Biological activities of four essential oils against Anopheles gambiae in Burkina Faso and their in vitro inhibition of acetylcholinesterase

The control of malaria is still a challenge partly due to mosquito’s resistance to current available insecticides. The aim of this work was to evaluate the ovicidal, larvicidal and repellent activities of Lantana camara, Hyptis suaveolens, Hyptis spicigera and Ocimum canum essential oils against  Anopheles gambiae s.l.  according to the World Health Organization standard method. The in vitro acetylcholinesterase inhibition activity of these oils was also evaluated. The repellent effect using the method of “separated arms” was evaluated. Lantana camara oil was more effective on both eggs and larvae. The LD50 and LD90 values observed in this oil solution were respectively 53.59 and 170.89 ppm on eggs whereas LD50 and LD90 were 61 and 125 ppm respectively on larvae. All oils exhibited repellent activities against adult mosquitoes. The most effective repellent was the oil of Hyptis suaveolens with a 50% efficacy dose value of 67 ppm. The highest  cetylcholinesterase inhibitory activity was observed with O. canum and H. suaveolens essential oils which IC50 was 0.21 and 0.55 μg/ml respectively. Results suggest that these essential oils have a potential for vector control and can be considered as a source of natural and ecofriendly substances for malaria vector control.Keywords: Anopheles gambiae s.l., malaria, essential oil, acetylcholinesterase, insecticidal activity

SNP Genotyping Defines Complex Gene-Flow Boundaries Among African Malaria Vector Mosquitoes

Mosquitoes in the Anopheles gambiae complex show rapid ecological and behavioral diversification, traits that promote malaria transmission and complicate vector control efforts. A high-density, genome-wide mosquito SNP-genotyping array allowed mapping of genomic differentiation between populations and species that exhibit varying levels of reproductive isolation. Regions near centromeres or within polymorphic inversions exhibited the greatest genetic divergence, but divergence was also observed elsewhere in the genomes. Signals of natural selection within populations were overrepresented among genomic regions that are differentiated between populations, implying that differentiation is often driven by population-specific selective events. Complex genomic differentiation among speciating vector mosquito populations implies that tools for genome-wide monitoring of population structure will prove useful for the advancement of malaria eradication

Efficacy of Olyset Duo, a bednet containing pyriproxyfen and permethrin, versus a permethrin-only net against clinical malaria in an area with highly pyrethroid-resistant vectors in rural Burkina Faso: a cluster-randomised controlled trial

Background Substantial reductions in malaria incidence in sub-Saharan Africa have been achieved with massive deployment of long-lasting insecticidal nets (LLINs), but pyrethroid resistance threatens control. Burkina Faso is an area with intense malaria transmission and highly pyrethroid-resistant vectors. We assessed the effectiveness of bednets containing permethrin, a pyrethroid, and pyriproxyfen, an insect growth regulator, versus permethrin-only (standard) LLINs against clinical malaria in children younger than 5 years in Banfora, Burkina Faso. Methods In this two-group, step-wedge, cluster-randomised, controlled, superiority trial, standard LLINs were incrementally replaced with LLINs treated with permethrin plus pyriproxyfen (PPF) in 40 rural clusters in Burkina Faso. In each cluster, 50 children (aged 6 months to 5 years) were followed up by passive case detection for clinical malaria. Cross-sectional surveys were done at the start and the end of the transmission seasons in 2014 and 2015. We did monthly collections from indoor light traps to estimate vector densities. Primary endpoints were the incidence of clinical malaria, measured by passive case detection, and the entomological inoculation rate. Analyses were adjusted for clustering and for month and health centre. This trial is registered as ISRCTN21853394. Findings 1980 children were enrolled in the cohort in 2014 and 2157 in 2015. At the end of the study, more than 99% of children slept under a bednet. The incidence of clinical malaria was 2·0 episodes per child-year in the standard LLIN group and 1·5 episodes per child-year in the PPF-treated LLIN group (incidence rate ratio 0·88 [95% CI 0·77–0·99; p=0·04]). The entomological inoculation rate was 85 (95% CI 63–108) infective bites per transmission season in the standard LLIN group versus 42 (32–52) infective bites per transmission season in the PPF-treated LLIN group (rate ratio 0·49, 95% CI 0·32–0·66; p<0·0001). Interpretation PPF-treated LLINs provide greater protection against clinical malaria than do standard LLINs and could be used as an alternative to standard LLINs in areas with intense transmission of Plasmodium falciparum malaria and highly pyrethroid-resistant vectors. Funding EU Seventh Framework Programme

Behavioural divergence of sympatric Anopheles funestus populations in Burkina Faso

Background: In Burkina Faso, two chromosomal forms of the malaria vector Anopheles funestus, Folonzo and Kiribina, are distinguished by contrasting frequencies of shared polymorphic chromosomal inversions. Sympatric and synchronous populations of Folonzo and Kiribina mate assortatively, as indicated by a significant deficit of heterokaryotypes, and genetic associations among inversions on independently segregating chromosome arms. The present study aimed to assess, by intensive longitudinal sampling, whether sympatric Folonzo and Kiribina populations are characterized by behavioural differences in key malaria vectorial parameters. Methods: The study was conducted in two adjacent villages near Ouagadougou, in the dry savanna of central Burkina Faso. Mosquito adult resting behaviour of both forms was compared based on parallel indoor/outdoor collections across six breeding seasons; 8,235 fully karyotyped samples of half-gravid females were analysed in total. Additionally, indoor/outdoor human biting behaviour, host selection, and Plasmodium falciparum sporozoite rate was assessed and compared between chromosomal forms. Results: The Kiribina form was numerically predominant in the area. However, the Folonzo form was significantly over-represented in indoor resting collections and showed stronger post-prandial endophily, while Kiribina predominated outdoors. Neither form was statistically distinguishable in human biting behaviour, and both were more likely to seek (> 89% and > 8%, respectively). Conclusions: Both Kiribina and Folonzo chromosomal forms are formidable malaria vectors in Burkina Faso. However, the significantly greater tendency for the Kiribina form to rest outdoors despite its pronounced anthropophily suggests that uniform exposure of the overall An. funestus population to indoor-based vector control tools cannot be expected; Kiribina is more likely to evade indoor interventions and escape unharmed outdoors, reducing the efficacy of malaria control. Accordingly, more efficient methods to detect Kiribina and Folonzo, and a more complete understanding of their distribution and behaviour in Africa are advocated

Effective population size of Anopheles funestus chromosomal forms in Burkina Faso - art. no. 115

Background: As Anopheles funestus is one of the principal Afro-tropical malaria vectors, a more complete understanding of its population structure is desirable. In West and Central Africa, An. funestus population structure is complicated by the coexistence of two assortatively mating chromosomal forms. Effective population size ( Ne) is a key parameter in understanding patterns and levels of intraspecific variation, as it reflects the role of genetic drift. Here, N-e was estimated from both chromosomal forms, Kiribina and Folonzo, in Burkina Faso. Methods: Short-term N-e was estimated by evaluating variation at 16 microsatellite loci across temporal samples collected annually from 2000 - 2002. Estimates were based on standardized variance in allele frequencies or a maximum likelihood method. Long-term N-e was estimated from genetic diversity estimates using mtDNA sequences and microsatellites. Results: For both forms, short-term and long-term N-e estimates were on the order of 103 and 105, respectively. Long-term N-e estimates were larger when based on loci from chromosome 3R ( both inside and outside of inversions) than loci outside of this arm. Conclusion: N-e values indicate that An. funestus is not subject to seasonal bottlenecks. Though not statistically different because of large and overlapping confidence intervals, short-term N-e estimates were consistently smaller for Kiribina than Folonzo, possibly due to exploitation of different breeding sites: permanent for Folonzo and intermittent for Kiribina. The higher long-term N-e estimates on 3R, the arm carrying the two inversions mainly responsible for defining the chromosomal forms, give natural selection broader scope and merit further study

To assess whether addition of pyriproxyfen to long-lasting insecticidal mosquito nets increases their durability compared to standard long-lasting insecticidal mosquito nets: study protocol for a randomised controlled trial

Background: The effectiveness of pyrethroid-treated bednets for malaria control in sub-Saharan Africa is under threat because of high levels of resistance to pyrethroid insecticides in the vectors. Here we assess the durability of polyethylene nets with a novel combination of permethrin, a pyrethroid, with pyriproxyfen, an insect juvenile mimic (PPF-LLIN), in comparison with a typical permethrin-treated long-lasting insecticidal net (LLIN). Methods: This is a cluster randomised controlled trial of net durability in Burkina Faso, with clustering at the level of the compound and includes entomological outcome measurements. Half the compounds in each village will be randomly allocated PPF-LLIN and half the LLIN. All sleeping places in a compound will be provided with one type of net. We will distribute the nets at the start of the first transmission season and follow net use at the start and end of each transmission season for 3 years. In one village, bio-efficacy and chemical content will be recorded immediately after net distribution and then at 6, 12, 18, 24, 30 and 36 months. In the other village net survivorship and fabric integrity will be recorded immediately after distribution, and then at 6, 12, 18, 24, 30 and 36 months. Routine measurements of indoor temperature and relative humidity will be made in both villages during the study. Residents will be followed for possible side effects of the PPF-LLIN by surveillance of known asthmatic subjects during the first month post-distribution and pregnancy outcomes will be monitored from antenatal clinic records. Discussion: The protocol is novel on two accounts. Firstly, it is the first to describe the procedure for measuring net durability following recent World Health Organisation (WHO) guidelines. Meeting the minimum requirements set in the guidelines is essential before a new type of net can be recommended by WHO’s Pesticide Evaluation Scheme (WHOPES). Secondly, it describes methods to monitor the persistence of an active ingredient that reduces vector fertility and fecundity. If the PPF-LLIN is both effective and persistent it will provide an alternative vector control strategy where pyrethroid-resistant vectors are present. Trial registration: ISRCTN30634670 webcite assigned 13 August 2014

Seasonal distribution of Anopheles funestus chromosomal forms from Burkina Faso

Background: Previous studies of Anopheles funestus chromosomal inversion polymorphisms in Burkina Faso showed large departures from Hardy-Weinberg equilibrium and linkage disequilibrium among inversions located on different chromosomes, implying the existence of two taxonomic units ("chromosomal forms") with limited genetic flow. One chromosomal form, named Folonzo, is highly polymorphic for alternative rearrangements of 3Ra, 3Rb, 2Ra, and 3La; the other, Kiribina, is predominantly characterized by the standard arrangement of these inversions. To investigate the temporal distribution of these chromosomal forms, further collections were carried out in two villages near Ouagadougou where they are found in sympatry. Methods: Chromosomal karyotypes were determined from indoor-resting, half-gravid females sampled within and across six breeding seasons, from December 1998 to April 2007. Results: As expected, the pattern of chromosomal polymorphism in An. funestus was consistent with assortatively mating Folonzo and Kiribina forms. When samples were assigned to each chromosomal form, their relative abundance varied within successive breeding seasons in a repeating pattern of temporal variability. Relative abundance of the Folonzo form was correlated with climatic variables related to temperature and rainfall. Conclusion: The relative abundance of Folonzo and Kiribina forms of An. funestus likely reflects different larval ecologies that are linked to varying climatic conditions. Further analysis of the bionomics of these vectors is recommended in light of its relevance to vector control

Malaria entomological inoculation rate in a village of Burkina Faso reveals high transmission risk both indoors and outdoors despite the large coverage of LLINs

Introduction – Long lasting insecticide treated bednets (LLINs) are considered among the most effective strategies in malaria vector control, leading to the prevention of 68% of the malaria cases in Africa in 15 years (Bhatt et al., 2015, Nature 526:207-211). Despite this success, the effectiveness of LLINs in sub-Saharan Africa seems to be heterogeneous, since in some hyperendemic countries the annual incidence and the entomological infection rates are still very high (Killeen et al., 2014, Mal. J. 13:330). In Burkina Faso the raising of LLIN coverage from 20% to 70% led to a significant reduction of malaria prevalence, without significantly affecting annual incidence. We here assessed the risk of malaria transmission in a LLIN-protected village of Burkina Faso where in previous surveys it has been detected a high SR rate (A. coluzzii, 7.6% in 2011, 9.3% in 2012) despite the high bednet coverage. Materials and methods – Host-seeking mosquitoes were collected in November 2015 by Human Landing Catch, both indoors and outdoors, in the village of Goden (Ouagadougou area). Collected mosquitoes were morphologically identified and Anopheles gambiae s.l. specimens were subsequently identified per species by PCR (Santolamazza et al., 2008, Mal. J. 7:163). Head+thorax of females were analysed for Plasmodium sporozoite presence by nested-PCR (Calzetta et al., 2018, Med Vet Entomol, in press). Results and conclusions – Among 695 out of 1955 Anopheles gambiae complex specimens (A. coluzzii 55%, A. arabiensis 44%, A. gambiae 1%) analysed so far for Plasmodium infection, the SR is 6.2% (6.9% A. coluzzii, 5.1% A. arabiensis), without significant differences between indoors and outdoors (6.6% and 5.7%, respectively; Chi-square= 0.27 P= 0.6). Considering an average of 83.3 host-seeking mosquitoes per person per night, the entomological inoculation rate (EIR) in the village can be estimated to 5.1 infective bites both indoors and outdoors. This results highlights that, despite the individual protection given by LLINs to the inhabitants from several years, the mosquito population in the area is still stably highly infected and present at high densities. This lead to a non-negligible risk of malaria transmission in the village indoors for people sleeping unprotected by a bednet and for all the population that exposed to mosquito bites outdoors

High Malaria transmission risk in Burkina Faso village despite bednets coverage

Long lasting insecticide treated bednets (LLINs) are considered by WHO among the most effective strategies in malaria control [1]; nevertheless, their impact in Africa seems to be heterogeneous, since in some hyperendemic countries (where LLIN coverage reached 70%) the malaria incidence and the entomological infection rates are still very high [2]. We assessed the risk of malaria transmission in Goden, a LLIN-protected village of Burkina Faso, where in a previous survey (2011) [3] it has been detected a high proportion of Plasmodium infective mosquitoes (7.1%). Host-seeking mosquitoes were sampled indoors and outdoors at three-night time points in November 2015 by Human Landing Catch. A total of 1996 Anopheles gambiae complex specimens was collected (A. coluzzii 55%, A. arabiensis 44%, A. gambiae 1%, with no significant differences in time and position) corresponding to a median number of 23.5 mosquitoes/hour/person. Among these 5.8% were Plasmodium infective with a rate of 1.4 infective bites/person/hour (9.5 per night), both inside and outside houses. Moreover, a high frequency of insecticide resistance was detected in sampled mosquitoes both indoors and outdoors (kdr allele 54%; N=298). These results confirm that, despite the use of LLINs in the village from several years, mosquito population is still highly infected. The high vector density, biting behavior and insecticide resistance, lead to a non-negligible risk of malaria transmission both to people unprotected by a bednet indoors and to those exposed outdoors. This suggest that LLINs, although effective for personal protection, do not significantly reduce the transmission risk at population level. Reference