Mosquito odour-baited mass trapping reduced malaria transmission intensity: a result from a controlled before-and-after intervention study.

BACKGROUND: Conventional vector control strategies have significantly reduced the malaria burden. The sustainability of these methods is currently challenged. Odour-based traps are emerging technologies that can complement the existing tools. Implementation of odour-based traps for mass trapping is limited due to the restricted range of vectors caught with available carbon dioxide-dependent lures, and the lack of comprehensive field studies. The objective of this study was to assess the impact of odour-mediated mass trapping targeting outdoor vectors, using a synthetic cattle urine lure that attracts a wide range of vector species in a variety of physiological states, on malaria prevalence and entomological parameters to determine malaria transmission intensities. METHODS: A controlled before-and-after study was conducted in two rural communities in southern Ethiopia. Baseline monthly entomological and seasonal cross-sectional malaria prevalence surveys were conducted in both communities for a year. Then, mass trapping of mosquitoes was conducted in one of the villages, while the monthly entomological surveillance and seasonal malaria prevalence surveys continued in both villages. Generalised linear mixed models were constructed and tested to determine which factors were significantly affected by the intervention. RESULTS: Mass trapping contributed to the reduction of the population of the principal malaria vector, Anopheles arabiensis, and the associated entomological indicators, the human bite rate (HBR) and the entomological inoculation rate (EIR), in the intervention village compared to the control village. The intervention village had an average HBR by An. arabiensis of 3.0 (95% CI 1.4-4.6) during the peak malaria transmission season, compared to 10.5 (95% CI - 0.5-21.5; P < 0.0001) in the control village. The intervention village (mean 0.02, 95% CI - 0.05-0.4.8) had a daily EIR eight times lower than the control village (mean 0.17, 95% CI), which likely contributed to the reduced malaria prevalence in the intervention community following its introduction by ca. 60% (95% CI 55-63). CONCLUSIONS: The combined use of odour-based mass trapping and conventional control strategies coincided with a reduction of human-vector contact and malaria prevalence, providing support for odour-baited technologies as a viable option for next-generation vector control tools. Further cluster-randomised control studies are recommended in different eco-epidemiological settings with varying malaria transmission intensities.

Malaria mosquitoes acquire and allocate cattle urine to enhance life history traits.

BACKGROUND: Nutrient acquisition and allocation integrate foraging and life-history traits in insects. To compensate for the lack of a particular nutrient at different life stages, insects may acquire these through supplementary feeding, for example, on vertebrate secretions, in a process known as puddling. The mosquito Anopheles arabiensis emerges undernourished, and as such, requires nutrients for both metabolism and reproduction. The purpose of this study was to assess whether An. arabiensis engage in puddling on cattle urine to obtain nutrients to improve life history traits. METHODS: To determine whether An. arabiensis are attracted to the odour of fresh, 24 h, 72 h and 168 h aged cattle urine, host-seeking and blood-fed (48 h post-blood meal) females were assayed in a Y-tube olfactometer, and gravid females assessed in an oviposition assay. Combined chemical and electrophysiological analyses were subsequently used to identify the bioactive compounds in all four age classes of cattle urine. Synthetic blends of bioactive compounds were evaluated in both Y-tube and field assays. To investigate the cattle urine, and its main nitrogenous compound, urea, as a potential supplementary diet for malaria vectors, feeding parameters and life history traits were measured. The proportion of female mosquitoes and the amount of cattle urine and urea imbibed, were assessed. Following feeding, females were evaluated for survival, tethered flight and reproduction. RESULTS: Host-seeking and blood-fed An. arabiensis were attracted to the natural and synthetic odour of fresh and aged cattle urine in both laboratory and field studies. Gravid females were indifferent in their response to cattle urine presence at oviposition sites. Host-seeking and blood-fed females actively imbibed cattle urine and urea, and allocated these resources according to life history trade-offs to flight, survival or reproduction, as a function of physiological state. CONCLUSIONS: Anopheles arabiensis acquire and allocate cattle urine to improve life history traits. Supplementary feeding on cattle urine affects vectorial capacity directly by increasing daily survival and vector density, as well as indirectly by altering flight activity, and thus should be considered in future models.

Malaria prevalence and associated risk factors in Dembiya district, North-western Ethiopia.

BACKGROUND: Ethiopia embarked on combating malaria with an aim to eliminate malaria from low transmission districts by 2030. A continuous monitoring of malaria prevalence in areas under elimination settings is important to evaluate the status of malaria transmission and the effectiveness of the currently existing malaria intervention strategies. The aim of this study was to assess the prevalence of malaria and associated risk factors in selected areas of Dembiya district. METHODS: A cross-sectional parasitological and retrospective survey was conducted in the two localities of Dembiya District, selected based on their long standing history of implementing malaria prevention and elimination strategies. Thin and thick blood smears collected from 735 randomly selected individuals between October and December, 2018 were microscopically examined for malaria parasites. Six years (2012-2017) retrospective malaria data was collected from the medical records of the health centres. Structured questionnaires were prepared to collect information about the socio-economic data of the population. Logistic regression analysis was used to determine a key risk factor explaining the prevalence of malaria. The data were analysed using SPSS version 20 and p ≤ 0.05 were considered statistically significant. RESULTS: The 6-year retrospective malaria prevalence trend indicates an overall malaria prevalence of 22.4%, out of which Plasmodium falciparum was the dominant species. From a total of 735 slides examined for the presence of malaria parasites, 3.5% (n = 26) were positive for malaria parasites, in which P. falciparum was more prevalent (n = 17; 2.3%), Plasmodium vivax (n = 5; 0.7%), and mixed infections (n = 4; 0.5%). Males were 2.6 times more likely to be infected with malaria than females (AOR = 2.6; 95% CI 1.0, 6.4), and individuals with frequent outdoor activity were 16.4 times more vulnerable than individuals with limited outdoor activities (AOR = 16.4, 95% CI 1.8, 147.9). Furthermore, awareness about malaria transmission was significantly associated with the prevalence of malaria. CONCLUSIONS: Malaria is still a public health problem in Dembiya district irrespective of the past and existing vector control interventions. Therefore, the authorities should work on designing alternative intervention strategies targeting outdoor malaria transmission and improving community awareness about malaria transmission and control methods in the study area. For this, continuous monitoring of vectors' susceptibility, density, and behaviour is very important in such areas.

Development of a chimeric odour blend for attracting gravid malaria vectors.

BACKGROUND: Odour-based tools targeting gravid malaria vectors may complement existing intervention strategies. Anopheles arabiensis are attracted to, and stimulated to oviposit by, natural and synthetic odours of wild and domesticated grasses associated with mosquito breeding sites. While such synthetic odour lures may be used for vector control, these may have limited efficacy when placed in direct competition with the natural source. In this study, workflows developed for plant-feeding pests was used to design and evaluate a chimeric odour blend based on shared attractive compounds found in domesticated grass odours. METHODS: Variants of a synthetic odour blend, composed of shared bioactive compounds previously identified in domesticated grasses, was evaluated sequentially in a two-choice olfactometer to identify a ratio-optimized attractive blend for malaria vectors. During this process, blends with ratios that were significantly more attractive than the previously identified synthetic rice blend were compared to determine which was most attractive in the two-choice olfactometer. To determine whether all volatile components of the most attractive blend were necessary for maximal attraction, subtractive assays were then conducted, in which individual components were removed for the most attractive blend, to define the final composition of the chimeric blend. Binary logistic regression models were used to determine significance in all two-choice assays. The chimeric blend was then assessed under field conditions in malaria endemic villages in Ethiopia, to assess the effect of dose, trap type, and placement relative to ground level. Field data were analyzed both descriptively and using a Welch-corrected t-test. RESULTS: A ratio-optimized chimeric blend was identified that significantly attracted gravid An. arabiensis under laboratory conditions. In the field, trap captures of An. arabiensis and Anopheles pharoensis were dependent on the presence of the lure, trap type (CDC, BG Sentinel and Suna traps), placement relevant to ground level, with low release rates generally luring more mosquitoes. CONCLUSIONS: The workflow designed for the development of chimeric lures provides an innovative strategy to target odour-mediated behaviours. The chimeric lure identified here can be used in existing trapping systems, and be customized to increase sustainability, in line with goals of the Global Vector Control Response Group.

Environmental Factors Associated with Larval Habitats of Anopheline Mosquitoes (Diptera: Culicidae) in Metema District, Northwestern Ethiopia.

BACKGROUND: Malaria is one of the major public health concerns in Ethiopia. Control options available for the management of malaria, include case detection, personal protection and larval source management. Effective control of Anopheles larvae largely depends on understanding of the habitats of the vectors. The aims of this study were to identify the breeding habitats of mosquitoes and characterize the larval habitats in Gende Wuha Town in northwestern Ethiopia. METHODS: Different aquatic habitats were sampled and characterized for anopheline larvae from November 2012 to June 2013. RESULTS: In total, 2784 larvae of Anopheles mosquitoes were collected from various breeding habitats. Microscopic identification of the III and IV instars revealed the presence of seven Anopheles species. Of the Anopheles spp, Anopheles gambiae s.l. (80%) was the most predominant species in the study area. Spearman correlation coefficient results also determined that the density of An. gambiae s.l. increased significantly with habitat temperature (r= 0.346, p< 0.01). Significantly higher An. gambiae s.l. larvae were obtained in non-shaded habitats (z= -3.120, p< 0.05) when compared with shaded habitats. CONCLUSION: The current study demonstrated An. gambiae s.l., the principal malaria vector in the country, is the predominant species in the larval sampling habitats. It was also noted the importance of edge of stream as larva breeding habitats for this species during the dry season of the year. Therefore, attention should be given for this breeding habitat for control of the vector during dry season.

Malaria hotspots explained from the perspective of ecological theory underlying insect foraging.

Hotspots constitute the major reservoir for residual malaria transmission, with higher malaria incidence than neighbouring areas, and therefore, have the potential to form the cornerstone for successful intervention strategies. Detection of malaria hotspots is hampered by their heterogenous spatial distribution, and the laborious nature and low sensitivity of the current methods used to assess transmission intensity. We adopt ecological theory underlying foraging in herbivorous insects to vector mosquito host seeking and modelling of fine-scale landscape features at the village level. The overall effect of environmental variables on the density of indoor mosquitoes, sporozoite infected mosquitoes, and malaria incidence, was determined using generalized linear models. Spatial analyses were used to identify hotspots for malaria incidence, as well as malaria vector density and associated sporozoite prevalence. We identify household occupancy and location as the main predictors of vector density, entomological inoculation rate and malaria incidence. We propose that the use of conventional vector control and malaria interventions, integrated with their intensified application targeting predicted hotspots, can be used to reduce malaria incidence in endemic and residual malaria settings.

Plasmodium falciparum gametocyte-induced volatiles enhance attraction of Anopheles mosquitoes in the field.

BACKGROUND: Plasmodium parasites manipulate the interaction between their mosquito and human hosts. Patients infected with gametocytes attract anopheline mosquitoes differentially compared to healthy individuals, an effect associated with an increased release of attractive volatile cues. This odour-driven manipulation is partly mediated by the gametocyte-specific metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which induces increased release of select aldehydes and terpenes from red blood cells and results in the enhanced attraction of host-seeking mosquitoes, which are vectors of malaria. This study investigates the effect of the HMBPP-induced volatiles on the attraction of wild Anopheles mosquitoes to humans under field conditions. METHODS: The efficacy of the HMBPP-induced odour blend to attract Anopheles was evaluated in a 4 × 6 Latin rectangular study design indoors using baited Suna traps. Furthermore, to assess the efficacy of the HMBPP-induced odour blend in (1) augmenting the attractiveness of human odour, and (2) attracting Anopheles mosquitoes in the absence of human odour, a two-choice assay using host decoy traps (HDTs) was used and evaluated using binomial generalized regression. RESULTS: Traps baited with the HMBPP-induced odour blend attracted and caught both Anopheles arabiensis and Anopheles pharoensis females in a dose-dependent manner in the presence of background human odour, up to 2.5 times that of an unbaited trap. Given a choice between human odour and human odour laden with the HMBPP-induced odour blend, mosquitoes differentially preferred traps augmented with the HMBPP-induced odour blend, which caught twice as many female An. arabiensis. Traps baited with the HMBPP-induced odour blend but lacking the background of human odour were not effective in attracting and catching mosquitoes. CONCLUSION: The findings of the present study revealed that the HMBPP-induced odour blend, when augmented with human body odour, is attractive to anopheline mosquitoes and could be used as a complementary vector control tool along with existing strategies.

Anopheles larval species composition and characterization of breeding habitats in two localities in the Ghibe River Basin, southwestern Ethiopia.

BACKGROUND: Documentation of the species composition of Anopheles mosquitoes and characterization of larval breeding sites is of major importance for the implementation of larval control as part of malaria vector control interventions in Ethiopia. The aims of this study were to determine the Anopheles larval species composition, larval density, available habitat types and the effects of related environmental and physico-chemical parameters of habitats in the Ghibe River basin of southwestern Ethiopia. METHODS: Anopheles larvae were sampled from November 2014 to October 2016 on a monthly basis and 3rd and 4th instars were identified microscopically to species. The larval habitats were characterized based on habitat perimeter, water depth, intensity of light, water current, water temperature, water pH, water turbidity, distance to the nearest house, vegetation coverage, permanence of the habitat, surface debris coverage, emergent plant coverage, habitat type and substrate type. RESULTS: In total, 9277 larvae of Anopheles mosquitoes and 494 pupae were sampled from borrow pits, hoof prints, rain pools, pools at river edges, pools in drying river beds, rock pools, tire tracks and swamps. Anopheles larval density was highest in pools in drying river beds (35.2 larvae per dip) and lowest in swamps (2.1 larvae per dip) at Darge, but highest in rain pools (11.9 larvae per dip), borrow pits (11.2 larvae per dip) and pools at river edges (7.9 larvae per dip), and lowest in swamps (0.5 larvae per dip) at Ghibe. A total of 3485 late instar Anopheles mosquito larvae were morphologically identified. Anopheles gambiae sensu lato was the primary Anopheles mosquito found in all larval habitats except in swamps. Temperature at the time of sampling and emergent vegetation, were the most important variables for Anopheles mosquito larval density. Anopheles gambiae density was significantly associated with habitats that had smaller perimeters, were sunlit, had low vegetation cover, and a lack of emergent plants. Generally, Anopheles mosquito larval density was not significantly associated with water pH, water temperature, water turbidity, algal content, and larval habitat depth. CONCLUSION: Different species of Anopheles larvae were identified including An. gambiae s.l., the main malaria vector in Ethiopia. Anopheles gambiae s.l. is the most abundant species that bred in most of the larval habitat types identified in the study area. The density of this species was high in sunlit habitat, absence of emergent plants, lack of vegetation near habitat and habitats closer to human habitation. Rainfall plays a great role in determining the availability of breeding habitats. The presence of rain enable to create some of the habitat types, but alter the habitats formed at the edge of the rivers due to over flooding. Controlling the occurrence of mosquito larvae through larval source management during the dry season, targeting the pools in drying river bed and pools formed at the edge of the rivers as the water receded can be very crucial to interrupt the re-emergence of malaria vectors on the onset of rainy season.

Species composition, blood meal hosts and Plasmodium infection rates of Anopheles mosquitoes in Ghibe River Basin, southwestern Ethiopia.

BACKGROUND: Vector control interventions using long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are commonly practiced tools for the control of malaria in Ethiopia. In order to evaluate the effectiveness of these control interventions, and understand the prevailing malaria vectors, their incrimination in disease transmission, and their resting and feeding behavior, we set out to identify the Anopheles species, their blood meal sources, and entomological inoculation rate (EIR) in Ghibe and Darge within the Ghibe River basin, southwestern Ethiopia. METHODS: Adult Anopheles mosquitoes were sampled both indoors and outdoors from January 2015 to October 2016 using Centers for Disease Control and Prevention (CDC) light traps, pyrethrum spray catch (PSC), artificial pit shelters and mouth aspirators. Mosquito species were morphologically identified, and their blood meal sources and malaria sporozoite rates were assessed using enzyme-linked immunosorbent assays. RESULTS: In total, 13 species of Anopheles mosquitoes were identified, among which Anopheles gambiae (s.l.) was the predominant species: 87.9 and 67.7% in Ghibe and Darge, respectively. The mean density of An. gambiae (s.l.) collected per night using CDC light traps was 1.8 and 0.7 outdoors and indoors, respectively, in Ghibe, and 0.125 and 0.07 indoors and outdoors, respectively, in Darge. Anopheles mosquito abundance was higher in houses near the river than in houses far from the river in both study sites. Among Anopheles mosquitoes sampled using CDC light trap catches, 67.6% were unfed and the indoor and outdoor human blood indices of An. gambiae (s.l.) were 58.4 and 15.8%, respectively in Ghibe, while in Darge, they were 57.1 and 50%, respectively. Sporozoite rates were 0.07% for P. vivax and 0.07% for P. falciparum in Ghibe and zero in Darge. In Ghibe, the overall EIRs for P. falciparum and P. vivax were zero and 8.4 infective bites/person/year, respectively, in 2015, while zero and 5.4 infective bites/person/year for P. vivax and P. falciparum, respectively, in 2016. No Plasmodium-positive Anopheles mosquitoes were identified from Darge. CONCLUSIONS: Anopheles gambiae (s.l.), the principal vector of malaria in Ethiopia was the most abundant species both indoors and outdoors, fed both on human and cattle blood and occurred at higher frequencies near rivers. Anopheles gambiae (s.l.) that were circumsporozoite-positive for Plasmodium species were collected from Ghibe, but not Darge.

Impact of combining indoor residual spraying and long-lasting insecticidal nets on Anopheles arabiensis in Ethiopia: results from a cluster randomized controlled trial.

BACKGROUND: Indoor residual house spraying (IRS) and long-lasting insecticidal nets (LLINs) are the key front-line malaria vector interventions against Anopheles arabiensis, the sole primary malaria vector in Ethiopia. Universal coverage of both interventions has been promoted and there is a growing demand in combinations of interventions for malaria control and elimination. This study compared the impact on entomological outcomes of combining IRS and LLINs with either intervention alone in Adami Tullu district, south-central Ethiopia. The epidemiological outcomes were recently published on a separate paper. METHODS: This factorial, cluster-randomized, controlled trial randomized villages to four study arms: IRS + LLIN, IRS, LLIN, and control. LLINs (PermaNet 2.0) were provided free of charge. IRS with propoxur was applied before the main malaria transmission season in 2014, 2015 and 2016. Adult mosquitoes were collected in randomly selected villages in each arm using CDC light trap catch (LTC) set close to a sleeping person, pyrethrum spray catch (PSC), and artificial pit shelter (PIT), for measuring mosquito host-seeking density (HSD), indoor resting density (IRD), and outdoor resting density (ORD), respectively. Human landing catch (HLC) was performed in a sub-set of villages to monitor An. arabiensis human biting rates (HBR). Mean vector densities and HBR were compared among study arms using incidence rate ratio (IRR) calculated by negative binomial regression. RESULTS: There were no significant differences in mean densities (HSD, IRD, ORD) and HBR of An. arabiensis between the IRS + LLIN arm and the IRS arm (p > 0.05). However, mean HSD, IRD, ORD, and HBR were significantly lower in the IRS + LLIN arm than in the LLIN alone arm (p < 0.05). All An. arabiensis tested for malaria infection were negative for Plasmodium species. For this reason, the entomological inoculation rate could not be determined. CONCLUSIONS: The IRS + LLIN were as effective as IRS alone in reducing densities and HBR of An. arabiensis. However, the effectiveness of the two interventions combined was higher than LLINs alone in reducing densities and HBR of the vector. Added impact of the combination intervention against malaria infectivity rates of An. arabiensis compared to either intervention alone remains unknown and warrants further research. Trial registration PACTR201411000882128. Registered 8 September 2014, https://trialsjournal.biomedcentral.com/articles/10.1186/s13063-016-1154-2.

Isolation and identification of microflora from the midgut and salivary glands of Anopheles species in malaria endemic areas of Ethiopia.

BACKGROUND: Anopheles mosquitoes are of great importance to human health. A number of studies have shown that midgut and salivary gland microflora have an impact on malaria parasite burden through colonization mechanisms, involving either direct Plasmodium microbiota interaction or bacterial-mediated induction of mosquito immune response. The objective of this study was to isolate and identify the microflora from the midgut and salivary glands of Anopheles species. METHODS: A total of 20 pools (ten per pool) from insectary-reared and 56 pools (five per pool) of field-collected Anopheles mosquitoes were anesthetized by chloroform and dissected. 70% of ethanol was used for surface sterilization of mosquitoes and laboratory equipment, followed by rinsing Anopheles mosquitoes four times with 1X PBS. Each pool of dissected midgut and salivary gland sample was transferred in 1X PBS and squashed, incubated in the water bath and enriched in tryptic soya broth for 24 h at 35 ± 2 °C. As a control, the PBS solutions used to rinse the mosquitoes were also incubated in tryptic soya broth in the same conditions as the sample. After enrichment, a loopful of each sample was taken and inoculated on Blood, Chocolate, MacConkey, and Sabouraud Dextrose agar. Finally, the microbiota was isolated by colony characteristics, biochemical tests, and automated VITEK 2 Compact Analyzer. RESULTS: From all field and laboratory mosquitoes, Pseudomonas was found to be the dominant microbiota identified from all species of Anopheles mosquitoes. Acinetobacter and Klebsiellapneumonia and other families of gram-positive and gram-negative bacteria were identified. CONCLUSIONS: A number of bacteria were isolated and identified. This is the first report on isolation and identification of microbiota from midgut and salivary glands of Anopheles species in Ethiopia. It can be used as a baseline for studying the relationship between microbiota and mosquitoes, and for the development of a new malaria biological control.

Environmental and meteorological factors linked to malaria transmission around large dams at three ecological settings in Ethiopia.

BACKGROUND: A growing body of evidence suggests that dams intensify malaria transmission in sub-Saharan Africa. However, the environmental characteristics underpinning patterns in malaria transmission around dams are poorly understood. This study investigated local-scale environmental and meteorological variables linked to malaria transmission around three large dams in Ethiopia. METHODS: Monthly malaria incidence data (2010-2014) were collected from health centres around three dams located at lowland, midland and highland elevations in Ethiopia. Environmental (elevation, distance from the reservoir shoreline, Normalized Difference Vegetation Index (NDVI), monthly reservoir water level, monthly changes in water level) and meteorological (precipitation, and minimum and maximum air temperature) data were analysed to determine their relationship with monthly malaria transmission at each dam using correlation and stepwise multiple regression analysis. RESULTS: Village distance to reservoir shoreline (lagged by 1 and 2 months) and monthly change in water level (lagged by 1 month) were significantly correlated with malaria incidence at all three dams, while NDVI (lagged by 1 and 2 months) and monthly reservoir water level (lagged by 2 months) were found to have a significant influence at only the lowland and midland dams. Precipitation (lagged by 1 and 2 months) was also significantly associated with malaria incidence, but only at the lowland dam, while minimum and maximum air temperatures (lagged by 1 and 2 months) were important factors at only the highland dam. CONCLUSION: This study confirmed that reservoir-associated factors (distance from reservoir shoreline, monthly average reservoir water level, monthly water level change) were important predictors of increased malaria incidence in villages around Ethiopian dams in all elevation settings. Reservoir water level management should be considered as an additional malaria vector control tool to help manage malaria transmission around dams.

Phosphite alters the behavioral response of potato tuber moth (Phthorimaea operculella) to field-grown potato.

BACKGROUND: The potato tuber moth (PTM) (Phthorimaea operculella) is a pest of solanaceous species that causes serious damage to potato tubers and tomato fruits. Control is mainly dependent on the use of synthetic chemicals, which pose a risk to the environment and health of farmers, especially in developing countries where application safety rules are often neglected. In this study we aimed at investigating the effects of a plant resistance inducer (PRI) potassium phosphite on PTM larval population density and PTM parasitoid levels, which can be used as biocontrol agents. We also tested whether intercropping with tomato, which is less attractive to PTM, provided a spatial border to further reduce PTM numbers. RESULTS: In two different locations over two seasons, we showed that foliar application of phosphite more than halved the PTM larval populations on potato, and that PTM parasitoid numbers were unaffected. No consistent reduction in PTM was achieved by intercropping potato with tomato. CONCLUSIONS: Phosphite reduced PTM numbers in the field without interfering with autochthonous parasitoids, indicating its suitability as part of an Integrated Pest Management strategy. Ex situ choice tests showed that phosphite-treated potato deterred PTM, which could be a reason for the control of PTM in the field. © 2018 Society of Chemical Industry.

Shady business: understanding the spatial ecology of exophilic Anopheles mosquitoes.

BACKGROUND: Understanding the ecology of exophilic anophelines is a key step toward developing outdoor control strategies to complement existing indoor control tools against malaria vectors. This study was conducted to assess the movement pattern of exophilic Anopheles mosquitoes between blood meal sources and resting habitats, and the landscape factors dictating their resting habitat choice. RESULTS: Resting clay pots were placed at 5 m, 25 m, 50 m, 75 m and 100 m away from isolated focal houses, radiating from them in four directions. The locations of the clay pots represent heterogeneous land cover types at a relatively fine spatial scale in the landscape. The effect of the landscape characters on the number of both female and male anophelines caught was modelled using zero-inflated negative binomial regression with a log link function. A total of 420 Anopheles mosquitoes (353 females and 67 males) belonging to three species; Anopheles arabiensis, Anopheles pharoensis, and Anopheles tenebrosus were caught in the resting clay pots, with An. arabiensis being the dominant species. Canopy cover, distance from the house, and land cover type were the significant landscape characters influencing the aggregation of resting mosquitoes. Both the count and binary models showed that canopy cover was the strongest predictor variable on the counts and the presence of Anopheles mosquitoes in the clay pots. Female Anopheles were most frequently found resting in the pots placed in banana plantations, and at sampling points that were at the greater distances (75 m and 100 m) from the focal house. CONCLUSIONS: This study showed that exophilic Anopheles mosquitoes tend to rest in shaded areas some distance away from human habitation. These findings are important when targeting mosquitoes outdoors, complementing the existing effort being made to control malaria vectors indoors.

Can water-level management reduce malaria mosquito abundance around large dams in sub-Saharan Africa?

BACKGROUND: Water level management has been suggested as a potential tool to reduce malaria around large reservoirs. However, no field-based test has been conducted to assess the effect of water level management on mosquito larval abundance in African settings. The objective of the present study is to evaluate the effects of water level drawdown rates on mosquito larval abundance. METHODS: Twelve experimental dams were constructed on the foreshore of the Koka Dam in Ethiopia. These were grouped into four daily water drawdown treatments, each with three replicates: no water-level drawdown (Group 1; Control), 10 mm.d-1 (Group 2), 15 mm.d-1 (Group 3) and 20 mm.d-1 (Group 4). Larval sampling was conducted weekly for a period of 6 weeks each in the main malaria transmission season (October to November 2013) and subsequent dry season (February to March 2014). Larval densities were compared among treatments over time using repeated measures Analysis of Variance (ANOVA). RESULTS: A total of 284 Anopheles mosquito larvae were collected from the experimental dams during the study period. Most (63.4%; n = 180) were collected during the main malaria transmission season while the remaining (36.6%; n = 104) were collected during the dry season. Larvae comprised four Anopheles species, dominated by Anopheles arabiensis (48.1% of total larval samples; n = 136) and An. pharoensis (33.2%; n = 94). Mean larval density was highest in control treatment dams with stable water levels throughout the study, and decreased significantly (P < 0.05) with increasing water drawdown rates in both seasons. During the main transmission season, anopheline larval density was generally lower by 30%, 70% and 84% in Groups 2, Group 3 and Group 4, respectively, compared with the control dams (Group 1). In the dry season, larval density was reduced by 45%, 70% and 84% in Groups 2, Group 3 and Group 4, respectively, when compared to the control dams. CONCLUSION: Increased water drawdown rates were associated with lower mosquito larval abundance. Water level management could thus serve as a potential control measure for malaria vectors around reservoirs by regulating the persistence of shallow shoreline breeding habitats. Dam operators and water resource managers should consider incorporating water level management as a malaria control mechanism into routine dam operations to manage the risk of malaria transmission to human populations around reservoirs.

Some aspects of entomological determinants of Phlebotomus orientalis in highland and lowland visceral leishmaniasis foci in northwestern Ethiopia.

Visceral leishmaniasis (VL) is one of the major public health problems in northwest Ethiopia, mainly in Libo-Kemkem and Metema districts, where Phlebotomus orientalis is the most probable vector of the disease. The aim of this study was to determine the physiological age, host preference and vectorial potential of P. orientalis in the highland and lowland foci of the region. Sand flies were collected using CDC light traps between May 2011 and April 2012 in Libo-Kemkem and October 2012 and September 2013 in Metema from household compounds, farm field and mixed forest. Females belonging to Phlebotomus were dissected for physiological age determination and Leishmania detection and isolation. Leishmania infections in sand flies were investigated using molecular methods. Freshly fed Phlebotomus females were tested to identify blood meal sources using PCR-RLB and ELISA. A total of 1149 (936 from Libo-Kemkem and 213 from Metema) blood unfed female P. orientalis were dissected for age determination. The parity rate was 45.6% and 66.2% in Libo-Kemkem and Metema, respectively. None of 798 female P. orientalis dissected (578 from Libo-Kemkem and 220 from Metema) was infected with Leishmania parasites. A total of 347 P. orientalis specimens collected from Libo-Kemkem were processed using PCR, of which 10 (2.8%) specimens were found with DNA of Leishmania spp. Of a total 491 freshly fed female P. orientalis analyzed for blood meal origins by RLB-PCR and ELISA, 57.6% (67.8% from Libo-Kemkem and 49.8% from Metema) were found to contain bovine blood while 4.9% (3.7% from Libo-Kemkem and 5.7% from Metema) were of human blood. In conclusion, the present study showed parity difference between the two populations of P. orientalis and that both populations have strong zoophilic behavior. Based on the presented evidences, the species is strongly implicated as a vector of kala-azar in both areas. Therefore, vector control should be a component of a strategy to manage visceral leishmaniasis in both study areas.

Sweet attraction: sugarcane pollen-associated volatiles attract gravid Anopheles arabiensis.

BACKGROUND: Anopheles arabiensis is a key vector for the transmission of human malaria in sub-Saharan Africa. Over the past 10,000 years, humans have successfully cultivated grasses and altered the landscape, creating An. arabiensis favourable environments that contain excellent habitats for both larvae and adults. Sugarcane is the most expanding agricultural system in sub-Saharan Africa, and is linked to the increased threat of malaria in rural communities. The prolific production and wind dispersal of sugarcane pollen, together with standing pools of water, often provide, as a result of irrigation, a nutrient-rich environment for the offspring of gravid malaria mosquitoes. RESULTS: In the present study, sugarcane pollen-associated volatiles from two cultivars are shown to attract gravid An. arabiensis in a still air two-port olfactometer and stimulate egg laying in an oviposition bioassay. Through combined gas chromatography and electroantennographic detection, as well as combined gas chromatography and mass spectrometric analyses, we identified the bioactive volatiles and generated a synthetic blend that reproduced the full behavioural repertoire of gravid mosquitoes in the Y-tube assay. Two subtractive odour blends, when compared with the full blend, were significantly more attractive. These three and four-component subtractive blends share the compounds (1R)-(+)-α-pinene, nonanal and benzaldehyde, of which, (1R)-(+)-α-pinene and nonanal are found in the attractive odour blends from rice plants and maize pollen. In pairwise comparisons, the rice synthetic odour blend was more attractive to gravid mosquitoes than either of the pollen blends, whereas the pollen blends did not differ in attraction. CONCLUSIONS: The attraction of gravid females to sugarcane pollen volatiles demonstrated in this study, together with the previously found grass-associated volatiles, raise the potential of developing a bioactive chimeric blend to attract gravid malaria mosquitoes. This is discussed in relation to the development of novel and cost-effective vector control measures.

Grass Pollen Affects Survival and Development of Larval Anopheles arabiensis (Diptera: Culicidae).

Nutrients in breeding sites are critical for the survival and development of malaria mosquitoes, having a direct impact on vectorial capacity. Yet, there is a limited understanding about the natural larval diet and its impact on the individual fitness of mosquitoes. Recent studies have shown that gravid Anopheles arabiensis Patton (Diptera: Culicidae) are attracted by and oviposit in grass-associated habitats. The pollen provided by these grasses is a potential source of nutrients for the larvae. Here, we assess the effect of Typha latifolia L. (Poales: Typhaceae), Echinochloa pyramidalis Lamarck, Pennisetum setaceum Forsskål, and Zea mays L. pollen on larval survival and rate of development in An. arabiensis under laboratory conditions. In addition, we characterize the carbon to nitrogen ratio and the size of pollen grains as a measure of diet quality. Carbon-rich pollen with a small grain size (T. latifolia and P. setaceum; 9.7 ± 0.3 × 103 and 5.5 ± 0.2 × 104 µm3, respectively) resulted in enhanced rates of development of An. arabiensis. In contrast, the larva fed on the nitrogen-rich control diet (TetraMin) was slower to develop, but demonstrated the highest larval survival. Larvae fed on carbon-rich and large-grained Z. mays pollen (4.1 ± 0.2 × 105 µm3) survived at similar levels as those fed on the control diet and also took a longer time to develop compared with larvae fed on the other pollens. While males and females did not appear to develop differently on the different pollen diets, males consistently emerged faster than their female counterparts. These results are discussed in relation to integrated vector management.

Comparative study on the nocturnal activity of phlebotomine sand flies in a highland and lowland foci of visceral leishmaniasis in north-western Ethiopia with special reference to Phlebotomus orientalis.

BACKGROUND: Leishmania donovani, the causative agent of visceral leishmaniasis (VL), is most probably vectored by Phlebotomus orientalis in north-western Ethiopia. The aim of this study was to determine and compare the nocturnal activity patterns of Ph. orientalis in VL endemic foci of Libo-Kemkem (highland) and Metema (lowland) districts of north-western Ethiopia. METHODS: Sampling of sand flies was conducted bimonthly from January-May 2012 in the highland and from March-June 2013 in the lowland. Sand flies were sampled using two CDC light traps placed in compounds occupied by both cattle and humans. Sampling of sand flies started at 18:00 h and ended at 06:00 h. Every hour, a cage was replaced by another cage. RESULTS: In total, 9479 nocturnally active sand flies were collected from both study areas. Six Phlebotomus species (Ph. orientalis, Ph. duboscqi, Ph. papatasi, Ph. bergeroti, Ph. rodhaini and Ph. martini) and several Sergentomyia spp. were identified. In both areas, of the six Phlebotomus spp., Ph. orientalis was the preponderate. In the highland, the hourly activity pattern of Ph. orientalis females was higher before midnight with a peak in density between 22:00-23:00 h, whereas in the lowland after midnight between 03:00-04:00 h. CONCLUSIONS: The present study showed that Ph. orientalis females exhibited different nocturnal activity patterns with a peak in the early part of the night in the highland and in the latter part of the night in the lowland areas. As the risk of acquiring L. donovani infections vary in the two areas, appropriate control strategies should be developed according to the activity of Ph. orientalis in the respective areas.

Preliminary study on investigation of zoonotic visceral leishmaniasis in endemic foci of Ethiopia by detecting Leishmania infections in rodents.

OBJECTIVE: To investigate the zoonotic visceral leishmaniasis (ZVL) by identification of the most probable reservoir hosts using parasite isolation and analysis of a possible transmission dynamics of the disease in extra-domestic agricultural fields and rural villages. METHODS: Rodents were collected from selected study sites in kala-azar endemic areas based on information for localities of kala-azar cases for screening of Leishmania infections using parasitological, serological and polymerase chain reaction (PCR) from March, 2013 to January, 2014. Ketamine (Clorketam Veterinary) was used to anaesthesize the rodents according the prescribed dosage (average 2 mg/kg for intra-venous route). The blood obtained using sterile needle was dropped into sterile filter paper and allowed to air dry before sealing in plastic bags. The tissues from liver, spleen and skin were macerated in Locke's solution before transferring them into NNN medium. Blood and touch smears of liver, spleen, skin and bone marrow were prepared for fixing using methanol and staining by Giemsa stain for microscopy. These tissues were also used for DNA extractions and PCR amplification of Leishmania infection. RESULTS: A total of 335 rodents (13 species) were analyzed by sampling internal organs. The infection rate by PCR was 11.1% (6/54) for Arvicanthis nilothicus compared to 17.6% (3/17) and 12.5% (2/16) for Acomys cahirinus and Tarera (G) robustus respectively. Almost all the infections were found from bone marrow samples (8/48 or 16.7%) compared with 1/91 (1.1%) liver, 2/87 (2.2%) spleen and 0/87 (0%) skin. In all study sites with past human VL cases, rodents and proved vectors shared similar habitats. CONCLUSIONS: Leishmania donovani might circulate among different species of rodents in kala-azar endemic lowlands and valleys of Ethiopia by Phlebotomus orientalis and Phlebotomus martini. Detailed studies to substantiate the preliminary data on the possible role of these rodents are urgently needed.