The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F₁ individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors.     

Malaria and the Anopheles mosquitoes of Tajikistan

Surveys of Anopheles mosquitoes were conducted in urban, rural, and natural areas of Tajikistan to obtain updated information on their distributions, especially in southern districts of the country where malaria is a prevalent disease. Nine species of Anopheles are found in Tajikistan. Anopheles superpictus, An. claviger, An. hyrcanus, and An. pulcherrimus are the most widespread and abundant species. Investigations in northern Tajikistan confirmed the presence of An. artemievi and the absence of An. martinius, both members of the An. maculipennis complex of malaria vectors. Anopheles barianensis, An. lindesayi, and An. marteri sogdianus, species previously recorded in the country, were not encountered during our surveys. The history of Anopheles and malaria research in Tajikistan is reviewed and bionomical and distributional information is provided for each of the nine species.     

An Integrated Genetic Map of the African Human Malaria Vector Mosquito, Anopheles Gambiae

We present a genetic map based on microsatellite polymorphisms for the African human malaria vector, Anopheles gambiae. Polymorphisms in laboratory strains were detected for 89% of the tested microsatellite markers. Genotyping was performed for individual mosquitoes from 13 backcross families that included 679 progeny. Three linkage groups were identified, corresponding to the three chromosomes. We added 22 new markers to the existing X chromosome map, for a total of 46 microsatellite markers spanning a distance of 48.9 cM. The second chromosome has 57 and the third 28 microsatellite markers spanning a distance of 72.4 and 93.7 cM, respectively. The overall average distance between markers is 1.6 cM (or 1.1, 1.2, and 3.2 cM for the X, second, and third chromosomes, respectively). In addition to the 131 microsatellite markers, the current map also includes a biochemical selectable marker, Dieldrin resistance (Dl), on the second chromosome and five visible markers, pink-eye (p) and white (w) on the X, collarless (c) and lunate (lu) on the second, and red-eye (r) on the third. The cytogenetic locations on the nurse cell polytene chromosomes have been determined for 47 markers, making this map an integrated tool for cytogenetic, genetic, and molecular analysis.     

Predicted Distribution of Major Malaria Vectors Belonging to the Anopheles dirus Complex in Asia: Ecological Niche and Environmental Influences

Methods derived from ecological niche modeling allow to define species distribution based on presence-only data. This is particularly useful to develop models from literature records such as available for the Anopheles dirus complex, a major group of malaria mosquito vectors in Asia. This research defines an innovative modeling design based on presence-only model and hierarchical framework to define the distribution of the complex and attempt to delineate sibling species distribution and environmental preferences. At coarse resolution, the potential distribution was defined using slow changing abiotic factors such as topography and climate representative for the timescale covered by literature records of the species. The distribution area was then refined in a second step using a mask of current suitable land cover. Distribution area and ecological niche were compared between species and environmental factors tested for relevance. Alternatively, extreme values at occurrence points were used to delimit environmental envelopes. The spatial distribution for the complex was broadly consistent with its known distribution and influencing factors included temperature and rainfall. If maps developed from environmental envelopes gave similar results to modeling when the number of sites was high, the results were less similar for species with low number of recorded presences. Using presence-only models and hierarchical framework this study not only predicts the distribution of a major malaria vector, but also improved ecological modeling analysis design and proposed final products better adapted to malaria control decision makers. The resulting maps can help prioritizing areas which need further investigation and help simulate distribution under changing conditions such as climate change or reforestation. The hierarchical framework results in two products one abiotic based model describes the potential maximal distribution and remains valid for decades and the other including a biotic mask easy to update with frequently available information gives current species distribution.     

Physical Map of the Malaria Vector Anopheles Gambiae

Random cDNA clones, cosmid clones and RAPD polymorphic fragments have been localized by in situ hybridization to the ovarian nurse cell polytene chromosomes of the malaria vector Anopheles gambiae. We thus established 85 molecular markers for 110 sites within the whole A. gambiae polytene chromosome complement. The cDNA clones analyzed were isolated at random, and their exact localizations were determined by in situ hybridization. For 15 of the cDNA clones, a partial nucleotide sequence has been obtained; for nine of them sequence searches in the GenBank database revealed high degrees of similarity with published sequences. The cosmid clones analyzed were obtained as the result of screening with a few of the aforementioned cDNA clones of particular interest, or taken from a small set of randomly isolated cosmid clones. The RAPD clones are polymorphic fragments, potentially diagnostic for the various chromosomal forms of A. gambiae that are currently being analyzed.     

Development of a Gravid Trap for Collecting Live Malaria Vectors Anopheles gambiae s.l.

Background

Effective malaria vector control targeting indoor host-seeking mosquitoes has resulted in fewer vectors entering houses in many areas of sub-Saharan Africa, with the proportion of vectors outdoors becoming more important in the transmission of this disease. This study aimed to develop a gravid trap for the outdoor collection of the malaria vector Anopheles gambiae s.l. based on evaluation and modification of commercially available gravid traps.

Methods

Experiments were implemented in an 80 m² semi-field system where 200 gravid Anopheles gambiae s.s. were released nightly. The efficacy of the Box, CDC and Frommer updraft gravid traps was compared. The Box gravid trap was tested to determine if the presence of the trap over water and the trap’s sound affected catch size. Mosquitoes approaching the treatment were evaluated using electrocuting nets or detergents added to the water in the trap. Based on the results, a new gravid trap (OviART trap) that provided an open, unobstructed oviposition site was developed and evaluated.

Results

Box and CDC gravid traps collected similar numbers (relative rate (RR) 0.8, 95% confidence interval (CI) 0.6–1.2; p = 0.284), whereas the Frommer trap caught 70% fewer mosquitoes (RR 0.3, 95% CI 0.2–0.5; p < 0.001). The number of mosquitoes approaching the Box trap was significantly reduced when the trap was positioned over a water-filled basin compared to an open pond (RR 0.7 95% CI 0.6–0.7; p < 0.001). This effect was not due to the sound of the trap. Catch size increased by 60% (RR 1.6, 1.2–2.2; p = 0.001) with the new OviART trap.

Conclusion

Gravid An. Gambiae s.s. females were visually deterred by the presence of the trapping device directly over the oviposition medium. Based on these investigations, an effective gravid trap was developed that provides open landing space for egg-laying Anopheles .     

Cryptic Genetic Diversity within the Anopheles nili group of Malaria Vectors in the Equatorial Forest Area of Cameroon (Central Africa)

Background

The Anopheles nili group of mosquitoes includes important vectors of human malaria in equatorial forest and humid savannah regions of sub-Saharan Africa. However, it remains largely understudied, and data on its populations’ bionomics and genetic structure are crucially lacking. Here, we used a combination of nuclear (i.e. microsatellite and ribosomal DNA) and mitochondrial DNA markers to explore and compare the level of genetic polymorphism and divergence among populations and species of the group in the savannah and forested areas of Cameroon, Central Africa.

Principal Findings

All the markers provided support for the current classification within the An. nili group. However, they revealed high genetic heterogeneity within An. nili s.s. in deep equatorial forest environment. Nuclear markers showed the species to be composed of five highly divergent genetic lineages that differed by 1.8 to 12.9% of their Internal Transcribed Spacer 2 (ITS2) sequences, implying approximate divergence time of 0.82 to 5.86 million years. However, mitochondrial data only detected three major subdivisions, suggesting different evolutionary histories of the markers.

Conclusions/Significance

This study enlightened additional cryptic genetic diversity within An. nili s.s. in the deep equatorial forest environment of South Cameroon, reflecting a complex demographic history for this major vector of malaria in this environment. These preliminary results should be complemented by further studies which will shed light on the distribution, epidemiological importance and evolutionary history of this species group in the African rainforest, providing opportunities for in-depth comparative studies of local adaptation and speciation in major African malaria vectors.     

Patterns of Mitochondrial Variation within and between African Malaria Vectors, Anopheles Gambiae and An. Arabiensis, Suggest Extensive Gene Flow

Anopheles gambiae and An. arabiensis are mosquito species responsible for most malaria transmission in sub-Saharan Africa. They are also closely related sibling species that share chromosomal and molecular polymorphisms as a consequence of incomplete lineage sorting or introgressive hybridization. To help resolve these processes, this study examined the partitioning of mtDNA sequence variation within and between species across Africa, from both population genetic and phylogeographic perspectives. Based on partial gene sequences from the cytochrome b, ND1 and ND5 genes, haplotype diversity was high but sequences were very closely related. Within species, little or no population subdivision was detected, and there was no evidence for isolation by distance. Between species, there were no fixed nucleotide differences, a high proportion of shared polymorphisms, and eight haplotypes in common over distances as great as 6000 km. Only one of 16 shared polymorphisms led to an amino acid difference, and there was no compelling evidence for nonneutral variation. Parsimony networks constructed of haplotypes from both species revealed no correspondence of haplotype with either geography or taxonomy. This trend of low intraspecific genetic divergence is consistent with evidence from allozyme and microsatellite data and is interpreted in terms of both extensive gene flow and recent range expansion from relatively large, stable populations. We argue that retention of ancestral polymorphisms is a plausible but insufficient explanation for low interspecific genetic divergence, and that extensive hybridization is a contributing factor.     

Malaria Vectors in Ecologically Heterogeneous Localities of the Colombian Pacific Region

The Colombian Pacific region is second nationally in number of malaria cases reported. This zone presents great ecological heterogeneity and Anopheles species diversity. However, little is known about the current spatial and temporal distribution of vector species. This study, conducted in three ecologically different localities of the Pacific region, aimed to evaluate the composition and distribution of Anopheles species and characterize transmission intensity. A total of 4,016 Anopheles mosquitoes were collected representing seven species. The composition and dominant species differed in each locality. Three species were infected with malaria parasites: Anopheles darlingi and An. calderoni were infected with Plasmodium falciparum and An. nuneztovari with Plasmodium vivax VK210 and VK247. Annual EIRs varied from 3.5–7.2 infective bites per year. These results confirm the importance of the primary vector An. nuneztovari in areas disturbed by human interventions, of An. darlingi in deforested margins of humid tropical rainforest and An. albimanus and the suspected vector An. calderoni in areas impacted by urbanization and large-scale palm oil agriculture close to the coast. This constitutes the first report in the Colombia Pacific region of naturally infected An. darlingi, and in Colombia of naturally infected An. calderoni. Further studies should evaluate the epidemiological importance of An. calderoni in the Pacific region.     

Anopheles aquasalis Infected by Plasmodium vivax Displays Unique Gene Expression Profiles when Compared to Other Malaria Vectors and Plasmodia

Malaria affects 300 million people worldwide every year and is endemic in 22 countries in the Americas where transmission occurs mainly in the Amazon Region. Most malaria cases in the Americas are caused by Plasmodium vivax, a parasite that is almost impossible to cultivate in vitro, and Anopheles aquasalis is an important malaria vector. Understanding the interactions between this vector and its parasite will provide important information for development of disease control strategies. To this end, we performed mRNA subtraction experiments using A. aquasalis 2 and 24 hours after feeding on blood and blood from malaria patients infected with P. vivax to identify changes in the mosquito vector gene induction that could be important during the initial steps of infection. A total of 2,138 clones of differentially expressed genes were sequenced and 496 high quality unique sequences were obtained. Annotation revealed 36% of sequences unrelated to genes in any database, suggesting that they were specific to A. aquasalis. A high number of sequences (59%) with no matches in any databases were found 24 h after infection. Genes related to embryogenesis were down-regulated in insects infected by P. vivax. Only a handful of genes related to immune responses were detected in our subtraction experiment. This apparent weak immune response of A. aquasalis to P. vivax infection could be related to the susceptibility of this vector to this important human malaria parasite. Analysis of some genes by real time PCR corroborated and expanded the subtraction results. Taken together, these data provide important new information about this poorly studied American malaria vector by revealing differences between the responses of A. aquasalis to P. vivax infection, in relation to better studied mosquito-Plasmodium pairs. These differences may be important for the development of malaria transmission-blocking strategies in the Americas.     

Spatial-Temporal Variation and Primary Ecological Drivers of Anopheles sinensis Human Biting Rates in Malaria Epidemic-Prone Regions of China

Background

Robust malaria vector surveillance is essential for optimally selecting and targeting vector control measures. Sixty-two vector surveillance sites were established between 2005 and 2008 by the national malaria surveillance program in China to measure Anopheles sinensis human biting rates. Using these data to determine the primary ecological drivers of malaria vector human biting rates in malaria epidemic-prone regions of China will allow better targeting of vector control resources in space and time as the country aims to eliminate malaria.

Methods

We analyzed data from 62 malaria surveillance sentinel sites from 2005 to 2008. Linear mixed effects models were used to identify the primary ecological drivers for Anopheles sinensis human biting rates as well as to explore the spatial-temporal variation of relevant factors at surveillance sites throughout China.

Results

Minimum semimonthly temperature (β = 2.99; 95% confidence interval (CI) 2.07- 3.92), enhanced vegetation index (β =1.07; 95% CI 0.11–2.03), and paddy index (the percentage of rice paddy field in the total cultivated land area of each site) (β = 0.86; 95% CI 0.17–1.56) were associated with greater An. Sinensis human biting rates, while increasing distance to the nearest river was associated with lower An. Sinensis human biting rates (β = −1.47; 95% CI −2.88, −0.06). The temporal variation (σt02=1.35) in biting rates was much larger than the spatial variation (σs02=0.83), with 19.3% of temporal variation attributable to differences in minimum temperature and enhanced vegetation index and 16.9% of spatial variance due to distance to the nearest river and the paddy index.

Discussion

Substantial spatial-temporal variation in An. Sinensis human biting rates exists in malaria epidemic-prone regions of China, with minimum temperature and enhanced vegetation index accounting for the greatest proportion of temporal variation and distance to nearest river and paddy index accounting for the greatest proportion of spatial variation amongst observed ecological drivers.

Conclusions

Targeted vector control measures based on these findings can support the ongoing malaria elimination efforts in China more effectively.     

Multigene Phylogenetics Reveals Temporal Diversification of Major African Malaria Vectors

The major vectors of malaria in sub-Saharan Africa belong to subgenus Cellia. Yet, phylogenetic relationships and temporal diversification among African mosquito species have not been unambiguously determined. Knowledge about vector evolutionary history is crucial for correct interpretation of genetic changes identified through comparative genomics analyses. In this study, we estimated a molecular phylogeny using 49 gene sequences for the African malaria vectors An. gambiae, An. funestus, An. nili, the Asian malaria mosquito An. stephensi, and the outgroup species Culex quinquefasciatus and Aedes aegypti. To infer the phylogeny, we identified orthologous sequences uniformly distributed approximately every 5 Mb in the five chromosomal arms. The sequences were aligned and the phylogenetic trees were inferred using maximum likelihood and neighbor-joining methods. Bayesian molecular dating using a relaxed log normal model was used to infer divergence times. Trees from individual genes agreed with each other, placing An. nili as a basal clade that diversified from the studied malaria mosquito species 47.6 million years ago (mya). Other African malaria vectors originated more recently, and independently acquired traits related to vectorial capacity. The lineage leading to An. gambiae diverged 30.4 mya, while the African vector An. funestus and the Asian vector An. stephensi were the most closely related sister taxa that split 20.8 mya. These results were supported by consistently high bootstrap values in concatenated phylogenetic trees generated individually for each chromosomal arm. Genome-wide multigene phylogenetic analysis is a useful approach for discerning historic relationships among malaria vectors, providing a framework for the correct interpretation of genomic changes across species, and comprehending the evolutionary origins of this ubiquitous and deadly insect-borne disease.