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Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance., Competing Interests: The authors have declared that no competing interests exist.

Second Internal Transcribed Spacer (ITS2) ribosomal DNA (rDNA) sequence is a widely used molecular marker for species-identification or -delimitation due to observed concerted evolution which is believed to homogenize rDNA copies in an interbreeding population. However, intra-specific differences in ITS2 of Anopheles stephensi have been reported. This study reports the presence of intragenomic sequence variation in the ITS2-rDNA of An. stephensi and hypothesizes that observed intra-specific differences in this species may have resulted due to ambiguous DNA sequence-chromatogram resulting from intragenomic heterogeneity. Anopheles stephensi collected from different parts of India were sequenced for complete ITS2 and the variable region of 28S-rDNA (d1-d3 domains). Intragenomic variations were found in ITS2 region of all An. stephensi sequenced, but no such variation was observed in d1 to d3 domains of 28S-rDNA. Cloning and sequencing of ITS2 through the d3 domain of the 28S region of rDNA from representative samples from northern, central, and southern India confirmed the presence of intragenomic variation in ITS2 due to transitions at three loci and two bp indel in a di-nucleotide microsatellite locus. Multiple haplotypes were observed in ITS2 raised from such variations. Due to the absence of detectable intragenomic sequence variation in the d1 to d3 domain of 28S rDNA of An. stephensi, this region can serve as an ideal reference sequence for taxonomic and phylogenetic studies. The presence of intragenomic variation in rDNA should be carefully examined before using this as a molecular marker for species delimitation or phylogenetic analyses., Competing Interests: The authors have declared that no competing interests exist.

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable production of mosquitoes is currently inhibited by the supply of fresh vertebrate blood. Alternatives to blood are required to promote efficient control strategies for malaria and other vector borne diseases that are transmitted by blood feeding insects. With this in mind, artificial liquid diets were formulated as substitutes for fresh vertebrate blood. Herein we report a blood-free artificial liquid diet that delivers feeding rates similar to blood and mimics the physiological effects of a fresh vertebrate blood meal. The diet induces ovarian and egg maturation of Anopheles mosquitoes and also produces good larval survival and development of functional adults. The formulated blood-free liquid diet is an important advance towards sustainable mosquito breeding in captivity and will reduce the maintenance costs of mosquito colonies and eliminate the need for fresh vertebrate blood.

Motivated by the One Health paradigm, we found the expected changes in temperature and UV radiation (UVR) to be a common trigger for enhancing the risk that viruses, vectors, and diseases pose to human and animal health. We compared data from the mosquito field collections and medical studies with regional climate model projections to examine the impact of climate change on the spreading of one malaria vector, the circulation of West Nile virus (WNV), and the incidence of melanoma. We analysed data obtained from ten selected years of standardised mosquito vector sampling with 219 unique location-year combinations, and 10 years of melanoma incidence. Trends in the observed data were compared to the climatic variables obtained by the coupled regional Eta Belgrade University and Princeton Ocean Model for the period 1961-2015 using the A1B scenario, and the expected changes up to 2030 were presented. Spreading and relative abundance of Anopheles hyrcanus was positively correlated with the trend of the mean annual temperature. We anticipated a nearly twofold increase in the number of invaded sites up to 2030. The frequency of WNV detections in Culex pipiens was significantly correlated to overwintering temperature averages and seasonal relative humidity at the sampling sites. Regression model projects a twofold increase in the incidence of WNV positive Cx. pipiens for a rise of 0.5°C in overwintering TOctober-April temperatures. The projected increase of 56% in the number of days with Tmax ≥ 30°C (Hot Days-HD) and UVR doses (up to 1.2%) corresponds to an increasing trend in melanoma incidence. Simulations of the Pannonian countries climate anticipate warmer and drier conditions with possible dominance of temperature and number of HD over other ecological factors. These signal the importance of monitoring the changes to the preparedness of mitigating the risk of vector-borne diseases and melanoma., Competing Interests: All authors except MP have declared that no competing interests exist. Commercial affiliation of MP is to Avia-GIS NV (commercial funder) in which she is under an employment contract since September 2016. Avia-GIS NV provided support in the form of salaries for author (MP) but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts whose expression is associated with insecticide resistance phenotypes in Anopheles gambiae mosquitoes. The application can be used online or downloaded and used locally by anyone. The local application can be modified to add new insecticide resistance datasets generated from multiple -omics platforms. This guide demonstrates how to add new datasets and handle missing data. Furthermore, IR-TEx can be completely and easily recoded to use-omics datasets from any experimental data, making it a valuable resource to many researchers. The protocol illustrates the utility of IR-TEx in identifying new insecticide resistance candidates using the the microsomal glutathione transferase, GSTMS1, as an example. This transcript is upregulated in multiple pyrethroid resistant populations from Côte D'Ivoire and Burkina Faso. The identification of co-correlated transcripts provides further insight into the putative roles of this gene.

Growing resistance is reported to carbamate insecticides in malaria vectors in Cameroon. However, the contribution of acetylcholinesterase ( Ace-1 ) to this resistance remains uncharacterised. Here, we established that the G119S mutation is driving resistance to carbamates in Anopheles gambiae populations from Cameroon. Insecticide bioassay on field-collected mosquitoes from Bankeng, a locality in southern Cameroon, showed high resistance to the carbamates bendiocarb (64.8% ± 3.5% mortality) and propoxur (55.71% ± 2.9%) but a full susceptibility to the organophosphate fenitrothion. The TaqMan genotyping of the G119S mutation in field-collected adults revealed the presence of this resistance allele (39%). A significant correlation was observed between the Ace-1 R and carbamate resistance at allelic ((bendiocarb; odds ratio (OR) = 75.9; p < 0.0001) and (propoxur; OR = 1514; p < 0.0001)) and genotypic (homozygote resistant vs. homozygote susceptible (bendiocarb; OR = 120.8; p < 0.0001) and (propoxur; OR = 3277; p < 0.0001)) levels. Furthermore, the presence of the mutation was confirmed by sequencing an Ace-1 portion flanking codon 119. The cloning of this fragment revealed a likely duplication of Ace-1 in Cameroon as mosquitoes exhibited at least three distinct haplotypes. Phylogenetic analyses showed that the predominant Ace-1 R allele is identical to that from West Africa suggesting a recent introduction of this allele in Central Africa from the West. The spread of this Ace-1 R represents a serious challenge to future implementation of indoor residual spraying (IRS)-based interventions using carbamates or organophosphates in Cameroon., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Competing Interests: The authors have declared that no competing interests exist.

Risk assessment regarding the distribution of malaria vectors and environmental variables underpinning their distribution under changing climates is crucial towards malaria control and eradication. On this basis, we used Maximum Entropy (MaxEnt) Model to estimate the potential future distribution of major transmitters of malaria in Nigeria-Anopheles gambiae sensu lato and its siblings: Anopheles gambiae sensu stricto, and Anopheles arabiensis under low and high emissions scenarios. In the model, we used mosquito occurrence data sampled from 1900 to 2010 alongside land use and terrain variables, and bioclimatic variables for baseline climate 1960-1990 and future climates of 2050s (2041-2060) and 2070s (2061-2080) that follow RCP2.6 and RCP8.5 scenarios. The Anopheles gambiae species are projected to experience large shift in potential range and population with increased distribution density, higher under high emissions scenario (RCP8.5) and 2070s than low emission scenario (RCP2.6) and 2050s. Anopheles gambiae sensu stricto and Anopheles arabiensis are projected to have highest invasion with 47-70% and 10-14% percentage increase, respectively in Sahel and Sudan savannas within northern states in 2041-2080 under RCP8.5. Highest prevalence is predicted for Humid forest and Derived savanna in southern and North Central states in 2041-2080; 91-96% and 97-99% for Anopheles gambiae sensu stricto, and 67-71% and 72-75% for Anopheles arabiensis under RCP2.6 and RCP8.5, respectively. The higher magnitude of change in species prevalence predicted for the later part of the 21st century under high emission scenario, driven mainly by increasing and fluctuating temperature, alongside longer seasonal tropical rainfall accompanied by drier phases and inherent influence of rapid land use change, may lead to more significant increase in malaria burden when compared with other periods and scenarios during the century; especially in Humid forest, Derived savanna, Sahel and Sudan savannas., Competing Interests: The authors have declared that no competing interests exist.

Conditional cell death systems are useful for various aspects of basic science with a wide range of applications, including genetic pest control. We recently demonstrated that expression of the mammalian pro-apoptotic factor, B-cell leukaemia/lymphoma 2-associated X protein (Bax), can induce apoptosis in specific tissues by using tissue specific promoters in silkworm and mosquito. Here, we newly identified a functional promoter in the Asian malaria vector, Anopheles stephensi, which enables gene expression specifically in the testis. We produced a transgenic mosquito line that expresses mouse Bax under the control of this testis-specific promoter. Transgenic mosquito males exhibited aberrant testes without functional sperm and complete sterility, whereas transgenic females maintained normal fecundity. Despite their abnormal testes, the transgenic males maintained normal function of male accessory glands and typical mating behaviour. As a result of mating with these males, females showed refractoriness to further mating. These results suggest that transgenic males induce female sterility via mating. The mosquito is one of the most important disease vectors, and the control of their population benefits global public health. Thus, this Bax-mediated synthetic male-specific sterilization system could be applied to population control of mosquitoes.

Olfaction is a key component of the multimodal approach used by mosquitoes to target and feed on humans, spreading various diseases. Current repellents have drawbacks, necessitating development of more effective agents. In addition to variable odorant specificity subunits, all insect odorant receptors (ORs) contain a conserved odorant receptor co-receptor (Orco) subunit which is an attractive target for repellent development. Orco directed antagonists allosterically inhibit odorant activation of ORs and we previously showed that an airborne Orco antagonist could inhibit insect olfactory behavior. Here, we identify novel, volatile Orco antagonists. We functionally screened 83 structurally diverse compounds against Orco from Anopheles gambiae. Results were used for training machine learning models to rank probable activity of a library of 1280 odorant molecules. Functional testing of a representative subset of predicted active compounds revealed enrichment for Orco antagonists, many structurally distinct from previously known Orco antagonists. Novel Orco antagonist 2-tert-butyl-6-methylphenol (BMP) inhibited odorant responses in electroantennogram and single sensillum recordings in adult Drosophila melanogaster and inhibited OR-mediated olfactory behavior in D. melanogaster larvae. Structure-activity analysis of BMP analogs identified compounds with improved potency. Our results provide a new approach to the discovery of behaviorally active Orco antagonists for eventual use as insect repellents/confusants.

Inversion polymorphisms are responsible for many ecologically important phenotypes and are often found under balancing selection. However, the same features that ensure their large role in local adaptation-especially reduced recombination between alternate arrangements-mean that uncovering the precise loci within inversions that control these phenotypes is unachievable using standard mapping approaches. Here, we take advantage of long-term balancing selection on a pair of inversions in the mosquito Anopheles gambiae to map desiccation tolerance via pool-GWAS. Two polymorphic inversions on chromosome 2 of this species (denoted 2La and 2Rb) are associated with arid and hot conditions in Africa and are maintained in spatially and temporally heterogeneous environments. After measuring thousands of wild-caught individuals for survival under desiccation stress, we used phenotypically extreme individuals homozygous for alternative arrangements at the 2La inversion to construct pools for whole-genome sequencing. Genomewide association mapping using these pools revealed dozens of significant SNPs within both 2La and 2Rb, many of which neighboured genes controlling ion channels or related functions. Our results point to the promise of similar approaches in systems with inversions maintained by balancing selection and provide a list of candidate genes underlying the specific phenotypes controlled by the two inversions studied here., (© 2018 John Wiley & Sons Ltd.)

BG-Malaria (BGM) trap is a simple adaptation of the widely-used BG-Sentinel trap (BGS). It is proven to be highly effective for trapping the Brazilian malaria vector, Anopheles darlingi, in field conditions, and the African vector, Anopheles arabiensis, under controlled semi-field environments, but has not been field-tested in Africa. Here, we validated the BGM for field sampling of malaria vectors in south-eastern Tanzania. Using a series of Latin-Square experiments conducted nightly (6pm-7am) in rural villages, we compared mosquito catches between BGM, BGS and human landing catches (HLC). We also compared BGMs baited with different attractants (Ifakara-blend, Mbita-blend, BG-Lure and CO2). Lastly, we tested BGMs baited with Ifakara-blend from three odour-dispensing methods (BG-Cartridge, BG-Sachet and Nylon strips). One-tenth of the field-collected female Anopheles gambiae s.l. and Anopheles funestus were dissected to assess parity. BGM captured more An. gambiae s.l. than BGS (p < 0.001), but HLC caught more than either trap (p < 0.001). However, BGM captured more An. funestus than HLC. Proportions of parous An. gambiae s.l. and An. funestus consistently exceeded 50%, with no significant difference between methods. While the dominant species caught by HLC was An. gambiae s.l. (56.0%), followed by Culex spp. (33.1%) and Mansonia spp. (6.0%), the BGM caught mostly Culex (81.6%), followed by An. gambiae s.l. (10.6%) and Mansonia (5.8%). The attractant-baited BGMs were all significantly superior to un-baited controls (p < 0.001), although no difference was found between the specific attractants. The BG-Sachet was the most efficient dispenser for capturing An. gambiae s.l. (14.5(2.75-42.50) mosquitoes/trap/night), followed by BG-Cartridge (7.5(1.75-26.25)). The BGM caught more mosquitoes than BGS in field-settings, but sampled similar species diversity and physiological states as BGS. The physiological states of malaria vectors caught in BGM and BGS were similar to those naturally attempting to bite humans (HLC). The BGM was most efficient when baited with Ifakara blend, dispensed from BG-Sachet. We conclude that though BGM traps have potential for field-sampling of host-seeking African malaria vectors with representative physiological states, both BGM and BGS predominantly caught more culicines than Anopheles, compared to HLC, which caught mostly An. gambiae s.l., Competing Interests: The authors have declared that no competing interests exist.

Background: Insecticides targeting adult mosquitoes are the main way of controlling malaria. They work not only by killing mosquitoes, but also by repelling and irritating them. Indeed their repellent action gives valuable personal protection against biting mosquitoes. In the context of malaria control this personal protection is especially relevant when mosquitoes are infectious, whereas to protect the community we would prefer that the mosquitoes that are not yet infectious are killed (so, not repelled) by the insecticide. As the infectious stage of malaria parasites increases the motivation of mosquitoes to bite, we predicted that it would also change their behavioural response to insecticides., Results: With two systems, a laboratory isolate of the rodent malaria Plasmodium berghei infecting Anopheles gambiae and several isolates of P. falciparum obtained from schoolchildren in Tanzania that infected Anopheles arabiensis, we found that mosquitoes harbouring the infectious stage (the sporozoites) of the parasite were less repelled by permethrin-treated nets than uninfected ones., Conclusions: Our results suggest that, at least in the laboratory, malaria infection decreases the personal protection offered by insecticide-treated nets at the stage where the personal protection is most valuable. Further studies must investigate whether these results hold true in the field and whether the less effective personal protection can be balanced by increased community protection.

Current evidence suggests that malarial infection could alter metabolites in the breath of patients, a phenomenon that could be exploited to create a breath-based diagnostic test. However, no study has explored this in a clinical setting. To investigate whether natural human malarial infection leads to a characteristic breath profile, we performed a field study in Malawi. Breath volatiles from children with and those without uncomplicated falciparum malaria were analyzed by thermal desorption-gas chromatography/mass spectrometry. Using an unbiased, correlation-based analysis, we found that children with malaria have a distinct shift in overall breath composition. Highly accurate classification of infection status was achieved with a suite of 6 compounds. In addition, we found that infection correlates with significantly higher breath levels of 2 mosquito-attractant terpenes, α-pinene and 3-carene. These findings attest to the viability of breath analysis for malaria diagnosis, identify candidate biomarkers, and identify plausible chemical mediators for increased mosquito attraction to patients infected with malaria parasites.

The efficiency of spatial repellents and long-lasting insecticide-treated nets (LLINs) is a key research topic in malaria control. Insecticidal nets reduce the mosquito-human contact rate and simultaneously decrease mosquito populations. However, LLINs demonstrate dissimilar efficiency against different species of malaria mosquitoes. Various factors have been proposed as an explanation, including differences in insecticide-induced mortality, flight characteristics, or persistence of attack. Here we present a discrete agent-based approach that enables the efficiency of LLINs, baited traps and Insecticide Residual Sprays (IRS) to be examined. The model is calibrated with hut-level experimental data to compare the efficiency of protection against two mosquito species: Anopheles gambiae and Anopheles arabiensis. We show that while such data does not allow an unambiguous identification of the details of how LLINs alter the vector behavior, the model calibrations quantify the overall impact of LLINs for the two different mosquito species. The simulations are generalized to community-scale scenarios that systematically demonstrate the lower efficiency of the LLINs in control of An. arabiensis compared to An. gambiae., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mosquito surveillance and control are at the heart of efforts to eliminate malaria, however, there remain significant gaps in our understanding of mosquito behaviour that impede innovation. We hypothesised that a combination of human-associated stimuli could be used to attract and kill malaria vectors more successfully than individual stimuli, and at least as well as a real human. To test this in the field, we quantified Anopheles responses to olfactory, visual and thermal stimuli in Burkina Faso using a simple adhesive trap. Traps baited with human odour plus high contrast visual stimuli caught more Anopheles than traps with odour alone, showing that despite their nocturnal habit, malaria vectors make use of visual cues in host-seeking. The best performing traps, however, combined odour and visual stimuli with a thermal signature in the range equivalent to human body temperature. When tested against a human landing catch during peak mosquito abundance, this "host decoy" trap caught nearly ten times the number of Anopheles mosquitoes caught by a human collector. Exploiting the behavioural responses of mosquitoes to the entire suite of host stimuli promises to revolutionise vector surveillance and provide new paradigms in disease control.

Malaria is an important public health problem in Thailand, especially along international borders. In this study, we conducted a longitudinal entomological survey in six villages and rubber plantation sites to address the spatio-temporal abundance and behavior of malaria vectors in Ubon Ratchathani Province along the Thailand-Laos border. Adult female mosquitoes were collected by human landing collections (indoor and outdoor) and by cattle bait collections twice per year, during rainy and dry seasons. Mosquitoes were morphologically identified and sibling species were determined by allele-specific PCR. Of the 10,024 Anopheles, 9,328 (93.1%) and 696 (6.9%) were collected during the rainy and dry seasons, respectively. A total of 9,769 (97.5%) and 255 (2.5%) was collected on cattle and human baits, respectively. Very few primary and secondary malaria vectors were collected, consisting of 12 specimens of An. dirus, eight An. minimus, and seven An. aconitus. Of the 152 specimens of the Maculatus Group, only three were identified to An sawadwongporni by molecular methods. The others were 112 An. rampae, a non-vector, that were not amplified or were misidentified as other non-vectors. The very low density of primary malaria vectors found in the study villages suggests that entomological risk and malaria transmission is higher in neighboring forest areas. Further studies on malaria vector distribution, as well as human behaviors, are needed to understand malaria transmission dynamics in the province and to develop suitable vector control methods., (© 2017 The Society for Vector Ecology.)

Understanding how phenotypic differences between males and females arise from the sex-biased expression of nearly identical genomes can reveal important insights into the biology and evolution of a species. Among Anopheles mosquito species, these phenotypic differences include vectorial capacity, as it is only females that blood feed and thus transmit human malaria. Here, we use RNA-seq data from multiple tissues of four vector species spanning the Anopheles phylogeny to explore the genomic and evolutionary properties of sex-biased genes. We find that, in these mosquitoes, in contrast to what has been found in many other organisms, female-biased genes are more rapidly evolving in sequence, expression, and genic turnover than male-biased genes. Our results suggest that this atypical pattern may be due to the combination of sex-specific life history challenges encountered by females, such as blood feeding. Furthermore, female propensity to mate only once in nature in male swarms likely diminishes sexual selection of post-reproductive traits related to sperm competition among males. We also develop a comparative framework to systematically explore tissue- and sex-specific splicing to document its conservation throughout the genus and identify a set of candidate genes for future functional analyses of sex-specific isoform usage. Finally, our data reveal that the deficit of male-biased genes on the X Chromosomes in Anopheles is a conserved feature in this genus and can be directly attributed to chromosome-wide transcriptional regulation that de-masculinizes the X in male reproductive tissues., (© 2017 Papa et al.; Published by Cold Spring Harbor Laboratory Press.)

The insect gut microbiota plays crucial roles in modulating the interactions between the host and intestinal pathogens. Unlike viruses, bacteria, and parasites, which need to be ingested to cause disease, entomopathogenic fungi infect insects through the cuticle and proliferate in the hemolymph. However, interactions between the gut microbiota and entomopathogenic fungi are unknown. Here we show that the pathogenic fungus Beauveria bassiana interacts with the gut microbiota to accelerate mosquito death. After topical fungal infection, mosquitoes with gut microbiota die significantly faster than mosquitoes without microbiota. Furthermore, fungal infection causes dysbiosis of mosquito gut microbiota with a significant increase in gut bacterial load and a significant decrease in bacterial diversity. In particular, the opportunistic pathogenic bacterium Serratia marcescens overgrows in the midgut and translocates to the hemocoel, which promotes fungal killing of mosquitoes. We further reveal that fungal infection down-regulates antimicrobial peptide and dual oxidase expression in the midgut. Duox down-regulation in the midgut is mediated by secretion of the toxin oosporein from B. bassiana Our findings reveal the important contribution of the gut microbiota in B. bassiana -killing activity, providing new insights into the mechanisms of fungal pathogenesis in insects., Competing Interests: The authors declare no conflict of interest.

The Anopheles gambiae complex contains a number of highly anthropophilic mosquito species that have acquired exceptional ability to thrive in complex human habitats. Thus, examining the evolutionary history of this Afrotropical mosquito may yield vital information on the selective processes that occurred during the adaptation to human-dominated environments. We performed reduced representation sequencing on 941 mosquitoes of the Anopheles gambiae complex collected across four ecogeographic zones in Cameroon. We find evidence for genetic and geographic subdivision within An. coluzzii and An. gambiae sensu stricto-the two most significant malaria vectors in the region. Importantly, in both species, rural and urban populations are genetically differentiated. Genome scans reveal pervasive signatures of selection centered on genes involved in xenobiotic resistance. Notably, a selective sweep containing detoxification enzymes is prominent in urban mosquitoes that exploit polluted breeding sites. Overall, our study suggests that recent anthropogenic environmental modifications and widespread use of insecticides are driving population differentiation and local adaptation in vectors with potentially significant consequences for malaria epidemiology., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Experimental huts are part of the WHO process for testing and evaluation of Insecticide Treated Nets (ITN) in semi-field conditions. Experimental Hut Trials (EHTs) mostly focus on two main indicators (i.e., mortality and blood feeding reduction) that serve as efficacy criteria to obtain WHO interim recommendation. However, several other outputs that rely on counts of vectors collected in the huts are neglected although they can give useful information about vectors' behavior and personal protection provided by ITNs. In particular, EHTs allow to measure the deterrent effect and personal protection of ITNs. To provide a better assessment of ITNs efficacy, we performed a retrospective analysis of the deterrence and the personal protection against malaria transmission for 12 unwashed and 13 washed ITNs evaluated through EHTs conducted in West Africa. A significant deterrent effect was shown for six of the 12 unwashed ITNs tested. When washed 20 times, only three ITNs had significant deterrent effect (Rate Ratios (RR)<1; p<0.05) and three showed an apparent "attractiveness" (RR>1; p<0.01). When compared to the untreated net, all unwashed ITNs showed lower number of blood-fed Anopheles indicating a significant personal protection (RR<1, p<0.05). However, when washed 20 times, three ITNs that were found to be attractive did not significantly reduce human-vector contact (p>0.05). Current WHO efficacy criteria do not sufficiently take into account the deterrence effect of ITNs. Moreover, the deterrence variability is rarely discussed in EHT's reports. Our findings highlighted the long-range effect (deterrent or attractive) of ITNs that may have significant consequences for personal/community protection against malaria transmission. Indicators measuring the deterrence should be further considered for the evaluation of ITNs., Competing Interests: The authors have declared that no competing interests exist.

The genome of the Neotropical malaria vector Anopheles albimanus was sequenced as part of the 16 Anopheles Genomes Project published in 2015. The draft assembly of this species consisted of 204 scaffolds with an N50 scaffold size of 18.1 Mb and a total assembly size of 170.5 Mb. It was among the smallest genomes with the longest scaffolds in the 16 Anopheles species cluster, making An. albimanus the logical choice for anchoring the genome assembly to chromosomes. In this study, we developed a high-resolution cytogenetic photomap with completely straightened polytene chromosomes from the salivary glands of the mosquito larvae. Based on this photomap, we constructed a chromosome-based genome assembly using fluorescent in situ hybridization of PCR-amplified DNA probes. Our physical mapping, assisted by an ortholog-based bioinformatics approach, identified and corrected nine misassemblies in five large genomic scaffolds. Misassemblies mostly occurred in junctions between contigs. Our comparative analysis of scaffolds with the An. gambiae genome detected multiple genetic exchanges between pericentromeric regions of chromosomal arms caused by partial-arm translocations. The final map consists of 40 ordered genomic scaffolds and corrected fragments of misassembled scaffolds. The An. albimanus physical map comprises 98.2% of the total genome assembly and represents the most complete genome map among mosquito species. This study demonstrates that physical mapping is a powerful tool for correcting errors in draft genome assemblies and for creating chromosome-anchored reference genomes., (Copyright © 2017 Artemov et al.)

To effectively control malaria, it is necessary to identify landscapes that are most favorable for its transmission. To achieve this goal, the authors developed landscape malariological zoning of Southern Uzbekistan as the most problematic area for malaria in the country. For landscape malariological zoning, the investigators applied the method developed by A.Ya.Lysenko et al. (1956), which allowed identification of different types of malariogenic landscapes, by using the existing scheme of physical and geographical zoning. The existing physical and geographical areas identified were assigned malariological characteristics, which permitted assessment of the landscapes of Southern Uzbekistan from the point of view of whether there is a risk of local malaria transmission. The zoning could identify 5 types of malariogenic landscapes. The most malariologically dangerous areas are the landscapes of lowland river valleys and irrigated (irrigation channel)-lands, where there is the larg- est area of anophelogenic reservoirs. The malariological situation in the low-hill landscapes depends on the situation in the lowland river and irrigation channel landscapes. An epidemic outbreak may occur in the mid-mountain landscapes if a large number of an infection carrier and source are present. The results can be used to optimize anti-malarial interventions, prognosis, and prevention of malaria resumption in the area under study.

Anopheles melas is a member of the recently diverged An. gambiae species complex, a model for speciation studies, and is a locally important malaria vector along the West-African coast where it breeds in brackish water. A recent population genetic study of An. melas revealed species-level genetic differentiation between three population clusters. An. melas West extends from The Gambia to the village of Tiko, Cameroon. The other mainland cluster, An. melas South, extends from the southern Cameroonian village of Ipono to Angola. Bioko Island, Equatorial Guinea An. melas populations are genetically isolated from mainland populations. To examine how genetic differentiation between these An. melas forms is distributed across their genomes, we conducted a genome-wide analysis of genetic differentiation and selection using whole genome sequencing data of pooled individuals (Pool-seq) from a representative population of each cluster. The An. melas forms exhibit high levels of genetic differentiation throughout their genomes, including the presence of numerous fixed differences between clusters. Although the level of divergence between the clusters is on a par with that of other species within the An. gambiae complex, patterns of genome-wide divergence and diversity do not provide evidence for the presence of pre- and/or postmating isolating mechanisms in the form of speciation islands. These results are consistent with an allopatric divergence process with little or no introgression., (Copyright © 2016 Deitz et al.)

Several studies suggest the potential for climate change to increase malaria incidence in cooler, marginal transmission environments. However, the effect of increasing temperature in warmer regions where conditions currently support endemic transmission has received less attention. We investigate how increases in temperature from optimal conditions (27 °C to 30 °C and 33 °C) interact with realistic diurnal temperature ranges (DTR: ± 0 °C, 3 °C, and 4.5 °C) to affect the ability of key vector species from Africa and Asia (Anopheles gambiae and An. stephensi) to transmit the human malaria parasite, Plasmodium falciparum. The effects of increasing temperature and DTR on parasite prevalence, parasite intensity, and mosquito mortality decreased overall vectorial capacity for both mosquito species. Increases of 3 °C from 27 °C reduced vectorial capacity by 51-89% depending on species and DTR, with increases in DTR alone potentially halving transmission. At 33 °C, transmission potential was further reduced for An. stephensi and blocked completely in An. gambiae. These results suggest that small shifts in temperature could play a substantial role in malaria transmission dynamics, yet few empirical or modeling studies consider such effects. They further suggest that rather than increase risk, current and future warming could reduce transmission potential in existing high transmission settings.

Background: In Zambia and other sub-Saharan African countries affected by ongoing malaria transmission, indoor residual spraying (IRS) for malaria prevention has typically been implemented over large areas, e.g., district-wide, and targeted to peri-urban areas. However, there is a recent shift in some countries, including Zambia, towards the adoption of a more strategic and targeted IRS approach, in coordination with increased emphasis on universal coverage of long-lasting insecticidal nets (LLINs) and effective insecticide resistance management. A true targeted approach would deliver IRS to sub-district areas identified as high-risk, with the goal of maximizing the prevention of malaria cases and deaths., Results: Together with the Government of the Republic of Zambia, a new methodology was developed applying geographic information systems and satellite imagery to support a targeted IRS campaign during the 2014 spray season using health management information system data., Discussion/conclusion: This case study focuses on the developed methodology while also highlighting the significant research gaps which must be filled to guide countries on the most effective strategy for IRS targeting in the context of universal LLIN coverage and evolving insecticide resistance.

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val109Ile, Asp335Glu and Asn384Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.

A cytogenetic analysis of Anopheles mosquitoes in the Astrakhan region was carried out. Three species of Anopheles were identified. An. messeae lives everywhere and prevails in all of the areas of research, An. hyrcanus is found in the southwest of the region, and An. maculipennis in the northern part of the region. The populations of An. messeae show a high level of inversion polymorphism for the sex chromosome and the third autosome. A clear clinal trend of an increase in chromosomal rearrangements XL1, 3R1, and 3L1 and a decrease in the frequency of evolutionary source alternatives was revealed in laraval hemipopulations of the species from south to north.

Malaria epidemiology is complex due to multiplicity of disease vectors, sibling species complex and variations in bionomical characteristics, vast varied terrain, various ecological determinants. There are six major mosquito vector taxa in India, viz. Anopheles culicifacies, An. fluviatilis, An. stephensi, An. minimus, An. dirus and An. sundaicus. Among these, An. culicifacies is widely distributed and considered the most important vector throughout the plains and forests of India for generating bulk of malaria cases (>60% annually). Major malaria epidemics are caused by An. culicifaices. It is also the vector of tribal malaria except parts of Odisha and Northeastern States of India. An. culicifacies has been the cause of perennial malaria transmission in forests, and over the years penetrated the deforested areas of Northeast. An. culicifacies participates in malaria transmission either alone or along with An. stephensi or An. fluviatilis. The National Vector Borne Disease Control Programme (NVBDCP) spends about 80 per cent malaria control budget annually in the control of An. culicifacies, yet it remains one of the most formidable challenges in India. With recent advances in molecular biology there has been a significant added knowledge in understanding the biology, ecology, genetics and response to interventions, requiring stratification for cost-effective and sustainable malaria control. Research leading to newer interventions that are evidence-based, community oriented and sustainable would be useful in tackling the emerging challenges in malaria control. Current priority areas of research should include in-depth vector biology and control in problem pockets, preparation of malaria-risk maps for focused and selective interventions, monitoring insecticide resistance, cross-border initiative and data sharing, and coordinated control efforts for achieving transmission reduction, and control of drug-resistant malaria. The present review on An. culicifacies provides updated information on vector biology and control outlining thrust areas of research.

Background and Objectives: Epidemiology and transmission of malaria vary within the tribal areas with the variation in topography, forest cover and type of forest. For the control of disease, understanding of the dynamics of transmission in the varied ecological situation is essential. This study was carried out in the two distinct tribal areas- Baiga Chak (thick forested area) of Dindori district and Bichhia block (forest fringe area) of Mandla district, Madhya Prasdesh, India, to understand the epidemiology and transmission dynamics of malaria., Methods: Mosquitoes were collected using hand catch and whole night collections to determine the proportion of vectors, their density and seasonality. Vector incrimination was done by sporozoite ELISA and feeding preferences of vector by gel diffusion method. Active fever surveys were carried out fortnightly to determine the age specific malaria parasite rates among the inhabitants of two areas., Results: Density of Anopheles culicifacies was significantly higher in Bichhia while the density of An. fluviatilis was higher in Baiga Chak. An. culicifacies was incriminated from both the areas while An. fluviatilis was incriminated from Baiga Chak only. Malaria slide positivity rate (SPR) was significantly higher (OR=3.7 95%CI, 3.1-4.4) in Baiga Chak (28.2%) than Bichhia (9.6%)., Interpretation & Conclusions: The features of malaria transmission in tribal areas differed from those reported in rural or semirural population. Site-specific and region-specific studies are required to develop appropriate intervention measures to control malaria.

Adult female mosquitoes need blood to develop their eggs and both sexes use nectar and honeydew as carbohydrate resources for flight, survival and to enhance reproduction. However, there are also a few reports in the literature of mosquitoes feeding on haemolymph of soft-bodied insects such as caterpillars. The frequency and significance of this entomophagous behavior is not well understood, but is thought to be a vestige of ancestral feeding behavior or an opportunistic behavior that has evolved over time. In our current paper we investigated the extent to which the malaria mosquito, Anopheles stephensi, is attracted to, and can successfully feed on, larvae of two common moth species, Manduca sexta and Heliothis subflexa. Using y-tube olfactometer assays we found that female An. stephensi readily flew upwind to and landed on the caterpillars of both moth species. The nature of the volatile cues used in host location remains unclear but respirometer studies suggest a possible role of CO2. Laboratory cage assays further showed that the female mosquitoes were able to actively feed on moth larvae and gain sufficient nutritional benefit to influence survival. The extent to which such an opportunistic behavior occurs in the field has yet to be explored but our results suggest that this haemolymph feeding behavior could play a role in malaria mosquito life history and could provide a novel mechanism for horizontal transmission of pathogens and other micro-organisms between hosts.

Background & Objectives: Anopheles minimus and An. fluviatilis were incriminated as the major malaria vectors in Keonjhar district of Odisha State recently. This study was carried out to elucidate the potential role of these two vector species in transmission of malaria during different seasons, and vectorial capacity of these species was also estimated., Methods: Three hilly and forested villages of Keonjhar district were randomly selected. Vectorial capacity (C) was calculated using the Macdonald's formula as modified by Garret-Jones. The human landing density of the vector species was obtained from all night human landing collections (bait protected by bed-net). Man feeding habit was estimated by multiplying the human blood index with feeding frequency, which was obtained on daily basis from the duration of gonotrophic cycle. The probability of survival through the extrinsic incubation cycle was calculated from the probability of survival through one day and duration of sporogonic cycle., Results: The estimated vectorial capacity of An. minimus varied between 0.014 and 1.09 for Plasmodium falciparum (Pf) and between 0.1 and 1.46 for P. vivax (Pv). The C of An. minimus for both Pf and Pv was higher during rainy season than the other two seasons. The estimated C of An. fluviatilis varied between 0.04 and 1.28 for Pf and between 0.20 and 1.54 for Pv., Interpretation & Conclusions: Based on the estimated values of vectorial capacity of the two vector species, the area could be stratified and such stratification would reflect the difference in the intensity of transmission between different strata and accordingly the appropriate control strategy could be adopted for each stratum.

Pyrethroid resistance is becoming a major problem for vector control programs, because at present, there are few suitable chemical substitutes for pyrethroids, as when used on bed nets the insecticide must have low mammalian toxicity as well as high activity to mosquitoes. Pyriproxyfen (PPF) is one of the most active chemicals among the juvenile hormone mimic (JHM) group. Sterilizing mosquitoes by using PPF could be a potential control measure for pyrethroid-resistant malaria vectors. We investigated the sterilizing effects of two types of PPF-impregnated bed nets - a 1% PPF-impregnated net and a 1% PPF +2% permethrin-impregnated net (Olyset Duo) - to pyrethroid-resistant wild population of Anopheles gambiae s.s. in western Kenya. High mortality of blood-fed mosquitos was observed 3 days post-collection, in the houses where PPF-impregnated nets were used, indicating the effect of PPF on the longevity of mosquitos that came in contact with the net. Reduction in the number of ovipositing females, number of eggs, and number of progeny per female were also observed in the houses in which both Olyset Duo and PPF-impregnated nets were used. This is the first field study showing the high sterilizing efficacy of PPF against wild pyrethroid-resistant An. gambiae s.s. population. In addition, we recognized the necessity of combined use of permethrin with PPF, in order to reduce the risk of mosquito bites and provide a level of personal protection. Further studies on wild pyrethroid-resistant mosquito populations such as An. arabiensis and An. funestus s.s. would provide more information on the practical use of the PPF-impregnated bed nets.

Background & Objectives: Selection of an insecticide and its appropriate formulation is a prerequisite of formulating any chemical control strategy against vectors. A hut scale field trial was carried out to study the effectiveness of house spraying with capsule suspension (CS) formulation of lambdacyhalothrin in comparison with its wettable powder (WP) formulation on mortality, density and behaviour of malaria vector in Malkangiri district, Odisha, India., Methods: The two formulations were tested at 20 and 30 mg (a.i.)/m 2 for their effectiveness in terms of deterrence, excito-repellency, blood-feeding success, mortality and residual activity against Anopheles fluviatilis, the major malaria vector, in experimental huts in Malkangiri district, Odisha State, India., Results: Both CS and WP formulations prevented the entry of An. fluviatilis in to the sprayed huts by >90 per cent for >6 months, the entire peak malaria transmission season in the area. The exit rate increased (90-99%) with different treatments and the feeding rate was reduced (91-97%). There was no significant difference between WP 30, CS 20 and CS 30 mg/m 2 in these respects. However, WP 20 mg/m 2 caused a lesser effect than the other three groups. The formulations or the dosages differed only in causing vector mortalities. Overall, the total mortality rate of An. fluviatilis was higher in the huts sprayed with CS 30 (58%) than the huts sprayed with CS 20 (48%), WP 20 (37%) and WP 30 mg/m 2 (47%)., Interpretation & Conclusions: Considering the duration of residual action and the effect on entry, exit, feeding and mortality of An. fluviatilis, the dosage 20 mg/m 2 of CS formulation of lambdacyhalothrin could be considered for further use.

A shift towards early morning biting behavior of the major malaria vector Anopheles funestus have been observed in two villages in south Benin following distribution of long-lasting insecticidal nets (LLINs), but the impact of these changes on the personal protection efficacy of LLINs was not evaluated. Data from human and An. funestus behavioral surveys were used to measure the human exposure to An. funestus bites through previously described mathematical models. We estimated the personal protection efficacy provided by LLINs and the proportions of exposure to bite occurring indoors and/or in the early morning. Average personal protection provided by using of LLIN was high (≥80% of the total exposure to bite), but for LLIN users, a large part of remaining exposure occurred outdoors (45.1% in Tokoli-V and 68.7% in Lokohoué) and/or in the early morning (38.5% in Tokoli-V and 69.4% in Lokohoué). This study highlights the crucial role of LLIN use and the possible need to develop new vector control strategies targeting malaria vectors with outdoor and early morning biting behavior. This multidisciplinary approach that supplements entomology with social science and mathematical modeling illustrates just how important it is to assess where and when humans are actually exposed to malaria vectors before vector control program managers, policy-makers and funders conclude what entomological observations imply.

Resurgence of malaria has been attributed, in part, to the development of resistance by Anopheles gambiae, a principal vector of the disease, to various insecticidal compounds such as Permethrin. Permethrin, a neurotoxicant, is widely used to impregnate mosquito nets. An alternative strategy to control mosquitoes is the use of Bacillus thuringiensis subsp. israelensis (Bti) because there is no observable resistance in the field to the bacterium. Bti kills mosquitoes by targeting cadherin molecules residing in the midgut epithelium of larvae of the insect. Cry proteins (Cry4A, Cry4B, Cry10A and Cry11A) produced by the bacterium during the sporulation phase of its life cycle bind to the cadherin molecules, which serve as receptors for the proteins. These Cry proteins have variable specificity to a variety of mosquitoes, including Culex and Aedes as well as Anopheles. Importantly, selective mosquitocidal action is occasioned by binding of the respective Cry toxins to cadherins distinctive to individual mosquito species. Differential fractionation of the four Cry proteins from a novel Bti isolate (M1) and cloning and expression of their genes in Escherichia coli revealed that Cry4B is the only Cry protein that exerts insecticidal action against An. gambiae. Indeed, it does so against a Permethrin-resistant strain of the mosquito. The other three Cry proteins are ineffective. Multiple sequence alignments of the four Cry proteins revealed a divergent sequence motif in the Cry4B toxin, which most likely determines binding of the toxin to its cognate receptor, BT-R3, in An. gambiae and to its specific toxicity. A model showing Cry4B toxin binding to BT-R3 is presented.

Background: Over the past decade, a sharp decline of malaria burden has been observed in several countries. Consequently, the conventional entomological methods have become insufficiently sensitive and probably under-estimate micro-geographical heterogeneity of exposure and subsequent risk of malaria transmission. In this study, we investigated whether the human antibody (Ab) response to Anopheles salivary gSG6-P1 peptide, known as a biomarker of Anopheles exposure, could be a sensitive and reliable tool for discriminating human exposure to Anopheles bites in area of low and seasonal malaria transmission., Methods: A multi-disciplinary survey was performed in Northern Senegal where An. gambiae s.l. is the main malaria vector. Human IgG Ab response to gSG6-P1 salivary peptide was compared according to the season and villages in children from five villages in the middle Senegal River valley, known as a low malaria transmission area., Results: IgG levels to gSG6-P1 varied considerably according to the villages, discriminating the heterogeneity of Anopheles exposure between villages. Significant increase of IgG levels to gSG6-P1 was observed during the peak of exposure to Anopheles bites, and decreased immediately after the end of the exposure season. In addition, differences in the season-dependent specific IgG levels between villages were observed after the implementation of Long-Lasting Insecticidal Nets by The National Malaria Control Program in this area., Conclusion: The gSG6-P1 salivary peptide seems to be a reliable tool to discriminate the micro-geographical heterogeneity of human exposure to Anopheles bites in areas of very low and seasonal malaria transmission. A biomarker such as this could also be used to monitor and evaluate the possible heterogeneous effectiveness of operational vector control programs in low-exposure areas.

Plasmodium falciparum is the causative agent of malaria, a disease that kills almost one million persons each year, mainly in sub-Saharan Africa. P. falciparum is transmitted to the human host by the bite of an Anopheles female mosquito, and Anopheles gambiae sensus stricto is the most tremendous malaria vector in Africa, widespread throughout the afro-tropical belt. An. gambiae s.s. is subdivided into two distinct molecular forms, namely M and S forms. The two molecular forms are morphologically identical but they are distinct genetically, and differ by their distribution and their ecological preferences. The epidemiological importance of the two molecular forms in malaria transmission has been poorly investigated so far and gave distinct results in different areas. We have developed a real-time quantitative PCR (qPCR) assay, and used it to detect P. falciparum at the oocyst stage in wild An. gambiae s.s. mosquitoes experimentally infected with natural isolates of parasites. Mosquitoes were collected at immature stages in sympatric and allopatric breeding sites and further infected at the adult stage. We next measured the infection prevalence and intensity in female mosquitoes using the qPCR assay and correlated the infection success with the mosquito molecular forms. Our results revealed different prevalence of infection between the M and S molecular forms of An. gambiae s.s. in Cameroon, for both sympatric and allopatric populations of mosquitoes. However, no difference in the infection intensity was observed. Thus, the distribution of the molecular forms of An. gambiae s.s. may impact on the malaria epidemiology, and it will be important to monitor the efficiency of malaria control interventions on the two M and S forms.

Malaria transmission is strongly influenced by climatic conditions which determine the abundance and seasonal dynamics of the Anopheles vector. In particular, water temperature influences larval development rates whereas air temperature determines adult longevity as well as the rate of parasite development within the adult mosquito. Although data on land surface temperature exist at a spatial resolution of approximately 1 km globally with four time steps per day, comparable data are not currently available for air temperature. In order to address this gap and demonstrate the importance of using the right type of temperature data, we fitted simple models of the relationship between land-surface and air temperature at lower resolution to obtain a high resolution estimate of air temperature across Africa. We then used these estimates to calculate some crucial malaria transmission parameters that strongly depend on air temperatures. Our results demonstrate substantial differences between air and surface temperatures that impact temperature-based maps of areas suitable for transmission. We present high resolution maps of the malaria transmission parameters driven by air temperature and their seasonal variation. The fitted air temperature datasets are made publicly available alongside this publication.

Background: The development of parasitological immunity against malaria affects the ability to detect infection, the efficiency of the local human parasite reservoir at infecting mosquitoes, and the response to reintroduction of parasites to previously cleared areas. Observations of similar age-trends in detected prevalence and mean parasitaemia across more than an order-of-magnitude of variation in baseline transmission complicate simple exposure-driven explanations., Methods: Mathematical models often employ age-dependent immune factors to match the observed trends, while the present model uses a new detailed mechanistic model of parasite transmission dynamics to explain age-trends through the mechanism of parasite diversity. Illustrative simulations are performed for multiple field sites in Tanzania and Nigeria, and observed age-trends and seasonality in parasite prevalence are recreated in silico, proffering possible mechanistic explanations of the observational data., Results: Observed temporal dynamics in measured parasitaemia are recreated for each location and age-prevalence outputs are studied. Increasing population-level diversity in malaria surface antigens delays development of broad parasitological immunity. A local parasite population with high diversity can recreate the observed trends in age-prevalence across more than an order of magnitude of variation in transmission intensities., Conclusions: Mechanistic models of human immunity and parasite antigen diversity can recreate the observed temporal patterns for the development of parasitological immunity across a wide range of transmission intensities. This has implications for the distribution of disease burden across the population, the human transmission reservoir, design of elimination campaigns, and development and roll-out of potential vaccines.

In areas of low malaria transmission, it is currently recommended that a single dose of primaquine (0.75 mg base/kg; 45 mg adult dose) be added to artemisinin combination treatment (ACT) in acute falciparum malaria to block malaria transmission. Review of studies of transmission-blocking activity based on the infectivity of patients or volunteers to anopheline mosquitoes, and of haemolytic toxicity in glucose 6-dehydrogenase (G6PD) deficient subjects, suggests that a lower primaquine dose (0.25 mg base/kg) would be safer and equally effective. This lower dose could be deployed together with ACTs without G6PD testing wherever use of a specific gametocytocide is indicated.

Background: Models of Plasmodium falciparum malaria epidemiology that provide realistic quantitative predictions of likely epidemiological outcomes of existing vector control strategies have the potential to assist in planning for the control and elimination of malaria. This work investigates the applicability of mathematical modelling of malaria transmission dynamics in Rachuonyo South, a district with low, unstable transmission in the highlands of western Kenya., Methods: Individual-based stochastic simulation models of malaria in humans and a deterministic model of malaria in mosquitoes as part of the OpenMalaria platform were parameterized to create a scenario for the study area based on data from ongoing field studies and available literature. The scenario was simulated for a period of two years with a population of 10,000 individuals and validated against malaria survey data from Rachuonyo South. Simulations were repeated with multiple random seeds and an ensemble of 14 model variants to address stochasticity and model uncertainty. A one-dimensional sensitivity analysis was conducted to address parameter uncertainty., Results: The scenario was able to reproduce the seasonal pattern of the entomological inoculation rate (EIR) and patent infections observed in an all-age cohort of individuals sampled monthly for one year. Using an EIR estimated from serology to parameterize the scenario resulted in a closer fit to parasite prevalence than an EIR estimated using entomological methods. The scenario parameterization was most sensitive to changes in the timing and effectiveness of indoor residual spraying (IRS) and the method used to detect P. falciparum in humans. It was less sensitive than expected to changes in vector biting behaviour and climatic patterns., Conclusions: The OpenMalaria model of P. falciparum transmission can be used to simulate the impact of different combinations of current and potential control interventions to help plan malaria control in this low transmission setting. In this setting and for these scenarios, results were highly sensitive to transmission, vector exophagy, exophily and susceptibility to IRS, and the detection method used for surveillance. The level of accuracy of the results will thus depend upon the precision of estimates for each. New methods for analysing and evaluating uncertainty in simulation results will enhance the usefulness of simulations for malaria control decision-making. Improved measurement tools and increased primary data collection will enhance model parameterization and epidemiological monitoring. Further research is needed on the relationship between malaria indices to identify the best way to quantify transmission in low transmission settings. Measuring EIR through mosquito collection may not be the optimal way to estimate transmission intensity in areas with low, unstable transmission.

Controlled human malaria infection by blood stage parasite (BSP) inoculation is an alternative to the well-established model of infection with Plasmodium falciparum sporozoites delivered by mosquito bites. The BSP model has been utilized less frequently, but its use is increasing. Advantages of BSP challenge include greater ease of administration, better standardization of the infecting dose per volunteer, and good inter-study reproducibility of in vivo parasite dynamics. Recently, a surprising reduction in clinical symptoms at microscopic patency in the BSP model has been identified, which has an undefined and intriguing pathophysiologic basis, but may make this approach more acceptable to volunteers. We summarize clinical, parasitologic, and immunologic data from all BSP challenges to date, explore differences between the BSP and sporozoite models, and propose future applications for BSP challenge.

As part of a field ecology study of arbovirus and malaria activity in the Amazon Basin, Loreto Department, Peru, we collected mosquitoes landing on humans at a forest site and inside and outside of residences and military barracks at periurban, rural, and village sites. We collected 11 Anopheles spp. from these four sites. An. darlingi, the principal malaria vector in the region, accounted for 98.7% of all Anopheles spp. collected at Puerto Almendra. Peaks in landing activity occurred during the December and April collection periods. However, the percent of sporozoite-positive Anopheles spp. was highest 1-2 months later, when landing activity decreased to approximately 10% of the peak activity periods. At all sites, peak landing activity occurred about 2 hours after sunset. These data provide a better understanding of the taxonomy, population density, and seasonal and habitat distribution of potential malaria vectors within the Amazon Basin region.

Climate change is expected to lead to latitudinal and altitudinal temperature increases. High-elevation regions such as the highlands of Africa and those that have temperate climate are most likely to be affected. The highlands of Africa generally exhibit low ambient temperatures. This restricts the distribution of Anopheles mosquitoes, the vectors of malaria, filariasis, and O'nyong'nyong fever. The development and survival of larval and adult mosquitoes are temperature dependent, as are mosquito biting frequency and pathogen development rate. Given that various Anopheles species are adapted to different climatic conditions, changes in climate could lead to changes in species composition in an area that may change the dynamics of mosquito-borne disease transmission. It is important to consider the effect of climate change on rainfall, which is critical to the formation and persistence of mosquito breeding sites. In addition, environmental changes such as deforestation could increase local temperatures in the highlands; this could enhance the vectorial capacity of the Anopheles. These experimental data will be invaluable in facilitating the understanding of the impact of climate change on Anopheles., (© 2012 New York Academy of Sciences.)

District of Jiroft is situated in south-east of Iran which is one of the malarious regions. Anopheles stephensi is considered as one of the main malaria vector in this region. Ecology of this species was studied in the area to understand its vector behavior for implementation of effective vector control measures. Different methods like total catch, pit shelter, night bite collection on human and animal, larval dipping methods were used for species identification, seasonal activity, anthropophilic index and egg morphological characteristics. Anthropophilicity index was assessed by ELISA test. Activity of Anopheles species started at the beginning of April, and its peak occurs in late spring. The larvae were found in the river bed with pools, stagnant streams, slow foothill streams, temporary pools, and slowly moving water with and without vegetation, drainage containers of air conditioner and palm irrigation canals. From different methods of adult collection, it was found that spray sheet collection is the appropriate method. ELISA testing of 144 blood meals of females revealed the anthropophilicity of 11.8% indicating host preference on animal, mainly cow. Ridge length and their number on the egg floats confirmed Anopheles stephensi mysorensis form. This study showed that Anopheles stephensi is the main vector of malaria in the region, although some other species may play a role. Our findings could provide a valuable clue for epidemiology and control of malaria in the southeast of Iran.

Background: The question of sampling and spatial aggregation of malaria vectors is central to vector control efforts and estimates of transmission. Spatial patterns of anopheline populations are complex because mosquitoes' habitats and behaviors are strongly heterogeneous. Analyses of spatially referenced counts provide a powerful approach to delineate complex distribution patterns, and contributions of these methods in the study and control of malaria vectors must be carefully evaluated., Methodology/principal Findings: We used correlograms, directional variograms, Local Indicators of Spatial Association (LISA) and the Spatial Analysis by Distance IndicEs (SADIE) to examine spatial patterns of Indoor Resting Densities (IRD) in two dominant malaria vectors sampled with a 5 × 5 km grid over a 2500 km(2) area in the forest domain of Cameroon. SADIE analyses revealed that the distribution of Anopheles gambiae was different from regular or random, whereas there was no evidence of spatial pattern in Anopheles funestus (Ia = 1.644, Pa<0.05 and Ia = 1.464, Pa>0.05, respectively). Correlograms and variograms showed significant spatial autocorrelations at small distance lags, and indicated the presence of large clusters of similar values of abundance in An. gambiae while An. funestus was characterized by smaller clusters. The examination of spatial patterns at a finer spatial scale with SADIE and LISA identified several patches of higher than average IRD (hot spots) and clusters of lower than average IRD (cold spots) for the two species. Significant changes occurred in the overall spatial pattern, spatial trends and clusters when IRDs were aggregated at the house level rather than the locality level. All spatial analyses unveiled scale-dependent patterns that could not be identified by traditional aggregation indices., Conclusions/significance: Our study illustrates the importance of spatial analyses in unraveling the complex spatial patterns of malaria vectors, and highlights the potential contributions of these methods in malaria control.

Introduction: High coverage of conventional and long-lasting insecticide treated nets (ITNs and LLINs) in parts of E Africa are associated with reductions in local malaria burdens. Shifts in malaria vector species ratio have coincided with the scale-up suggesting that some species are being controlled by ITNs/LLINs better than others., Methods: Between 2005-2006 six experimental hut trials of ITNs and LLINs were conducted in parallel at two field stations in northeastern Tanzania; the first station was in Lower Moshi Rice Irrigation Zone, an area where An. arabiensis predominates, and the second was in coastal Muheza, where An. gambiae and An. funestus predominate. Five pyrethroids and one carbamate insecticide were evaluated on nets in terms of insecticide-induced mortality, blood-feeding inhibition and exiting rates., Results: In the experimental hut trials mortality of An. arabiensis was consistently lower than that of An. gambiae and An. funestus. The mortality rates in trials with pyrethroid-treated nets ranged from 25-52% for An. arabiensis, 63-88% for An. gambiae s.s. and 53-78% for An. funestus. All pyrethroid-treated nets provided considerable protection for the occupants, despite being deliberately holed, with blood-feeding inhibition (percentage reduction in biting rates) being consistent between species. Veranda exiting rates did not differ between species. Percentage mortality of mosquitoes tested in cone bioassays on netting was similar for An. gambiae and An. arabiensis., Conclusions: LLINs and ITNs treated with pyrethroids were more effective at killing An. gambiae and An. funestus than An. arabiensis. This could be a major contributing factor to the species shifts observed in East Africa following scale up of LLINs. With continued expansion of LLIN coverage in Africa An. arabiensis is likely to remain responsible for residual malaria transmission, and species shifts might be reported over larger areas. Supplementary control measures to LLINs may be necessary to control this vector species.

The increasing usage of long-lasting insecticide-treated nets allows protection of millions of people from malaria infection. Monitoring studies should be planned during any wide-scale malaria control program integrating insecticide-treated materials, to evaluate their effects and effectiveness on epidemiologically relevant parameters. Such operational control interventions may be challenged by insecticide resistance spread within vector populations, as a result of wide insecticide pressure. A nationwide distribution of long-lasting insecticidal nets was implemented throughout Niger in 2005. We studied the population genetic structure of major malaria vectors across Nigerien Sahel, and investigated potential effects of this large malaria control intervention. Wild-caught Anopheles gambiae sensu lato females from seven villages and two wet seasons were genotyped at 12 microsatellite loci. The genetic diversity within both species appeared homogenous between villages and years. The estimated genetic differentiation among samples was very low within both species, indicating high gene flow across the area. An absence of differentiation was also found between 2005 and 2006 wet seasons, for all samples but one, showing that the net distribution did not impact significantly the genetic diversity and structure of vector populations in a single year. We provide valuable results participating to document effects of large malaria control programs, to maximize the efficiency of available tools in future interventions.