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Introduction: Longitudinal monitoring of outdoor-biting malaria vector populations is becoming increasingly important in understanding the dynamics of residual malaria transmission. However, the human landing catch (HLC), the gold standard for measuring human biting rates indoors and outdoors, is costly and raises ethical concerns related to increased risk of infectious bites among collectors. Consequently, routine data on outdoor-feeding mosquito populations are usually limited because of the lack of a scalable tool with similar sensitivity to outdoor HLC., Methodology: The Anopheles trapping sensitivity of four baited proxy outdoor trapping methods-Furvela tent trap (FTT), host decoy trap (HDT), mosquito electrocuting traps (MET) and outdoor CDC light traps (OLT)-was assessed relative to HLC in a 5 × 5 replicated Latin square conducted over 25 nights in two villages of western Kenya. Indoor CDC light trap (ILT) was run in one house in each of the compounds with outdoor traps, while additional non-Latin square indoor and outdoor HLC collections were performed in one of the study villages., Results: The MET, FTT, HDT and OLT sampled approximately 4.67, 7.58, 5.69 and 1.98 times more An. arabiensis compared to HLC, respectively, in Kakola Ombaka. Only FTT was more sensitive relative to HLC in sampling An. funestus in Kakola Ombaka (RR = 5.59, 95% CI 2.49-12.55, P < 0.001) and Masogo (RR = 4.38, 95% CI 1.62-11.80, P = 0.004) and in sampling An. arabiensis in Masogo (RR = 5.37, 95% CI 2.17-13.24, P < 0.001). OLT sampled significantly higher numbers of An. coustani in Kakola Ombaka (RR = 3.03, 95% CI 1.65-5.56, P < 0.001) and Masogo (RR = 2.88, 95% CI 1.15-7.22, P = 0.02) compared to HLC. OLT, HLC and MET sampled mostly An. coustani, FTT had similar proportions of An. funestus and An. arabiensis, while HDT sampled predominantly An. arabiensis in both villages. FTT showed close correlation with ILT in vector abundance for all three species at both collection sites., Conclusion: FTT and OLT are simple, easily scalable traps and are potential replacements for HLC in outdoor sampling of Anopheles mosquitoes. However, the FTT closely mirrored indoor CDC light trap in mosquito indices and therefore may be more of an indoor mimic than a true outdoor collection tool. HDT and MET show potential for sampling outdoor host-seeking mosquitoes. However, the traps as currently designed may not be feasible for large-scale, longitudinal entomological monitoring. Therefore, the baited outdoor CDC light trap may be the most appropriate tool currently available for assessment of outdoor-biting and malaria transmission risk.

Background: Many indigenous villages in the Amazon basin still suffer from a high malaria burden. Despite this health situation, there are few studies on the bionomics of anopheline larvae in such areas. This publication aims to identify the main larval habitats of the most abundant anopheline species and to assess their associations with some environmental factors., Methods: We conducted a 19-month longitudinal study from January 2013 to July 2014, sampling anopheline larvae in two indigenous Yanomami communities, comprised of four villages each. All natural larval habitats were surveyed every two months with a 350 ml manual dipper, following a standardized larval sampling methodology. In a third study area, we conducted two field expeditions in 2013 followed by four systematic collections during the long dry season of 2014-2015., Results: We identified 177 larval habitats in the three study areas, from which 9122 larvae belonging to 13 species were collected. Although species abundance differed between villages, An. oswaldoi (s.l.) was overall the most abundant species. Anopheles darlingi, An. oswaldoi (s.l.), An. triannulatus (s.s.) and An. mattogrossensis were primarily found in larval habitats that were partially or mostly sun-exposed. In contrast, An. costai-like and An. guarao-like mosquitoes were found in more shaded aquatic habitats. Anopheles darlingi was significantly associated with proximity to human habitations and larval habitats associated with river flood pulses and clear water., Conclusions: This study of anopheline larvae in the Brazilian Yanomami area detected high heterogeneities at micro-scale levels regarding species occurrence and densities. Sun exposure was a major modulator of anopheline occurrence, particularly for An. darlingi. Lakes associated with the rivers, and particularly oxbow lakes, were the main larval habitats for An. darlingi and other secondary malaria vectors. The results of this study will serve as a basis to plan larval source management activities in remote indigenous communities of the Amazon, particularly for those located within low-order river-floodplain systems.

Background: The 2La chromosomal inversion, a genetic polymorphism in An. gambiae (sensu stricto) (s.s.), is associated with adaptation to microclimatic differences in humidity and desiccation resistance and mosquito behaviors. Ownership of insecticide-treated bed nets (ITNs) for malaria control has increased markedly in western Kenya in the last 20 years. An increase in the frequency of ITNs indoors could select against house entering or indoor resting of Anopheles mosquitoes. Thus, the frequency of the 2La inversion is postulated to change in An. gambiae (s.s.) with the increase of ITN ownership over time., Methods: Anopheles gambiae mosquitoes were sampled between 1994 and 2011 using pyrethrum knockdown, bednet traps and human landing catches (HLC) from Asembo and Seme, western Kenya. The 2La inversion was detected by a PCR assay with primers designed for proximal breakpoints of the 2La/a and 2L+(a)/+(a) chromosomal conformation. Mosquitoes were tested for malaria parasite infection by sporozoite ELISA., Results: The frequency of the 2La chromosomal inversion declined from 100 % of all chromosomes in 1994 to 17 % in 2005 and remained low through 2011 (21 %). ITN ownership increased from 0 to > 90 % of houses in the study area during this interval. The decline in the frequency of the 2La chromosomal inversion was significantly, negatively correlated with year (r = -0.93) and with increase in ITN ownership (r = -0.96). The frequency of the homo- and heterokaryotypes departed significantly from Hardy-Weinberg equilibrium, suggesting that 2La/a karyotype was under selection, earlier in its favor and later, against it. Precipitation and maximum monthly temperature did not vary over time, therefore there was no trend in climate that could account for the decline. There was no significant difference in frequency of the 2La inversion in An. gambiae (s.s.) females sampled indoors or outdoors in HCL in 2011, nor was there an association between the 2La inversion and infection with Plasmodium falciparum sporozoites., Conclusions: The increase in ITN ownership in the study area was negatively correlated with the frequency of 2La inversion. The decline in 2La frequency in western Kenya is postulated to be due to differential impacts of ITNs on mosquitoes with different 2La karyotypes, possibly mediated by differences in behavior associated with the 2La karyotypes. Further research is required to determine if this is a widespread phenomenon, to further determine the association of the 2La karyotypes with mosquito behavior, and to assess whether ITNs are exerting selection mediated by differences in behavior on the different karyotypes.

Background: It has been speculated that widespread and sustained use of insecticide treated bed nets (ITNs) for over 10 years in Asembo, western Kenya, may have selected for changes in the location (indoor versus outdoor) and time (from late night to earlier in the evening) of biting of the predominant species of human malaria vectors (Anopheles funestus, Anopheles gambiae sensu stricto, and Anopheles arabiensis)., Methods: Mosquitoes were collected by human landing catches over a six week period in June and July, 2011, indoors and outdoors from 17 h to 07 h, in 75 villages in Asembo, western Kenya. Collections were separated by hour of the night, and mosquitoes were identified to species and tested for sporozoite infection with Plasmodium falciparum. A subset was dissected to determine parity. Human behavior (time going to bed and rising, time spent indoors and outdoors) was quantified by cross-sectional survey. Data from past studies of a similar design and in nearby settings, but conducted before the ITN scale up commenced in the early 2000s, were compared with those from the present study., Results: Of 1,960 Anopheles mosquitoes collected in 2011, 1,267 (64.6%) were morphologically identified as An. funestus, 663 (33.8%) as An. gambiae sensu lato (An. gambiae s.s. and An. arabiensis combined), and 30 (1.5%) as other anophelines. Of the 663 An. gambiae s.l. collected, 385 were successfully tested by PCR among which 235 (61.0%) were identified as An. gambiae s.s. while 150 (39.0%) were identified as An. arabiensis. Compared with data collected before the scale-up of ITNs, daily entomological inoculation rates (EIRs) were consistently lower for An. gambiae s.l. (indoor EIR = 0.432 in 1985-1988, 0.458 in 1989-1990, 0.023 in 2011), and An. arabiensis specifically (indoor EIR = 0.532 in 1989-1990, 0.039 in 2009, 0.006 in 2011) but not An. funestus (indoor EIR = 0.029 in 1985-1988, 0.147 in 1989-1990, 0.010 in 2009 and 0.103 in 2011). Sporozoite rates were lowest in 2009 but rose again in 2011. Compared with data collected before the scale-up of ITNs, An. arabiensis and An. funestus were more likely to bite outdoors and/or early in the evening (p < 0.001 for all comparisons). However, when estimates of human exposure that would occur indoors (πi) or while asleep (πs) in the absence of an ITN were generated based on human behavioral patterns, the changes were modest with >90% of exposure of non-ITN users to mosquito bites occurring while people were indoors in all years. The proportion of bites occurring among non-ITN users while they were asleep was ≥90% for all species except for An. arabiensis. For this species, 97% of bites occurred while people were asleep in 1989-1990 while in 2009 and 2011, 80% and 84% of bites occurred while people were asleep for those not using ITNs. Assuming ITNs prevent a theoretical maximum of 93.7% of bites, it was estimated that 64-77% of bites would have occurred among persons using nets while they were asleep in 1989-1990, while 20-52% of bites would have occurred among persons using nets while they were asleep in 2009 and 2011., Conclusions: This study found no evidence to support the contention that populations of Anopheles vectors of malaria in Asembo, western Kenya, are exhibiting departures from the well-known pattern of late night, indoor biting characteristic of these typically highly anthropophilic species. While outdoor, early evening transmission likely does occur in western Kenya, the majority of transmission still occurs indoors, late at night. Therefore, malaria control interventions such as ITNs that aim to reduce indoor biting by mosquitoes should continue to be prioritized.

Malaria vectors which predominantly feed indoors upon humans have been locally eliminated from several settings with insecticide treated nets (ITNs), indoor residual spraying or larval source management. Recent dramatic declines of An. gambiae in east Africa with imperfect ITN coverage suggest mosquito populations can rapidly collapse when forced below realistically achievable, non-zero thresholds of density and supporting resource availability. Here we explain why insecticide-based mosquito elimination strategies are feasible, desirable and can be extended to a wider variety of species by expanding the vector control arsenal to cover a broader spectrum of the resources they need to survive. The greatest advantage of eliminating mosquitoes, rather than merely controlling them, is that this precludes local selection for behavioural or physiological resistance traits. The greatest challenges are therefore to achieve high biological coverage of targeted resources rapidly enough to prevent local emergence of resistance and to then continually exclude, monitor for and respond to re-invasion from external populations.

Background: Understanding the relationship between Plasmodium falciparum malaria transmission and health outcomes requires accurate estimates of exposure to infectious mosquitoes. However, measures of exposure such as mosquito density and entomological inoculation rate (EIR) are generally aggregated over large areas and time periods, biasing the outcome-exposure relationship. There are few studies examining the extent and drivers of local variation in malaria exposure in endemic areas., Methods: We describe the spatio-temporal dynamics of malaria transmission intensity measured by mosquito density and EIR in the KEMRI/CDC health and demographic surveillance system using entomological data collected during 2002-2004. Geostatistical zero inflated binomial and negative binomial models were applied to obtain location specific (house) estimates of sporozoite rates and mosquito densities respectively. Model-based predictions were multiplied to estimate the spatial pattern of annual entomological inoculation rate, a measure of the number of infective bites a person receive per unit of time. The models included environmental and climatic predictors extracted from satellite data, harmonic seasonal trends and parameters describing space-time correlation., Results: Anopheles gambiae s.l was the main vector species accounting for 86% (n=2309) of the total mosquitoes collected with the remainder being Anopheles funestus. Sixty eight percent (757/1110) of the surveyed houses had no mosquitoes. Distance to water bodies, vegetation and day temperature were strongly associated with mosquito density. Overall annual point estimates of EIR were 6.7, 9.3 and 9.6 infectious bites per annum for 2002, 2003 and 2004 respectively. Monthly mosquito density and EIR varied over the study period peaking in May during the wet season each year. The predicted and observed densities of mosquitoes and EIR showed a strong seasonal and spatial pattern over the study area., Conclusions: Spatio-temporal maps of malaria transmission intensity obtained in this study are not only useful in understanding variability in malaria epidemiology over small areas but also provide a high resolution exposure surface that can be used to analyse the impact of transmission on malaria related and all-cause morbidity and mortality.