Aquaporin water channel AgAQP1 in the malaria vector mosquito Anopheles gambiae during blood feeding and humidity adaptation

Altered patterns of malaria endemicity reflect, in part, changes in feeding behavior and climate adaptation of mosquito vectors. Aquaporin (AQP) water channels are found throughout nature and confer high-capacity water flow through cell membranes. The genome of the major malaria vector mosquito Anopheles gambiae contains at least seven putative AQP sequences. Anticipating that transmembrane water movements are important during the life cycle of A. gambiae, we identified and characterized the A. gambiae aquaporin 1 (AgAQP1) protein that is homologous to AQPs known in humans, Drosophila, and sap-sucking insects. When expressed in Xenopus laevis oocytes, AgAQP1 transports water but not glycerol. Similar to mammalian AQPs, water permeation of AgAQP1 is inhibited by HgCl(2) and tetraethylammonium, with Tyr185 conferring tetraethylammonium sensitivity. AgAQP1 is more highly expressed in adult female A. gambiae mosquitoes than in males. Expression is high in gut, ovaries, and Malpighian tubules where immunofluorescence microscopy reveals that AgAQP1 resides in stellate cells but not principal cells. AgAQP1 expression is up-regulated in fat body and ovary by blood feeding but not by sugar feeding, and it is reduced by exposure to a dehydrating environment (42% relative humidity). RNA interference reduces AgAQP1 mRNA and protein levels. In a desiccating environment (<20% relative humidity), mosquitoes with reduced AgAQP1 protein survive significantly longer than controls. These studies support a role for AgAQP1 in water homeostasis during blood feeding and humidity adaptation of A. gambiae, a major mosquito vector of human malaria in sub-Saharan Africa.     

Molecular basis of odor coding in the malaria vector mosquito Anopheles gambiae

A systematic functional analysis across much of the conventional Anopheles gambiae odorant receptor (AgOR) repertoire was carried out in Xenopus oocytes using two-electrode, voltage-clamp electrophysiology. The resulting data indicate that each AgOR manifests a distinct odor-response profile and tuning breadth. The large diversity of tuning responses ranges from AgORs that are responsive to a single or small number of odorants (specialists) to more broadly tuned receptors (generalists). Several AgORs were identified that respond robustly to a range of human volatiles that may play a critical role in anopheline host selection. AgOR responses were analyzed further by constructing a multidimensional odor space representing the relationships between odorants and AgOR responses. Within this space, the distance between odorants is related to both chemical class and concentration and may correlate with olfactory discrimination. This study provides a comprehensive overview of olfactory coding mechanisms of An. gambiae that ultimately may aid in fostering the design and development of olfactory-based strategies for reducing the transmission of malaria and other mosquito-borne diseases.     

Olfactory responses in a gustatory organ of the malaria vector mosquito Anopheles gambiae

The proboscis is an important head appendage in insects that has primarily been thought to process gustatory information during food intake. Indeed, in Drosophila and other insects in which they have been identified, most gustatory receptors are expressed in proboscis neurons. Our previous characterization of the expression of AgOR7, a highly conserved odorant receptor (OR) of the Afrotropical malaria vector mosquito Anopheles gambiae in the labellum at the tip of the proboscis was suggestive of a potential olfactory function in this mosquito appendage. To test this hypothesis, we used electrophysiological recording and neuronal tracing, and carried out a molecular characterization of candidate OR expression in the labellum of A. gambiae. These studies have uncovered a set of labial olfactory responses to a small spectrum of human-related odorants, such as isovaleric acid, butylamine, and several ketones and oxocarboxylic acids. Molecular analyses indicated that at least 24 conventional OR genes are expressed throughout the proboscis. Furthermore, to more fully examine AgOR expression within this tissue, we characterized the AgOR profile within a single labial olfactory sensillum. This study provides compelling data to support the hypothesis that a cryptic set of olfactory neurons that respond to a small set of odorants are present in the mouth parts of hematophagous mosquitoes. This result is consistent with an important role for the labellum in the close-range discrimination of bloodmeal hosts that directly impacts the ability of A. gambiae to transmit malaria and other diseases.     

Low-resolution genome map of the malaria mosquito Anopheles gambiae.

We have microdissected divisions of the Anopheles gambiae polytene chromosomes, digested the DNAs with a restriction enzyme, and PCR-amplified the DNA fragments to generate a set of pooled probes, each corresponding to approximately 2% of the mosquito genome. These divisional probes were shown to have high complexity. Except for those derived from near the centromeres, they hybridize specifically with their chromosomal sites of origin. Thus, they can be used to map cloned DNAs by a dot blot procedure, which is much more convenient than in situ hybridization to polytene chromosomes. We discuss additional potential uses of these probes, such as easier isolation of molecular markers and genes, including those that cross-hybridize with clones available from other insects. It is expected that the probes will substantially accelerate molecular genetic analysis of this most important malaria vector.     

From the Cover: Visual arrestins in olfactory pathways of Drosophila and the malaria vector mosquito Anopheles gambiae

Arrestins are important components for desensitization of G protein-coupled receptor cascades that mediate neurotransmission as well as olfactory and visual sensory reception. We have isolated AgArr1, an arrestin-encoding cDNA from the malaria vector mosquito, Anopheles gambiae, where olfaction is critical for vectorial capacity. Analysis of AgArr1 expression revealed an overlap between chemosensory and photoreceptor neurons. Furthermore, an examination of previously identified arrestins from Drosophila melanogaster exposed similar bimodal expression, and Drosophila arrestin mutants demonstrate impaired electrophysiological responses to olfactory stimuli. Thus, we show that arrestins in Drosophila are required for normal olfactory physiology in addition to their previously described role in visual signaling. These findings suggest that individual arrestins function in both olfactory and visual pathways in Dipteran insects; these genes may prove useful in the design of control strategies that target olfactory-dependent behaviors of insect disease vectors.     

First report of knockdown mutations in the malaria vector Anopheles gambiae from Cameroon

We report the first finding of the knockdown Leu-Phe and Leu-Ser mutations associated with resistance to pyrethroids and DDT insecticides in the malaria mosquito Anopheles gambiae from Cameroon. The Leu-Phe mutation was found in both the M and S molecular forms of An. gambiae. Importantly, two specimens of the S molecular form were found to carry both mutations in a heterozygous state.     

Genetic variation in the malaria mosquito vector Anopheles maculatus from Peninsular Malaysia

Starch-gel electrophoretic studies on nine gene-enzyme systems comprising 14 loci revealed a fair level of genetic variation in two population samples of Anopheles maculatus from Peninsular Malaysia. The proportion of polymorphic loci was 0.36 for the Fort Bertau sample and 0.29 for the Gua Musang sample, while the mean heterozygosity value was 0.09 for Fort Bertau and 0.07 for Gua Musang. The values of genetic similarity (I = 0.98) and genetic distance (D = 0.02) were of the rank of geographical populations.     

Gambicin: a novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae

A novel mosquito antimicrobial peptide, gambicin, and the corresponding gene were isolated in parallel through differential display-PCR, an expressed sequence tag (EST) project, and characterization of an antimicrobial activity in a mosquito cell line by reverse-phase chromatography. The 616-bp gambicin ORF encodes an 81-residue protein that is processed and secreted as a 61-aa mature peptide containing eight cysteines engaged in four disulfide bridges. Gambicin lacks sequence homology with other known proteins. Like other Anopheles gambiae antimicrobial peptide genes, gambicin is induced by natural or experimental infection in the midgut, fatbody, and hemocyte-like cell lines. Within the midgut, gambicin is predominantly expressed in the anterior part. Both local and systemic gambicin expression is induced during early and late stages of natural malaria infection. In vitro experiments showed that the 6.8-kDa mature peptide can kill both Gram-positive and Gram-negative bacteria, has a morphogenic effect on a filamentous fungus, and is marginally lethal to Plasmodium berghei ookinetes. An oxidized form of gambicin isolated from the cell line medium was more active against bacteria than the nonoxidized form from the same medium.     

Evolutionary lability of odour-mediated host preference by the malaria vector Anopheles gambiae

Many species of disease‐vector mosquitoes display vertebrate host specificity. Despite considerable progress in recent years in understanding the proximate and ultimate factors related to non‐random host selection at the interspecific level, the basis of this selection remains only partially understood. Anopheles gambiae sensu stricto, the main malaria vector in Africa, is considered a highly anthropophilic mosquito, and host odours have been shown to play a major role in the host‐seeking process of this species. Studies on host preference of An. gambiae have been either conducted in controlled conditions using laboratory reared mosquitoes and worn stockings as host‐related stimuli, or have been done in the field with methods that do not account for internal (e.g. age of sampled mosquitoes) and/or environmental effects. We explored differential behavioural responses to host odours between two populations of the same sibling species, An. gambiae in semi‐field conditions in Burkina Faso. The behavioural responses (i.e. degree of activation and strength of anemotaxis) were investigated using a Y‐olfactometer designed to accommodate whole hosts as a source of odour stimuli. Two strains of An. gambiae (3 to 4‐day‐old female) from laboratory Kisumu strain, and from field‐collected individuals were confronted to combinations of stimuli comprising calf odour, human odour and outdoor air. In dual‐choice tests, field mosquitoes chose human odour over calf odour, outdoor air over calf odour and responded equally to human and outdoor air, while laboratory mosquitoes responded equally to human and calf odour, human odour over outdoor air and calf odour over outdoor air. Overall, no effect of CO₂ exhaled by humans and calves neither on the proportion of activated mosquitoes nor on the relative attractiveness to odour stimuli was found. We report for the first time an intraspecific variation in host‐odour responses. This study clearly suggests that there may be genetic polymorphism underlying host preference and emphasizes that the highly anthropophilic label given to An. gambiae s.s. must be carefully interpreted and refer to populations rather than the whole sibling species.     

A cell surface mucin specifically expressed in the midgut of the malaria mosquito Anopheles gambiae

An invertebrate intestinal mucin gene, AgMuc1, was isolated from the malaria vector mosquito Anopheles gambiae. The predicted 122-residue protein consists of a central core of seven repeating TTTTVAP motifs flanked by hydrophobic N- and C-terminal domains. This structure is similar to that of mucins that coat the protozoan parasite Trypanosoma cruzi. Northern blot analysis indicated that the gene is expressed exclusively in the midgut of adult mosquitoes. A length polymorphism and in situ hybridization were used to genetically and cytogenetically map AgMuc1 to division 7A of the right arm of the second chromosome. The subcellular localization of the encoded protein in tissue culture cells was examined by using a baculovirus vector to express AgMuc1 protein tagged with the green fluorescent protein (GFP). The results indicated that this protein is found at the cell surface and that both hydrophobic domains are required for cell surface targeting. We propose that AgMuc1 is an abundant mucin-like protein that lines the surface of the midgut microvilli, potentially protecting the intestinal epithelium from the proteinase-rich environment of the gut lumen. An intriguing possibility is that, as an abundant surface protein, AgMuc1 may also interact with the malaria parasite during its invasion of the mosquito midgut.     

传疟按蚊抗药性研究进展

我国卫生部于2010年5月印发《中国消除疟疾行动计划（2010-2020年）》,提出在2020年,将全面彻底消除疟疾.控制及消除疟疾的关键在于传播媒介的控制,而按蚊为疟疾主要传播媒介,传疟媒介对杀虫剂的抗药性直接导致了疟疾发病的死灰复燃.为了全面了解疟疾蚊媒的抗药性现状和产生机制,该文对国内外传疟按蚊抗药性现状、产生机制和检测方法进行综述.     

Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.