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Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution. Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila. Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover but instead diversify through protein-sequence changes. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts.
Copyright © 2015, American Association for the Advancement of Science.

The principal Afrotropical malaria vector mosquito, Anopheles gambiae remains a significant threat to human health. In this anthropophagic species, females detect and respond to a range of human-derived volatile kairomones such as ammonia, lactic acid, and other carboxylic acids in their quest for blood meals. While the molecular underpinnings of mosquito olfaction and host seeking are becoming better understood, many questions remain unanswered. In this study, we have identified and characterized two candidate ammonium transporter genes, AgAmt and AgRh50 that are expressed in the mosquito antenna and may contribute to physiological and behavioral responses to ammonia, which is an important host kairomone for vector mosquitoes. AgAmt transcripts are highly enhanced in female antennae while a splice variant of AgRh50 appears to be antennal-specific. Functional expression of AgAmt in Xenopus laevis oocytes facilitates inward currents in response to both ammonium and methylammonium, while AgRh50 is able to partially complement a yeast ammonium transporter mutant strain, validating their conserved roles as ammonium transporters. We present evidence to suggest that both AgAmt and AgRh50 are in vivo ammonium transporters that are important for ammonia sensitivity in An. gambiae antennae, either by clearing ammonia from the sensillar lymph or by facilitating sensory neuron responses to environmental exposure. Accordingly, AgAmt and AgRh50 represent new and potentially important targets for the development of novel vector control strategies.

Insects, such as the malaria vector mosquito, Anopheles gambiae, depend upon chemoreceptors to respond to volatiles emitted from a range of environmental sources, most notably blood meal hosts and oviposition sites. A subset of peripheral signaling pathways involved in these insect chemosensory-dependent behaviors requires the activity of heteromeric odorant receptor (OR) ion channel complexes and ligands for numerous A. gambiae ORs (AgOrs) have been identified. Although AgOrs are expressed in nonhead appendages, studies characterizing potential AgOr function in nonolfactory tissues have not been conducted. In the present study, we explore the possibility that AgOrs mediate responses of spermatozoa to endogenous signaling molecules in A. gambiae. In addition to finding AgOr transcript expression in testes, we show that the OR coreceptor, AgOrco, is localized to the flagella of A. gambiae spermatozoa where Orco-specific agonists, antagonists, and other odorant ligands robustly activate flagella beating in an Orco-dependent process. We also demonstrate Orco expression and Orco-mediated activation of spermatozoa in the yellow fever mosquito, Aedes aegypti. Moreover, we find Orco localization in testes across distinct insect taxa and posit that OR-mediated responses in spermatozoa may represent a general characteristic of insect reproduction and an example of convergent evolution.

Thermosensation provides vital inputs for the malaria vector mosquito, Anopheles gambiae which utilizes heat-sensitivity within a broad spectrum of behaviors, most notably, the localization of human hosts for blood feeding. In this study, we examine thermosensory behaviors in larval-stage An. gambiae, which as a result of their obligate aquatic habitats and importance for vectorial capacity, represents an opportunistic target for vector control as part of the global campaign to eliminate malaria. As is the case for adults, immature mosquitoes respond differentially to a diverse array of external heat stimuli. In addition, larvae exhibit a striking phenotypic plasticity in thermal-driven behaviors that are established by temperature at which embryonic development occurs. Within this spectrum, RNAi-directed gene-silencing studies provide evidence for the essential role of the Transient Receptor Potential sub-family A1 (TRPA1) channel in mediating larval thermal-induced locomotion and thermal preference within a discrete upper range of ambient temperatures.

Wolbachia pipientis is the most common bacterial infection in the animal world and wields a vast influence on invertebrate reproduction, sex determination, speciation, and behavior worldwide. These avenues of research have made seminal gains, including the latest use of Wolbachia to alter mosquito populations and a strengthened focus on using anti-Wolbachia therapies against filarial nematode infections. This work is further bolstered by a more refined knowledge of Wolbachia biology spanning mechanisms to relevance. Here we tally the most up-to-date knowledge in the field and review the immense implications that this global infection has for the basic and applied life sciences.
Copyright © 2013 Elsevier Ltd. All rights reserved.

BACKGROUND - Malaria parasites undergo complex developmental transitions within the mosquito vector. A commonly used laboratory model for studies of mosquito-malaria interaction is the rodent parasite, P. berghei. Anopheles funestus is a major malaria vector in sub-Saharan Africa but has received less attention than the sympatric species, Anopheles gambiae. The imminent completion of the A. funestus genome sequence will provide currently lacking molecular tools to describe malaria parasite interactions in this mosquito, but previous reports suggested that A. funestus is not permissive for P. berghei development.
METHODS - An A. funestus population was generated in the laboratory by capturing female wild mosquitoes in Mali, allowing them to oviposit, and rearing the eggs to adults. These F1 progeny of wild mosquitoes were allowed to feed on mice infected with a fluorescent P. berghei strain. Fluorescence microscopy was used to track parasite development inside the mosquito, salivary gland sporozoites were tested for infectivity to mice, and parasite development in A. funestus was compared to A. gambiae.
RESULTS - P. berghei oocysts were detectable on A. funestus midguts by 7 days post-infection. By 18-20 days post-infection, sporozoites had invaded the median and distal lateral lobes of the salivary glands, and hemocoel sporozoites were observed in the hemolymph. Mosquitoes were capable of infecting mice via bite, demonstrating that A. funestus supports the complete life cycle of P. berghei. In a random sample of wild mosquito genotypes, A. funestus prevalence of infection and the characteristics of parasite development were similar to that observed in A. gambiae-P. berghei infections.
CONCLUSIONS - The data presented in this study establish an experimental laboratory model for Plasmodium infection of A. funestus, an important vector of human malaria. Studying A. funestus-Plasmodium interactions is now feasible in a laboratory setting. This information lays the groundwork for exploitation of the awaited genome sequence of A. funestus.

BACKGROUND - Human Malaria is transmitted by mosquitoes of the genus Anopheles. Transmission is a complex phenomenon involving biological and environmental factors of humans, parasites and mosquitoes. Among more than 500 anopheline species, only a few species from different branches of the mosquito evolutionary tree transmit malaria, suggesting that their vectorial capacity has evolved independently. Anopheles albimanus (subgenus Nyssorhynchus) is an important malaria vector in the Americas. The divergence time between Anopheles gambiae, the main malaria vector in Africa, and the Neotropical vectors has been estimated to be 100 My. To better understand the biological basis of malaria transmission and to develop novel and effective means of vector control, there is a need to explore the mosquito biology beyond the An. gambiae complex.
RESULTS - We sequenced the transcriptome of the An. albimanus adult female. By combining Sanger, 454 and Illumina sequences from cDNA libraries derived from the midgut, cuticular fat body, dorsal vessel, salivary gland and whole body, we generated a single, high-quality assembly containing 16,669 transcripts, 92% of which mapped to the An. darlingi genome and covered 90% of the core eukaryotic genome. Bidirectional comparisons between the An. gambiae, An. darlingi and An. albimanus predicted proteomes allowed the identification of 3,772 putative orthologs. More than half of the transcripts had a match to proteins in other insect vectors and had an InterPro annotation. We identified several protein families that may be relevant to the study of Plasmodium-mosquito interaction. An open source transcript annotation browser called GDAV (Genome-Delinked Annotation Viewer) was developed to facilitate public access to the data generated by this and future transcriptome projects.
CONCLUSIONS - We have explored the adult female transcriptome of one important New World malaria vector, An. albimanus. We identified protein-coding transcripts involved in biological processes that may be relevant to the Plasmodium lifecycle and can serve as the starting point for searching targets for novel control strategies. Our data increase the available genomic information regarding An. albimanus several hundred-fold, and will facilitate molecular research in medical entomology, evolutionary biology, genomics and proteomics of anopheline mosquito vectors. The data reported in this manuscript is accessible to the community via the VectorBase website (http://www.vectorbase.org/Other/AdditionalOrganisms/).

Over the last 20 years, ecological immunology has provided much insight into how environmental factors shape host immunity and host-parasite interactions. Currently, the application of this thinking to the study of mosquito immunology has been limited. Mechanistic investigations are nearly always conducted under one set of conditions, yet vectors and parasites associate in a variable world. We highlight how environmental temperature shapes cellular and humoral immune responses (melanization, phagocytosis and transcription of immune genes) in the malaria vector, Anopheles stephensi. Nitric oxide synthase expression peaked at 30°C, cecropin expression showed no main effect of temperature and humoral melanization, and phagocytosis and defensin expression peaked around 18°C. Further, immune responses did not simply scale with temperature, but showed complex interactions between temperature, time and nature of immune challenge. Thus, immune patterns observed under one set of conditions provide little basis for predicting patterns under even marginally different conditions. These quantitative and qualitative effects of temperature have largely been overlooked in vector biology but have significant implications for extrapolating natural/transgenic resistance mechanisms from laboratory to field and for the efficacy of various vector control tools.

BACKGROUND - Chemosensory signal transduction guides the behavior of many insects, including Anopheles gambiae, the major vector for human malaria in sub-Saharan Africa. To better understand the molecular basis of mosquito chemosensation we have used whole transcriptome RNA sequencing (RNA-seq) to compare transcript expression profiles between the two major chemosensory tissues, the antennae and maxillary palps, of adult female and male An. gambiae.
RESULTS - We compared chemosensory tissue transcriptomes to whole body transcriptomes of each sex to identify chemosensory enhanced genes. In the six data sets analyzed, we detected expression of nearly all known chemosensory genes and found them to be highly enriched in both olfactory tissues of males and females. While the maxillary palps of both sexes demonstrated strict chemosensory gene expression overlap, we observed acute differences in sensory specialization between male and female antennae. The relatively high expression levels of chemosensory genes in the female antennae reveal its role as an organ predominately assigned to chemosensation. Remarkably, the expression of these genes was highly conserved in the male antennae, but at much lower relative levels. Alternatively, consistent with a role in mating, the male antennae displayed significant enhancement of genes involved in audition, while the female enhancement of these genes was observed, but to a lesser degree.
CONCLUSIONS - These findings suggest that the chemoreceptive spectrum, as defined by gene expression profiles, is largely similar in female and male An. gambiae. However, assuming sensory receptor expression levels are correlated with sensitivity in each case, we posit that male and female antennae are perceptive to the same stimuli, but possess inverse receptive prioritizations and sensitivities. Here we have demonstrated the use of RNA-seq to characterize the sensory specializations of an important disease vector and grounded future studies investigating chemosensory processes.

Pathogenic protozoa threaten lives of several hundred million people throughout the world and are responsible for large numbers of deaths globally. The parasites are transmitted to humans by insect vectors, more than a hundred of infected mammalian species forming reservoir. With human migrations, HIV-coinfections, and blood bank contamination the diseases are now spreading beyond the endemic tropical countries, being found in all parts of the world including the USA, Canada and Europe. In spite of the widely appreciated magnitude of this health problem, current treatment for sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi) and leishmaniasis (Leishmania spp.) remains unsatisfactory. The drugs are decades old, their efficacy and safety profiles are unacceptable. This review describes sterol 14α-demethylase, an essential enzyme in sterol biosynthesis in eukaryotes and clinical target for antifungal azoles, as a promising target for antiprotozoan chemotherapy. While several antifungal azoles have been proven active against Trypanosomatidae and are under consideration as antiprotozoan agents, crystal structures of sterol 14α-demethylases from three protozoan pathogens, Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum provide the basis for the development of new, highly potent and pathogen-specific drugs with rationally optimized pharmacological properties.