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The current study aimed to screen the efficacy of F. succisae against two types of mosquitoes (Culex pipiens and Anopheles stephensi). The results revealed the effectiveness of gradual concentrations of fungal suspension on different stages of life cycles of two mosquitoes. The ratioof egg mortality for Culex pipiens and Anopheles stephensi is (36.72, 48.97%) respectively when using the higher concentration 3×106 Spore/ml after 24 hours and the ratio of LC50 of egg mortality (3.06×106, 2.778×106) Spore/ml for the two Culicidae, respectively. As for the sensitivity of larvae phases, the first phase was the highest sensitive one for An. stephensi Culicidae and it is more sensitive than Cx. pipiens with a mortality rate of (70, 67.5, 60, 57.5% and (75, 70, 65,60)% for the two Culicidae respectively after 120 hours and the use of high concentration 3×106 Spore/ml of F. succisae suspension and LC50 for the fourth phases mortality of the two Culicidae respectively after 120 hours were (9.184×105, 1.417×106, 1.423×106, 1.773×106) and (1.497×105, 3.115×105, 4.118×105, 1.243×106) Spore/ml. Whereas the ratio mortality amongthe pupae was (28.94, 33.33)% and (23, 68, 25.64)% for the two kinds of Culicidae respectively within 72 hours. LC50 has the range of (7.411×106, 7.373×106), (9.473×106, 9.371×106) Spore/ml. As for the adults, the ratio was (65, 67.5)%, (60, 65)% and (52.5, 55)%, (47.5, 55)% for both male and female mosquitos and both kinds respectively in the same circumstances, time duration and the same concentricity. LC50 was (4.18 ×105, 2.927 ×105) Spore/ml and (1.055 ×106, 4.43×105) Spore/ml and (2.315×106, 1.435×106) Spore/ml and (3.375×106, 2.042×106) Spore / ml. [ABSTRACT FROM AUTHOR]

Understanding the population structure and mechanisms of taxa diversification is important for organisms responsible for the transmission of human diseases. Two vectors of West Nile virus, Culex pipiens pipiens and Cx. p. molestus, exhibit epidemiologically important behavioral and physiological differences, but the whole-genome divergence between them was unexplored. The goal of this study is to better understand the level of genomic differentiation and population structures of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared the whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Principal Component, ADMIXTURE, and neighbor joining analyses of the nuclear genomes identified two major intercontinental, monophyletic clusters of Cx. p. pipiens and Cx. p. molestus. The level of genomic differentiation between the subspecies was uniform along chromosomes. The ADMIXTURE analysis determined signatures of admixture in Cx. p. pipens populations but not in Cx. p. molestus populations. Comparison of mitochondrial genomes among the specimens showed a paraphyletic origin of the major haplogroups between the subspecies but a monophyletic structure between the continents. Thus, our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units with monophyletic origin that have undergone incipient ecological speciation. [ABSTRACT FROM AUTHOR]

The introduction of exotic disease vectors into a new habitat can drastically change the local epidemiological situation. During 2012–2015, larvae and an adult of the yellow-fever mosquito, Aedes aegypti, were captured alive at two international airports serving the Greater Tokyo Area, Japan. Because this species does not naturally distribute in this country, those mosquitoes were considered to be introduced from overseas via air-transportation. To infer the places of origin of those mosquitoes, we genotyped the 12 microsatellite loci for which the most comprehensive population genetic reference is currently available. Although clustering by Bayesian and multivariate methods both suggested that all those mosquitoes captured at the airports in Japan belonged to the Asia/Pacific populations, they were not clustered into a single cluster. Moreover, there was variation in mitochondrial cytochrome oxidase I gene (CoxI) haplotypes among mosquitoes collected in different incidents of discovery which indicated the existence of multiple maternal origins. We conclude there is little evidence to support the overwintering of Ae. aegypti at the airports; nevertheless, special attention is still needed to prevent the invasion of this prominent arbovirus vector. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Aedes cantans and Ae. cinereus are Wolbachia infected in Eastern Europe. • The MLST haplotype of w AlbB is recombinant of A and B supergroups. • The genetic content of mosquitoes' Wolbachia is found among different host taxa especially in Lepidoptera. • The MLST content of w AlbA strain is linked with Wolbachia haplotypes from ants. Abstract Many mosquitoes harbour Wolbachia symbionts that could affect the biology of their host in different ways. Evolutionary relationships of mosquitoes' Wolbachia infection, geographical distribution and symbiont prevalence in many mosquito species are not yet clear. Here, we present the results of Wolbachia screening of 17 mosquito species of four genera— Aedes, Anopheles, Coquillettidia and Culex collected from five regions of Eastern Europe and the Caucasus in 2012–2016. Based on multilocus sequence typing (MLST) data previously published and generated in this study, we try to reveal genetic links between mosquitoes' and other hosts' Wolbachia. The Wolbachia symbionts are found in Culex pipiens , Aedes albopictus and Coquillettidia richiardii and for the first time in Aedes cinereus and Aedes cantans , which are important vectors of human pathogens. Phylogenetic analysis demonstrated multiple origins of infection in mosquitoes although the one-allele-criterion approach revealed links among B-supergroup mosquito Wolbachia with allele content of lepidopteran hosts. The MLST gene content of strain w AlbA from the A-supergroup is linked with different ant species. Several cases of intersupergroup recombinations were found. One of them occurred in the w AlbaB strain of Aedes albopictus , which contains the coxA allele of the A-supergroup, whereas other loci, including wsp , belong to supergroup B. Other cases are revealed for non-mosquito symbionts and they exemplified genetic exchanges of A, B and F supergroups. We conclude that modern Wolbachia diversity in mosquitoes and in many other insect taxa is a recent product of strain recombination and symbiont transfers. [ABSTRACT FROM AUTHOR]

Subterranean cavities serve as resting places and hibernation shelters for mosquitoes. In Europe, members of the genus Culex are often the most abundant insects on cave walls. Culex pipiens L., the common house mosquito, exists in two physically very similar, yet genetically and ecologically distinct biotypes (or forms, 'f.'), namely Cx. pipiens f. pipiens and Cx. pipiens f. molestus. Autogeny and stenogamy of the latter form have been interpreted as adaptations to underground habitats. The epigean occurrence of the two biotypes and their hybrids was recently examined in Eastern Austria, but the hypogean distribution of the Cx. pipiens complex and morphologically similar non-members such as Cx. torrentium is unknown. Considering the key role of Culex mosquitoes in the epidemiology of certain zoonotic pathogens, the general paucity of data on species composition and relative abundance in subterranean shelters appears unfortunate. For a first pertinent investigation in Austria, we collected mosquitoes in four eastern federal states. Based on analyses of the ACE2 gene and the CQ11 microsatellite locus, 150 female and three male mosquitoes of the genus Culex, two females of the genus Culiseta and a single female of the genus Anopheles were determined to species level or below. In our catches, Cx. pipiens f. pipiens exceeded the apparent abundance of the purportedly cave-adapted Cx. pipiens f. molestus many times over. Records of Cx. hortensis and Cx. territans, two species rarely collected in Austria, lead us to infer that underground habitats host a higher diversity of culicine mosquitoes than previously thought. [ABSTRACT FROM AUTHOR]

Background: Traditional vector control approaches such as source reduction and insecticide spraying have limited effect on reducing Aedes aegypti population. The endosymbiont Wolbachia is pointed as a promising tool to mitigate arbovirus transmission and has been deployed worldwide. Models predict a rapid increase on the frequency of Wolbachia-positive Aedes aegypti mosquitoes in local settings, supported by cytoplasmic incompatibility (CI) and high maternal transmission rate associated with the wMelBr strain. Methodology/principle findings: Wolbachia wMelBr strain was released for 20 consecutive weeks after receiving >87% approval of householders of the isolated community of Tubiacanga, Rio de Janeiro. wMelBr frequency plateued~40% during weeks 7–19, peaked 65% but dropped as releases stopped. A high (97.56%) maternal transmission was observed. Doubling releases and deploying mosquitoes with large wing length and low laboratory mortality produced no detectable effects on invasion trend. By investigating the lab colony maintenance procedures backwardly, pyrethroid resistant genotypes in wMelBr decreased from 68% to 3.5% after 17 generations. Therefore, we initially released susceptible mosquitoes in a local population highly resistant to pyrethroids which, associated with the over use of insecticides by householders, ended jeopardizing Wolbachia invasion. A new strain (wMelRio) was produced after backcrossing wMelBr females with males from field to introduce mostly pyrethroid resistance alleles. The new strain increased mosquito survival but produced relevant negative effects on Ae. aegypti fecundity traits, reducing egg clutche size and egg hatch. Despite the cost on fitness, wMelRio successful established where wMelBr failed, revealing that matching the local population genetics, especially insecticide resistance background, is critical to achieve invasion. Conclusions/significance: Local householders support was constantly high, reaching 90% backing on the second release (wMelRio strain). Notwithstanding the drought summer, the harsh temperature recorded (daily average above 30°C) did not seem to affect the expression of maternal transmission of wMel on a Brazilian background. Wolbachia deployment should match the insecticide resistance profile of the wild population to achieve invasion. Considering pyrethroid-resistance is a widely distributed phenotype in natural Ae. aegypti populations, future Wolbachia deployments must pay special attention in maintaining insecticide resistance in lab colonies for releases. [ABSTRACT FROM AUTHOR]

Pesticide resistance is normally associated with genetic changes, resulting in varied responses to insecticides between different populations. There is little evidence of resistance to plant allelochemicals; it is likely that their efficacy varies between genetically diverse populations, which may lead to the development of resistance in the future. This study evaluated the response of Anopheles gambiae (larvae and adults) from spatially different populations to acetone extracts of two botanicals, Piper guineense and Eugenia aromatica. Mosquito samples from 10 locations within Akure metropolis in Southwest Nigeria were tested for variation in susceptibility to the toxic effect of botanical extracts. The spatial distribution of the tolerance magnitude (T.M.) of the mosquito populations to the botanicals was also mapped. The populations of An. gambiae manifested significant differences in their level of tolerance to the botanicals. The centre of the metropolis was the hot spot of tolerance to the botanicals. There was a significant positive correlation between the adulticidal activities of both botanicals and initial knockdown. Hence, knockdown by these botanicals could be a predictor of their subsequent mortality. In revealing variation in response to botanical pesticides, our work has demonstrated that any future use of botanicals as alternative environmentally friendly vector control chemicals needs to be closely monitored to ensure that resistance does not develop. [ABSTRACT FROM AUTHOR]

Mosquitoes are widely despised for their exasperating buzzing and irritating bites, and more poignantly because, during blood-feeding, females may transmit pathogens that cause devastating diseases. However, the ability to transmit such viruses, filarial worms, or malaria parasites varies greatly amongst the ∼3500 recognised mosquito species. Applying omics technologies to sample this diversity and explore the biology underlying these variations is bringing increasingly greater resolution that enhances our understanding of mosquito evolution. Here we review the current status of mosquito omics, or 'mozomics', resources and recent advances in their applications to characterise mosquito biology and evolution, with a focus on the intersection of evolutionary and functional genomics to understand the putative links between gene and genome dynamism and mosquito diversity. Highlights Genomic sampling of mosquito diversity has greatly improved in recent years and looks set to take advantage of emerging technologies to explore even further. Evolutionary genomics analyses have unveiled dynamic patterns of gene and genome evolution likely linked to mosquito adaptability that will guide future research and control efforts. Functional genomics assays have helped to characterise biological roles of thousands of genes, albeit with condition-patchy and species-biased coverage that is now starting to be remedied. Comparative genomics approaches are increasingly being applied to contextualise and enhance the interpretation of results from multispecies studies with an evolutionary perspective. These trends mean that effective data sharing will be critical to facilitate future integrative meta-analyses and fully harness the benefits of combined evolutionary and functional analyses. [ABSTRACT FROM AUTHOR]

Diapause is an alternative life-history strategy that allows organisms to enter developmental arrest in anticipation of unfavorable conditions. Diapause is widespread among insects and plays a key role in enhancing overwinter survival as well as defining the seasonal and geographic distributions of populations. Next-generation sequencing has greatly advanced our understanding of the transcriptional basis for this crucial adaptation but less is known about the regulation of embryonic diapause physiology at the metabolite level. Here, we characterized the lipid and metabolite profiles of embryonic diapause in the Asian tiger mosquito, Aedes albopictus. We used an untargeted approach to capture the relative abundance of 250 lipids and 241 metabolites. We observed adjustments associated with increased energy storage, including an accumulation of lipids, the formation of larger lipid droplets and increased lipogenesis, as well as metabolite shifts suggesting reduced energy utilization.We also found changes in neuroregulatory- and insulin-associated metabolites with potential roles in diapause regulation. Finally, we detected a group of unidentified, diapause-specific metabolites which have physical properties similar to those of steroids/steroid derivatives and may be associated with the ecdysteroidal regulation of embryonic diapause in A. albopictus. Together, these results deepen our understanding of the metabolic regulation of embryonic diapause and identify key targets for future investigations. [ABSTRACT FROM AUTHOR]

Highlights • Two engineered underominance systems for mosquito control are investigated. • Male-only releases must be larger than bi-sex releases to trigger the gene drive. • Female-specific lethality averts more disease cases than bi-sex lethality. • Genetic lethality efficiency is less important than ambient transgene fitness cost. • Mating success and longevity of engineered insects strongly governs drive success. Abstract Underdominance gene drives are frequency-dependent drives that aim to spread a desired homozygote genotype within a population. When the desired homozygote is released above a threshold frequency, heterozygote fitness disadvantage acts to drive the desired trait to fixation. Underdominance drives have been proposed as a way to control vector-borne disease through population suppression and replacement in a spatially contained and reversible way—benefits that directly address potential safety concerns with gene drives. Here, ecological and epidemiological dynamics are coupled to a model of mosquito genetics to investigate theoretically the impact of different types of underdominance gene drive on disease prevalence. We model systems with two engineered alleles carried either on the same pair of chromosomes at the same locus or homozygously on different pairs at different loci, genetic lethality that affects both sexes or only females, and bi-sex or male-only releases. Further, the different genetic and ecological fitness costs that can arise from genetic modification and artificial rearing are investigated through their effect on the population threshold frequency that is required to trigger the drive mechanism. We show that male-only releases must be significantly larger than bi-sex releases to trigger the underdominance drive. In addition, we find that female-specific lethality averts a higher percentage of disease cases over a control period than does bi-sex lethality. Decreases in the genetic fitness of the engineered homozygotes can increase the underdominance threshold substantially, but we find that the mating success of transgenic mosquitoes with wild-type females (influenced by a lack of competitiveness or the evolution of behavioural resistance in the form of active female mate preference) and the longevity of artificially-reared mosquitoes are vitally important to the success chances of underdominance based gene drive control efforts. [ABSTRACT FROM AUTHOR]

Mosquito-borne diseases, such as malaria, dengue and chikungunya, cause morbidity and mortality around the world. Recent advances in gene drives have produced control methods that could theoretically modify all populations of a disease vector, from a single release, making whole species less able to transmit pathogens. This ability has caused both excitement, at the prospect of global eradication of mosquito-borne diseases, and concern around safeguards. Drive mechanisms that require individuals to be released at high frequency before genes will spread can therefore be desirable as they are potentially localised and reversible. These include underdominance-based strategies and use of the reproductive parasite Wolbachia. Here, we review recent advances in practical applications and mathematical analyses of these threshold-dependent gene drives with a focus on implementation in Aedes aegypti, highlighting their mechanisms and the role of fitness costs on introduction frequencies. Drawing on the parallels between these systems offers useful insights into practical, controlled application of localised drives, and allows us to assess the requirements needed for gene drive reversal. [ABSTRACT FROM AUTHOR]

Aedes albopictus has been described as one of the 100 worst invasive species in the world. This mosquito originated from southeastern Asia and currently has a widespread presence in every continent except Antarctica. The rapid global expansion of Ae. albopictus has increased public health concerns about arbovirus-related disease threats. Adaptation, adaption to novel areas is a biological challenge for invasive species, and the underlying processes can be studied at the molecular level. In this study, genetic analysis was performed using mitochondrial gene NADH dehydrogenase subunit 5 (ND5), based on both native and invasive populations. Altogether, 38 haplotypes were detected with H1 being the dominant and widely distributed in 21 countries. Both phylogenetic and network analyses supported the existence of five clades, with only clade I being involved in the subsequent global spread of Asian tiger mosquito. The other four clades (II, III, IV and V) were restricted to their original regions, which could be ancestral populations that had diverged from clade I in the early stages of evolution. Neutrality tests suggested that most of the populations had experienced recent expansion. Analysis of molecular variance and the population-pair statistic F ST revealed that most populations lacked genetic structure, while high variability was detected within populations. Multiple and independent human-mediated introductions may explain the present results. [ABSTRACT FROM AUTHOR]

Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti , and Hsu cells from Culex quinquefasciatus , for the presence of adventitious (i.e. exogenous) viruses. All four cell lines stained positive for double-stranded RNA, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems. [ABSTRACT FROM AUTHOR]

Background: Genomes rearrangements carry valuable information for phylogenetic inference or the elucidation of molecular mechanisms of adaptation. However, the detection of genome rearrangements is often hampered by current deficiencies in data and methods: Genomes obtained from short sequence reads have generally very fragmented assemblies, and comparing multiple gene orders generally leads to computationally intractable algorithmic questions. Results: We present a computational method, ADSEQ, which, by combining ancestral gene order reconstruction, comparative scaffolding and de novo scaffolding methods, overcomes these two caveats. ADSEQ provides simultaneously improved assemblies and ancestral genomes, with statistical supports on all local features. Compared to previous comparative methods, it runs in polynomial time, it samples solutions in a probabilistic space, and it can handle a significantly larger gene complement from the considered extant genomes, with complex histories including gene duplications and losses. We use ADSEQ to provide improved assemblies and a genome history made of duplications, losses, gene translocations, rearrangements, of 18 complete Anopheles genomes, including several important malaria vectors. We also provide additional support for a differentiated mode of evolution of the sex chromosome and of the autosomes in these mosquito genomes. Conclusions: We demonstrate the method's ability to improve extant assemblies accurately through a procedure simulating realistic assembly fragmentation. We study a debated issue regarding the phylogeny of the Gambiae complex group of Anopheles genomes in the light of the evolution of chromosomal rearrangements, suggesting that the phylogenetic signal they carry can differ from the phylogenetic signal carried by gene sequences, more prone to introgression. [ABSTRACT FROM AUTHOR]

The ability to manipulate the Anopheles gambiae genome and alter gene expression effectively and reproducibly is a prerequisite for functional genetic analysis and for the development of novel control strategies in this important disease vector. However, in vivo transgenic analysis in mosquitoes is limited by the lack of promoters active ubiquitously. To address this, we used the GAL4/UAS system to investigate the promoter of the An. gambiae Polyubiquitin-c (PUBc) gene and demonstrated its ability to drive expression in mosquito cell culture before incorporation into An. gambiae transgenic driver lines. To generate such lines, piggyBac-mediated insertion was used to identify genomic regions able to sustain widespread expression and to create φC31 docking lines at these permissive sites. Patterns of expression induced by PUBc-GAL4 drivers carrying single intergenic insertions were assessed by crossing with a novel responder UAS-mCD8:mCherry line that was created by φC31-mediated integration. Amongst the drivers created at single, unique chromosomal integration loci, two were isolated that induced differential expression levels in a similar multiple-tissue spatial pattern throughout the mosquito life cycle. This work expands the tools available for An. gambiae functional analysis by providing a novel promoter for investigating phenotypes resulting from widespread multi-tissue expression, as well as identifying and tagging genomic sites that sustain broad transcriptional activity. [ABSTRACT FROM AUTHOR]

Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age. Gene stability was inferred from obtained quantification data through three widely cited evaluation algorithms i.e. BestKeeper, geNorm, and NormFinder. No single gene showed a satisfactory degree of stability throughout all developmental stages. Therefore, we propose combinations of PGK and ILK for early embryos; RPL32 and RPS17 for late embryos, all four larval instars, and pupae samples; eEF1-γ with STK for adult males; eEF1-γ with RPS17 for non-blood fed females; and eEF1-γ with eIF2α for both blood-fed females and cell culture. The results from this study should be able to provide a more informed selection of normalizing genes during qPCR in Ae.albopictus. [ABSTRACT FROM AUTHOR]

Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Anopheles albimanus, A. coluzzii, and A. funestus. When guide RNAs (gRNAs) and Cas9 protein are injected at high concentration, germline mutations are common and usually biallelic, allowing for the rapid creation of stable mutant lines for reverse genetic analysis. Our protocol should enable researchers to dissect the molecular and cellular basis of anopheline traits critical to successful disease transmission, potentially exposing new targets for malaria control. [ABSTRACT FROM AUTHOR]

Abstract: Gene duplications occur at a high rate. Although most appear detrimental, some homogeneous duplications (identical gene copies) can be selected for beneficial increase in produced proteins. Heterogeneous duplications, which combine divergent alleles of a single locus, are seldom studied due to the paucity of empirical data. We investigated their role in an ongoing adaptive process at the ace‐1 locus in Culex pipiens mosquitoes. We assessed the worldwide diversity of the ace‐1 alleles (single‐copy, susceptible S and insecticide‐resistant R, and duplicated D that pair one S and one R copy), analysed their phylogeography and measured their fitness to understand their early dynamics using population genetics models. It provides a coherent and comprehensive evolutionary scenario. We show that D alleles are present in most resistant populations and display a higher diversity than R alleles (27 vs. 4). Most appear to result from independent unequal crossing‐overs between local single‐copy alleles, suggesting a recurrent process. Most duplicated alleles have a limited geographic distribution, probably resulting from their homozygous sublethality (HS phenotype). In addition, heterozygotes carrying different HS D alleles showed complementation, indicating different recessive lethal mutations. Due to mosaic insecticide control practices, balancing selection (overdominance) plays a key role in the early dynamics of heterogeneous duplicated alleles; it also favours a high local polymorphism of HS D alleles in natural populations (overdominance reinforced by complementation). Overall, our study shows that the evolutionary fate of heterogeneous duplications (and their long‐term role) depends on finely balanced selective pressures due to the environment and to their genomic structure. [ABSTRACT FROM AUTHOR]

Background: Anopheles (Kerteszia) cruzii is the primary vector of human and simian malarias in Brazilian regions covered by the Atlantic Rainforest. Previous studies found that An. cruzii presents high levels of behavioural, chromosomal and molecular polymorphisms, which led to the hypothesis that it may be a complex of cryptic species. Here, An. cruzii specimens were collected in five sites in South-East Brazil located at different altitudes on the inner and coastal slopes of two mountain ranges covered by Atlantic Rainforest, known as Serra do Mar and Serra da Mantiqueria. Partial sequences for two genes (Clock and cpr) were generated and compared with previously published sequences from Florianópolis (southern Brazil). Genetic diversity was analysed with estimates of population structure (FST) and haplotype phylogenetic trees in order to understand how many species of the complex may occur in this biome and how populations across the species distribution are related. Results: The sequences from specimens collected at sites located on the lower coastal slopes of Serra do Mar (Guapimirim, Tinguá and Sana) clustered together in the phylogenetic analysis, while the major haplotypes from sites located on higher altitude and at the continental side of the same mountains (Bocaina) clustered with those from Serra da Mantiqueira (Itatiaia), an inner mountain range. These two An. cruzii lineages showed statistically significant genetic differentiation and fixed characters, and have high FST values typical of between species comparisons. Finally, in Bocaina, where the two lineages occur in sympatry, we found deviations from Hardy-Weinberg equilibrium due to a deficit of heterozygotes, indicating partial reproductive isolation. These results strongly suggest that at least two distinct lineages of An. cruzii (provisorily named "Group 1" and "Group 2") occur in the mountains of South-East Brazil. Conclusions: At least two genetically distinct An. cruzii lineages occur in the Atlantic Forest covered mountains of South-East Brazil. The co-occurrence of distinct lineages of An. cruzii (possibly incipient species) in those mountains is an interesting biological phenomenon and may have important implications for malaria prevalence, Plasmodium transmission dynamics and control. [ABSTRACT FROM AUTHOR]

Background: Ionotropic glutamate receptors (iGluRs) are conserved ligand-gated ion channel receptors, and ionotropic receptors (IRs) were revealed as a new family of iGluRs. Their subdivision was unsettled, and their characteristics are little known. Anopheles sinensis is a major malaria vector in eastern Asia, and its genome was recently well sequenced and annotated. Methods: We identified iGluR genes in the An. sinensis genome, analyzed their characteristics including gene structure, genome distribution, domains and specific sites by bioinformatic methods, and deduced phylogenetic relationships of all iGluRs in An. sinensis, Anopheles gambiae and Drosophila melanogaster. Based on the characteristics and phylogenetics, we generated the classification of iGluRs, and comparatively analyzed the intron number and selective pressure of three iGluRs subdivisions, iGluR group, Antenna IR and Divergent IR subfamily. Results: A total of 56 iGluR genes were identified and named in the whole-genome of An. sinensis. These genes were located on 18 scaffolds, and 31 of them (29 being IRs) are distributed into 10 clusters that are suggested to form mainly from recent gene duplication. These iGluRs can be divided into four groups: NMDA, non-NMDA, Antenna IR and Divergent IR based on feature comparison and phylogenetic analysis. IR8a and IR25a were suggested to be monophyletic, named as Putative in the study, and moved from the Antenna subfamily in the IR family to the non-NMDA group as a sister of traditional non-NMDA. The generated iGluRs of genes (including NMDA and regenerated non-NMDA) are relatively conserved, and have a more complicated gene structure, smaller ω values and some specific functional sites. The iGluR genes in An. sinensis, An. gambiae and D. melanogaster have amino-terminal domain (ATD), ligand binding domain (LBD) and Lig_Chan domains, except for IR8a that only has the LBD and Lig_Chan domains. However, the new concept IR family of genes (including regenerated Antenna IR, and Divergent IR), especially for Divergent IR are more variable, have a simpler gene structure (intron loss phenomenon) and larger ω values, and lack specific functional sites. These IR genes have no other domains except for Antenna IRs that only have the Lig_Chan domain. Conclusions: This study provides a comprehensive information framework for iGluR genes in An. sinensis, and generated the classification of iGluRs by feature and bioinformatics analyses. The work lays the foundation for further functional study of these genes. [ABSTRACT FROM AUTHOR]

The known mosquito mitogenomes, containing a total of 34 species, which belong to five genera, were collected from GenBank, and the practicality and effectiveness of the variation in the complete mitochondrial DNA genome and portions of mitochondrial COI gene were assessed to reconstruct the phylogeny of mosquitoes. Phylogenetic trees were reconstructed on the basis of parsimony, maximum likelihood, and Bayesian (BI) methods. It is concluded that: (1) Both mitogenomes and COI gene support the monophly of following taxa: Subgenus Nyssorhynchus, Subgenus Cellia, Anopheles albitarsis complex, Anopheles gambiae complex, and Anopheles punctulatus group; (2) Genus Aedes is not monophyletic relative to Ochlerotatus vigilax; (3) The mitogenome results indicate a close relationship between Anopheles epiroticus and Anopheles gambiae complex, Anopheles dirus complex and Anopheles punctulatus group, respectively; (4) The Bayesian posterior probability (BPP) within phylogenetic tree reconstructed by mitogenomes is higher than COI tree. The results show that phylogenetic relationships reconstructed using the mitogenomes were more similar to those based on morphological data. [ABSTRACT FROM AUTHOR]

Explaining how and why reproductive isolation evolves and determining which forms of reproductive isolation have the largest impact on the process of population divergence are major goals in the study of speciation. By studying recent adaptive radiations in incompletely isolated taxa, it is possible to identify barriers involved at early divergence before other confounding barriers emerge after speciation is complete. Sibling species of the Anopheles gambiae complex offer opportunities to provide insights into speciation mechanisms. Here, we studied patterns of reproductive isolation among three taxa, Anopheles coluzzii, An. gambiae s.s. and Anopheles arabiensis, to compare its strength at different spatial scales, to dissect the relative contribution of pre- versus postmating isolation, and to infer the involvement of ecological divergence on hybridization. Because F1 hybrids are viable, fertile and not uncommon, understanding the dynamics of hybridization in this trio of major malaria vectors has important implications for how adaptations arise and spread across the group, and in planning studies of the safety and efficacy of gene drive as a means of malaria control. We first performed a systematic review and meta-analysis of published surveys reporting on hybrid prevalence, showing strong reproductive isolation at a continental scale despite geographically restricted exceptions. Second, we exploited our own extensive field data sets collected at a regional scale in two contrasting environmental settings, to assess: (i) levels of premating isolation; (ii) spatio/temporal and frequency-dependent dynamics of hybridization, (iii) relationship between reproductive isolation and ecological divergence and (iv) hybrid viability penalty. Results are in accordance with ecological speciation theory predicting a positive association between the strength of reproductive isolation and degree of ecological divergence, and indicate that postmating isolation does contribute to reproductive isolation among these species. Specifically, only postmating isolation was positively associated with ecological divergence, whereas premating isolation was correlated with phylogenetic distance. [ABSTRACT FROM AUTHOR]

The effective population size ( N e) is a fundamental parameter in population genetics that determines the relative strength of selection and random genetic drift, the effect of migration, levels of inbreeding, and linkage disequilibrium. In many cases where it has been estimated in animals, N e is on the order of 10%-20% of the census size. In this study, we use 12 microsatellite markers and 14,888 single nucleotide polymorphisms ( SNPs) to empirically estimate N e in Aedes aegypti, the major vector of yellow fever, dengue, chikungunya, and Zika viruses. We used the method of temporal sampling to estimate N e on a global dataset made up of 46 samples of Ae. aegypti that included multiple time points from 17 widely distributed geographic localities. Our N e estimates for Ae. aegypti fell within a broad range (~25-3,000) and averaged between 400 and 600 across all localities and time points sampled. Adult census size (Nc) estimates for this species range between one and five thousand, so the N e/ N c ratio is about the same as for most animals. These N e values are lower than estimates available for other insects and have important implications for the design of genetic control strategies to reduce the impact of this species of mosquito on human health. [ABSTRACT FROM AUTHOR]

Aedes aegypti is a model species in which the endogenous regulation of odor-mediated host seeking behavior has received some attention. Sugar feeding and host seeking in female A. aegypti are transiently inhibited following a blood meal. This inhibition is partially mediated by short neuropeptide F (sNPF). The paired antennal lobes (ALs), as the first processing centers for olfactory information, has been shown to play a significant role in the neuropeptidergic regulation of odor-mediated behaviors in insects. The expression of sNPF, along with other peptides in the ALs of A. aegypti, indicate parallel neuromodulatory systems that may affect olfactory processing. To identify neuropeptides involved in regulating the odor-mediated host seeking behavior in A. aegypti, we use a semi-quantitative neuropeptidomic analysis of single ALs to analyze changes in the levels of five individual neuropeptides in response to different feeding regimes. Our results show that the level of sNPF-2, allatostatin-A-5 (AstA-5) and neuropeptide-like precursor-1-5 (NPLP-1-5), but not of tachykinin-related-peptides and SIFamide (SIFa), in the AL of female mosquitoes, changes 24 h and 48 h post-blood meal, and are dependent on prior access to sugar. To assess the role of these neuropeptides in modulating host seeking behavior, when systemically injected individually, sNPF-2 and AstA-5 significantly reduced host seeking behavior. However, only the injection of the binary mixture of the two neuropeptides lead to a host seeking inhibition similar to that observed in blood fed females. We conclude that modulation of the odor mediated host seeking behavior of A. aegypti is likely regulated by a dual neuropeptidergic pathway acting in concert in the ALs. [ABSTRACT FROM AUTHOR]

Aedes albopictus is a highly invasive disease vector with an expanding worldwide distribution. Genetic assays using low to medium resolution markers have found little evidence of spatial genetic structure even at broad geographic scales, suggesting frequent passive movement along human transportation networks. Here we analysed genetic structure of Aedes albopictus collected from 12 sample sites in Guangzhou, China, using thousands of genome-wide single nucleotide polymorphisms (SNPs). We found evidence for passive gene flow, with distance from shipping terminals being the strongest predictor of genetic distance among mosquitoes. As further evidence of passive dispersal, we found multiple pairs of full-siblings distributed between two sample sites 3.7 km apart. After accounting for geographical variability, we also found evidence for isolation by distance, previously undetectable in Ae. albopictus. These findings demonstrate how large SNP datasets and spatially-explicit hypothesis testing can be used to decipher processes at finer geographic scales than formerly possible. Our approach can be used to help predict new invasion pathways of Ae. albopictus and to refine strategies for vector control that involve the transformation or suppression of mosquito populations. [ABSTRACT FROM AUTHOR]

Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes. [ABSTRACT FROM AUTHOR]

Mosquitoes (Diptera, Culicidae) occur in natural, urban, and peri-urban areas throughout the globe. Although the characteristics of urban and peri-urban habitats differ from those of natural habitats in many ways (e.g., fragmentation, pollution, noise, and light), few studies have examined the population connectivity of mosquitoes in urban areas. To obtain an overview of the species composition, we sampled mosquitoes from 23 sites in and around the city of Berlin, Germany. Of 23 species, five occurred in urban, 10 in peri-urban, and 20 in rural areas. Culex pipiens Linnaeus (Diptera: Culicidae) was the most common species collected (75% of all individuals) and occurred in all habitats. Hence this species was selected to be analysed at 10 microsatellite markers. There was no significant differentiation (FST = 0.016, P = 0.9) or isolation by distance (P = 0.06) among Cx. pipiens populations along an urban-rural gradient. The only significant differences detected were between Cx. pipiens and a laboratory population of Cx. pipiens f. molestus (pairwise FST = 0.114-0.148, P = 0.001 in all comparisons), suggesting that the markers chosen were suitable for the identification of population differentiation. Our results indicate that Cx. pipiens gene flow is widespread within and among urban, peri-urban, and rural areas and that urban habitat does not necessarily impede or enhance gene flow among these populations. [ABSTRACT FROM AUTHOR]

Background: There is emerging evidence that mosquito anti-Plasmodium immunity is multimodal with distinct mechanisms for killing malaria parasites at either the ookinete or oocyst stages. Early-phase responses targeting the ookinete require complement-like components circulating in the mosquito hemolymph that result in TEP1-mediated lysis or melanization. Additional responses mediated by the LL3 and STAT pathways limit oocyst survival through unknown mechanisms that require mosquito hemocyte function. While previous experiments argue that these mechanisms of parasite killing are independent, the transient nature of gene-silencing has rendered these experiments inconclusive. To address this issue, we outline experiments using a TALEN-derived TEP1 mutant line to examine the role of TEP1 in the Anopheles gambiae late-phase immune response. Results: Despite higher early oocyst numbers in the TEP1 mutant line, no differences in oocyst survival were observed when compared to control mosquitoes, suggesting that TEP1 function is independent of the late-phase immune response. To further validate this phenotype in the TEP1 mutant, oocyst survival was evaluated in the TEP1 mutant background by silencing either LL3 or STAT-A. Surprisingly, only STAT-A silenced mosquitoes were able to reconstitute the late-phase immune phenotype increasing oocyst survival in the TEP1 mutant line. Additional experiments highlight significant differences in LL3 expression in the M/S hybrid genetic background of the TEP1 mutant line compared to that of the Keele strain (M form) of An. gambiae, and demonstrate that LL3 is not required for granulocyte differentiation in the M/S hybrid G3 genetic background in response to malaria parasite infection. Conclusions: Through the combination of genetic experiments utilizing genetic mutants and reverse genetic approaches, new information has emerged regarding the mechanisms of mosquito late-phase immunity. When combined with previously published experiments, the body of evidence argues that Plasmodium oocyst survival is TEP1 independent, thus establishing that the mechanisms of early- and late-phase immunity are distinct. Moreover, we identify that the known components that mediate oocyst survival are susceptible to strain-specific differences depending on their genetic background and provide further evidence that the signals that promote hemocyte differentiation are required to limit oocyst survival. Together, this study provides new insights into the mechanisms of oocyst killing and the importance of genetics in shaping mosquito vector competence. [ABSTRACT FROM AUTHOR]

Information regarding the infection rate and genotype shifts for Japanese encephalitis virus (JEV) are important for JE vaccine application. In Jiangsu province, China, which is one of the provinces with a high prevalence of JE, JEV infection in swine and mosquitoes in certain cities has only been investigated in 2008-2009. Lianyungang City has one of the highest numbers of JE cases in Jiangsu province, and it has a high risk of JEV invasion via migrant birds. JEV infection in vectors in Lianyungang City, which has urban and rural parts, has not been investigated. In 2015-2016, we collected mosquitoes in cowsheds with ultraviolet light traps and detected JEV by reverse transcription-polymerase chain reaction (RT-PCR) method in Culex tritaeniorhynchus in Xintan village, Xuzhuang village, and Xiaogaozhuang village in Lianyungang City, China. The proportion of positive pools, which is calculated by the number of infected pools to the total number of pools tested in these villages, were 16.67%, 20.00%, and 4.17%, respectively, and the minimum infection rates, which is calculated as the ratio of the number of positive pools to the total number of mosquitoes tested, were 3.33‰, 4.00‰, and 0.83‰, respectively. Four JEV strains from positive samples were coded as LYG-1, LYG-2, LYG-3, and LYG-4, and the complete E genes were sequenced. Furthermore, the complete genome of LYG-3 was sequenced. The phylogenetic analysis indicated that all the four JEV strains belonged to genotype I-b. This is the first report of genotype I JEV strain in Jiangsu province. The high JEV infection rate in Culex tritaeniorhynchus indicated a high risk of JE reemergence in Lianyungang. The detected JEV strains may have similar antigenicity to that of SA14-14-2 according to molecular characters. These findings suggest that the vaccine can still be effective in Lianyungang. [ABSTRACT FROM AUTHOR]

This paper discusses the ethical issues associated with genetic modification of mosquito species that are human disease vectors. The Oxitec genetically changed mosquito--a variant of a species called Aedes aegypti, OX513A, is taken as an example. The benefits and risks are discussed, and questions need to be discussed in public prior to release of this gene drive system. [ABSTRACT FROM AUTHOR]

Mosquitoes are insects belonging to the order Diptera and family Culicidae. They are distributed worldwide and include approximately 3500 species, of which about 300 have medical and veterinary importance. The evolutionary success of mosquitoes, in both tropical and temperate regions, is due to the various survival strategies these insects have developed throughout their life histories. Of the many adaptive mechanisms, diapause and quiescence, two different types of dormancy, likely contribute to the establishment, maintenance and spread of natural mosquito populations. This review seeks to objectively and coherently describe the terms diapause and quiescence, which can be confused in the literature because the phenotypic effects of these mechanisms are often similar. [ABSTRACT FROM AUTHOR]

The combination of drug resistance, lack of an effective vaccine, and ongoing conflict and poverty means that malaria remains a major global health crisis. Understanding metabolic pathways at all parasite life stages is important in prioritising and targeting novel anti-parasitic compounds. The unusual heme synthesis pathway of the rodent malaria parasite, Plasmodium berghei, requires eight enzymes distributed across the mitochondrion, apicoplast and cytoplasm. Deletion of the ferrochelatase (FC) gene, the final enzyme in the pathway, confirms that heme synthesis is not essential in the red blood cell stages of the life cycle but is required to complete oocyst development in mosquitoes. The lethality of FC deletions in the mosquito stage makes it difficult to study the impact of these mutations in the subsequent liver stage. To overcome this, we combined locus-specific fluorophore expression with a genetic complementation approach to generate viable, heterozygous oocysts able to produce a mix of FC expressing and FC deficient sporozoites. These sporozoites show normal motility and can invade liver cells, where FC deficient parasites can be distinguished by fluorescence and phenotyped. Parasites lacking FC exhibit a severe growth defect within liver cells, with development failure detectable in the early to mid stages of liver development in vitro. FC deficient parasites could not complete liver stage development in vitro nor infect naïve mice, confirming liver stage arrest. These results validate the heme pathway as a potential target for prophylactic drugs targeting liver stage parasites. In addition, we demonstrate that our simple genetic approach can extend the phenotyping window beyond the insect stages, opening considerable scope for straightforward reverse genetic analysis of genes that are dispensable in blood stages but essential for completing mosquito development. [ABSTRACT FROM AUTHOR]

Background: Genus Anopheles is a major mosquito group of interest in Sri Lanka as it includes vectors of malaria and its members exist as species complexes. Taxonomy of the group is mainly based on morphological features, which are not conclusive and can be easily erased while handling the specimens. A combined effort, using morphology and DNA barcoding (using the markers cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) region, was made during the present study to recognize anophelines collected from eight districts of Sri Lanka for the first time. Methods: Cytochrome c oxidase subunit I and ITS2 regions of morphologically identified anopheline mosquitoes from Sri Lanka were sequenced. These sequences together with GenBank sequences were used in phylogenetic tree construction and molecular characterization of mosquitoes. Results: According to morphological identification, the field-collected adult mosquitoes belonged to 15 species, i.e., Anopheles aconitus, Anopheles annularis, Anopheles barbirostris, Anopheles culicifacies, Anopheles jamesii, Anopheles karwari, Anopheles maculatus, Anopheles nigerrimus, Anopheles pallidus, Anopheles peditaeniatus, Anopheles pseudojamesi, Anopheles subpictus, Anopheles tessellatus, Anopheles vagus, and Anopheles varuna. However, analysis of 123 COI sequences (445 bp) (16 clades supported by strong bootstrap value in the neighbour joining tree and inter-specific distances of >3%) showed that there are 16 distinct species. Identity of the morphologically identified species, except An. subpictus, was comparable with the DNA barcoding results. COI sequence analysis showed that morphologically identified An. subpictus is composed of two genetic entities: An. subpictus species A and species B (inter-specific K2P distance 0.128). All the four haplotypes of An. culicifacies discovered during the present study belonged to a single species. ITS2 sequences (542 bp) were obtained for all the species except for An. barbirostris, An. subpictus species B, An. tessellatus, and An. varuna. Each of these sequences was represented by a single species-specific haplotype. Conclusions: The present study reflects the importance and feasibility of COI and ITS2 genetic markers in identifying anophelines and their sibling species, and the significance of integrated systematic approach in mosquito taxonomy. Wide distribution of malaria vectors in the country perhaps indicates the potential for re-emergence of malaria in the country. [ABSTRACT FROM AUTHOR]

Background: Phenol oxidases (POs) catalyze the oxidation of dopa and dopamine to melanin, which is crucial for cuticle formation and innate immune maintenance in insects. Although, Laccase 2, a member of the PO family, has been reported to be a requirement for melanin-mediated cuticle tanning in the development stages of some insects, whether it participates in cuticle construction and other physiological processes during the metamorphosis of mosquito pupae is unclear. Methods: The association between the phenotype and the expression profile of Anopheles sinensis Laccase 2 (AsLac2) was assessed from pupation to adult eclosion. Individuals showing an expression deficiency of AsLac2 that was produced by RNAi and their phenotypic defects and physiological characterizations were compared in detail with the controls. Results: During the dominant expression period, knockdown of AsLac2 in pupae caused the cuticle to be unpigmented, and produced thin and very soft cuticles, which further impeded the eclosion rate of adults as well as their fitness. Moreover, melanization immune responses in the pupae were sharply decreased, leading to poor resistance to microorganism infection. Both the high conservation among Laccase 2 homologs and a very similar genomic synteny of the neighborhood in Anopheles genus implies a conservative function in the pupal stage. Conclusions: To our knowledge, this is the first study to report the serious phenotypic defects in mosquito pupae caused by the dysfunction of Laccase 2. Our findings strongly suggest that Laccase 2 is crucial for Anopheles cuticle construction and melanization immune responses to pathogen infections during pupal metamorphosis. This irreplaceability provides valuable information on the application of Lacccase 2 and/or other key genes in the melanin metabolism pathway for developing mosquito control strategies. [ABSTRACT FROM AUTHOR]

Testes-biased genes evolve rapidly and are important in the establishment, solidification, and maintenance of reproductive isolation between incipient species. The Anopheles gambiae complex, a group of at least eight isomorphic mosquito species endemic to Sub-Saharan Africa, is an excellent system to explore the evolution of testes-biased genes. Within this group, the testes are an important tissue in the diversification process because hybridization between species results in sterile hybrid males, but fully fertile females. We conducted RNA sequencing of A. gambiae and A. merus carcass and testes to explore tissueand species-specific patterns of gene expression. Our data provides support for transcriptional repression of X-linked genes in the male germline, which likely drives demasculinization of the X chromosome. Testesbiased genes predominately function in cellular differentiation and show a number of interesting patterns indicative of their rapid evolution, including elevated dN/dS values, low evolutionary conservation, poor annotation in existing reference genomes, and a high likelihood of differential expression between species. [ABSTRACT FROM AUTHOR]

Background: Opsins are light sensitive receptors associated with visual processes. Insects typically possess opsins that are stimulated by ultraviolet, short and long wavelength (LW) radiation. Six putative LW-sensitive opsins predicted in the yellow fever mosquito, Aedes aegypti and malaria mosquito, Anopheles gambiae, and eight in the southern house mosquito, Culex quinquefasciatus, suggest gene expansion in the Family Culicidae (mosquitoes) relative to other insects. Here we report the first detailed molecular and evolutionary analyses of LW opsins in three mosquito vectors, with a goal to understanding the molecular basis of opsin-mediated visual processes that could be exploited for mosquito control. Results: Time of divergence estimates suggest that the mosquito LW opsins originated from 18 or 19 duplication events between 166.9/197.5 to 1.07/0.94 million years ago (MY) and that these likely occurred following the predicted divergence of the lineages Anophelinae and Culicinae 145-226 MY. Fitmodel analyses identified nine amino acid residues in the LW opsins that may be under positive selection. Of these, eight amino acids occur in the N and C termini and are shared among all three species, and one residue in TMIII was unique to culicine species. Alignment of 5' non-coding regions revealed potential Conserved Non-coding Sequences (CNS) and transcription factor binding sites (TFBS) in seven pairs of LW opsin paralogs. Conclusions: Our analyses suggest opsin gene duplication and residues possibly associated with spectral tuning of LW-sensitive photoreceptors. We explore two mechanisms - positive selection and differential expression mediated by regulatory units in CNS - that may have contributed to the retention of LW opsin genes in Culicinae and Anophelinae. We discuss the evolution of mosquito LW opsins in the context of major Earth events and possible adaptation of mosquitoes to LW-dominated photo environments, and implications for mosquito control strategies based on disrupting vision-mediated behaviors. [ABSTRACT FROM AUTHOR]

Insecticide resistance in mosquito populations threatens recent successes in malaria prevention. Elucidating patterns of genetic structure in malaria vectors to predict the speed and direction of the spread of resistance is essential to get ahead of the ‘resistance curve’ and to avert a public health catastrophe. Here, applying a combination of microsatellite analysis, whole genome sequencing and targeted sequencing of a resistance locus, we elucidated the continent-wide population structure of a major African malaria vector, Anopheles funestus. We identified a major selective sweep in a genomic region controlling cytochrome P450-based metabolic resistance conferring high resistance to pyrethroids. This selective sweep occurred since 2002, likely as a direct consequence of scaled up vector control as revealed by whole genome and fine-scale sequencing of pre- and post-intervention populations. Fine-scaled analysis of the pyrethroid resistance locus revealed that a resistance-associated allele of the cytochrome P450 monooxygenase CYP6P9a has swept through southern Africa to near fixation, in contrast to high polymorphism levels before interventions, conferring high levels of pyrethroid resistance linked to control failure. Population structure analysis revealed a barrier to gene flow between southern Africa and other areas, which may prevent or slow the spread of the southern mechanism of pyrethroid resistance to other regions. By identifying a genetic signature of pyrethroid-based interventions, we have demonstrated the intense selective pressure that control interventions exert on mosquito populations. If this level of selection and spread of resistance continues unabated, our ability to control malaria with current interventions will be compromised. [ABSTRACT FROM AUTHOR]

Mosquitoes host communities of microbes in their digestive tract that consist primarily of bacteria. We previously reported that Aedes aegypti larvae colonized by a native community of bacteria and gnotobiotic larvae colonized by only Escherichia coli develop very similarly into adults, whereas axenic larvae never molt and die as first instars. In this study, we extended these findings by first comparing the growth and abundance of bacteria in conventional, gnotobiotic, and axenic larvae during the first instar. Results showed that conventional and gnotobiotic larvae exhibited no differences in growth, timing of molting, or number of bacteria in their digestive tract. Axenic larvae in contrast grew minimally and never achieved the critical size associated with molting by conventional and gnotobiotic larvae. In the second part of the study we compared patterns of gene expression in conventional, gnotobiotic and axenic larvae by conducting an RNAseq analysis of gut and nongut tissues (carcass) at 22 h post-hatching. Approximately 12% of Ae. aegypti transcripts were differentially expressed in axenic versus conventional or gnotobiotic larvae. However, this profile consisted primarily of transcripts in seven categories that included the down-regulation of select peptidases in the gut and up-regulation of several genes in the gut and carcass with roles in amino acid transport, hormonal signaling, and metabolism. Overall, our results indicate that axenic larvae exhibit alterations in gene expression consistent with defects in acquisition and assimilation of nutrients required for growth. [ABSTRACT FROM AUTHOR]

The genome of the Neotropical malaria vector Anopheles albimanus was sequenced as part of the 16 Anopheles Genomes Project published in 2015. The draft assembly of this species consisted of 204 scaffolds with an N50 scaffold size of 18.1 Mb and a total assembly size of 170.5 Mb. It was among the smallest genomes with the longest scaffolds in the 16 Anopheles species cluster, making An. albimanus the logical choice for anchoring the genome assembly to chromosomes. In this study, we developed a high-resolution cytogenetic photomap with completely straightened polytene chromosomes from the salivary glands of the mosquito larvae. Based on this photomap, we constructed a chromosome-based genome assembly using fluorescent in situ hybridization of PCR-amplified DNA probes. Our physical mapping, assisted by an ortholog-based bioinformatics approach, identified and corrected nine misassemblies in five large genomic scaffolds. Misassemblies mostly occurred in junctions between contigs. Our comparative analysis of scaffolds with the An. gambiae genome detected multiple genetic exchanges between pericentromeric regions of chromosomal arms caused by partial-arm translocations. The finalmap consists of 40 ordered genomic scaffolds and corrected fragments of misassembled scaffolds. The An. albimanus physical map comprises 98.2% of the total genome assembly and represents the most complete genome map among mosquito species. This study demonstrates that physical mapping is a powerful tool for correcting errors in draft genome assemblies and for creating chromosome-anchored reference genomes. [ABSTRACT FROM AUTHOR]

Mosquitoes pose a major threat to human health as they spread many deadly diseases like malaria, dengue, chikungunya, filariasis, Japanese encephalitis and Zika. Identification and use of novel molecular tools are essential to combat the spread of vector borne diseases. Genome editing tools have been used for the precise alterations of the gene of interest for producing the desirable trait in mosquitoes. Deletion of functional genes or insertion of toxic genes in vector mosquitoes will produce either knock-out or knock-in mutants that will check the spread of vector-borne diseases. Presently, three types of genome editing tools viz., zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) are widely used for the editing of the genomes of diverse organisms. These tools are also applied in vector mosquitoes to control the spread of vector-borne diseases. A few studies have been carried out on genome editing to control the diseases spread by vector mosquitoes and more studies need to be performed with the utilization of more recently invented tools like CRISPR/Cas9 to combat the spread of deadly diseases by vector mosquitoes. The high specificity and flexibility of CRISPR/Cas9 system may offer possibilities for novel genome editing for the control of important diseases spread by vector mosquitoes. In this review, we present the current status of genome editing research on vector mosquitoes and also discuss the future applications of vector mosquito genome editing to control the spread of vectorborne diseases. [ABSTRACT FROM AUTHOR]

Mosquitoes are insects of interest because several species vector disease-causing pathogens to humans and other vertebrates. We previously reported that mosquitoes from long-term laboratory cultures require living bacteria in their gut to develop, but development does not depend on particular species of bacteria. Here, we focused on three distinct but interrelated areas of study to better understand the role of bacteria in mosquito development by studying field and laboratory populations of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from the southeastern United States. Sequence analysis of bacterial 16S rRNA gene amplicons showed that bacterial community composition differed substantially in larvae from different collection sites, whereas larvae from the same site shared similarities. Although previously unknown to be infected by Wolbachia, results also indicated that Ae. aegypti from one field site hosted a dual infection. Regardless of collection site or factors like Wolbachia infection, however, each mosquito species required living bacteria in their digestive tract to develop. Results also identified several concerns in using antibiotics to eliminate the bacterial community in larvae in order to study its developmental consequences. Altogether, our results indicate that several mosquito species require living bacteria for development. We also hypothesize these species do not rely on particular bacteria because larvae do not reliably encounter the same bacteria in the aquatic habitats they develop in. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) is an RNA virus showing a high degree of genetic variation as a consequence of its proofreading inability. This variation plays an important role in virus evolution and pathogenesis. Although levels of within-host genetic variation are similar following equilibrium, variation among different hosts is frequently different. To identify dengue quasispecies present among two hosts, we collected patient samples from six acute DENV cases and two pools of Aedes aegypti mosquitoes and analyzed the genetic variation of regions of the viral envelope gene. Among human and mosquito samples, we found three major clusters originating from two subpopulations. Although several shared lineages were observed in the two hosts, only one lineage showing evidence of neutral selection was observed among two hosts. Taken together, our data provide evidence for the existence of a DENV quasispecies, with less genetic variation observed in mosquitoes than humans and with circulating lineages found in both host types. [ABSTRACT FROM AUTHOR]

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co-occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub-Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans-Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations. [ABSTRACT FROM AUTHOR]

Alpha esterase is one of the major contributing genes of carboxylesterase family involved in insecticide resistance mechanism. The alpha esterase gene of Anopheles gambiae was blasted against the 20 mosquito genome of VectorBase database that resulted in 254 gene hits. The resulted sequences were validated and 163 genes were obtained. The number of genes in the 20 mosquito vector in order as follows: Culex quinquefasciatus > Anopheles gambiae > An. sinensis (china strain) > An. Melas > Aedes aegypti > An. farauti > An. atroparvus > An. darlingi > An. Coluzii > Ae. albopictus > An. Dirus > An. christyi > An. quadriannulatus > An. stephensi (SDA 500 strain) > An. arabiensis > An. Funestus > An. Epiroticus > An. minimus > An. Albimanus > An. maculatus. This study concluded the presence of alpha esterase genes in the mosquito vector and showed species specific expansion of genes. Organization of phylogenetic tree showed that the alpha esterase genes are diverged among the species. This result gives the information on the organisation of alpha esterase genes and will further provide the possible ways for suggesting strategies for insecticide resistance management using insecticides. [ABSTRACT FROM AUTHOR]

Background: Around half of the global population is living in areas at risk of malaria infection. Plasmodium vivax malaria has become increasingly prevalent and responsible for a high health and socio-economic burden in Ethiopia. The availability of gametocyte carriers and mosquito species susceptible to P. vivax infection are vital for malaria transmission. Determining the susceptibility of vector species to parasite infection in space and time is important in vector control programs. This study assesses the susceptibility of Anopheles arabiensis, An. pharoensis and An. coustani group to Plasmodium vivax infection in Ethiopia. Methods: Larvae of An. arabiensis, An. pharoensis and An. coustani group were collected from an array of breeding sites and reared to adult under controlled conditions. Batches of adult female mosquitoes of the three species were allowed to feed in parallel on the same infected blood with gametocytes drawn from Plasmodium vivax infected patients by Direct Membrane Feeding Assays (DMFA). Fed mosquitoes were kept in an incubator under controlled laboratory conditions. Seven days after each feeding assay, mosquitoes were dissected for midgut oocyst microscopy and enumeration. Data were analysed using R statistical software package version 3.1.0. Results: Over all, 8,139 adult female mosquitoes were exposed to P. vivax infection. Of the exposed mosquitoes 16. 64 % (95 % CI: 1,354-8,139) were properly fed and survived until dissection. The infection rate in An. arabiensis and An. pharoensis was 31.72 % (95 % CI: 28.35-35.08) and 28.80 % (95 % CI: 25.31-32.28), respectively. The intensity of infection for An. arabiensis and An. pharoensis was 2.5 (95 % CI: 1.9-3.2) and 1.4 (95 % CI: 1.1-1.8), respectively. Gametocyte density was positively correlated to infection rate and intensity of infection in An. arabiensis as well as An. pharoensis. No An. coustani group mosquitoes were found infected, though almost four hundred mosquitoes were successfully fed and dissected. All groups received blood from the same infected blood source containing gametocytes in parallel. There was no significant difference in susceptibility rates between An. arabiensis and An. pharoensis (P = 0.215). Conclusions: Anopheles arabiensis and An. pharoensis showed similar susceptibility to P. vivax infection. However, An. coustani group was not permissive for the development of P. vivax parasites. [ABSTRACT FROM AUTHOR]

Nucleotide sequences of the circadian rhythm genes, period and timeless, were studied for the first time in mosquitoes Culex pipiens Linnaeus, 1758. In this work we evaluated variations of the studied genome fragments for the two forms of C. pipiens (forma "pipiens" - mosquitoes common for aboveground habitats, forma "molestus" - underground mosquitoes). We compared C. pipiens from Russia with transatlantic C. pipiens and subtropical Culex quinquefasciatus Say, 1823. Our results show that intraspecies variability is higher for the gene period than for the gene timeless. The revealed substitutions in nucleotide sequences and especially in amino acid sequences grouped the individuals of the two forms into distinct clusters with high significance. The detected fixed amino acid substitutions may appear essential for functioning of the circadian rhythm proteins in C. pipiens, and may be correlated with adaptations of the taxa within the group C. pipiens. Our results suggest that natural selection favors fixed mutations and the decrease in diversity of the genes period and timeless in mosquitoes of the C. pipiens f. "molestus" compared with the C. pipiens f. "pipiens", is probably correlated with adaptive features of C. pipiens f. "molestus". The studied genome regions may be considered as promising molecular-genetic markers for identification, population and phylogenetic analysis of similar species and forms of the Culex pipiens complex. [ABSTRACT FROM AUTHOR]

Background: Determination of the interactions between hematophagous arthropods and their hosts is a necessary component to understanding the transmission dynamics of arthropod-vectored pathogens. Current molecular methods to identify hosts of blood-fed arthropods require the preservation of host DNA to serve as an amplification template. During transportation to the laboratory and storage prior to molecular analysis, genetic samples need to be protected from nucleases, and the degradation effects of hydrolysis, oxidation and radiation. Preservation of host DNA contained in field-collected blood-fed specimens has an additional caveat: suspension of the degradative effects of arthropod digestion on host DNA. Unless effective preservation methods are implemented promptly after blood-fed specimens are collected, host DNA will continue to degrade. Preservation methods vary in their efficacy, and need to be selected based on the logistical constraints of the research program. Methods: We compared four preservation methods (cold storage at -20 °C, desiccation, ethanol storage of intact mosquito specimens and crushed specimens on filter paper) for field storage of host DNA from blood-fed mosquitoes across a range of storage and post-feeding time periods. The efficacy of these techniques in maintaining host DNA integrity was evaluated using a polymerase chain reaction (PCR) to detect the presence of a sufficient concentration of intact host DNA templates for blood meal analysis. We applied a logistic regression model to assess the effects of preservation method, storage time and post-feeding time on the binomial response variable, amplification success. Results: Preservation method, storage time and post-feeding time all significantly impacted PCR amplification success. Filter papers and, to a lesser extent, 95 % ethanol, were the most effective methods for the maintenance of host DNA templates. Amplification success of host DNA preserved in cold storage at -20 °C and desiccation was poor. Conclusions: Our data suggest that, of the methods tested, host DNA template integrity was most stable when blood meals were preserved using filter papers. Filter paper preservation is effective over short- and long-term storage, while ethanol preservation is only suitable for short-term storage. Cold storage at -20 °C, and desiccation of blood meal specimens, even for short time periods, should be avoided. [ABSTRACT FROM AUTHOR]