Behavioral response of the malaria mosquito, Anopheles gambiae, to human sweat inoculated with axilla bacteria and to volatiles composing human axillary odor.

The responses of Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) to odors from male and female axillary sweat incubated with human axilla bacteria were recorded in a dual-choice olfactometer. Staphylococcus epidermidis was selected for its low odor-producing pattern, Corynebacterium jeikeium for its strong Nα-acylglutamine aminoacylase activity liberating carboxylic acids including (R)/(S)-3-hydroxy-3-methylhexanoic acid (HMHA) and Staphylococcus haemolyticus for its capacity to liberate sulfur-containing compounds including (R/S)-3-methyl-3-sulfanylhexan-1-ol (MSH). Anopheles gambiae behavioral responses were evaluated under (i) its responsiveness to take off and undertake sustained upwind flight and (ii) its discriminating capacity between the two olfactometer arms bearing a test odor in either one or both arms. Experiments were conducted in the presence of carbon dioxide pulses as a behavioral sensitizer. Anopheles gambiae clearly discriminated for the olfactometer arm conveying odor generated by incubating any of the three bacteria species with either male or female sweat. Whereas An. gambiae did not discriminate between male and female sterile sweat samples in the olfactometer, the mosquito consistently showed a preference for male sweat over female sweat incubated with the same bacterium, independent of the species used as inoculum. Sweat incubated with C. jeikeium rendered mosquitoes particularly responsive and this substrate elicited the strongest preference for male over female sweat. Tested on their own, neither HMHA nor MSH elicited a clear discriminating response but did affect mosquito responsiveness. These findings serve as a basis for further research on the odor-mediated anthropophilic host-seeking behavior of An. gambiae.

Contemporary evolution of resistance at the major insecticide target site gene Ace-1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae.

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine-serine substitution at codon 119 of the Ace-1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace-1 119S haplotype, whereas 119G diversity was high overall but very low at non-synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace-1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace-1 gene, whereas 119G alleles were unduplicated. Ace-1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace-1, emphasizing the need to integrate CNV analysis into genome scans for selection.

A standard photomap of ovarian nurse cell chromosomes in the European malaria vector Anopheles atroparvus.

Anopheles atroparvus (Diptera: Culicidae) is one of the main malaria vectors of the Maculipennis group in Europe. Cytogenetic analysis based on salivary gland chromosomes has been used in taxonomic and population genetic studies of mosquitoes from this group. However, a high-resolution cytogenetic map that could be used in physical genome mapping in An. atroparvus is still lacking. In the present study, a high-quality photomap of the polytene chromosomes from ovarian nurse cells of An. atroparvus was developed. Using fluorescent in situ hybridization, 10 genes from the five largest genomic supercontigs on the polytene chromosome were localized and 28% of the genome was anchored to the cytogenetic map. The study established chromosome arm homology between An. atroparvus and the major African malaria vector Anopheles gambiae, suggesting a whole-arm translocation between autosomes of these two species. The standard photomap constructed for ovarian nurse cell chromosomes of An. atroparvus will be useful for routine physical mapping. This map will assist in the development of a fine-scale chromosome-based genome assembly for this species and will also facilitate comparative and evolutionary genomics studies in the genus Anopheles.

Polymorphic chromosomal inversions in Anopheles moucheti, a major malaria vector in Central Africa.

Anopheles moucheti Evans (Diptera: Culicidae) is a major vector of malaria in forested areas of Central Africa. However, few genetic tools are available for this species. The present study represents the first attempt to characterize chromosomes in An. moucheti females collected in Cameroon. Ovarian nurse cells contained polytene chromosomes, which were suitable for standard cytogenetic applications. The presence of three polymorphic chromosomal inversions in An. moucheti was revealed. Two of these inversions were located on the 2R chromosome arm. The homology between the 2R chromosome arms of An. moucheti and Anopheles gambiae Giles was established by fluorescent in situ hybridization of six An. gambiae genic sequences. Mapping of the probes on chromosomes of An. moucheti detected substantial gene order reshuffling between the two species. The presence of polytene chromosomes and polymorphic inversions in An. moucheti provides a new basis for further population genetic, taxonomic and ecological studies of this neglected malaria vector.

The Legacy of Sir Ronald Ross: From Malaria Research to Multifaceted Achievements.

Sir Ronald Ross (13th May 1857 - 16th September 1932), a British doctor, was awarded the Nobel Prize in Physiology/Medicine in 1902 for research on the spread of malaria. This article highlights the multifaceted and significant scientific work by Ross. In 1897, he demonstrated that malaria is transmitted via mosquito bites and that malaria parasites exist in the gastrointestinal tract of the mosquito. Ross elucidated the transmission cycle in mosquitoes and birds infected with Plasmodium. His 25-year career in the Indian Medical Service laid the foundation for his ground-breaking work in malaria. Besides medicine, Ross excelled in poetry, music, and mathematics. The London School of Hygiene & Tropical Medicine has a frieze dedicated to 23 people chosen for their accomplishments in the field of public health, one of whom is Sir Ronald Ross. His legacy lives on through various honors and institutions, like the Ross Institute.

A chromosomal reference genome sequence for the malaria mosquito, Anopheles moucheti, Evans, 1925.

We present a genome assembly from an individual male Anopheles moucheti (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), from a wild population in Cameroon. The genome sequence is 271 megabases in span. The majority of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.5 kilobases in length.

A chromosomal reference genome sequence for the malaria mosquito, Anopheles gambiae, Giles, 1902, Ifakara strain.

We present a genome assembly from an individual female Anopheles gambiae (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), Ifakara strain. The genome sequence is 264 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

The genome sequence of the malaria mosquito, Anopheles funestus, Giles, 1900.

We present a genome assembly from an individual female Anopheles funestus (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae). The genome sequence is 251 megabases in span. The majority of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

Chromosome-level genome assemblies of the malaria vectors Anopheles coluzzii and Anopheles arabiensis.

BACKGROUND: Anopheles coluzzii and Anopheles arabiensis belong to the Anopheles gambiae complex and are among the major malaria vectors in sub-Saharan Africa. However, chromosome-level reference genome assemblies are still lacking for these medically important mosquito species. FINDINGS: In this study, we produced de novo chromosome-level genome assemblies for A. coluzzii and A. arabiensis using the long-read Oxford Nanopore sequencing technology and the Hi-C scaffolding approach. We obtained 273.4 and 256.8 Mb of the total assemblies for A. coluzzii and A. arabiensis, respectively. Each assembly consists of 3 chromosome-scale scaffolds (X, 2, 3), complete mitochondrion, and unordered contigs identified as autosomal pericentromeric DNA, X pericentromeric DNA, and Y sequences. Comparison of these assemblies with the existing assemblies for these species demonstrated that we obtained improved reference-quality genomes. The new assemblies allowed us to identify genomic coordinates for the breakpoint regions of fixed and polymorphic chromosomal inversions in A. coluzzii and A. arabiensis. CONCLUSION: The new chromosome-level assemblies will facilitate functional and population genomic studies in A. coluzzii and A. arabiensis. The presented assembly pipeline will accelerate progress toward creating high-quality genome references for other disease vectors.

Characterisation of anopheline larval habitats in southern Malawi.

INTRODUCTION: Increasing the knowledgebase of anopheline larval ecology could enable targeted deployment of malaria control efforts and consequently reduce costs of implementation. In Malawi, there exists a knowledge gap in anopheline larval ecology and, therefore, basis for targeted deployment of larval source management (LSM) for malaria control, specifically larvicides. We set out to characterize anopheline larval habitats in the Majete area of Malawi on the basis of habitat ecology and anopheline larval productivity to create a basis for larval control initiatives in the country. METHODS: Longitudinal surveys were conducted in randomly selected larval habitats over a period of fifteen months in Chikwawa district, southern Malawi. Biotic and abiotic parameters of the habitats were modelled to determine their effect on the occurrence and densities of anopheline larvae. RESULTS: Seventy aquatic habitats were individually visited between 1-7 times over the study period. A total of 5,123 immature mosquitoes (3,359 anophelines, 1,497 culicines and 267 pupae) were collected. Anopheline and culicine larvae were observed in sympatry in aquatic habitats. Of the nine habitat types followed, dams, swamps, ponds, borehole runoffs and drainage channels were the five most productive habitat types for anopheline mosquitoes. Anopheline densities were higher in aquatic habitats with bare soil making up part of the surrounding land cover (p<0.01) and in aquatic habitats with culicine larvae (p<0.01) than in those surrounded by vegetation and not occupied by culicine larvae. Anopheline densities were significantly lower in highly turbid habitats than in clearer habitats (p<0.01). Presence of predators in the aquatic habitats significantly reduced the probability of anopheline larvae being present (p=0.04). CONCLUSIONS: Anopheline larval habitats are widespread in the study area. Presence of bare soil, culicine larvae, predators and the level of turbidity of water are the main determinants of anopheline larval densities in aquatic habitats in Majete, Malawi. While the most productive aquatic habitats should be prioritised, for the most effective control of vectors in the area all available aquatic habitats should be targeted, even those that are not characterized by the identified predictors. Further research is needed to determine whether targeted LSM would be cost-effective when habitat characterisation is included in cost analyses and to establish what methods would make the characterisation of habitats easier.

Three-dimensional Organization of Polytene Chromosomes in Somatic and Germline Tissues of Malaria Mosquitoes.

Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism's development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of Anopheles gambiae, An. coluzzii, and An. merus. We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome-nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome-chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.

A chordwise offset of the wing-pitch axis enhances rotational aerodynamic forces on insect wings: a numerical study.

Most flying animals produce aerodynamic forces by flapping their wings back and forth with a complex wingbeat pattern. The fluid dynamics that underlies this motion has been divided into separate aerodynamic mechanisms of which rotational lift, that results from fast wing pitch rotations, is particularly important for flight control and manoeuvrability. This rotational force mechanism has been modelled using Kutta-Joukowski theory, which combines the forward stroke motion of the wing with the fast pitch motion to compute forces. Recent studies, however, suggest that hovering insects can produce rotational forces at stroke reversal, without a forward motion of the wing. We have conducted a broad numerical parametric study over a range of wing morphologies and wing kinematics to show that rotational force production depends on two mechanisms: (i) conventional Kutta-Joukowski-based rotational forces and (ii) a rotational force mechanism that enables insects with an offset of the pitch axis relative to the wing's chordwise symmetry axis to generate rotational forces in the absence of forward wing motion. Because flying animals produce control actions frequently near stroke reversal, this pitch-axis-offset dependent aerodynamic mechanism may be particularly important for understanding control and manoeuvrability in natural flyers.

Commonly Used Insect Repellents Hide Human Odors from Anopheles Mosquitoes.

The mode of action for most mosquito repellents is unknown. This is primarily due to the difficulty in monitoring how the mosquito olfactory system responds to repellent odors. Here, we used the Q-system of binary expression to enable activity-dependent Ca2+ imaging in olfactory neurons of the African malaria mosquito Anopheles coluzzii. This system allows neuronal responses to common insect repellents to be directly visualized in living mosquitoes from all olfactory organs, including the antenna. The synthetic repellents N,N-diethyl-meta-toluamide (DEET) and IR3535 did not activate Anopheles odorant receptor co-receptor (Orco)-expressing olfactory receptor neurons (ORNs) at any concentration, and picaridin weakly activated ORNs only at high concentrations. In contrast, natural repellents (i.e. lemongrass oil and eugenol) strongly activated small numbers of ORNs in the Anopheles mosquito antennae at low concentrations. We determined that DEET, IR3535, and picaridin decrease the response of Orco-expressing ORNs when these repellents are physically mixed with activating human-derived odorants. We present evidence that synthetic repellents may primarily exert their olfactory mode of action by decreasing the amount of volatile odorants reaching ORNs. These results suggest that synthetic repellents disruptively change the chemical profile of host scent signatures on the skin surface, rendering humans invisible to Anopheles mosquitoes.

Photoperiodic responses of Sahelian malaria mosquitoes Anopheles coluzzii and An. arabiensis.

BACKGROUND: Throughout large parts of sub-Saharan Africa, seasonal malaria transmission follows mosquito density, approaching zero during the dry season and peaking during the wet season. The mechanisms by which malaria mosquitoes survive the long dry season, when no larval sites are available remain largely unknown, despite being long recognized as a critical target for vector control. Previous work in the West African Sahel has led to the hypothesis that Anopheles coluzzii (formerly M-form Anopheles gambiae) undergoes aestivation (dry-season diapause), while Anopheles gambiae (s.s.) (formerly S-form An. gambiae) and Anopheles arabiensis repopulate each wet season via long-distance migration. The environmental cues used by these species to signal the oncoming dry season have not been determined; however, studies, mostly addressing mosquitoes from temperate zones, have highlighted photoperiod and temperature as the most common token stimuli for diapause initiation. We subjected newly established colonies of An. coluzzii and An. arabiensis from the Sahel to changes in photoperiod to assess and compare their responses in terms of longevity and other relevant phenotypes. RESULTS: Our results showed that short photoperiod alone and to a lesser extent, lower nightly temperature (representing the early dry season), significantly increased longevity of An. coluzzii (by ~30%, P < 0.001) but not of An. arabiensis. Further, dry season conditions increased body size but not relative lipid content of An. coluzzii, whereas body size of An. arabiensis decreased under these conditions. CONCLUSIONS: These species-specific responses underscore the capacity of tropical anophelines to detect mild changes (~1 h) in photoperiod and thus support the role of photoperiod as a token stimulus for An. coluzzii in induction of aestivation, although, these responses fall short of a complete recapitulation of aestivation under laboratory conditions.

Comparative pharmacological characterization of D1-like dopamine receptors from Anopheles gambiae, Aedes aegypti and Culex quinquefasciatus suggests pleiotropic signaling in mosquito vector lineages.

BACKGROUND: Small molecule antagonists of mosquito dopamine receptors (DARs) are under investigation as a new class of vector-selective insecticides. Antagonists that inhibit the D1-like DARs AaDOP2 and CqDOP2 from the mosquitoes Aedes aegypti L. and Culex quinquefasciatus Say, respectively, also cause larval mortality in bioassays. Here, we report on the orthologous DAR, AgDOP2, from the malaria mosquito Anopheles gambiae Giles that was cloned and pharmacologically characterized in HEK293 cells. Larval bioassays were then conducted to examine the potential of DAR antagonist insecticides against Anopheles vectors. FINDINGS: Previous in vitro cAMP accumulation assays demonstrated Gαs coupling for AaDOP2 and CqDOP2 and dose-dependent inhibition by DAR antagonists. We observed a negligible response of AgDOP2 in the cAMP assay, which prompted an investigation of alternative coupling for mosquito DARs. In an in vitro IP-One Gαq second messenger assay of calcium signaling, dopamine stimulation increased IP1 accumulation in AaDOP2-, CqDOP2- and AgDOP2-expressing cells, and DAR antagonists inhibited IP1 signaling in a dose-dependent manner. In larval bioassays, DAR antagonists caused considerable mortality of An. gambiae larvae within 24 h post-exposure. CONCLUSIONS: In vitro data reveal pleiotropic coupling of AaDOP2 and CqDOP2 to Gαq and Gαs. In contrast, AgDOP2 appeared to selectively couple to Gαq signaling. In vitro antagonist studies revealed general conservation in pharmacology between mosquito DARs. In vivo data suggest potential for DAR antagonist insecticides against An. gambiae. Sequence conservation among the DOP2 receptors from 15 Anopheles species indicates utility of antagonists to control residual malaria transmission. AgDOP2 Gαq-dependent signaling could be exploited for An. gambiae control via pathway specific antagonists.