African Malaria Vector Anopheles Gambiae Research Articles

Rapid and non-destructive identification of Anopheles gambiae and Anopheles arabiensis mosquito species using Raman spectroscopy via machine learning classification models

BackgroundIdentification of malaria vectors is an important exercise that can result in the deployment of targeted control measures and monitoring the susceptibility of the vectors to control strategies. Although known to possess distinct biting behaviours and habitats, the African malaria vectors Anopheles gambiae and Anopheles arabiensis are morphologically indistinguishable and are known to be discriminated by molecular techniques. In this paper, Raman spectroscopy is proposed to complement the tedious and time-consuming Polymerase Chain Reaction (PCR) method for the rapid screening of mosquito identity.MethodsA dispersive Raman microscope was used to record spectra from the legs (femurs and tibiae) of fresh anaesthetized laboratory-bred mosquitoes. The scattered Raman intensity signal peaks observed were predominantly centered at approximately 1400 cm−1, 1590 cm−1, and 2067 cm−1. These peaks, which are characteristic signatures of melanin pigment found in the insect cuticle, were important in the discrimination of the two mosquito species. Principal Component Analysis (PCA) was used for dimension reduction. Four classification models were built using the following techniques: Linear Discriminant Analysis (LDA), Logistic Regression (LR), Quadratic Discriminant Analysis (QDA), and Quadratic Support Vector Machine (QSVM).ResultsPCA extracted twenty-one features accounting for 95% of the variation in the data. Using the twenty-one principal components, LDA, LR, QDA, and QSVM discriminated and classified the two cryptic species with 86%, 85%, 89%, and 93% accuracy, respectively on cross-validation and 79%, 82%, 81% and 93% respectively on the test data set.ConclusionRaman spectroscopy in combination with machine learning tools is an effective, rapid and non-destructive method for discriminating and classifying two cryptic mosquito species, Anopheles gambiae and Anopheles arabiensis belonging to the Anopheles gambiae complex.

Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell–based screening platform, we identified and characterized several antagonists of the odorant receptor coreceptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds. Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.

Reduced human-biting preferences of the African malaria vectors Anopheles arabiensis and Anopheles gambiae in an urban context: controlled, competitive host-preference experiments in Tanzania

BackgroundHost preference is a critical determinant of human exposure to vector-borne infections and the impact of vector control interventions. Widespread use of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) across sub-Saharan Africa, which protect humans against mosquitoes, may select for altered host preference traits of malaria vectors over the long term. Here, the host preferences of Anopheles arabiensis and Anopheles gambiae sensu stricto (s.s.) were experimentally assessed in the field, using direct host-preference assays in two distinct ecological settings in Tanzania.MethodsEight Ifakara Tent Trap (ITT), four baited with humans and four with bovine calves, were simultaneously used to catch malaria vectors in open field sites in urban and rural Tanzania. The numbers of mosquitoes collected in human-baited traps versus calf-baited traps were used to estimate human feeding preference for each site's vector species.ResultsThe estimated proportion [95% confidence interval (CI)] of mosquitoes attacking humans rather than cattle was 0.60 [0.40, 0.77] for An. arabiensis in the rural setting and 0.61 [0.32, 0.85] for An. gambiae s.s. in the urban setting, indicating no preference for either host in both cases (P = 0.32 and 0.46, respectively) and no difference in preference between the two (Odds Ratio (OR) [95%] = 0.95 [0.30, 3.01], P = 0.924). However, only a quarter of An. arabiensis in the urban setting attacked humans (0.25 [0.09, 0.53]), indicating a preference for cattle that approached significance (P = 0.08). Indeed, urban An. arabiensis were less likely to attack humans rather than cattle when compared to the same species in the rural setting (OR [95%] = 0.21 [0.05, 0.91], P = 0.037).ConclusionUrban An. arabiensis had a stronger preference for cattle than the rural population and urban An. gambiae s.s. showed no clear preference for either humans or cattle. In the urban setting, both species exhibited stronger tendencies to attack cattle than previous studies of the same species in rural contexts. Cattle keeping may, therefore, particularly limit the impact of human-targeted vector control interventions in Dar es Salaam and perhaps in other African towns and cities.

Ecological drivers of genetic connectivity for African malaria vectors Anopheles gambiae and An. arabiensis

Anopheles gambiae and An. arabiensis are major malaria vectors in sub-Saharan Africa. Knowledge of how geographical factors drive the dispersal and gene flow of malaria vectors can help in combatting insecticide resistance spread and planning new vector control interventions. Here, we used a landscape genetics approach to investigate population relatedness and genetic connectivity of An. gambiae and An. arabiensis across Kenya and determined the changes in mosquito population genetic diversity after 20 years of intensive malaria control efforts. We found a significant reduction in genetic diversity in An. gambiae, but not in An. arabiensis as compared to prior to the 20-year period in western Kenya. Significant population structure among populations was found for both species. The most important ecological driver for dispersal and gene flow of An. gambiae and An. arabiensis was tree cover and cropland, respectively. These findings highlight that human induced environmental modifications may enhance genetic connectivity of malaria vectors.

Derivatization increases mosquito larvicidal activity of the sesquiterpene lactone parthenin isolated from the invasive weed Parthenium hysterophorus.

Extracts of the invasive weed Parthenium hysterophorus (Asteraceae) have been shown to possess larvicidal activity against a wide range of disease vectors. However, the phytochemicals responsible for the larvicidal activity from this plant remain unidentified. Here, we isolated the major sesquiterpene lactone, parthenin (1) from the plant and synthesized two derivatives [ethylene glycol (2) and azide (3) derivatives] targeting the α,β-unsaturated carbonyl group, previously known to account for its biological activity such as toxicity towards cells and microorganism. All three compounds were screened for larvicidal activity against the African malaria vector Anopheles gambiae. The larval mortality of ethylene glycol derivative (2) and 2α-azidocoronopilin (3) were approximately two-four-fold higher than that of parthenin (1) and neem oil with LC50 values of 37 and 66 mg L-1 , respectively. Parthenin (1) and the positive control, neem oil, had comparable median lethal concentration (LC50 ) values of 154 and 121 mg L-1 , respectively. In assays with binary combinations of the three compounds, larvicidal activity followed the order: parthenin (1) + 2α-azidocoronopilin (3) (LC50 = 14 mg L-1 ) > parthenin (1) + ethylene glycol derivative (2) (LC50 = 109 mg L-1 ), > blend of 2α-azidocoronopilin (3) and ethylene glycol derivative (2) (LC50 = 200 mg L-1 ). Structural modification of parthenin (1) through addition of hydroxyl groups increases its larvicidal effects. These findings advance the use of structural modification approach in the development of lead chemical molecules for potential exploitation in larval source management.

Assessing connectivity despite high diversity in island populations of a malaria mosquito.

Documenting isolation is notoriously difficult for species with vast polymorphic populations. High proportions of shared variation impede estimation of connectivity, even despite leveraging information from many genetic markers. We overcome these impediments by combining classical analysis of neutral variation with assays of the structure of selected variation, demonstrated using populations of the principal African malaria vector Anopheles gambiae. Accurate estimation of mosquito migration is crucial for efforts to combat malaria. Modeling and cage experiments suggest that mosquito gene drive systems will enable malaria eradication, but establishing safety and efficacy requires identification of isolated populations in which to conduct field testing. We assess Lake Victoria islands as candidate sites, finding one island 30 km offshore is as differentiated from mainland samples as populations from across the continent. Collectively, our results suggest sufficient contemporary isolation of these islands to warrant consideration as field‐testing locations and illustrate shared adaptive variation as a useful proxy for connectivity in highly polymorphic species.

Preparation and Use of a Yeast shRNA Delivery System for Gene Silencing in Mosquito Larvae.

The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the dengue and Zika virus vector Aedes aegypti and the primary African malaria vector Anopheles gambiae. RNA interference (RNAi) has facilitated gene silencing experiments in both of these disease vector mosquito species and could one day be applied as a new method of vector control. Here, we describe a procedure for the genetic engineering of Saccharomyces cerevisiae (baker's yeast) that express short hairpin RNA (shRNA) corresponding to mosquito target genes of interest. Following cultivation, which facilitates inexpensive propagation of shRNA, the yeast is inactivated and prepared in a ready-to-use dry tablet formulation that is fed to mosquito larvae. Ingestion of the yeast tablets results in effective larval target gene silencing. This technically straightforward and affordable technique may be applicable to a wide variety of mosquito species and potentially to other arthropods that feed on yeast.

Systems genetic analysis of inversion polymorphisms in the malaria mosquito Anopheles gambiae

Inversion polymorphisms in the African malaria vector Anopheles gambiae segregate along climatic gradients of aridity. Despite indirect evidence of their adaptive significance, little is known of the phenotypic targets of selection or the underlying genetic mechanisms. Here we adopt a systems genetics approach to explore the interaction of two inversions on opposite arms of chromosome 2 with gender, climatic conditions, and one another. We measure organismal traits and transcriptional profiles in 8-d-old adults of both sexes and four alternative homokaryotypic classes reared under two alternative climatic regimes. We show that karyotype strongly influences both organismal traits and transcriptional profiles but that the strength and direction of the effects depend upon complex interactions with gender and environmental conditions and between inversions on independent arms. Our data support the suppressed recombination model for the role of inversions in local adaptation, and-supported by transcriptional and physiological measurements following perturbation with the drug rapamycin-suggest that one mechanism underlying their adaptive role may be the maintenance of energy homeostasis.

Evolution of host preference in anthropophilic mosquitoes

BackgroundInsecticide-treated bed nets (ITNs) have played a large role in reducing the burden of malaria. There is concern however regarding the potential of the mass distributions and use of ITNs to select for insecticide and behavioural resistance in mosquito populations. A key feature of the vectorial capacity of the major sub-Saharan African malaria vector Anopheles gambiae sensu stricto (s.s.) is its tendency to feed almost exclusively on humans. Here, an evolutionary model is used to investigate the potential for ITNs to select for increased zoophily in this highly anthropophilic species and how this is influenced by ecological and operational conditions.ResultsThe evolution of a single trait, namely the tendency to accept cattle as hosts, is modelled in mosquito populations which initially only bite humans. Thus, the conditions under which a resource specialist would broaden its diet and become a generalist are investigated. The results indicate that in the absence of insecticide-treated nets, host specialization in mosquitoes is either driven toward human specialization (when humans are more abundant than alternative hosts), or displays evolutionary bistability. The latter implies that the evolutionary endpoint relies on the initial trait value of the population. Bed nets select for increased zoophily while in use. When ITNs are removed, whether or not the population reverts to anthropophagic or zoophagic behaviour depends on whether the intervention had been maintained sufficiently long to drive the population past the evolutionarily unstable point.ConclusionsThe use of ITNs is likely to select for an increase in the biting preference for cattle. Bed nets may thus alter the population composition of major vector species in a manner that has positive epidemiological ramifications. Whether populations are set on a trajectory toward increased zoophily following the cessation of intense bed net usage in an area depends on the composition of host communities as well as operational conditions. This has potential implications for bed net campaigns, particularly with an eye toward scaling down interventions following interruption of transmission. Further research on malaria mosquito feeding behaviour is warranted to explore the conditions under which such adaptive shifts may actually occur in the field.

A selective αvβ5 integrin antagonist hidden into the anophelin family protein cE5 from the malaria vector Anopheles gambiae

AbstractA RGD motif was identified in the N‐terminal region of cE5, a potent salivary thrombin inhibitor from the African malaria vector Anopheles gambiae. A peptide (APQ30) encompassing the first 30 amino acids residues of the protein and including the RGD tripeptide was tested in cell adhesion assays and found to inhibit αvβ3 and αvβ5 mediated adhesion. A shorter peptide (APQ16), strongly conserved among members of the A. gambiae species complex and including only the first 16 residues, retained adhesion inhibitory properties, however with enhanced specificity toward αvβ5. In addition, migration and invasion assays showed its capacity to inhibit the invasiveness of the malignant cell lines HepG2 and MDA‐MB231. Altogether our data point to APQ16 as a new promising candidate as theranostic agent.

Mosquito larvicidal activity of Cassia tora seed extract and its key anthraquinones aurantio-obtusin and obtusin

BackgroundThe edible and medicinal leguminous plant Cassia tora L. (Fabaceae) is known to possess insecticidal properties against a wide range of plant-feeding insects. However, the bioactivity of extracts of this plant and their constituents against vectors of medical importance has been largely unexplored. We investigated the mosquito larvicidal activity of the seed extract and its major anthraquinones against larvae of the African malaria vector Anopheles gambiae (s.s.).MethodsThird-fourth instar larval mortality was observed after 24, 48, 72 and 96 h of exposure to varying doses of the extracts, and two anthraquinones isolates identified using liquid chromatography- quadrupole time of flight mass spectrometry (LC-QtoF-MS). The mosquito larval mortality was evaluated relative to the natural insecticide azadirachtin.ResultsFractionation of the crude extract decreased mosquito larvicidal activity, however, larvicidal activity increased with increasing dose of the treatment and exposure time. The known anthraquinones aurantio-obtusin and obtusin were identified as key larvicidal compounds. Aurantio-obtusin and obtusin, exhibited similar toxicity to larvae of A. gambiae (s.s.) with LD50 values of 10 and 10.2 ppm, respectively. However, the two anthraquinones were four- and ~ six-fold less potent than that of the crude seed extract and azadirachtin, which had comparable LD50 values of 2.5 and 1.7 ppm, respectively.ConclusionBoth aurantio-obtusin and obtusin showed mosquito larvicidal activity which were comparable to their respective fractions although they were less potent relative to the crude extract and azadirachtin. Further studies need to be conducted on C. tora for its exploitation as a potential eco-friendly tool in mosquito larval source reduction.

Mapping insecticide resistance and characterization of resistance mechanisms in Anopheles arabiensis (Diptera: Culicidae) in Ethiopia

BackgroundThe emergence and spread of insecticide resistance in the major African malaria vectors Anopheles gambiae (s.s.) and An. arabiensis may compromise the current vector control interventions and threatens the global malaria control and elimination efforts.MethodsInsecticide resistance was monitored in several study sites in Ethiopia from 2013 to 2015 using papers impregnated with discriminating concentrations of DDT, deltamethrin, bendiocarb, propoxur, malathion, fenitrothion and pirimiphos-methyl, following the WHO insecticide susceptibility test procedure. Mosquitoes sampled from different localities for WHO bioassay were morphologically identified as An. gambiae (s.l.) using standard taxonomic keys. Samples were identified to species using species-specific polymerase chain reaction (PCR) and screened for the presence of target site mutations L1014F, L1014S and N1575Y in the voltage gated sodium channel (VGSC) gene and G119S in the acethylcholinesterase (AChE) gene using allele-specific PCR. Biochemical assays were performed to assess elevated levels of acetylcholinesterases, carboxylcholinesterases, glutathione-S-transferases (GSTs) and cytochrome P450s monooxygenases in wild populations of An. arabiensis, compared to the fully susceptible Sekoru An. arabiensis laboratory strain.ResultsPopulations of An. arabiensis were resistant to DDT and deltamethrin but were susceptible to fenitrothion in all the study sites. Reduced susceptibility to malathion, pirimiphos-methyl, propoxur and bendiocarb was observed in some of the study sites. Knockdown resistance (kdr L1014F) was detected in all mosquito populations with allele frequency ranging from 42 to 91%. Elevated levels of glutathione-S-transferases (GSTs) were detected in some of the mosquito populations. However, no elevated levels of monooxygenases and esterases were detected in any of the populations assessed.ConclusionsAnopheles arabiensis populations from all surveyed sites in Ethiopia exhibited resistance against DDT and pyrethroids. Moreover, some mosquito populations exhibited resistance to propoxur and possible resistance to bendiocarb. Target site mutation kdr L1014F was detected in all mosquito populations while elevated levels of glutathione-S-transferases (GSTs) was detected in some mosquito populations. The reduced susceptibility of An. arabiensis to propoxur and bendiocarb, which are currently used for indoor residual spraying (IRS) in Ethiopia, calls for continuous resistance monitoring, in order to plan and implement evidence based insecticide resistance management.

The transcription factor Maf-S regulates metabolic resistance to insecticides in the malaria vector Anopheles gambiae

BackgroundMalaria control in Africa is dependent upon the use insecticides but intensive use of a limited number of chemicals has led to resistance in mosquito populations. Increased production of enzymes that detoxify insecticides is one of the most potent resistance mechanisms. Several metabolic enzymes have been implicated in insecticide resistance but the processes controlling their expression have remained largely elusive.ResultsHere, we show that the transcription factor Maf-S regulates expression of multiple detoxification genes, including the key insecticide metabolisers CYP6M2 and GSTD1 in the African malaria vector Anopheles gambiae. Attenuation of this transcription factor through RNAi induced knockdown reduced transcript levels of these effectors and significantly increased mortality after exposure to the pyrethroid insecticides and DDT (permethrin: 9.2% to 19.2% (p = 0.015), deltamethrin: 3.9% to 21.6% (p = 0.036) and DDT: 1% to 11.7% (p = <0.01), whilst dramatically decreasing mortality induced by the organophosphate malathion (79.6% to 8.0% (p = <0.01)). Additional genes regulated by Maf-S were also identified providing new insight into the role of this transcription factor in insects.ConclusionMaf-S is a key regulator of detoxification genes in Anopheles mosquitoes. Disrupting this transcription factor has opposing effects on the mosquito’s response to different insecticide classes providing a mechanistic explanation to the negative cross resistance that has been reported between pyrethroids and organophosphates.

Functional analyses yield detailed insight into the mechanism of thrombin inhibition by the antihemostatic salivary protein cE5 from Anopheles gambiae

Saliva of blood-feeding arthropods carries several antihemostatic compounds whose physiological role is to facilitate successful acquisition of blood. The identification of novel natural anticoagulants and the understanding of their mechanism of action may offer opportunities for designing new antithrombotics disrupting blood clotting. We report here an in-depth structural and functional analysis of the anophelin family member cE5, a salivary protein from the major African malaria vector Anopheles gambiae that specifically, tightly, and quickly binds and inhibits thrombin. Using calorimetry, functional assays, and complementary structural techniques, we show that the central region of the protein, encompassing amino acids Asp-31-Arg-62, is the region mainly responsible for α-thrombin binding and inhibition. As previously reported for the Anopheles albimanus orthologue anophelin, cE5 binds both thrombin exosite I with segment Glu-35-Asp-47 and the catalytic site with the region Pro-49-Arg-56, which includes the highly conserved DPGR tetrapeptide. Moreover, the N-terminal Ala-1-Ser-30 region of cE5 (which includes an RGD tripeptide) and the additional C-terminal serine-rich Asn-63-Glu-82 region (absent in orthologues from anophelines of the New World species A. albimanus and Anopheles darlingi) also played some functionally relevant role. Indeed, we observed decreased thrombin binding and inhibitory properties even when using the central cE5 fragment (Asp-31-Arg-62) alone. In summary, these results shed additional light on the mechanism of thrombin binding and inhibition by this family of salivary anticoagulants from anopheline mosquitoes.

The evolutionary divergence of STAT transcription factor in different Anopheles species

Anopheles mosquito transmits Plasmodium, the malaria causing parasite. Different species of Anopheles mosquito dominate in a particular geographical location and are capable of transmitting specific strains of Plasmodium. It is important to understand the biology of different anophelines to control the parasite transmission. STAT is an evolutionary conserved transcription factor that regulates the parasite development in African malaria vector Anopheles gambiae. Unlike Drosophila and Aedes aegypti, where a single STAT gene plays an important role in immunity, An. gambiae contains one evolutionary conserved STAT-A and another retro-duplicated, introns-less STAT-B gene. To find out whether other species of Anopheles also have two STATs, the available genomic data of different anophelines were used to annotate their STATs through in silico analyses. Our results revealed that Indian malaria vector An. stephensi genome contains two STATs, AsSTAT-A and AsSTAT-B genes. These genes were cloned and confirmed by sequencing. Both AsSTATs were found to be expressed in different development stages of mosquito. However, the relative mRNA levels of evolutionary conserved AsSTAT-A gene were always higher than the retroduplicated AsSTAT-B gene. STAT pathway was activated upon Plasmodium berghei infection, indicated its role in immunity. Furthermore, comparative in silico analysis of eighteen Anopheles species revealed that five species: An. sinensis, An. albimanus, An. darlingi, An. dirus andAn. farauti do not contain STAT-B gene in their genome. Interestingly, thirteen species of the subgenus Anopheles and Cellia that contain both STATs were also mutually diverged. This consequence leads to sequence variability in some significant protein motifs within the STAT-B genes. Phylogenetic analyses indicated that an independent, lineage-specific duplication occurred in the subgenus Cellia after the diversification of series Neomyzomyia from its last common ancestor. In An. atroparvus (subgenus Anopheles), STAT gene underwent recent lineage-specific duplication and give rise to a highly similar STAT-B gene. This suggested that the genetic divergence in various Anopheles species might appeared due to their adaptations to the altered environmental conditions or pathogen encounters.

Delayed mortality effects cut the malaria transmission potential of insecticide-resistant mosquitoes

Malaria transmission has been substantially reduced across Africa through the distribution of long-lasting insecticidal nets (LLINs). However, the emergence of insecticide resistance within mosquito vectors risks jeopardizing the future efficacy of this control strategy. The severity of this threat is uncertain because the consequences of resistance for mosquito fitness are poorly understood: while resistant mosquitoes are no longer immediately killed upon contact with LLINs, their transmission potential may be curtailed because of longer-term fitness costs that persist beyond the first 24 h after exposure. Here, we used a Bayesian state-space model to quantify the immediate (within 24 h of exposure) and delayed (>24 h after exposure) impact of insecticides on daily survival and malaria transmission potential of moderately and highly resistant laboratory populations of the major African malaria vector Anopheles gambiae Contact with LLINs reduced the immediate survival of moderately and highly resistant An. gambiae strains by 60-100% and 3-61%, respectively, and delayed mortality impacts occurring beyond the first 24 h after exposure further reduced their overall life spans by nearly one-half. In total, insecticide exposure was predicted to reduce the lifetime malaria transmission potential of insecticide-resistant vectors by two-thirds, with delayed effects accounting for at least one-half of this reduction. The existence of substantial, previously unreported, delayed mortality effects within highly resistant malaria vectors following exposure to insecticides does not diminish the threat of growing resistance, but posits an explanation for the apparent paradox of continued LLIN effectiveness in the presence of high insecticide resistance.

Increased production of piRNAs from euchromatic clusters and genes in Anopheles gambiae compared with Drosophila melanogaster.

BackgroundSpecific genomic loci, termed Piwi-interacting RNA (piRNA) clusters, manufacture piRNAs that serve as guides for the inactivation of complementary transposable elements (TEs). The piRNA pathway has been accurately detailed in Drosophila melanogaster, while it remains poorly examined in other insects. This pathway is increasingly recognized as critical for germline development and reproduction. Understanding of the piRNA functions in mosquitoes could offer an opportunity for disease vector control by the reduction of their reproductive potential.ResultsTo analyze the similarities and differences in this pathway between Drosophila and mosquito, we performed an in-depth analysis of the genomic loci producing piRNAs and their targets in the African malaria vector Anopheles gambiae. We identified 187 piRNA clusters in the An. gambiae genome and 155 piRNA clusters in the D. melanogaster genome. We demonstrate that many more piRNA clusters in the mosquito compared with the fruit fly are uni-directionally transcribed and are located outside pericentromeric heterochromatin. About 11 % of the An. gambiae piRNA population map to gene transcripts. This is a noticeable increase compared with the ~6 % of the piRNA population mapped to genes in D. melanogaster. A subset of the piRNA-enriched genes in An. gambiae has functions related to reproduction and development. At least 24 and 65 % of the mapped piRNAs correspond to genomic TE sequences in An. gambiae and D. melanogaster, respectively. DNA transposons and non-LTR retrotransposons are more abundant in An. gambiae, while LTR retrotransposons are more abundant in D. melanogaster. Yet, piRNAs predominantly target LTR retrotransposons in both species, which may point to a distinct feature of these elements compared to the other classes of TEs concerning their silencing by the piRNA pathway.ConclusionsHere, we demonstrate that piRNA-producing loci have more ubiquitous distribution in the An. gambiae genome than in the genome of D. melanogaster. Also, protein-coding genes have an increased role in production of piRNAs in the germline of this mosquito. Genes involved in germline and embryonic development of An. gambiae generate a substantial portion of piRNAs, suggesting a role of the piRNA pathway in the epigenetic regulation of the reproductive processes in the African malaria vector.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0041-5) contains supplementary material, which is available to authorized users.

Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae.

Vector mosquitoes inflict more human suffering than any other organism-and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

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.

Inhibition of Anopheles gambiae Odorant Receptor Function by Mosquito Repellents

The identification of molecular targets of insect repellents has been a challenging task, with their effects on odorant receptors (ORs) remaining a debatable issue. Here, we describe a study on the effects of selected mosquito repellents, including the widely used repellent N,N-diethyl-meta-toluamide (DEET), on the function of specific ORs of the African malaria vector Anopheles gambiae. This study, which has been based on quantitative measurements of a Ca(2+)-activated photoprotein biosensor of recombinant OR function in an insect cell-based expression platform and a sequential compound addition protocol, revealed that heteromeric OR (ORx/Orco) function was susceptible to strong inhibition by all tested mosquito repellents except DEET. Moreover, our results demonstrated that the observed inhibition was due to efficient blocking of Orco (olfactory receptor coreceptor) function. This mechanism of repellent action, which is reported for the first time, is distinct from the mode of action of other characterized insect repellents including DEET.