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.

Effect of aqueous extract of lemon (Citrus limon) on Anopheles gambiae sensu lato (Diptera: Culicidae) larvae tolerance in malaria vector control in Dogbo district in south-western Republic of Benin, West Africa

The use of chemical insecticides causes important damages to environment and human health and there is a need to search for alternative solutions. This study aims to investigate on the effect of aqueous extract of lemon in Anopheles gambiae s.l. larvae tolerance in couffo department in south-western Republic of Benin, West Africa. Larvae of Anopheles gambiae s.l mosquitoes were collected from breeding sites using the dipping method in July 2020 during the rainy season in Dogbo district. A batch of 15 larvae of fourth instar were exposed to a mixture of aqueous extract of lemon with distilled water saturated with oxygen containing in each of five glass jars or test cups of same dimensions contained each 48 ml distilled water saturated with oxygen plus 2 ml of aqueous extract of lemon (Citrus limon) and one control jar containing no trace of aqueous extract of lemon. Larval mortality was recorded after 24hours, 48hours and 72hours exposure. The results show that the use of aqueous extract of lemon causes full-grown Anopheles larvae to die by suffocation. After the application of this mixture, the larvae of four instars cannot breathe. The use of aqueous extract of lemon is effective method for disturbing the siphonal respiration of mosquito larvae. Aqueous extract of lemon is effective method for mosquito larvae control.

Swarming Behavior in Anopheles gambiae (sensu lato): Current Knowledge and Future Outlook

Effective management of insect disease vectors requires a detailed understanding of their ecology and behavior. In Anopheles gambiae sensu lato (s.l.) (Diptera: Culicidae) mating occurs during swarming, but knowledge of their mating behavior under natural conditions is limited. Mosquitoes mate in flight over specific landmarks, known as swarm markers, at particular locations. Swarms consist of males; the females usually approach the swarm and depart following copulation. The number of mating pairs per swarm is closely associated with swarm size. The shape and height of swarm markers vary and may depend on the environmental conditions at the swarm’s location. Male–male interactions in mosquito swarms with similar levels of attractive flight activity can offer a mating advantage to some individuals. Flight tone is used by mosquitoes to recognize the other sex and choose a desirable mate. Clarifying these and other aspects of mosquito reproductive behavior can facilitate the development of population control measures that target swarming sites. This review describes what is currently known about swarming behavior in Anopheles gambiae s.l., including swarm characteristics; mating within and outside of swarms, insemination in females, and factors affecting and stimulating swarming.

Pyrethroid and Etofenprox Resistance in Anopheles gambiae and Anopheles coluzzii from Vegetable Farms in Yaoundé, Cameroon: Dynamics, Intensity and Molecular Basis

Previous studies have indicated widespread insecticide resistance in malaria vector populations from Cameroon. However, the intensity of this resistance and underlying mechanisms are poorly known. Therefore, we conducted three cross-sectional resistance surveys between April 2018 and October 2019, using the revised World Health Organization protocol, which includes resistance incidences and intensity assessments. Field-collected Anopheles gambiae s.l. populations from Nkolondom, Nkolbisson and Ekié vegetable farms in the city of Yaoundé were tested with deltamethrin, permethrin, alpha-cypermethrin and etofenprox, using 1× insecticide diagnostic concentrations for resistance incidence, then 5× and 10× concentrations for resistance intensity. Subsamples were analyzed for species identification and the detection of resistance-associated molecular markers using TaqMan® qPCR assays. In Nkolbisson, both An. coluzzii (96%) and An. gambiae s.s. (4%) were found together, whereas only An. gambiae s.s. was present in Nkolondom, and only An. coluzzii was present in Ekié. All three populations were resistant to the four insecticides (<75% mortality rates―MR1×), with intensity generally fluctuating over the time between mod-erate (<98%―MR5×; ≥98%―MR10×) and high (76–97%―MR10×). The kdr L995F, L995S, and N1570Y, and the Ace-1 G280S-resistant alleles were found in An. gambiae from Nkolondom, at 73%, 1%, 16% and 13% frequencies, respectively, whereas only the kdr L995F was found in An. gambiae s.s. from Nkolbisson at a 50% frequency. In An. coluzzii from Nkolbisson and Ekié, we detected only the kdr L995F allele at 65% and 60% frequencies, respectively. Furthermore, expression levels of Cyp6m2, Cyp9k1, and Gste2 metabolic genes were highly upregulated (over fivefold) in Nkolondom and Nkolbisson. Pyrethroid and etofenprox-based vector control interventions may be jeopardized in the prospected areas, due to high resistance intensity, with multiple mechanisms in An. gambiae s.s. and An. coluzzii.

Dynamics of organophosphate and carbamate resistance in Anopheles gambiae s. l. populations from south and north Benin, West Africa

The current study was aimed to investigate on dynamics of propoxur resistance in Anopheles gambiae s.l. populations from N’ dali district in northern Benin (West Africa) and also to investigate on dynamics of malathion resistance in Anopheles gambiae s. l. populations from Toffo district in southern Benin. Larvae and pupae of Anopheles gambiae s. l . mosquitoes were collected from the breeding sites in Borgou and Atlantic departments in 2015 and 2019. WHO susceptibility tests were conducted on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers with propoxur 0.1% and with malathion 5%. PCR techniques were used to detect species and Ace-1 mutations in 2015. Anopheles gambiae s. l. populations from N’dali were resistant to propoxur in 2015 and were still remained resistant to this product in 2019. Regarding Anopheles gambiae s. l. populations from Toffo, they were susceptible to malathion in 2015 whereas the malathion resistance status of these mosquitoes requires further investigation in 2019. PCR revealed that all specimens tested were Anopheles gambiae s. s. The presence of Ace-1R at very low frequency (0.01) was observed in Anopheles gambiae s. l. populations from both districts. This study shows that propoxur resistance detected in An. gambiae s. l. populations from N’ dali needs to be monitored for insecticide resistance in this area.

Evaluation of Yahe® and Panda® 2.0 long-lasting insecticidal nets against wild pyrethroid-resistant Anopheles gambiae s.l. from Côte d’Ivoire: an experimental hut trial

Background Long-lasting insecticidal nets (LLINs) have played an important role in reducing the global malaria burden since 2000. They are a core prevention tool used widely by people at risk of malaria. The Vector Control Prequalification mechanism of the Word Health Organization (WHO-Vector Control PQ) established the testing and evaluation guidelines for LLINs before registration for public use. In the present study, two new brands of deltamethrin-impregnated nets (Yahe® LN and Panda® Net 2.0) were evaluated in an experimental hut against wild pyrethroid-resistant Anopheles gambiae s.l. in M’Bé nearby Bouaké, central Côte d’Ivoire. Methods The performance of Yahe® LN and Panda® Net 2.0 was compared with that of PermaNet 2.0, conventionally treated nets (CTN), and untreated net to assess the blood-feeding inhibition, deterrence, induced exophily, and mortality. Results Cone bioassay results showed that Panda® Net 2.0, PermaNet 2.0 and Yahe® LN (both unwashed and washed 20 times) induced > 95% knockdown or > 80% mortality of the susceptible Anopheles gambiae Kisumu strain. With the pyrethroid-resistant M’Bé strain, mortality rate for all treated nets did not exceed 70%. There was a significant reduction in entry and blood feeding (p < 0.05) and an increase in exophily and mortality rates (p < 0.05) with all treatments compared to untreated nets, except the CTNs. However, the personal protection induced by these treated nets decreased significantly after 20 washes. The performance of Panda® Net 2.0 was equal to PermaNet® 2.0 in terms of inhibiting blood feeding, but better than PermaNet® 2.0 in terms of mortality. Conclusion This study showed that Yahe® LN and Panda® Net 2.0 met the WHO Pesticide Evaluation Scheme (WHOPES) criteria to undergo phase III trial at the community level. Due to an increasing spread and development of pyrethroid resistance in malaria vectors, control of malaria transmission must evolve into an integrated vector management relying on a large variety of efficient control tools. Graphical Abstract

Dynamics of organophosphate and carbamate resistance in Anopheles gambiae s. l. populations from south and north Benin, West Africa

The current study was aimed to investigate on dynamics of propoxur resistance in Anopheles gambiae s.l. populations from N’ dali district in northern Benin (West Africa) and also to investigate on dynamics of malathion resistance in Anopheles gambiae s. l. populations from Toffo district in southern Benin. Larvae and pupae of Anopheles gambiae s. l . mosquitoes were collected from the breeding sites in Borgou and Atlantic departments in 2015 and 2019. WHO susceptibility tests were conducted on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers with propoxur 0.1% and with malathion 5%. PCR techniques were used to detect species and Ace-1 mutations in 2015. Anopheles gambiae s. l. populations from N’dali were resistant to propoxur in 2015 and were still remained resistant to this product in 2019. Regarding Anopheles gambiae s. l. populations from Toffo, they were susceptible to malathion in 2015 whereas the malathion resistance status of these mosquitoes requires further investigation in 2019. PCR revealed that all specimens tested were Anopheles gambiae s. s. The presence of Ace-1R at very low frequency (0.01) was observed in Anopheles gambiae s. l. populations from both districts. This study shows that propoxur resistance detected in An. gambiae s. l. populations from N’ dali needs to be monitored for insecticide resistance in this area.

Mesocosm Experiments to Quantify Predation of Mosquito Larvae by Aquatic Predators to Determine Potential of Ecological Control of Malaria Vectors in Ethiopia

Malaria parasites are transmitted to humans by infectious female Anopheles mosquitoes. Chemical-insecticide-based mosquito control has been successful in reducing the burden of malaria. However, the emergence of insecticide resistance in malaria vectors and concerns about the effect of the chemicals on the environment, human health, and non-target organisms present a need for new or alternative vector control intervention tools. Biocontrol methods using aquatic invertebrate predators have emerged as a potential alternative and additional tool to control mosquito populations. Ecological control specifically makes use of species insights for improving the physical habitat conditions of competitors and predators of vectors. A first step towards this is to gain knowledge on the predation potential of several typically present macroinvertebrates. Hence, this study aimed at (1) examining the influence of the predation of hemipterans on the number of emerging adult mosquitoes and (2) detecting Anopheles mosquito DNA in the gut of those predators. The prey and predators were collected from a range of water bodies located in the Gilgel Gibe watershed, southwest Ethiopia. A semi-field study was carried out using mesocosms which were constructed using plastic containers mimicking the natural aquatic habitat of immature Anopheles mosquitoes. Adult mosquitoes that emerged from the mesocosms were collected using a mechanical aspirator. At the end of the experiment, predators were withdrawn from the mesocosms and identified to genus level. Polymerase Chain Reaction (PCR) was employed to identify sibling species of Anopheles gambiae s.l. and to detect Anopheles mosquito DNA in the gut of the predators. Data were analysed using R software. Giant water bugs (belostomatids) were the most aggressive predators of Anopheles larvae, followed by backswimmers (notonectids) and water boatmen (corixids). All female Anopheles gambiae s.l. emerged from the mesocosms were identified as Anopheles arabiensis. Anopheles arabiensis DNA was detected in the gut content of hemipteran specimens analysed from the three families. The number of the adult mosquitoes emerging from the mesocosms was affected by the presence of predators. The findings of this study provide evidence of the potential use of aquatic macroinvertebrate predators as biocontrol agents against immature Anopheles mosquitoes and their potential to be considered as a component of integrated vector management for insecticide resistance and the combined restoration of aquatic ecosystems via smart ecological engineering.