Detection of a reduced susceptibility to chlorfenapyr in the malaria vector Anopheles gambiae contrasts with full susceptibility in Anopheles funestus across Africa.

New insecticides have recently been produced to help control pyrethroid-resistant malaria vectors including the pyrrole, chlorfenapyr. Monitoring the susceptibility of mosquito populations against this new product and potential cross-resistance with current insecticides is vital for better resistance management. In this study, we assessed the resistance status of the major malaria vectors Anopheles gambiae and Anopheles funestus to chlorfenapyr across Africa and explored potential cross-resistance with known pyrethroid resistance markers. Efficacy of chlorfenapyr 100 µg/ml against An. gambiae and An. funestus from five Cameroonian locations, the Democratic Republic of Congo, Ghana, Uganda, and Malawi was assessed using CDC bottle assays. Synergist assays were performed with PBO (4%), DEM (8%) and DEF (0.25%) and several pyrethroid-resistant markers were genotyped in both species to assess potential cross-resistance between pyrethroids and chlorfenapyr. Resistance to chlorfenapyr was detected in An. gambiae populations from DRC (Kinshasa) (mortality rate: 64.3 ± 7.1%) Ghana (Obuasi) (65.9 ± 7.4%), Cameroon (Mangoum; 75.2 ± 7.7% and Nkolondom; 86.1 ± 7.4). In contrast, all An. funestus populations were fully susceptible. A negative association was observed between the L1014F-kdr mutation and chlorfenapyr resistance with a greater frequency of homozygote resistant mosquitoes among the dead mosquitoes after exposure compared to alive (OR 0.5; P = 0.02) whereas no association was found between GSTe2 (I114T in An. gambiae; L119F in An. funestus) and resistance to chlorfenapyr. A significant increase of mortality to chlorfenapyr 10 µg/ml was observed in An. funestus after to PBO, DEM and DEF whereas a trend for a decreased mortality was observed in An. gambiae after PBO pre-exposure. This study reveals a greater risk of chlorfenapyr resistance in An. gambiae populations than in An. funestus. However, the higher susceptibility in kdr-resistant mosquitoes points to higher efficacy of chlorfenapyr against the widespread kdr-based pyrethroid resistance.

Escalating pyrethroid resistance in two major malaria vectors Anopheles funestus and Anopheles gambiae (s.l.) in Atatam, Southern Ghana.

BACKGROUND: Aggravation of insecticide resistance in malaria vectors is threatening the efforts to control malaria by reducing the efficacy of insecticide-based interventions hence needs to be closely monitored. This study investigated the intensity of insecticide resistance of two major malaria vectors An. funestus sensu stricto (s.s.) and An. gambiae sensu lato (s.l.) collected in southern Ghana and assessed the bio-efficacy of several long-lasting insecticidal nets (LLINs) against these mosquito populations. METHODS: The insecticide susceptibility profiles of Anopheles funestus s.s. and Anopheles gambiae s.l. populations from Obuasi region (Atatam), southern Ghana were characterized and the bio-efficacy of some LLINs was assessed to determine the impact of insecticide resistance on the effectiveness of these tools. Furthermore, molecular markers associated with insecticide resistance in both species were characterized in the F0 and F1 populations using PCR and qPCR methods. RESULTS: Anopheles funestus s.s. was the predominant species and was resistant to pyrethroids, organochlorine and carbamate insecticides, but fully susceptible to organophosphates. An. gambiae s.l. was resistant to all four insecticide classes. High intensity of resistance to 5 × and 10 × the discriminating concentration (DC) of pyrethroids was observed in both species inducing a considerable loss of efficacy of long-lasting insecticidal nets (LLINs). Temporal expression analysis revealed a massive 12-fold increase in expression of the CYP6P4a cytochrome P450 gene in An. funestus s.s., initially from a fold change of 41 (2014) to 500 (2021). For both species, the expression of candidate genes did not vary according to discriminating doses. An. gambiae s.l. exhibited high frequencies of target-site resistance including Vgsc-1014F (90%) and Ace-1 (50%) while these mutations were absent in An. funestus s.s. CONCLUSIONS: The multiple and high intensity of resistance observed in both malaria vectors highlights the need to implement resistance management strategies and the introduction of new insecticide chemistries.

Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa.

BACKGROUND: New insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health 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 (Obuasi), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, 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 odds ratio based on Fisher exact test were used to evaluate potential cross-resistance between pyrethroids and clothianidin. RESULTS: Lower mortality was observed when using absolute ethanol or acetone alone as solvent for clothianidin (11.4‒51.9% mortality in Nkolondom, 31.7‒48.2% in Mangoum, 34.6‒56.1% in Mayuge, 39.4‒45.6% in Obuasi, 83.7‒89.3% in Congo and 71.1‒95.9% in Njombe pendja) compared to acetone + MERO for which 100% mortality were observed for all the populations. Similar observations were done for imidacloprid and acetamiprid. Synergist assays (PBO, DEM and DEF) with clothianidin 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 to lower mortality, thus possibly overestimating resistance, whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development. Therefore, we recommend monitoring the susceptibility using both acetone alone and acetone + MERO (4 µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.

Trapping Spodoptera frugiperda (Lepidoptera: Noctuidae) Moths in Different Crop Habitats in Togo and Ghana.

The economic impact of the invasion of Spodoptera frugiperda (J.E. Smith, Lepidoptera: Noctuidae) into Africa has so far been limited to maize agriculture but could potentially impact many other crops. Trapping based on pheromone lures provides a cost-effective method for detecting this important pest (commonly known as fall armyworm) and will be essential for large-scale monitoring of populations to determine its geographical distribution and migration behavior as the species equilibrates to its new environment. However, the effective use of pheromone trapping requires optimization for a given location. An earlier report demonstrated that two commercial lures (one 3-component and the other 4-component) that were effective for trapping S. frugiperda in maize fields in Togo, Africa. The current study extends these findings to agricultural areas that differ in plant host composition (maize, pasture grasses, rice, and sorghum) in multiple locations in Ghana and Togo. In two seasons, significantly higher numbers of moths were found in maize, and in one season, higher numbers were found in rice than in sorghum and pasture grass systems. The results confirm the effectiveness of pheromone trapping and identify pheromone lures and trapping methods best suited for the different agroecosystems common to West Africa and that are at risk of infestation by S. frugiperda.

Trypanosome prevalence in pigs and tsetse flies from selected areas of Jomoro district of the western region of Ghana.

Detection of trypanosomes in tsetse or domestic livestock is a basic requirement for epidemiological studies as well as for planning and implementing control measures against tsetse and trypanosomiasis. This epidemiological study aimed at assessing the prevalence of trypanosomes in pigs and tsetse flies in the Jomoro district of the western region of Ghana using molecular techniques. Blood was collected from pigs and biconical traps were used to collect tsetse flies. DNA was isolated from 300 pig blood samples and 300 flies for trypanosome detection and identification by PCR. Packed Cell Volume (PCV) of blood samples from 300 pigs was measured using a micro-haematocrit reader. Glossina palpalis palpalis was the only tsetse species found in the area with fly apparent density of 18.4 fly/trap/day. An overall prevalence of trypanosomes in the study area was 4.3% and 0.8% in pigs and tsetse flies respectively. Mixed infection with Trypanosoma (T.) congolense forest and T. vivax was most prevalent 46.2% followed by single infection of T. vivax 15.4%, T. congolense and a mixed infection of T. congolense, T. vivax and T. brucei sl. were the least with 7.7% each. There were no significant differences in trypanosome prevalence among different age groups and between both sexes of the studied pigs (p > 0.05). Trypanosome prevalence was lower in healthy looking 1.9% than the sick looking 20%, pigs (P < 0.05). Mean PCV of parasitaemic pigs 29.3% was significantly lower than that of aparasitaemic pigs 37.8%. Two out of the five species-specific primers used could not identify any trypanosome species from the total blood samples examined. This could possibly mean that those species are not found in the present study area. These results provide useful background information for further study and justification to extend tsetse control to the Jomoro district.

Maize Infestation of Fall Armyworm (Lepidoptera: Noctuidae) Within Agro-Ecological Zones of Togo and Ghana in West Africa 3 Yr After Its Invasion.

The fall armyworm Spodoptera frugiperda (J. E. Smith) invaded several West African countries in 2016 causing severe injury to maize plants and economic damage. This study assesses variations in the occurrence of this species in different Agro-Ecological Zones (AEZs) in Togo and Ghana during the 3 yr following its discovery. The surveys were conducted on 120 farms in Togo and 94 farms in Ghana by collecting larvae from 200 maize plants per hectare. Infestation levels were 68.46% in 2016, 55.82% in 2017, and 17.76% in 2018. The number of larvae recorded per hectare and infestation levels were higher in Togo than in Ghana. The lowest number of collected larvae and infestation levels of S. frugiperda were in 2018, compared to the other 2 yr. Larvae per hectare and the infestation level varied regionally inside the two countries. The southern part of Togo (AEZ five) contained higher numbers of larvae and higher infestation levels during the 2 yr following the invasion of the pest. We concluded that infestation levels of S. frugiperda are much lower in 2018 than the two previous years and it is therefore necessary to determine the factors that affect the population dynamics of S. frugiperda in the field, which is a perquisite for developing management interventions.

The Efficacy of Alternative, Environmentally Friendly Plant Protection Measures for Control of Fall Armyworm, Spodoptera Frugiperda, in Maize.

The invasive fall armyworm (FAW) is threatening maize production and the livelihoods of millions of smallholder farmers in the newly invaded areas in Africa and Asia. To control this new key pest and to overcome health, environmental, and resistance problems related to the indiscriminate use of insecticides, effective and sustainable alternative pest control approaches are needed. Here, we report on field trials that tested maltodextrin, neem-based products, ash, and soil, as well as the locally produced alata samina soap, in the Upper West and Greater Accra regions, Ghana. Significant reductions of larval numbers and crop damage, together with increased yields, were mostly achieved by applying the insecticide emamectin benzoate, which was considered the positive control in this set of trials. However, high efficiency and cost-benefit ratios were also achieved with two neem-based products. Maltodextrin was only efficient at one of the two sites, with a clear dose-dependent effect, while the higher dosage was nearly as effective as emamectin benzoate. Due to its relatively high product cost, maltodextrin is generally less cost-efficient. Ash and soil, as well as alata samina soap treatments, did not efficiently reduce FAW larval numbers or crop damage at the dosages tested; thus, they also did not significantly increase maize yields.

Multiple insecticide resistance in the major malaria vector Anopheles funestus in southern Ghana: implications for malaria control.

BACKGROUND: Understanding the dynamics of insecticide resistance in African malaria vectors is crucial for successful implementation of resistance management strategies in the continent. This study reports a high and multiple insecticide resistance in Anopheles funestus from southern Ghana which could compromise the Malaria Operational Plan in this country, if not tackled. Adult Anopheles mosquitoes were collected in Obuasi and Adawukwa, in southern Ghana. Plasmodium infection rates, susceptibility to the main insecticides used in public health and the molecular basis of insecticide resistance were established. RESULTS: An. funestus (sensu stricto) (s.s.) was the predominant mosquito species found resting inside the houses in Obuasi, while at Adawukwa it was found together with An. coluzzii. Parasite rates were high in An. funestus (s.s.) populations from both localities, with Plasmodium infection rates greater than 12.5 %. Both, An. funestus (s.s.) and An. coluzzii, from the two sites exhibited high resistance to the insecticide from various classes including the pyrethroids, carbamates and DDT, but remained fully susceptible to the organophosphates. A preliminary characterization of the underlying molecular mechanisms of resistance in An. funestus (s.s.) populations from both sites revealed that CYP6P9a, CYP6P9b, CYP6M7 and GSTe2 genes are upregulated, markedly higher in Obuasi (between 3.35 and 1.83 times) than in Adawukwa population. The frequency of L119F-GSTe2 and A296S-RDL resistance markers were also higher in Obuasi (42.5 and 68.95 % higher), compared with An. funestus (s.s.) populations from Adawukwa. These findings suggest that the similar resistance pattern observed in both An. funestus (s.s.) populations are driven by different mechanisms. CONCLUSIONS: Resistance to multiple insecticides in public health use is present in malaria vectors from Ghana with major resistance genes already operating in the field. This should be taken into consideration in the design of resistance management strategies to avoid operational failure.

Storage and persistence of a candidate fungal biopesticide for use against adult malaria vectors.

BACKGROUND: New products aimed at augmenting or replacing chemical insecticides must have operational profiles that include both high efficacy in reducing vector numbers and/or blocking parasite transmission and be long lasting following application. Research aimed at developing fungal spores as a biopesticide for vector control have shown considerable potential yet have not been directly assessed for their viability after long-term storage or following application in the field. METHODS: Spores from a single production run of the entomopathogenic fungi Beauveria bassiana were dried and then stored under refrigeration at 7°C. After 585 days these spores were sub-sampled and placed at either 22°C, 26°C or 32°C still sealed in packaging (closed storage) or in open beakers and exposed to the 80% relative humidity of the incubator they were kept in. Samples were subsequently taken from these treatments over a further 165 days to assess viability. Spores from the same production run were also used to test their persistence following application to three different substrates, clay, cement and wood, using a hand held sprayer. The experiments were conducted at two different institutes with one using adult female Anopheles stephensi and the other adult female Anopheles gambiae. Mosquitoes were exposed to the treated substrates for one hour before being removed and their survival monitored for the next 14 days. Assays were performed at monthly intervals over a maximum seven months. RESULTS: Spore storage under refrigeration resulted in no loss of spore viability over more than two years. Spore viability of those samples kept under open and closed storage was highly dependent on the incubation temperature with higher temperatures decreasing viability more rapidly than cooler temperatures. Mosquito survival following exposure was dependent on substrate type. Spore persistence on the clay substrate was greatest achieving 80% population reduction for four months against An. stephensi and for at least five months against Anopheles gambiae. Cement and wood substrates had more variable mortality with the highest spore persistence being two to three months for the two substrates respectively. CONCLUSIONS: Spore shelf-life under refrigeration surpassed the standard two year shelf-life expected of a mosquito control product. Removal to a variety of temperatures under either closed or open storage indicated that samples sent out from refrigeration should be deployed rapidly in control operations to avoid loss of viability. Spore persistence following application onto clay surfaces was comparable to a number of chemical insecticides in common use. Persistence on cement and wood was shorter but in one assay still comparable to some organophosphate and pyrethroid insecticides. Optimized formulations could be expected to improve spore persistence still further.