Funestus Group Research Articles

Outdoor malaria vector species profile in dryland ecosystems of Kenya

Outdoor biting by anopheline mosquitoes is one of the contributors to residual malaria transmission, but the profile of vectors driving this phenomenon is not well understood. Here, we studied the bionomics and genetically characterized populations of An. gambiae and An. funestus complexes trapped outdoors in three selected dryland areas including Kerio Valley, Nguruman and Rabai in Kenya. We observed a higher abundance of Anopheles funestus group members (n = 639, 90.6%) compared to those of the An. gambiae complex (n = 66, 9.4%) with An. longipalpis C as the dominant vector species with a Plasmodium falciparum sporozoite rate (Pfsp) of 5.2% (19/362). The known malaria vectors including An. funestus s.s. (8.7%, 2/23), An. gambiae (14.3%, 2/14), An. rivulorum (14.1%, 9/64), An. arabiensis (1.9%, 1/52) occurred in low densities and displayed high Pfsp rates, which varied with the site. Additionally, six cryptic species found associated with the An. funestus group harbored Pf sporozoites (cumulative Pfsp rate = 7.2%, 13/181). We detected low frequency of resistant 119F-GSTe2 alleles in An. funestus s.s. (15.6%) and An. longipalpis C (3.1%) in Kerio Valley only. Evidence of outdoor activity, emergence of novel and divergent vectors and detection of mutations conferring metabolic resistance to pyrethroid/DDT could contribute to residual malaria transmission posing a threat to effective malaria control.

Community structure and insecticide resistance of malaria vectors in northern-central Myanmar

BackgroundMyanmar is one of the six countries in the Greater Mekong Subregion (GMS) of Southeast Asia. Malaria vectors comprise many Anopheles species, which vary in abundance and importance in malaria transmission among different geographical locations in the GMS. Information about the species composition, abundance, and insecticide resistance status of vectorial systems in Myanmar is scarce, hindering our efforts to effectively control malaria vectors in this region.MethodsDuring October and November 2019, larvae and adult females of Anopheles mosquitoes were collected in three sentinel villages of Banmauk township in northern Myanmar. Adult female mosquitoes collected by cow-baited tent collection (CBTC) and adults reared from field-collected larvae (RFCL) were used to determine mortality rates and knockdown resistance (kdr) against deltamethrin using the standard WHO susceptibility test. Molecular species identification was performed by multiplex PCR and ITS2 PCR, followed by DNA sequencing. The kdr mutation at position 1014 of the voltage-gated sodium channel gene was genotyped by DNA sequencing for all Anopheles species tested.ResultsA total of 1596 Anopheles mosquitoes from seven morphologically identified species groups were bioassayed. Confirmed resistance to deltamethrin was detected in the populations of An. barbirostris (s.l.), An. hyrcanus (s.l.), and An. vagus, while possible resistance was detected in An. annularis (s.l.), An. minimus, and An. tessellatus. Anopheles kochi was found susceptible to deltamethrin. Compared to adults collected by CBTC, female adults from RFCL had significantly lower mortality rates in the four species complexes. A total of 1638 individuals from 22 Anopheles species were molecularly identified, with the four most common species being An. dissidens (20.5%) of the Barbirostris group, An. peditaeniatus (19.4%) of the Hyrcanus group, An. aconitus (13.4%) of the Funestus group, and An. nivipes (11.5%) of the Annularis group. The kdr mutation L1014F was only detected in the homozygous state in two An. subpictus (s.l.) specimens and in a heterozygous state in one An. culicifacies (s.l.) specimen.ConclusionsThis study provides updated information about malaria vector species composition and insecticide resistance status in northern Myanmar. The confirmed deltamethrin resistance in multiple species groups constitutes a significant threat to malaria vector control. The lack or low frequency of target-site resistance mutations suggests that other mechanisms are involved in resistance. Continual monitoring of the insecticide resistance of malaria vectors is required for effective vector control and insecticide resistance management.Graphical

<i>Anopheles leesoni</i> Evans 1931, a Member of the <i>Anopheles funestus</i> Group, Is a Potential Malaria Vector in Cameroon

Background: Understanding the biology of Anopheles malaria vector species is essential to planning effective and sustainable malaria control strategies in endemic countries. This study reported the implication of Anopheles leesoni in malaria transmission in Cameroon, Central Africa. Methods: Mosquitoes were collected in three localities from May 2015 to March 2018 using electric aspirators and Centers for Disease Control light traps (CDC-LT). Anopheles funestus sensu lato (s.l.) mosquitoes were identified as species using polymerase chain reaction assay (PCR). Furthermore, Plasmodium falciparum infection status was determined using the enzyme-linked immunosorbent assay (ELISA) method. Results: A total of 12,744 Anopheles mosquitoes were collected by electric aspirator (N = 4844) and CDC-LT (N = 7900). Anopheles funestus s.l. (86.95%) was the major species and the main malaria vector in rural savannah and rural forest sites followed by A. gambiae s.l. (13.05%) whereas in urban areas, A. gambiae s.l. was by far the most abundant representing 91.45% of Anopheles mosquitoes collected. Two members of the A. funestus group were identified among 1389 analysed by PCR: 1307 A. funestus sensu stricto (s.s.) (94.10%) and 82 A. leesoni (5.9%). Plasmodium falciparum infection rate was 21.04% in A. funestus s.s. For the first time, A. leesoni was found positive for P. falciparum (infection rate: 10.98%) in Cameroon. Conclusion: A very high P. falciparum infection rate was observed in this study in A. funestus s.s., highlighting its high implication in malaria transmission in Cameroon. Furthermore, the detection of P. falciparum infection in A. leesoni calls for more attention towards this neglected vector species.

The effect of irrigation on malaria vector bionomics and transmission intensity in western Ethiopia

BackgroundIrrigation schemes may result in subsequent changes in malaria disease dynamics. Understanding the mechanisms and effects of irrigation on malaria vector bionomics and transmission intensity is essential to develop new or alternative surveillance and control strategies to reduce or control malaria risk. This study was designed to assess the effect of rice irrigation on malaria vector bionomics and transmission intensity in the Gambella Region, Ethiopia.MethodsComparative cross-sectional study was conducted in Abobo District of the Gambella Region, Ethiopia. Accordingly, clusters (kebeles) were classified into nearby and faraway clusters depending on their proximity to the irrigation scheme. Adult mosquito survey was conducted in February, August and November 2018 from three nearby and three faraway clusters using Centers for Disease Control and Prevention (CDC) light traps (LTs). During the November survey, human landing catch (HLC) and pyrethrum spray catch (PSC) were also conducted. The collected mosquitoes were morphologically identified to species and tested for Plasmodium infection using circumsporozoite protein enzyme-linked immunosorbent assay (CSP-ELISA). Furthermore, species-specific polymerase chain reaction (PCR) was performed to identify member species of the Anopheles gambiae complex. Chi-square and t-tests were used to analyze the data using the SPSS version 20 software package.ResultsA total of 4319 female anopheline mosquitoes comprising An. gambiae sensu lato, An. funestus group, An. pharoensis, An. coustani complex and An. squamosus were collected. Overall, 84.5% and 15.5% of the anopheline mosquitoes were collected from the nearby and faraway clusters, respectively. Anopheles gambiae s.l. was the predominant (56.2%) anopheline species in the area followed by An. pharoensis (15.7%). The density of anopheline mosquitoes was significantly higher in the nearby clusters in both HLCs [t(3) = 5.14, P = 0.0143] and CDC LT catches [t(271.97) = 7.446, P < 0.0001). The overall sporozoite rate of anopheline species from the nearby clusters was 10-fold higher compared to the faraway clusters.ConclusionsSignificantly higher mosquito population density was observed in areas close to the irrigation sites. Sporozoite infection rate in the mosquito population was also markedly higher from the nearby clusters. Therefore, the irrigation scheme could increase the risk of malaria in the area.Graphical abstract

Mosquitoes (Diptera: Culicidae) from Villages and Forest Areas of Rural Communes in Khanh Hoa and Binh Phuoc Provinces, Vietnam.

This study presents the diversity of mosquitoes collected from communes, endemic with malaria and dengue, located in Khanh Hoa and Binh Phuoc Provinces, Vietnam. A total of 10,288 mosquitoes were collected in the village and forested sites using standard larval dippers, cow-baited traps, ultra-violet light traps, and mechanical aspirators. Mosquito taxa were identified morphologically and species complexes/groups were further characterized molecularly. Five genera of mosquitoes were morphologically identified: Anopheles Meigen (21 species), Aedes Meigen (2 species), Culex Linnaeus (5 species), Mansonia Blanchard sp., and Armigeres Theobald sp. The PCR-based identification methods allowed the distinction of members of Maculatus Group, Funestus Group, and Dirus Complex; and DNA barcodes enabled the further identification of the Barbirostris Complex. Data reported here include the first report of An. saeungae Taai & Harbach and An. wejchoochotei Taai & Harbach from Vietnam, and re-emphasizes the significance of using molecular data in an integrated systematic approach to identify cryptic species and better understand their role in disease transmission.

Piperonyl butoxide (PBO) combined with pyrethroids in insecticide-treated nets to prevent malaria in Africa.

Background: Public health strategies that target mosquito vectors, particularly pyrethroid long-lasting insecticidal nets (LLINs), have been largely responsible for the substantial reduction in the number of people in Africa developing malaria. The spread of insecticide resistance in Anopheles mosquitoes threatens these impacts. One way to control insecticide-resistant populations is by using insecticide synergists. Piperonyl butoxide (PBO) is a synergist that inhibits specific metabolic enzymes within mosquitoes and has been incorporated into pyrethroid-LLINs to form pyrethroid-PBO nets. Pyrethroid-PBO nets are currently produced by four LLIN manufacturers and,following a recommendation from the World Health Organization (WHO) in 2017, are being included in distribution campaigns incountries. This review examines epidemiological and entomological evidence on whether the addition of PBO to LLINs improves theirefficacy. Objectives1. Evaluate whether adding PBO to pyrethroid LLINs increasesthe epidemiological and entomological effectiveness of the nets.2. Compare the effects of pyrethroid-PBO nets currently in commercial development or on the market with their non-PBO equivalentin relation to:a. malaria infection (prevalence or incidence);b. entomological outcomes. Search methods . We searched the Cochrane Infectious Diseases Group (CIDG) Specialized Register; CENTRAL, MEDLINE, Embase, Web of Science,CAB Abstracts, and two clinical trial registers (ClinicalTrials.gov and WHO International Clinical Trials Registry Platform) up to 24August 2018. We contacted organizations for unpublished data. We checked the reference lists of trials identified by the above methods.

Microbiota identified from preserved Anopheles

BackgroundMosquito species from the Anopheles gambiae complex and the Anopheles funestus group are dominant African malaria vectors. Mosquito microbiota play vital roles in physiology and vector competence. Recent research has focused on investigating the mosquito microbiota, especially in wild populations. Wild mosquitoes are preserved and transported to a laboratory for analyses. Thus far, microbial characterization post-preservation has been investigated in only Aedes vexans and Culex pipiens. Investigating the efficacy of cost-effective preservatives has also been limited to AllProtect reagent, ethanol and nucleic acid preservation buffer. This study characterized the microbiota of African Anopheles vectors: Anopheles arabiensis (member of the An. gambiae complex) and An. funestus (member of the An. funestus group), preserved on silica desiccant and RNAlater® solution.MethodsMicrobial composition and diversity were characterized using culture-dependent (midgut dissections, culturomics, MALDI-TOF MS) and culture-independent techniques (abdominal dissections, DNA extraction, next-generation sequencing) from laboratory (colonized) and field-collected mosquitoes. Colonized mosquitoes were either fresh (non-preserved) or preserved for 4 and 12 weeks on silica or in RNAlater®. Microbiota were also characterized from field-collected An. arabiensis preserved on silica for 8, 12 and 16 weeks.ResultsElizabethkingia anophelis and Serratia oryzae were common between both vector species, while Enterobacter cloacae and Staphylococcus epidermidis were specific to females and males, respectively. Microbial diversity was not influenced by sex, condition (fresh or preserved), preservative, or preservation time-period; however, the type of bacterial identification technique affected all microbial diversity indices.ConclusionsThis study broadly characterized the microbiota of An. arabiensis and An. funestus. Silica- and RNAlater®-preservation were appropriate when paired with culture-dependent and culture-independent techniques, respectively. These results broaden the selection of cost-effective methods available for handling vector samples for downstream microbial analyses.

Estimates of the population size and dispersal range of Anopheles arabiensis in Northern KwaZulu-Natal, South Africa: implications for a planned pilot programme to release sterile male mosquitoes

BackgroundAnopheles arabiensis is a major malaria vector, recently implicated as contributing to ongoing residual malaria transmission in South Africa, which feeds and rests both indoors and outdoors. This species is, therefore, not effectively targeted using core malaria vector control interventions alone. Additionally, increasing resistance to available insecticides necessitates investigations into complementary non-insecticide-based vector control methods for outdoor-resting mosquitoes. The feasibility of the sterile insect technique (SIT) as a complementary vector control intervention is being investigated in South Africa. Successful implementation of an SIT programme largely depends on inundating a target insect population with sterilized laboratory-bred males. Therefore, knowledge of the native population size and dispersal ability of released sterile laboratory-reared males is critical. In this study, we estimated the male An. arabiensis population size and the dispersal of released males in an area targeted for a pilot sterile male release programme.MethodsThree separate releases were performed within a 2-year period. Approximately 5000–15,000 laboratory-reared male An. arabiensis (KWAG) were produced and marked for mark–release–recapture experiments. To recapture released mosquitoes, cloth tubes were deployed in widening concentric circles. The average dispersal distance of released males was calculated and the wild male An. arabiensis population size was estimated using two Lincoln index formulae. The natural population was sampled concurrently and Anopheles species diversity examined.ResultsThe Anopheles gambiae complex and An. funestus group species made up the majority of wild collections along with other anophelines. The An. arabiensis population size was estimated to be between 550 and 9500 males per hectare depending on time of year, weather conditions and method used. Average dispersal distance of marked males ranged from 58 to 86 m. Marked males were found in swarms with wild males, indicating that laboratory-reared males are able to locate and participate in mating swarms.ConclusionsIt was logistically feasible to conduct mark–release–recapture studies at the current scale. The population size estimates obtained may provide a guideline for the initial number of males to use for a pending SIT pilot trial. It is promising for future SIT trials that laboratory-reared marked males participated in natural swarms, appearing at the right place at the right time.Graphical

Lymphatic filariasis, infection status in Culex quinquefasciatus and Anopheles species after six rounds of mass drug administration in Masasi District, Tanzania

BackgroundLymphatic filariasis (LF) elimination program in Tanzania started in 2000 in response to the Global program for the elimination of LF by 2020. Evidence shows a persistent LF transmission despite more than a decade of mass drug administration (MDA). It is advocated that, regular monitoring should be conducted in endemic areas to evaluate the progress towards elimination and detect resurgence of the disease timely. This study was therefore designed to assess the status of Wuchereria bancrofti infection in Culex quinqefasciatus and Anopheles species after six rounds of MDA in Masasi District, South Eastern Tanzania.MethodsMosquitoes were collected between June and July 2019 using Center for Diseases Control (CDC) light traps and gravid traps for indoor and outdoor respectively. The collected mosquitoes were morphologically identified into respective species. Dissections and PCR were carried out to detect W. bancrofti infection. Questionnaire survey and checklist were used to assess vector control interventions and household environment respectively. A Poisson regression model was run to determine the effects of household environment on filarial vector density.ResultsOverall, 12 452 mosquitoes were collected of which 10 545 (84.7%) were filarial vectors. Of these, Anopheles gambiae complex, An. funestus group and Cx. quinquefasciatus accounted for 0.1%, 0.7% and 99.2% respectively. A total of 365 pools of Cx. quinquefasciatus (each with 20 mosquitoes) and 46 individual samples of Anopheles species were analyzed by PCR. For Cx. quinquefasciatus pools, 33 were positive for W. bancrofti, giving an infection rate of 0.5%, while the 46 samples of Anopheles species were all negative. All 1859 dissected mosquitoes analyzed by microscopy were also negative. Households with modern latrines had less mosquitoes than those with pit latrines [odds ratio (OR) = 0.407, P < 0.05]. Houses with unscreened windows had more mosquitoes as compared to those with screened windows (OR = 2.125, P < 0.05). More than 80% of the participants own bednets while 16.5% had no protection.ConclusionsLF low transmission is still ongoing in Masasi District after six rounds of MDA and vector control interventions. The findings also suggest that molecular tools may be essential for xenomonitoring LF transmission during elimination phase.

Fine-scale distribution of malaria mosquitoes biting or resting outside human dwellings in three low-altitude Tanzanian villages

While malaria transmission in Africa still happens primarily inside houses, there is a substantial proportion of Anopheles mosquitoes that bite or rest outdoors. This situation may compromise the performance of indoor insecticidal interventions such as insecticide-treated nets (ITNs). This study investigated the distribution of malaria mosquitoes biting or resting outside dwellings in three low-altitude villages in south-eastern Tanzania. The likelihood of malaria infections outdoors was also assessed. Nightly trapping was done outdoors for 12 months to collect resting mosquitoes (using resting bucket traps) and host-seeking mosquitoes (using odour-baited Suna® traps). The mosquitoes were sorted by species and physiological states. Pooled samples of Anopheles were tested to estimate proportions infected with Plasmodium falciparum parasites, estimate proportions carrying human blood as opposed to other vertebrate blood and identify sibling species in the Anopheles gambiae complex and An. funestus group. Environmental and anthropogenic factors were observed and recorded within 100 meters from each trapping positions. Generalised additive models were used to investigate relationships between these variables and vector densities, produce predictive maps of expected abundance and compare outcomes within and between villages. A high degree of fine-scale heterogeneity in Anopheles densities was observed between and within villages. Water bodies covered with vegetation were associated with 22% higher densities of An. arabiensis and 51% lower densities of An. funestus. Increasing densities of houses and people outdoors were both associated with reduced densities of An. arabiensis and An. funestus. Vector densities were highest around the end of the rainy season and beginning of the dry seasons. More than half (14) 58.3% of blood-fed An. arabiensis had bovine blood, (6) 25% had human blood. None of the Anopheles mosquitoes caught outdoors was found infected with malaria parasites. Outdoor densities of both host-seeking and resting Anopheles mosquitoes had significant heterogeneities between and within villages, and were influenced by multiple environmental and anthropogenic factors. Despite the high Anopheles densities outside dwellings, the substantial proportion of non-human blood-meals and absence of malaria-infected mosquitoes after 12 months of nightly trapping suggests very low-levels of outdoor malaria transmission in these villages.

Patterns of human exposure to early evening and outdoor biting mosquitoes and residual malaria transmission in Ethiopia

Ethiopia has shown a notable progress in reducing malaria burden over the past decade, mainly due to the scaleup of vector control interventions such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). Based on the progress, the country has set goals to eliminate malaria by 2030. However, residual malaria transmission due to early evening and outdoor biting vectors could pose a challenge to malaria elimination efforts. This study assessed vector behavior, patterns of human exposure to vector bites and residual malaria transmission in southwestern Ethiopia. Anopheles mosquitoes were collected monthly from January to December 2018 using human landing catches (HLCs), human-baited double net traps, CDC light traps and pyrethrum spray catches. Human behavior data were collected using questionnaire to estimate the magnitude of human exposure to mosquito bites occurring indoors and outdoors at various times of the night. Enzyme-linked immunosorbent assay (ELISA) was used to determine mosquito blood meal sources and sporozoite infections. A total of 2,038 female Anopheles mosquitoes comprising Anopheles arabiensis (30.8%), An. pharoensis (40.5%), An. coustani (28.1%), An. squamosus (0.3%) and An. funestus group (0.2%) were collected. Anopheles arabiensis and An. pharoensis were 2.4 and 2.5 times more likely to seek hosts outdoors than indoors, respectively. However, 66% of human exposure to An. arabiensis and 39% of exposure to An. pharoensis bites occurred indoors for LLIN non-users. For LLIN users, 75% of residual exposure to An. arabiensis bites occurred outdoors while 23% occurred indoors before bed time. Likewise, 84% of residual exposure to An. pharoensis bites occurred outdoors while 15% occurred indoors before people retired to bed. Anopheles arabiensis and An. pharoensis were 4.1 and 4.8 times more likely to feed on bovine than humans, respectively. Based on the HLC, an estimated indoor and outdoor EIR of An. arabiensis was 6.2 and 1.4 infective bites/person/year, respectively, whereas An. pharoensis had an estimated outdoor EIR of 3.0 infective bites/person/year. In conclusion, An. arabiensis and An. pharoensis showed exophagic and zoophagic behavior. Human exposure to An. arabiensis bites occurred mostly indoors for LLIN non-users, while most of the exposure to both An. arabiensis and An. pharoensis bites occurred outdoors for LLIN users. Malaria transmission by An. arabiensis occurred both indoors and outdoors, whereas An. pharoensis contributed exclusively to outdoor transmission. Additional control tools targeting early-evening and outdoor biting malaria vectors are required to complement the current control interventions to control residual transmission and ultimately achieve malaria elimination.

&lt;i&gt;Anopheles&lt;/i&gt; species composition and some climatic factors that influence their survival and population abundance in Anambra East LGA, Anambra State, Nigeria

Malaria parasites are transmitted in nature through the bites of female Anopheles mosquitoes. However, the vector distribution varies in time and space. This study aimed to determine the species composition, abundance and climatic factors influencing Anopheles mosquitoes in Anambra East Local Government Area of Anambra State, Nigeria from October 2016 to September 2017. Adult Anopheles mosquitoes were collected from indoor and outdoor locations using Pyrethrum Knockdown Collection (PKC) and Human Landing Catch (HLC) Methods respectively. Anopheles mosquito larvae were collected using dipping method. Morphological identification of Anopheles species was carried out using standard identification keys. Climatic data was obtained from Nigerian Meteorological Agency Office in Anambra State. A total of 8181 female Anopheles mosquitoes which comprised 4127 (50.4%) larvae and 4054 (49.6%) adults were collected (P &gt; 0.05) in the study. Four Anopheles species: An. gambiae s. l (70.1%), An. funestus group (18.2%), An. moucheti (6.3%) and An. nili (5.4%) were identified (P &lt; 0.05). In the study area, 2608 (31.9%), 3025 (37.0%) and 2548 (31.1%) Anopheles mosquitoes were collected from Aguleri, Igbariam and Nsugbe respectively; with 100% species overlap. In each selected selected town, the Simpson’s index of diversity was ~2 and Shannon-wiener diversity was ~1. Only An. gambiaes l showed varied seasonal abundance with wet season contributing 67.9% and dry season 32.1% of the overall An. gambiaes. l collection (P &lt; 0.05). The correlation between rainfall and An. gambiae s. l. abundance was significantly strong (r = 0.66; P &lt; 0.05). No significant correlation was found between Anopheles species abundance and temperature as well as relative humidity. The study revealed the preponderance of four Anopheles species: An. gambiae s. l., An. funestus, An. moucheti and An. nili; and rainfall is the only climatic factor that causes temporal change in the abundance of one of the species, An. gambiaes. l. in the study area.&#x0D; Keywords: Anopheles mosquitoes, abundance, temperature, rainfall, relative humidity

Species diversity and distribution of Anopheles mosquitoes in Bure district, Northwestern Ethiopia

Malaria is one the leading health problem of the Ethiopia. Previously, areas above 2,000 m elevation were considered as malaria free areas. However, the major malaria epidemics were seen in areas at an altitude up to 3,000 m above sea level. These epidemics were due to climate and land-use changes (ecological changes) and still malaria is a growing health problem in highland parts of Ethiopia. This study aimed to investigate the species diversity, abundance and distribution of Anopheles mosquitoes in highland fringe of Bure district, Northwestern Ethiopia. It was done in the three different agroecological villages, Bukta (Irrigated), Workimdr (non-irrigated with few dry season breeding habitats) and Shnebekuma (non-irrigated with many dry season breeding habitats). Anopheles mosquitoes were collected by the Centers for Disease Control and Prevention Light Traps Catches, Pyrethrum Spray Catches, and Artificial Pit Shelters (APSs) from twenty-seven houses, thirty houses, and six APSs, respectively. Anopheles mosquitoes were identified morphologically to species using standard keys. Furthermore, molecular identification of Anopheles gambiae s.l was carried out using species-specific Polymerase Chain Reaction. Independent T-Test and One-way- ANOVA were employed to compare the mean mosquito's density between villages and species, indoor and outdoor host seeking mosquitoes. Descriptive statistic was used to calculate the proportion of each Anopheles species. Nine Anopheles mosquito species were identified in the study area which includes: Anopheles demeilloni, An. arabiensis, An. funestus group, An. coustani, An. squamosus, An. cinereus, An. pharoensis, An. rupicolus, and An. natalensis. Of the 4,703 Anopheles mosquitoes collected, An. demeilloni was the most prominent (50.7%, n = 2383) whereas An. rupicolus (0.03%, n = 3), and An. natalensis (0.02%, n = 1) were the least abundant. Higher mean density of Anopheles mosquitoes was collected from the non-irrigated village (2.395 ± 0.100) than irrigated (1.351 ± 0.109) (p = 0.001). In conclusion, three of the most important malaria vectors (An. arabiensis, An. funestus group and An. pharoensis) of Ethiopia were recorded in the study sites, especially the first two was found thought-out the year. Most of the Anopheles mosquitoes were collected from non-irrigated villages. Thus, breeding habitat management must be practiced throughout the year together with long-lasting insecticide-treated nets and insecticide residual sprays.

Anopheles bionomics, insecticide resistance and malaria transmission in southwest Burkina Faso: A pre-intervention study.

BackgroundTwenty-seven villages were selected in southwest Burkina Faso to implement new vector control strategies in addition to long lasting insecticidal nets (LLINs) through a Randomized Controlled Trial (RCT). We conducted entomological surveys in the villages during the dry cold season (January 2017), dry hot season (March 2017) and rainy season (June 2017) to describe malaria vectors bionomics, insecticide resistance and transmission prior to this trial.MethodsWe carried out hourly catches (from 17:00 to 09:00) inside and outside 4 houses in each village using the Human Landing Catch technique. Mosquitoes were identified using morphological taxonomic keys. Specimens belonging to the Anopheles gambiae complex and Anopheles funestus group were identified using molecular techniques as well as detection of Plasmodium falciparum infection and insecticide resistance target-site mutations.ResultsEight Anopheles species were detected in the area. Anopheles funestus s.s was the main vector during the dry cold season. It was replaced by Anopheles coluzzii during the dry hot season whereas An. coluzzii and An. gambiae s.s. were the dominant species during the rainy season. Species composition of the Anopheles population varied significantly among seasons. All insecticide resistance mechanisms (kdr-w, kdr-e and ace-1 target site mutations) investigated were found in each members of the An. gambiae complex but at different frequencies. We observed early and late biting phenotypes in the main malaria vector species. Entomological inoculation rates were 2.61, 2.67 and 11.25 infected bites per human per month during dry cold season, dry hot season and rainy season, respectively.ConclusionThe entomological indicators of malaria transmission were high despite the universal coverage with LLINs. We detected early and late biting phenotypes in the main malaria vector species as well as physiological insecticide resistance mechanisms. These data will be used to evaluate the impact of complementary tools to LLINs in an upcoming RCT.

Malaria Vectors and Vector Surveillance in Limpopo Province (South Africa): 1927 to 2018.

Despite the annual implementation of a robust and extensive indoor residual spraying programme against malaria vectors in Limpopo Province (South Africa), significant transmission continues and is a serious impediment to South Africa’s malaria elimination objectives. In order to gain a better understanding regarding possible causes of this residual malaria, we conducted a literature review of the historical species composition and abundance of malaria vector mosquitoes in the Limpopo River Valley region of the Vhembe District, northern Limpopo Province, the region with the highest remaining annual malaria cases in South Africa. In addition, mosquito surveys were carried out in the same region between October 2017 and October 2018. A total of 2225 adult mosquitoes were collected using CO2-baited tent and light traps, human landing catches and cow-baited traps. Of the 1443 Anopheles collected, 516 were members of the An. gambiae complex and 511 An. funestus group. In the malaria endemic rural areas outside the Kruger National Park, one specimen each of An. gambiae s.s. and An. funestus and only three of An. arabiensis were collected. The latter species was abundant at a remote hot spring in the neighboring Kruger National Park. Eighteen other species of Anopheles were collected. Our survey results support the historical findings that An. arabiensis, the species widely held to be the prime malaria vector in South Africa, is a rare species in the malaria endemic Limpopo River Valley. The implications of the mosquito surveys for malaria transmission, elimination and vector control in northern Limpopo Province and neighboring regions are discussed.

Loop-mediated isothermal amplification (LAMP) assays for Anopheles funestus group and Anopheles gambiae complex species.

Species of the genus Anopheles vary with regard to their vector capacity for Plasmodium spp., the causative agent of malaria, and their accurate identification is often required. Loop-mediated isothermal amplification (LAMP) is a rapid, simple and low-cost method for specific DNA amplification. Primers for LAMP assays specific for the Anopheles funestus group and Anopheles gambiae complex species as well as for the species Anopheles arabiensis, An. funestus, An. gambiae s.s/Anopheles coluzzii (major vectors) and Anopheles rivulorum (minor vector) were designed targeting specific genome or rDNA internal transcribed spacer regions. Reaction conditions (buffer composition, primer concentrations, incubation time) were evaluated and the specificities of the assays confirmed with DNA from non-target Anopheles species. DNA release from the mosquitoes is achieved simply by heating them for 5 min in water. An aliquot of the DNA solutions is transferred to the reaction tube using disposable inoculation loops. The outcome of the LAMP amplifications after 1 h incubation at 65 °C can easily be visualized by a colour change visible to the naked eye. The assays are operable under field conditions requiring only basic equipment (portable heat block programmable at 65 and 80 °C, cooler for master mixes).

Member species of the Anopheles gambiae complex can be misidentified as Anopheles leesoni

BackgroundAccurate Anopheles species identification is key for effective malaria vector control. Identification primarily depends on morphological analysis of field samples as well as molecular species-specific identifications. During an intra-laboratory assessment (proficiency testing) of the Anopheles funestus group multiplex PCR assay, it was noted that Anopheles arabiensis can be misidentified as Anopheles leesoni, a zoophilic member of the An. funestus group. The aim of this project was, therefore, to ascertain whether other members of the Anopheles gambiae complex can also be misidentified as An. leesoni when using the standard An. funestus multiplex PCR.MethodsThe An. funestus multiplex PCR was used to amplify DNA from An. gambiae complex specimens. These included specimens from the laboratory colonies and field samples from the Democratic Republic of Congo. Amplified DNA from these specimens, using the universal (UV) and An. leesoni species-specific primers (LEES), were sequence analysed. Additionally, An. leesoni DNA was processed through the diagnostic An. gambiae multiplex PCR to determine if this species can be misidentified as a member of the An. gambiae complex.ResultsLaboratory-colonized as well as field-collected samples of An. arabiensis, An. gambiae, Anopheles merus, Anopheles quadriannulatus, Anopheles coluzzii as well as Anopheles moucheti produced an amplicon of similar size to that of An. leesoni when using an An. funestus multiplex PCR. Sequence analysis confirmed that the UV and LEES primers amplify a segment of the ITS2 region of members of the An. gambiae complex and An. moucheti. The reverse was not true, i.e. the An. gambiae multiplex PCR does not amplify DNA from An. leesoni.ConclusionThis investigation shows that An. arabiensis, An. gambiae, An. merus, An. quadriannulatus, An. coluzzii and An. moucheti can be misidentified as An. leesoni when using An. funestus multiplex PCR. This shows the importance of identifying specimens using standard morphological dichotomous keys as far as possible prior to the use of appropriate PCR-based identification methods. Should there be doubt concerning field-collected specimens molecularly identified as An. leesoni, the An. gambiae multiplex PCR and sequencing of the internal transcribed spacer 2 (ITS2) can be used to eliminate false identifications.

Distribution of Anopheles mosquito species, their vectorial role and profiling of knock-down resistance mutations in Botswana.

Knowledge of vector species composition and monitoring their change over time is critical to evaluate malaria transmission and assess control interventions. This is especially important in countries such as Botswana, where malaria transmission is subjected to fluctuations due to climate variability. Another important aspect that impacts vector populations is the insecticide resistance. In order to assess species composition and the presence of mutations associated with insecticide resistance, Anopheles specimens from larval samplings and indoor pyrethrum spray sheet collections were analysed. A total of 349 Anopheles were screened by morphology and PCR as belonging to the An. gambiae complex and An. funestus group. Specimens were subsequently analysed for human blood meal and Plasmodium index. Finally, knock-down resistance polymorphisms were assessed. Anopheles arabiensis accounted for the majority of specimens collected through larval (96.7%) and pyrethrum spray sheet collection (87.4%) across all sampling sites, and this species was the only one found positive for human blood and for P. falciparum. Other Anopheles species were collected in small numbers by pyrethrum spray catches, namely An. quadriannulatus, An. longipalpis type C and An. parensis. The authors speculate on changing climate patterns and their possible impact on species composition. The kdr assay revealed that Anopheles mosquitoes were homozygous wild type for both L1014F (kdr-w) and L1014S (kdr-e) mutations. These results highlight the unique vectorial role of An. arabiensis in Botswana and indicated that even with prolonged use of pyrethroids and DDT, the mosquito population has not developed kdr mutations, despite some in vivo evidence of insecticide resistance.

Anopheline Mosquito Species Composition, Kdr Mutation Frequency, and Parasite Infectivity Status in Northern Tanzania.

The scaling-up of malaria control interventions in northern Tanzania has resulted in a decline in malaria prevalence and vector species composition. Despite this achievement, residual malaria transmission remains a concern in the area. The main aim of this study was to investigate malaria vector species composition, parasite infectivity rates, and the presence of insecticide knockdown resistance (kdr) mutations in three sites that have experienced a significant decline in malaria in northern Tanzania. Adult mosquitoes were sampled using light traps in houses and hand-aspirators in cowsheds, whereas the standard dipping method was used for sampling mosquito larvae. Adult mosquitoes identified as Anopheles gambiae s.l. and An. funestus s.l. and larval stages III and IV of An. gambiae s.l. were stored in absolute ethanol for further laboratory molecular identification. The identified species in the An. gambiae complex were An. gambiae s.s., An. merus, An. quadriannulatus, and An. arabiensis, whereas the An. funestus group comprised An. funestus s.s., An. rivulorum, and An. leesoni. For An. gambiae s.s. analyzed from Zeneth, 47.6% were kdr-East homozygous susceptible, 35.7% kdr-East heterozygous resistant, 9.6% kdr-East homozygous resistant, and 7.1% undefined, whereas specimens from Kwakibuyu were 45.5% kdr-East homozygous susceptible, 32.7% kdr-East heterozygous resistant, 16.3% kdr-East homozygous resistant, and 5.5% undefined. There were no kdr-West alleles identified from any specimen. The overall malaria parasite infectivity rate was 0.75%. No infections were found in Moshi. The findings indicate that populations of the major malaria vector mosquitoes are still present in the study area, with An. funestus taking a lead in malaria transmission.

Implication of Anopheles funestus in malaria transmission in the city of Yaoundé, Cameroon

The contribution of Anopheles funestus to malaria transmission in the urban environment is still not well documented. The present study assesses the implication of An. funestus in malaria transmission in two districts, Nsam and Mendong, in the city of Yaoundé. Adult mosquitoes were collected using Centers for Disease Control and Prevention miniature light traps (CDC-LT) and human landing catches from April 2017 to March 2018 and were identified morphologically to the species level. Those belonging to the Anopheles gambiae complex and to the Anopheles funestus group were further processed by PCR to identify members of each complex/group. Anopheline mosquitoes were analysed to determine their infection status using an enzyme-linked immunosorbent assay. Bioassays were conducted with 2–5-day-old female Anopheles funestus and An. gambiae s.l. to determine their susceptibility to permethrin, deltamethrin and dichlorodiphenyltrichloroethane (DDT). Six anopheline species were collected in the peri-urban district of Mendong: Anopheles gambiae, An. coluzzii, An. funestus, An. leesoni, An. ziemanni and An. marshallii; only four out of the six were recorded in Nsam. Of the two members of the Anopheles gambiae complex collected, An. coluzzii was the most prevalent. Anopheles coluzzii was the most abundant species in Nsam, while An. funestus was the most abundant in Mendong. Both Anopheles funestus and An. gambiae s.l. were found to be infected with human Plasmodium at both sites, and both were found to be resistant to DDT, permethrin, and deltamethrin. This study confirms the participation of An. funestus in malaria transmission in Yaoundé and highlights the need to also target this species for sustainable control of malaria transmission.