoids have recently been recommended for public health use by WHO. Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations. In this study, we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.Methods: Mosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon (Njombe-Penja, Nkolondom, and Mangoum), the Democratic Republic of Congo (Ndjili-Brasserie), Ghana (Atatam), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, acetone, acetone+MERO) on the efficacy of neonicotinoids against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. gambiae and to evaluate potential cross-resistance between pyrethroids and clothianidin.Results: Lower mortalities were observed for all populations when using absolute ethanol or acetone alone as solvent (11.4- 51.9% mortality for Nkolondom, 31.7- 48.2% for Mangoum, 34.6- 56.1% for Mayuge, 39.4- 45.6% for Atatam, 83.7- 89.3% for Congo and 71.05- 95.9% for Njombe pendja) compared to acetone + MERO for which 100% mortality was observed for all the populations. Synergist assays (PBO, DEM and DEF) revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility. A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors (OR=0.5; P=0.02). However, the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes (OR=2.10; P=0.013). Conclusions: This study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting in lower mortality, thus possibly overestimating resistance, whereas the addition of MERO consistently increased the efficacy of neonicotinoids in terms of percentage mortalities and time to final mortality. The addition of MERO could however prevent the early detection of resistance development. We therefore recommend monitoring susceptibility using both acetone alone and acetone+MERO (8-10µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.

First report of the L1014F kdr mutation in wild populations of Anopheles arabiensis in Cabo Verde, West Africa

Background Due to the lack of vaccines, malaria control mainly involves the control of anopheline vectors (Anopheles spp.) using chemical insecticides. However, the prolonged and indiscriminate use of these compounds has led to the emergence of resistance in Anopheles populations in Africa. Insecticide resistance surveillance programs are less frequent in Cabo Verde than in other African countries. This study aimed to investigate the circulation of the L1014F and L1014S alleles in natural populations of Anopheles arabiensis collected from two sampling sites in the city of Praia, Cabo Verde. Methods Anopheles larvae were collected from the two sampling sites and reared in the laboratory until the adult stage. Mosquitoes were first morphologically identified by classical taxonomy and then by molecular species identification using molecular markers. All Anopheles arabiensis were subjected to PCR analysis to screen for mutations associated to resistance in the Nav gene. Results A total of 105 mosquitoes, all belonging to the Anopheles gambiae complex, were identified by classical taxonomy as well as by molecular taxonomy. Molecular identification showed that 100% of the An. gambiae senso lato specimens analyzed corresponded to An. arabiensis. Analysis of the Nav gene revealed the presence of L1014S and L1014F alleles with frequencies of 0.10 and 0.19, respectively. Conclusions Our data demonstrated, for the first time, the presence of the L1014F allele in the An. arabiensis population from Cabo Verde, as well as an increase in the frequency of the kdr L1014S allele reported in a previous study. The results of this study demonstrate the need to establish new approaches in vector control programs in Cabo Verde. Graphical Abstract

First Report of the L1014F kdr Mutation in Wild Populations of Anopheles Arabiensis in Cabo Verde, West Africa

BackgroundDue to the lack of vaccines, malaria control mainly involves the control of the incriminated vector, Anopheles spp, using chemical insecticides. However, the prolonged and indiscriminate use of these compounds has led to the emergence of resistance in Anopheles populations from African continent. The surveillance of this resistance is more frequent in other African countries; however, Cabo Verde does not carry out this surveillance in the same way. Thus, this study aimed to investigate the circulation of the L1014F and L1014S alleles in natural populations of Anopheles arabiensis from the city of Praia, Cabo Verde. MethodsLarvae of Anopheles genus were collected in two areas of Praia and reared in the laboratory until maturity. Mosquitoes were morphologically identified by classical taxonomy and then, subjected to molecular species identification using molecular markers. All Anopheles arabiensis were subjected to PCR to screen for mutations associated to resistance in the Nav gene. ResultsA total of 105 mosquitoes, belonging to the Anopheles gambiae complex, were identified by classical taxonomy were also identified by molecular taxonomy. The molecular identification showed that 100% of the An. gambiae s.l. analyzed were An. arabiensis.. Analysis of the Nav gene revealed the presence of L1014S and L1014F alleles with frequencies of 0.14 and 0.23, respectively. ConclusionsOur data demonstrate, for the first time, the presence of the L1014F allele in the An. arabiensis population from Cabo Verde and an increase of the previously found Kdr L1014S allele. The results presented in this study demonstrate the need to establish new approaches in vector control programs in Cabo Verde.

First Report of Target Site Insensitivity in Pyrethroid Resistant Anopheles gambiae from Southern Guinea Savanna, Northern-Nigeria

Background: Malaria is a major public health problem and life threatening parasitic vector-borne disease. For the first time, we established and report the molecular mechanism responsible for Anopheles gambiae s.l. resistance to pyrethroids and DDT from Yamaltu Deba, Southern Guinea Savanna, Northern-Nigeria. Methods: The susceptibility profile of An. gambiae s.l. to four insecticides (DDT 4%, bendiocarb 0.1%, malathion 5% and deltamethrin 0.05%) using 2–3 days old females from larvae collected from study area between August and November, 2018 was first established. Genomic DNA was then extracted from 318 mosquitoes using Livak DNA extraction protocol for specie identification and kdr genotyping. The mosquitoes were identified to species level and then 96 genotyped for L1014F and L1014S kdr target site mutations. Results: The mosquitoes were all resistant to DDT, bendiocarb and deltamethrin but fully susceptible to malathion. An. coluzzii was found to be the dominant sibling species (97.8%) followed by An. arabiensis (1.9%) and An. gambiae s.s (0.3%). The frequency of the L1014F kdr mutation was relatively higher (83.3%) than the L1014S (39%) in the threespecies studied. The L1014F showed a genotypic frequency of 75% resistance (RR), 17% heterozygous (RS) and 8% susceptible (SS) with an allelic frequency of 87% RR and 13% SS while the L1014S showed a genotypic frequency of RR (16%), RS (38%) and SS (46%) with an allelic frequency of 40% RR and 60% SS, respectively. Conclusion: This study reveals that both kdr mutations present simultaneously in Northern-Nigeria, however contribution of L1014F which is common in West Africa was more than twice of L1014S mutation found in East Africa.

Susceptibility to Pyrethroids and the First Report of L1014F kdr Mutation in Culex quinquefasciatus (Diptera: Culicidae) in Colombia

The use of insecticides for the control of Aedes aegypti (L.) (Diptera: Culicidae) in Colombia has indirectly influenced the susceptibility status of Culex quinquefasciatus Say populations. We evaluated pyrethroid susceptibility in two populations of Cx. quinquefasciatus in the Atlantico Department of Colombia and its possible resistance mechanism (kdr mutation). Bottle bioassays were performed for permethrin, deltamethrin, and λ-cyhalothrin in female mosquitoes of Cx. quinquefasciatus. The resistance ratios (RRs) for KC50 and LC50 for each insecticide in the field populations examined were determined, using the Cartagena strain as the susceptible control. The L1014F kdr mutation was identified in the para gene of the voltage-gated sodium channel (vgsc), along with its allelic and genotypic frequency. Low knockdown resistance (RRKC50) to deltamethrin was found in Puerto Colombia and Soledad populations as well as low resistance to λ-cyalothrin in this latter population. Moderate knockdown resistance to permethrin was found in both populations. At 24 h post-exposure on the other hand, there was low resistance (RRLC50) to permethrin in Puerto Colombia and moderate resistance in Soledad. Moderate resistance to deltamethrin was found in Puerto Colombia and low resistance in Soledad. Low resistance to λ-cyhalothrin was seen in Puerto Colombia and moderate resistance in Soledad. Variability was found in the susceptibility to the pyrethroids in the populations of Cx. quinquefasciatus evaluated, and the L1014F kdr mutation is reported for the first time as a possible pyrethroid resistance mechanism in this species in Colombia.

The double-mutation（M918I + L1014F）kdr allele is fixed in Cimex hemipterus populations in Guangxi, China

Four putative knockdown resistance (kdr) mutations have been documented in the voltage-gated sodium channel (VGSC) gene of Cimex hemipterus from several countries. However, no information regarding kdr mutations in any Chinese tropical bed bug population is available to date. In this study, a double-mutation（M918I + L1014F）kdr allele was identified in six C. hemipterus populations across Guangxi Zhuang Autonomous Region of China. The frequency of this allele was 100% in all the six examined populations. In addition, only two cytochrome c oxidase I (COI) gene haplotypes, with one synonymous nucleotide variation, were identified in a total of 48 individuals from six locations. The fixation and broad geographic distribution of this resistant allele questions the continued use of pyrethroids in the treatment of tropical bed bug infestations. The very low genetic diversity within and among these populations indicates that these bed bugs may have a single origin.

Surveillance of a kdr Resistance Mutation in Culex pipiens (Diptera: Culicidae) and Culex quinquefasciatus in California

Culex pipiens Linnaeus and Culex quinquefasciatus Say are the primary vectors of West Nile and St. Louis encephalitis viruses in California. Pyrethrins and pyrethroids (synthetic pyrethrins) are the most widely used insecticides to control adult stage mosquitoes to prevent disease transmission. The most abundant and widespread mutation associated with pyrethroid resistance is the L1014F mutation of the voltage-sensitive sodium channel gene. Statewide, based on the testing of almost 2,000 mosquitoes from 14 counties, the resistant allele frequency was 71%. Although the L1014F mutation was found in all counties assessed, the resistance allele profiles differed between regions of California. The highest resistant allele frequency occurred in the Central region and lowest frequencies were from the Northern and Southern regions. Resistance allele frequencies observed in 2014–2016 are nearly 1.5 times higher than those from pre-2012, indicating that resistance profiles can change over time. Regular monitoring of the L1014F kdr mutation will help aid in operational decisions.