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.

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.

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.

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.

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.

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.

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.

The role of grass volatiles on oviposition site selection by Anopheles arabiensis and Anopheles coluzzii.

BACKGROUND: The reproductive success and population dynamics, of Anopheles malaria mosquitoes is strongly influenced by the oviposition site selection of gravid females. Mosquitoes select oviposition sites at different spatial scales, starting with selecting a habitat in which to search. This study utilizes the association of larval abundance in the field with natural breeding habitats, dominated by various types of wild grasses, as a proxy for oviposition site selection by gravid mosquitoes. Moreover, the role of olfactory cues emanating from these habitats in the attraction and oviposition stimulation of females was analysed. METHODS: The density of Anopheles larvae in breeding sites associated with Echinochloa pyramidalis, Echinochloa stagnina, Typha latifolia and Cyperus papyrus, was sampled and the larvae identified to species level. Headspace volatile extracts of the grasses were collected and used to assess behavioural attraction and oviposition stimulation of gravid Anopheles arabiensis and Anopheles coluzzii mosquitoes in wind tunnel and two-choice oviposition assays, respectively. The ability of the mosquitoes to differentiate among the grass volatile extracts was tested in multi-choice tent assays. RESULTS: Anopheles arabiensis larvae were the most abundant species found in the various grass-associated habitats. The larval densities described a hierarchical distribution, with Poaceae (Echinochloa pyramidalis and Echinochloa stagnina)-associated habitat sites demonstrating higher densities than that of Typha-associated sites, and where larvae were absent from Cyperus-associated sites. This hierarchy was maintained by gravid An. arabiensis and An. coluzzii mosquitoes in attraction, oviposition and multi-choice assays to grass volatile extracts. CONCLUSIONS: The demonstrated hierarchical preference of gravid An. coluzzii and An. arabiensis for grass volatiles indicates that vegetation cues associated with larval habitats are instrumental in the oviposition site choice of the malaria mosquitoes. Identifying volatile cues from grasses that modulate gravid malaria mosquito behaviours has distinct potential for the development of tools to be used in future monitoring and control methods.

A(maize)ing attraction: gravid Anopheles arabiensis are attracted and oviposit in response to maize pollen odours.

BACKGROUND: Maize cultivation contributes to the prevalence of malaria mosquitoes and exacerbates malaria transmission in sub-Saharan Africa. The pollen from maize serves as an important larval food source for Anopheles mosquitoes, and females that are able to detect breeding sites where maize pollen is abundant may provide their offspring with selective advantages. Anopheles mosquitoes are hypothesized to locate, discriminate among, and select such sites using olfactory cues, and that synthetic volatile blends can mimic these olfactory-guided behaviours. METHODS: Two-port olfactometer and two-choice oviposition assays were used to assess the attraction and oviposition preference of gravid Anopheles arabiensis to the headspace of the pollen from two maize cultivars (BH-660 and ZM-521). Bioactive compounds were identified using combined gas chromatography and electroantennographic detection from the headspace of the cultivar found to be most attractive (BH-660). Synthetic blends of the volatile compounds were then assessed for attraction and oviposition preference of gravid An. arabiensis, as above. RESULTS: Here the collected headspace volatiles from the pollen of two maize cultivars was shown to differentially attract and stimulate oviposition in gravid An. arabiensis. Furthermore, a five-component synthetic maize pollen odour blend was identified, which elicited the full oviposition behavioural repertoire of the gravid mosquitoes. CONCLUSIONS: The cues identified from maize pollen provide important substrates for the development of novel control measures that modulate gravid female behaviour. Such measures are irrespective of indoor or outdoor feeding and resting patterns, thus providing a much-needed addition to the arsenal of tools that currently target indoor biting mosquitoes.

Comparison of two adult mosquito sampling methods with human landing catches in south-central Ethiopia.

BACKGROUND: The human landing catch (HLC) is the standard reference method for measuring human exposure to mosquito bites. However, HLC is labour-intensive, exposes collectors to infectious mosquito bites and is subjected to collector bias. These necessitate local calibration and application of alternative methods. This study was undertaken to determine the relative sampling efficiency (RSE) of light traps with or without yeast-produced carbon dioxide bait vs. HLC in south-central Ethiopia. METHODS: The experiment was conducted for 39 nights in a 3 × 3 Latin square randomized design with Anopheles arabiensis as the target species in the period between July and November 2014 in Edo Kontola village, south-central Ethiopia. Center for Disease Control and Prevention light trap catches (LTC) and yeast-generated carbon dioxide-baited light trap catches (CB-LTC) were each evaluated against HLC. The total nightly mosquito catches for each Anopheles species in either method was compared with HLC by Pearson correlation and simple linear regression analysis on log-transformed [log10(x + 1)] values. To test if the RSE of each alternative method was affected by mosquito density, the ratio of the number of mosquitoes in each method to the number of mosquitoes in HLC was plotted against the average mosquito abundance. RESULTS: Overall, 7606 Anopheles females were collected by the three sampling methods. Among these 5228 (68.7%) were Anopheles ziemanni, 1153 (15.2%) An. arabiensis, 883 (11.6%) Anopheles funestus s.l., and 342 (4.5%) Anopheles pharoensis. HLC yielded 3392 (44.6%), CB-LTC 2150 (28.3%), and LTC 2064 (27.1%) Anopheles females. The RSEs of LTC and HLC for An. arabiensis were significantly correlated (p < 0.001) and density independent (p = 0.65). However, for outdoor collection of the same species, RSEs of LTC and CB-LTC were density dependent (p < 0.001). It was estimated that on average, indoor LTC and CB-LTC each caught 0.35 and 0.44 times that of indoor HLC for An. arabiensis respectively. CONCLUSIONS: Results showed that HLC was the most efficient method for sampling An. arabiensis. LTC can be used for large-scale indoor An. arabiensis surveillance and monitoring when it is difficult to use HLC. CB-LTC does not substantially improve sampling of this major vector compared to LTC in this setting. Trial registration PACTR201411000882128 (retrospectively registered 8 September, 2014).

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.

Chicken volatiles repel host-seeking malaria mosquitoes.

BACKGROUND: Anopheles arabiensis is a dominant vector of malaria in sub-Saharan Africa, which feeds indoors and outdoors on human and other vertebrate hosts, making it a difficult species to control with existing control methods. Novel methods that reduce human-vector interactions are, therefore, required to improve the impact of vector control programmes. Investigating the mechanisms underlying the host discrimination process in An. arabiensis could provide valuable knowledge leading to the development of novel control technologies. In this study, a host census and blood meal analysis were conducted to determine the host selection behaviour of An. arabiensis. Since mosquitoes select and discriminate among hosts primarily using olfaction, the volatile headspace of the preferred non-human host and non-host species, were collected. Using combined gas chromatography and electroantennographic detection analysis followed by combined gas chromatography and mass spectrometry, the bioactive compounds in the headspace collections were identified. The efficiency of the identified non-host compounds to repel host-seeking malaria mosquitoes was tested under field conditions. RESULTS: The host census and blood meal analyses demonstrated that An. arabiensis strongly prefers human blood when host seeking indoors, while it randomly feeds on cattle, goats and sheep when found outdoors. However, An. arabiensis avoids chickens despite their relatively high abundance, indicating that chickens are a non-host species for this vector. Eleven bioactive compounds were found in the headspace of the non-host species. Six of these were species-specific, out of which four were identified using combined gas chromatography and mass spectrometry. When tested in the field, the chicken-specific compounds, isobutyl butyrate, naphthalene, hexadecane and trans-limonene oxide, and the generic host compounds, limonene, cis-limonene oxide and ß-myrcene, significantly reduced trap catches within the house compared to a negative control. A significant reduction in trap catch was also observed when suspending a caged chicken next to the trap. CONCLUSIONS: Non-host volatiles repel host-seeking An. arabiensis and thus play a significant role in host discrimination. As such, this study demonstrates that non-host volatiles can provide protection to humans at risk of mosquito-vectored diseases in combination with established control programmes.

Sero-prevalence of Leishmania donovani infection in labour migrants and entomological risk factors in extra-domestic habitats of Kafta-Humera lowlands - kala-azar endemic areas in the northwest Ethiopia.

BACKGROUND: Visceral leishmaniasis (VL) or kala-azar cases in seasonal labour migrants from highland areas are addressed to travel history to the Metema-Humera lowlands, northwestern Ethiopia. Factors that affect the incidence of VL in extra-domestic habitats were not evaluated. The aim of this study was to evaluate sero-prevalence of Leishmania donovani infection in randomly selected labour migrant workers and entomological risk factors which might affect the incidence of kala-azar. METHODS: Sero-prevalence of L. donovani infection in labour migrants was obtained from Direct Agglutination Test (DAT) using blood samples. Logistic regression analysis was used to correlate the possible risk factors with L. donovani infection. The season for L. donovani infection or Phlebotomus orientalis bite was estimated from the study of population dynamic of P. orientalis in areas where the blood was sampled. RESULT: A total of 7, 443 P. orientalis (1,748 female and 5,695 male) were collected from agricultural fields and thickets of Acacia seyal using 461 CDC light traps. The highest mean number of P. orientalis/trap in the thickets of A. seyal and agricultural fields were 46.9 and 43.9 in March and April respectively. The mean P. orientalis/trap for November - May dry season in agricultural fields (11.39) and thickets of A. seyal (25.30) were higher compared to 0.66 in fields and 3.92 in thickets during June - August weeding season. Of the total 359 labour migrants screened using DAT, 45 (12.5%) were DAT-positive (≥1:800) for L. donovani infections. Very high titers (1:12800) were found in 3 (0.8%) individuals who had the risk of kala-azar development. Statistically significant p-values and odd ratio (OR) for staying in the areas both in the weeding and harvesting seasons (p = 0.035; OR = 2.83) and sleeping in the agricultural fields (p = 0.01; OR = 15.096) were positively correlated with L. donovani infection. Night harvest (p = 0.028; OR = 0.133) and knowledge about sign or symptoms (p = 0.042; OR = 0.383) were negatively associated with this infection. CONCLUSIONS: Sleeping in open agricultural fields was related with L. donovani infections in labour migrants during June-August weeding season.

Increased malaria transmission around irrigation schemes in Ethiopia and the potential of canal water management for malaria vector control.

BACKGROUND: Irrigation schemes have been blamed for the increase in malaria in many parts of sub-Saharan Africa. However, proper water management could help mitigate malaria around irrigation schemes in this region. This study investigates the link between irrigation and malaria in Central Ethiopia. METHODS: Larval and adult mosquitoes were collected fortnightly between November 2009 and October 2010 from two irrigated and two non-irrigated (control) villages in the Ziway area, Central Ethiopia. Daily canal water releases were recorded during the study period and bi-weekly correlation analysis was done to determine relationships between canal water releases and larval/adult vector densities. Blood meal sources (bovine vs human) and malaria sporozoite infection were tested using enzyme-linked immunosorbent assay (ELISA). Monthly malaria data were also collected from central health centre of the study villages. RESULTS: Monthly malaria incidence was over six-fold higher in the irrigated villages than the non-irrigated villages. The number of anopheline breeding habitats was 3.6 times higher in the irrigated villages than the non-irrigated villages and the most common Anopheles mosquito breeding habitats were waterlogged field puddles, leakage pools from irrigation canals and poorly functioning irrigation canals. Larval and adult anopheline densities were seven- and nine-fold higher in the irrigated villages than in the non-irrigated villages, respectively, during the study period. Anopheles arabiensis was the predominant species in the study area. Plasmodium falciparum sporozoite rates of An. arabiensis and Anopheles pharoensis were significantly higher in the irrigated villages than the non-irrigated villages. The annual entomological inoculation rate (EIR) calculated for the irrigated and non-irrigated villages were 34.8 and 0.25 P. falciparum infective bites per person per year, respectively. A strong positive correlation was found between bi-weekly anopheline larval density and canal water releases. Similarly, there was a strong positive correlation between bi-weekly vector density and canal water releases lagged by two weeks. Furthermore, monthly malaria incidence was strongly correlated with monthly vector density lagged by a month in the irrigated villages. CONCLUSION: The present study revealed that the irrigation schemes resulted in intensified malaria transmission due to poor canal water management. Proper canal water management could reduce vector abundance and malaria transmission in the irrigated villages.

Agro-ecosystems impact malaria prevalence: large-scale irrigation drives vector population in western Ethiopia.

BACKGROUND: Development strategies in Ethiopia have largely focused on the expansion of irrigated agriculture in the last decade to reduce poverty and promote economic growth. However, such irrigation schemes can worsen the socio-economic state by aggravating the problem of mosquito-borne diseases. In this study, the effect of agro-ecosystem practices on malaria prevalence and the risk of malaria transmission by the primary vector mosquito, Anopheles arabiensis, in Ethiopia were investigated. METHODS: In three villages in western Ethiopia practising large-scale sugarcane irrigation, traditional smallholder irrigation and non-irrigated farming, cross-sectional parasitological surveys were conducted during the short rains, after the long rains and during the dry season. Entomological surveys were undertaken monthly (February 2010-January 2011) in each village using light traps, pyrethrum spray collections and artificial pit shelters. RESULTS: Malaria prevalence and the risk of transmission by An. arabiensis assessed by the average human biting rate, mean sporozoite rate and estimated annual entomological inoculation rate were significantly higher in the irrigated sugarcane agro-ecosystem compared to the traditionally irrigated and non-irrigated agro-ecosystems. The average human biting rate was significantly elevated by two-fold, while the mean sporozoite rate was 2.5-fold higher, and the annual entomological inoculation rate was 4.6 to 5.7-fold higher in the irrigated sugarcane compared to the traditional and non-irrigated agro-ecosystems. Active irrigation clearly affected malaria prevalence by increasing the abundance of host seeking Anopheles mosquitoes year-round and thus increasing the risk of infective bites. The year-round presence of sporozoite-infected vectors due to irrigation practices was found to strengthen the coupling between rainfall and risk of malaria transmission, both on- and off-season. CONCLUSION: This study demonstrates the negative impact of large-scale irrigation expansion on malaria transmission by increasing the abundance of mosquito vectors and indicates the need for effective vector monitoring and control strategies in the implementation of irrigation projects.

Fresh, dried or smoked? Repellent properties of volatiles emitted from ethnomedicinal plant leaves against malaria and yellow fever vectors in Ethiopia.

BACKGROUND: In the search for plant-based mosquito repellents, volatile emanations were investigated from five plant species, Corymbia citriodora, Ocimum suave, Ocimum lamiifolium, Olea europaea and Ostostegia integrifolia, traditionally used in Ethiopia as protection against mosquitoes. METHODS: The behaviour of two mosquitoes, the malaria vector Anopheles arabiensis and the arbovirus vector Aedes aegypti, was assessed towards volatiles collected from the headspace of fresh and dried leaves, and the smoke from burning the dried leaves in a two-choice landing bioassay and in the background of human odour. RESULTS: Volatile extracts from the smoke of burning dried leaves were found to be more repellent than those from fresh leaves, which in turn were more repellent to mosquitoes than volatiles from dried leaves. Of all smoke and fresh volatile extracts, those from Co. citriodora (52-76%) and Oc. suave (58-68%) were found to be the most repellent, Os. integrifolia (29-56%) to be intermediate while Ol. europaea (23-40%) and Os. integrifolia (19-37%) were the least repellent. One volatile present in each of the fresh leaf extracts of Co. citriodora, Oc. suave and Os. integrifolia was ß-ocimene. The levels of ß-ocimene reflected the mosquito repellent activity of these three fresh leaf extracts. Female host-seeking mosquitoes responded dose-dependently to ß-ocimene, both physiologically and behaviourally, with a maximal behavioural repulsion at 14% ß-ocimene. ß-ocimene (14%) repels mosquitoes in our 6-minute landing assays comparable to the synthetic insect repellent N,N-diethyl-m-toluamide (10% DEET). CONCLUSIONS: Volatiles in the smoke of burning as well as fresh leaves of Co. citriodora and Oc. suave have significant repellent properties against host seeking An. arabiensis and Ae. aegypti mosquitoes. ß-ocimene, present in the fresh leaf headspace of Co. citriodora, Oc. suave and Os. integrifolia, is a significantly effective volatile mosquito repellent in the laboratory. In addition to its repellent properties, ß-ocimene has long approved safe for use in food and cosmetics, making this volatile an intriguing compound to pursue in further tests in the laboratory and field to validate its mosquito repellent activity and potential for use in a commercial product. Also, the landing bioassay with humanised membranes is a potentially useful repellent screening technique that does not require the exposure of humans to the vectors, however further tests in parallel with conventional techniques are advised.