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Background Most malaria vectors belong to species complexes. Sibling species often exhibit divergent behaviors dictating the measures that can be deployed effectively in their control. Despite the importance of the Anopheles funestus complex in malaria transmission in sub-Saharan Africa, sibling species have rarely been identified in the past and their vectoring potential remains understudied. Methods We analyzed 1149 wild-caught An. funestus ( senso lato ) specimens from 21 sites in Kenya, covering the major malaria endemic areas including western, central and coastal areas. Indoor and outdoor collection tools were used targeting host-seeking and resting mosquitoes. The identity of sibling species, infection with malaria Plasmodium parasites, and the host blood meal sources of engorged specimens were analyzed using PCR-based and sequencing methods. Results The most abundant sibling species collected in all study sites were Anopheles funestus (59.8%) and Anopheles rivulorum (32.4%) among the 1062 successfully amplified specimens of the An. funestus complex. Proportionally, An. funestus dominated in indoor collections whilst An. rivulorum dominated in outdoor collections. Other species identified were Anopheles leesoni (4.6%), Anopheles parensis (2.4%), Anopheles vaneedeni (0.1%) and for the first time in Kenya, Anopheles longipalpis C (0.7%). Anopheles funestu s had an overall Plasmodium infection rate of 9.7% (62/636), predominantly Plasmodium falciparum (59), with two infected with Plasmodium ovale and one with Plasmodium malariae . There was no difference in the infection rate between indoor and outdoor collections. Out of 344 An. rivulorum , only one carried P. falciparum. We also detected P. falciparum infection in two non-blood-fed An. longipalpis C (2/7) which is the first record for this species in Kenya. The mean human blood indices for An. funestus and An. rivulorum were 68% (93/136) and 64% (45/70), respectively, with feeding tendencies on a broad host range including humans and domestic animals such as cow, goat, sheep, chicken and pig. Conclusions Our findings underscore the importance of active surveillance through application of molecular approaches to unravel novel parasite-vector associations possibly contributed by cryptic species with important implications for effective malaria control and elimination.

Anopheles mosquitoes present a major public health challenge in sub-Saharan Africa; notably, as vectors of malaria that kill over half a million people annually. In parts of the east and southern Africa region, one species in the Funestu s group, Anopheles funestus, has established itself as an exceptionally dominant vector in some areas, it is responsible for more than 90% of all malaria transmission events. However, compared to other malaria vectors, the species is far less studied, partly due to difficulties in laboratory colonization and the unresolved aspects of its taxonomy and systematics. Control of An. funestus is also increasingly difficult because it has developed widespread resistance to public health insecticides. Fortunately, recent advances in molecular techniques are enabling greater insights into species identity, gene flow patterns, population structure, and the spread of resistance in mosquitoes. These advances and their potential applications are reviewed with a focus on four research themes relevant to the biology and control of An. funestus in Africa, namely: (i) the taxonomic characterization of different vector species within the Funestu s group and their role in malaria transmission; (ii) insecticide resistance profile; (iii) population genetic diversity and gene flow, and (iv) applications of genetic technologies for surveillance and control. The research gaps and opportunities identified in this review will provide a basis for improving the surveillance and control of An. funestus and malaria transmission in Africa.

Background & objectives: Malaria remains a public health problem in Côte d'Ivoire. To cope with this issue, the Ministry of Health established strategies through Long-Lasting Insecticidal Nets (LLINs) and artemisinin-based medicines. To better understand the influence of periodic mass distribution of LLINs on malaria transmission, this entomological survey was conducted in three regions of Côte d'Ivoire. Methods: Mosquitoes were sampled by Human Landing Catches (HLC) in urban and rural settings of Korhogo, Man and Abengourou. Mosquitoes were identified morphologically and by molecular methods. Plasmodium falciparum infection was assessed by ELISA, and the Entomological Inoculation Rates (EIR) were calculated for each species and setting. Results: Only An. gambiae s.l. was identified in Korhogo and in Abengourou while An. gambiae s.l. and An. funestus s.l. was reported in Man. An. coluzzii was the most abundant species of the An. gambiae siblings collected in Abengourou, and in Man while An. gambiae was most abundant in Korhogo. In urban settings, malaria vectors showed high aggressiveness (>11 bites per person per night) and the annual EIR was high (83.22-438.44 infectious bites per person per year). In rural settings, malaria vectors showed also high aggressiveness (19-52 b/p/n). The annual EIR is very high (>94 ib/p/yr). However, the weakest EIR was recorded in the northern region with 94.90 ib/p/yr. Interpretation & conclusion: This work indicates that malaria transmission remains high and heterogeneous across Côte d'Ivoire, despite repeated mass distribution of LLINs. Also, in Man, malaria transmission is more intense with the involvement of two main vectors. Furthermore, in the village of Korhogo, the EIR remained relatively low.

Background The human landing catch (HLC) is the gold standard method for sampling host-seeking malaria vectors. However, the HLC is ethically questionable because it requires exposure of humans to potentially infectious mosquito bites. Methods Two exposure-free methods for sampling host-seeking mosquitoes were evaluated using electrocuting surfaces as potential replacements for HLC: (1) a previously evaluated, commercially available electrocuting grid (CA-EG) designed for killing flies, and (2) a custom-made mosquito electrocuting trap (MET) designed to kill African malaria vectors. The MET and the CA-EG were evaluated relative to the HLC in a Latin Square experiment conducted in the Kilombero Valley, Tanzania. The sampling consistency of the traps across the night and at varying mosquito densities was investigated. Estimates of the proportion of mosquitoes caught indoors (P i ), proportion of human exposure occurring indoors (π i ), and proportion of mosquitoes caught when most people are likely to be indoors (P fl ) were compared for all traps. Results Whereas the CA-EG performed poorly (<10 % of catch of HLC), sampling efficiency of the MET for sampling Anopheles funestus s.l. was indistinguishable from HLC indoors and outdoors. For Anopheles gambiae s.l. , sampling sensitivity of MET was 20.9 % (95 % CI 10.3–42.2) indoors and 58.5 % (95 % CI 32.2–106.2) outdoors relative to HLC. There was no evidence of density-dependent sampling by the MET or CA-EG. Similar estimates of P i were obtained for An. gambiae s.l . and An. funestus s.l. from all trapping methods. The proportion of mosquitoes caught when people are usually indoors (P fl ) was underestimated by the CA-EG and MET for An. gambiae s.l ., but similar to the HLC for An. funestus . Estimates of the proportion of human exposure occurring indoors (π i ) obtained from the CA-EG and MET were similar to the HLC for An. gambiae s.l ., but overestimated for An. funestus. Conclusions The MET showed promise as an outdoor sampling tool for malaria vectors where it achieved >50 % sampling sensitivity relative to the HLC. The CA-EG had poor sampling sensitivity outdoors and inside. With further modification, the MET could provide an efficient and safer alternative to the HLC for the surveillance of mosquito vectors outdoors.

Background Chemical-based malaria vector control interventions are threatened by the development of insecticide resistance and changes in the behavior of the vectors, and thus require the development of alternative control methods. Bacterial-based larvicides have the potential to target both insecticide resistant and outdoor-biting mosquitoes and are safe to use in the environment. However, the currently available microbial larvicide formulations have a short duration of activity requiring frequent re-applications which increase the cost of control interventions. This study was designed to evaluate the efficacy and duration of activity of two long-lasting formulations of Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) (LL3 and FourStar®) under field conditions in western Kenya highlands. Methods Three sites were selected for this study in the highlands of western Kenya. In each site, one hundred anopheline larval habitats were selected and assigned to one of three arms: (i) LL3; (ii) FourStar®; and (iii) untreated control larval habitats. Four types of larval habitats were surveyed: abandoned gold mines, drainage canals, fish ponds and non-fish ponds. The habitats were sampled for mosquito larvae by using a standard dipping technique and collected larvae were recorded according to the larval stages of the different Anopheles species. The larvicides were applied at manufacturers’ recommended dosage of 1 briquette per 100 square feet. Both treatment and control habitats were sampled for mosquito larvae immediately before treatment (day 0), and then at 24 hours, 3 days and weekly post-treatment for 5 months. Results Overall larval density in treatment habitats was significantly reduced after application of the two microbial larvicides as compared to the control habitats. Post-intervention reduction in anopheline larval density by LL3 was 65, 71 and 84% for 1 day, 2 weeks and 4 weeks, respectively. FourStar® reduced anopheline larval density by 60, 66 and 80% for 1 day, 2 weeks and 4 weeks, respectively. Comparisons between the treatments reveal that LL3 and FourStar® were similar in efficacy. A higher reduction in Anopheles larval density was observed in the abandoned goldmines, while drainage canals had the lowest reduction. Conclusions Both LL3 and FourStar® long-lasting microbial larvicides were effective in reducing immature stages of An. gambiae complex and An. funestus group species, with significant reductions lasting for three months post-application.

The biological activities of essential oils from three plants grown in Cameroon: Ocimum basilicum, Ocimum canum, and Cymbopogon citratus were tested against Plasmodium falciparum and mature-stage larvae of Anopheles funestus. Gas chromatography and gas chromatography – mass spectrometry analyses showed that the main compounds are geranial, 1,8-cineole and linalool in C. citratus, O. canum and O. basilicum, respectively. Larvicidal tests carried out according to the protocol recommended by the World Health Organization showed that the essential oil of leaves of C. citratus is the most active against larvae of An. funestus (LC50 values = 35.5 ppm and 34.6 ppm, respectively, for larval stages III and IV after 6 h of exposure). Besides, the in vitro anti-plasmodial activity evaluated by the radioisotopic method showed that the C. citratus oil is the most active against P. falciparum, with an IC50 value of 4.2 ± 0.5 μg/mL compared with O. canum (20.6 ± 3.4 μg/mL) and O. basilicum (21 ± 4.6 μg/mL). These essential oils can be recommended for the development of natural biocides for fighting the larvae of malaria vectors and for the isolation of natural products with anti-malarial activity. Les activités biologiques des huiles essentielles de trois plantes cultivées au Cameroun, Ocimum basilicum, Ocimum canum et Cymbopogon citratus ont été testées contre Plasmodium falciparum et les stades mûrs des larves d’Anopheles funestus s.s. Les analyses par chromatographie en phase gazeuse et chromatographie en phase gazeuse–spectrométrie de masse ont montré que les principaux composés sont le géranial, le 1,8-cineole et le linalool, respectivement, dans les huiles essentielles de C. citratus, O. canum et O. basilicum. Les tests larvicides réalisés selon le protocole recommandé par l’Organisation Mondiale de la Santé ont montré que l’huile essentielle des feuilles de C. citratus est la plus active vis-à-vis des larves d’An. funestus s.s. (CL50 = 35,5 ppm et 34,6 ppm respectivement pour les larves de stades III et IV après 6 heures d’exposition). En outre, l’activité antiplasmodiale in vitro évaluée par la méthode radio-isotopique a montré que l’huile essentielle de C. citratus est la plus active contre P. falciparum avec une CL50 de 4,2 ± 0,5 μg/ml comparée à celles d’O. canum (20,6 ± 3,4 μg/ml) et d’O. basilicum (21 ± 4,6 μg/ml). Ces huiles essentielles peuvent être recommandées pour le développement de biocides naturels contre les larves des vecteurs du paludisme et pour l’isolement de molécules naturelles ayant une activité anti-plasmodiale.

Malaria is the major cause of morbidity and mortality in sub-Saharan Africa. A child below five years dies after every 30 min. Highland areas under land use change impact on malaria transmission by altering the microclimate of the immature stages and adult mosquitoes. Adult vector population dynamics is important because it is an indicator of transmission risk of the disease. This study was to investigate the effects of microclimatic changes on the mosquito indoor-resting behavior. The study was conducted at a highland site of Marani and at a lowland site of Kombewa where 30 houses were randomly selected at either site. Outdoor and indoor weather conditions were monitored throughout the study period. Indoor mosquitoes were collected using the pyrethrum spray catch method, gonotrophic stage of the females determined and species identified to species level using rDNA polymerase chain reaction method. ELISA was carried out to determine the Plasmodium sporozoites in mosquitoes. Anopheles gambiae s.s. was more abundant at the highland site whereas An. funestus at the lowland site. Indoor densities were highest in June 2003 at both the sites: An. gambiae at the highland site and An. funestus at the lowland site. There was an association between An. gambiae s.s. abundance and relative humidity at the highland site. Combined entomological inoculation rate (EIR) for both the vector species was 0.4 infected bite per year (ib/yr) at the highland site and 31.1 ib/yr at the lowland site. Prolonged indoor spraying with insecticide decreased vector indoor abundance.

Background & objectives: Biting behaviour of Anopheles funestus in Mutare and Mutasa districts, Zimbabwe, is little understood. An investigation was conducted to primarily compare indoor and outdoor biting behaviour of the mosquito, as well as blood meal sources and sporozoite rates. Methods: Monthly adult anopheline sampling was conducted from October 2013 to September 2014 using Centers for Disease Control light-traps, pyrethrum spray catch and artificial pit shelter methods. Mosquitoes sampled by light-traps were divided into two cohorts. In one cohort, traps were left overnight and mosquitoes were collected the following morning, while in the other set, mosquitoes were collected hourly from 1800-0600 hrs . Collected females were identified using morphological characters and categorised according to their abdominal status. Polymerase chain reaction was used to identify An. funestus sibling species and blood meal sources. Infection rate was tested by enzyme-linked immunosorbent assay. Results: Morphological identification showed that indoor and outdoor catches comprised Anopheles funestus (98.3%) and Anopheles gambiae s.l. (1.7%). Of the 2268 mosquitoes collected, 66.2% were caught by light-traps, and 33.8% were caught resting indoors and outdoors. Anopheles funestus and An. gambiae s.l. were trapped more abundantly indoors (68%) than outdoors (32%). Both indoor and outdoor An. funestus densities were higher in wet (4.3) than dry season (1.8). In Burma Valley and Zindi areas, An. funestus demonstrated variable nocturnal indoor and outdoor flight activity rhythms, with two peaks during the night; between 2200-2300 hrs and 0200- 0400 hrs. Human blood index in An. funestus was 0.64, with Plasmodium falciparum infection rate of 1.8%. Interpretation & conclusion: The present work highlighted important information on the host-seeking behaviour, blood meal sources and infection rates in An. funestus. The information would be helpful in improving the vector control strategies.

In Africa, malaria is predominantly a rural disease where agriculture forms the backbone of the economy. Various agro-ecosystems and crop production systems have an impact on mosquito productivity, and hence malaria transmission intensity. This study was carried out to determine spatial and temporal variations in anopheline mosquito population and malaria transmission intensity in five villages, representing different agro-ecosystems in Mvomero district, Tanzania, so as to provide baseline information for malaria interventions. The agro-ecosystems consisted of irrigated sugarcane, flooding rice irrigation, non-flooding rice irrigation, wet savannah and dry savannah. In each setting, adult mosquitoes were sampled monthly using light traps recommended by the Centers for Disease Control and Prevention (CDC) from August 2004 to July 2005. A total of 35,702 female mosquitoes were collected. Anopheles gambiae sensu lato was the most abundant (58.9%) mosquito species. An. funestus accounted for 12.0% of the mosquitoes collected. There was a substantial village to village variation and seasonality in the density of Anopheles mosquito population, with peaks in May towards the end of the warm and rainy season. Significantly larger numbers of anophelines were collected from traditional flooding rice irrigation ecosystem (70.7%) than in non-flooding rice irrigation (8.6%), sugarcane (7.0%), wet savannah (7.3%) and dry savannah (6.4%). The overall sporozoite rates for An. gambiae and An. funestus were 3.4% and 2.3%, respectively. The combined overall sporozoite rate (An. gambiae+An. funestus) was 3.2%. The mean annual entomological inoculation rate (EIR) for An. gambiae s.l. was 728 infective bites per person per year and this was significantly higher in traditional flooding rice irrigation (1351) than in other agro-ecosystems. The highest EIRs for An. gambiae s.l. and An. funestus were observed during May 2005 (long rainy season) and December 2004 (short rainy season), respectively. The findings support the evidence that malaria transmission risk varies even between neighbouring villages and is influenced by agro-ecosystems. This study therefore, demonstrates the need to generate spatial and temporal data on transmission intensity on smaller scales taking into consideration agro-ecosystems that will identify area-specific transmission intensity to guide targeted control of malaria operations.

Background In Kenya, malaria remains a major public health menace equally affecting the semi-arid to arid ecologies. However, entomologic knowledge of malaria vectors in such areas remains poor. Methods Morphologically-identified wild-caught Anopheles funestus ( s.l. ) specimens trapped outdoors from the semi-arid to arid area of Kacheliba, West Pokot County, Kenya, were analysed by PCR and sequencing for species identification, malaria parasite infection and host blood-meal sources. Results Three hundred and thirty specimens were analysed to identify sibling species of the An. funestus group, none of which amplified using the available primers; two were infected with Plasmodium falciparum and Plasmodium ovale , separately, while 84% ( n = 25) of the blood-fed specimens had fed on humans. Mitochondrial cytochrome c oxidase subunit 1 ( cox 1) and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences of 55 specimens ( Plasmodium -positive, blood-fed and Plasmodium -negative) did not match reference sequences, possibly suggesting a previously unreported species, resolving as two clades. Conclusions Our findings indicate the existence of yet-to-be identified and described anopheline species with a potential as malaria vectors in Kenya.