Expanding the diversity of the IS630-Tc1-mariner superfamily: discovery of a unique DD37E transposon and reclassification of the DD37D and DD39D transposons.

A novel transposon named ITmD37E was discovered in a wide range of mosquito species. Sequence analysis of multiple copies in three Aedes species showed similar terminal inverted repeats and common putative TA target site duplications. The ITmD37E transposases contain a conserved DD37E catalytic motif, which is unique among reported transposons of the IS630-Tc1-mariner superfamily. Sequence comparisons and phylogenetic analyses suggest that ITmD37E forms a novel family distinct from the widely distributed Tc1 (DD34E), mariner (DD34D), and pogo (DDxD) families in the IS630-Tc1-mariner superfamily. The inclusion in the phylogenetic analysis of recently reported transposons and transposons uncovered in our database survey provided revisions to previous classifications and identified two additional families, ITmD37D and ITmD39D, which contain DD37D and DD39D motifs, respectively. The above expansion and reorganization may open the doors to the discovery of related transposons in a broad range of organisms and help illustrate the evolution and structure-function relationships among these distinct transposases in the IS630-Tc1-mariner superfamily. The presence of intact open reading frames and highly similar copies in some of the newly characterized transposons suggests recent transposition. Studies of these novel families may add to the limited repertoire of transgenesis and mutagenesis tools for a wide range of organisms, including the medically important mosquitoes.     

Next-generation sequencing reveals recent horizontal transfer of a DNA transposon between divergent mosquitoes

Horizontal transfer of genetic material between complex organisms often involves transposable elements (TEs). For example, a DNA transposon mariner has been shown to undergo horizontal transfer between different orders of insects and between different phyla of animals. Here we report the discovery and characterization of an ITmD37D transposon, MJ1, in Anopheles sinensis. We show that some MJ1 elements in Aedes aegypti and An. sinensis contain intact open reading frames and share nearly 99% nucleotide identity over the entire transposon, which is unexpectedly high given that these two genera had diverged 145-200 million years ago. Chromosomal hybridization and TE-display showed that MJ1 copy number is low in An. sinensis. Among 24 mosquito species surveyed, MJ1 is only found in Ae. aegypti and the hyrcanus group of anopheline mosquitoes to which An. sinensis belongs. Phylogenetic analysis is consistent with horizontal transfer and provides the basis for inference of its timing and direction. Although report of horizontal transfer of DNA transposons between higher eukaryotes is accumulating, our analysis is one of a small number of cases in which horizontal transfer of nearly identical TEs among highly divergent species has been thoroughly investigated and strongly supported. Horizontal transfer involving mosquitoes is of particular interest because there are ongoing investigations of the possibility of spreading pathogen-resistant genes into mosquito populations to control malaria and other infectious diseases. The initial indication of horizontal transfer of MJ1 came from comparisons between a 0.4x coverage An. sinensis 454 sequence database and available TEs in mosquito genomes. Therefore we have shown that it is feasible to use low coverage sequencing to systematically uncover horizontal transfer events. Expanding such efforts across a wide range of species will generate novel insights into the relative frequency of horizontal transfer of different TEs and provide the evolutionary context of these lateral transfer events.     

Study of sequence variation of dengue type 3 virus in naturally infected mosquitoes and human hosts: implications for transmission and evolution

Dengue virus is an arbovirus that replicates alternately in the mosquito vector and human host. We investigated sequences of dengue type 3 virus in naturally infected Aedes aegypti mosquitoes and in eight patients from the same outbreak and reported that the extent of sequence variation seen with the mosquitoes was generally lower than that seen with the patients (mean diversity, 0.21 versus 0.38% and 0.09 versus 0.23% for the envelope [E] and capsid [C] genes, respectively). This was further verified with five experimentally infected mosquitoes (mean diversity, 0.09 and 0.10% for the E and C genes, respectively). Examination of the quasispecies structures of the E sequences of the mosquitoes and of the patients revealed that the sequences of the major variants were the same, suggesting that the major variant was transmitted. These findings support our hypothesis that mosquitoes contribute to the evolutionary conservation of dengue virus by maintaining a more homogenous viral population and a dominant variant during transmission.     

Broad surveys of DNA viral diversity obtained through viral metagenomics of mosquitoes

Viruses are the most abundant and diverse genetic entities on Earth; however, broad surveys of viral diversity are hindered by the lack of a universal assay for viruses and the inability to sample a sufficient number of individual hosts. This study utilized vector-enabled metagenomics (VEM) to provide a snapshot of the diversity of DNA viruses present in three mosquito samples from San Diego, California. The majority of the sequences were novel, suggesting that the viral community in mosquitoes, as well as the animal and plant hosts they feed on, is highly diverse and largely uncharacterized. Each mosquito sample contained a distinct viral community. The mosquito viromes contained sequences related to a broad range of animal, plant, insect and bacterial viruses. Animal viruses identified included anelloviruses, circoviruses, herpesviruses, poxviruses, and papillomaviruses, which mosquitoes may have obtained from vertebrate hosts during blood feeding. Notably, sequences related to human papillomaviruses were identified in one of the mosquito samples. Sequences similar to plant viruses were identified in all mosquito viromes, which were potentially acquired through feeding on plant nectar. Numerous bacteriophages and insect viruses were also detected, including a novel densovirus likely infecting Culex erythrothorax. Through sampling insect vectors, VEM enables broad survey of viral diversity and has significantly increased our knowledge of the DNA viruses present in mosquitoes.     

冈比亚按蚊嗅觉结合蛋白候选基因cDNA的克隆、鉴定及其表达型分析

疟蚊主要依靠嗅觉发现寄主。非洲疟蚊冈比亚按蚊Anopheles gambiae是一种嗜吸人血的疟疾传播媒介昆虫。该文作者基于其全基因组序列,采用RT-PCR和标准分子克隆技术获得2个嗅觉结合蛋白候选基因agLZ3788和agLZ9988。测序分析结果表明,它们具有嗅觉结合蛋白的标志性结构域。进一步采用半定量RT-PCR技术研究了它们的空间表达型,结果发现它们不但在雌蚊触角中表达,也在其他部位(尤其是蚊虫足部)有强的表达。这一发现说明疟蚊嗅觉结合蛋白可能具有更广的功能,也为进一步重组表达和功能研究提供了重要依据。     

Mosquito phytophagy – sources exploited,ecological function,and evolutionary transition to haematophagy

For a very long time, mosquitoes have been known or suspected to consume plant liquids. Recently eclosed mosquitoes cannot survive long without consuming sugary plant liquids that provide fuel for flight and enable blood-feeding and mating. Populations of even highly synanthropic mosquitoes may not be able to persist without phytophagy, even when vertebrate blood is readily available. Phytophagy is a key element of mosquito ecology, and understanding it is critical to combat mosquito-borne diseases. In this review, we summarize the current knowledge about mosquito phytophagy and outline future research needs. Specifically, we review the many plant-derived food sources mosquitoes exploit, study the pollination function of mosquitoes, highlight the predation risks of plant-foraging mosquitoes, investigate the role of microbes in the sugar-foraging ecology of mosquitoes, and shed light on the evolution of haematophagy.     

Effects of global warming on mosquitoes & mosquito‐borne diseases and the new strategies for mosquito control

Global warming has shortened mosquitoes’ lifecycle period and increased the disease transmission rates by mosquito vectors. We reviewed only three mosquito‐borne diseases: malaria, dengue fever, and the Japanese encephalitis. Billions of people get infected with those diseases and millions of people die every year. Although we struggle to find the most effective way to control mosquitoes using various methods (including pesticides), mosquito‐borne diseases are still among the most serious problems being faced. This paper, therefore, reviews the strategies for controlling mosquitoes. The use of pesticides to control mosquitoes might have more negative effects on humans and environments than benefits. Although the development of genetically modified (GM) mosquitoes raises new hopes for effective mosquito control, it will take longer to assess the risks to humans and environments. Furthermore, there has been concern about the possible adverse effects from the release of GM mosquitoes into the environment. The various mosquito traps may not be as effective at controlling only female mosquito populations. Therefore, new strategies for the control of mosquitoes are vital. The smart mosquito counter device was developed by Korean Centre for Disease Control (KCDC) in 2013. The mosquito pest control office is able to set up the appropriate mosquito control strategies by using quantitative mosquito information. The smart device will bring mosquito control in line with modern smart generation technology and the device will also soon be able to identify different mosquito species. This new strategy will change the methods of mosquito control and will provide beneficial effects toward sustainable nature and human health.     

Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome.

The Escherichia coli K-12 chromosome contains six copies of insertion element IS1 at loci is1A-is1F. We determined their nucleotide (nt) sequences and found that they were classified into four types. Two copies of IS1 which flank a chromosomal segment containing the argF gene (IS1B and IS1C) have identical nt sequences. Another identical pair are IS1A and IS1E. Comparison of their nt sequences with the IS1 in plasmid R100 revealed seven nt mismatches for IS1A (or IS1E), two for IS1B (or IS1C), four for IS1D, and 75 for IS1F. The fact that the IS1s flanking the argF segment are identical supports the idea that the segment, together with the particular pair of IS1s, has constituted a composite transposon and transposed after genetic transfer from another bacterial species into E. coli K-12. Duplicated sequences were not observed in the regions flanking each of four copies of IS1, indicating that rearrangements have occurred in these chromosomal regions after IS1 elements had been inserted into several target sites. The four types of IS1 present in the E. coli K-12 chromosome were essentially similar to IS1s in plasmid R100 and in the chromosomes of Shigella strains. This and the above results suggest that they have been transferred horizontally from other Enterobacteriaceae, including Shigella, into E. coli K-12.     

Production and characterization of monoclonal antibodies to two new mosquito Aedes aegypti salivary proteins

Mosquito salivary proteins, which are fundamental to the process of blood feeding, also facilitate disease transmission and cause allergic reactions. The identification and characterisation of these proteins have been hampered by the difficulty of obtaining them in purified form. In this report, we describe the production of mouse monoclonal antibodies (mAbs) against mosquito salivary proteins. BALB/c mice were immunised with Aedes aegypti saliva proteins. Hybridomas were produced by fusion of spleen cells with a mouse myeloma cell line. Positive clones were selected using a saliva-capture ELISA and further identified using immunoblotting. Three mAbs reacted with a 44 kDa protein (Aed a X1) in the saliva-immunoblotting, and did not react with 2 recombinant salivary proteins, rAed a 1 (apyrase) and rAed a 2 (D7), in both immunoblotting and ELISA. Two other mAbs reacted with a 37 kDa protein in saliva-immunoblotting, but failed to react with the 37 kDa rAed a 2 in either immunoblotting or ELISA, suggesting that there is a second 37 kDa protein (Aed a X2) which is recognised by the two mAbs. The 44 kDa and 37 kDa proteins have not been previously identified. These mAbs provide a means to purify proteins, to isolate new genes from the salivary gland cDNA library, and to standardise mosquito extracts, facilitating studies of disease transmission by mosquitoes and of mosquito allergy.     

Identification of Swedish mosquitoes based on molecular barcoding of the COI gene and SNP analysis

Mosquito‐borne infectious diseases are emerging in many regions of the world. Consequently, surveillance of mosquitoes and concomitant infectious agents is of great importance for prediction and prevention of mosquito‐borne infectious diseases. Currently, morphological identification of mosquitoes is the traditional procedure. However, sequencing of specified genes or standard genomic regions, DNA barcoding, has recently been suggested as a global standard for identification and classification of many different species. Our aim was to develop a genetic method to identify mosquitoes and to study their relationship. Mosquitoes were captured at collection sites in northern Sweden and identified morphologically before the cytochrome c oxidase subunit I (COI) gene sequences of 14 of the most common mosquito species were determined. The sequences obtained were then used for phylogenetic placement, for validation and benchmarking of phenetic classifications and finally to develop a hierarchical PCR‐based typing scheme based on single nucleotide polymorphism sites (SNPs) to enable rapid genetic identification, circumventing the need for morphological characterization. The results showed that exact phylogenetic relationships between mosquito taxa were preserved at shorter evolutionary distances, but at deeper levels, they could not be inferred with confidence using COI gene sequence data alone. Fourteen of the most common mosquito species in Sweden were identified by the SNP/PCR‐based typing scheme, demonstrating that genetic typing using SNPs of the COI gene is a useful method for identification of mosquitoes with potential for worldwide application.     

A Metagenomic Survey of Viral Abundance and Diversity in Mosquitoes from Hubei Province

Mosquitoes as one of the most common but important vectors have the potential to transmit or acquire a lot of viruses through biting, however viral flora in mosquitoes and its impact on mosquito-borne disease transmission has not been well investigated and evaluated. In this study, the metagenomic techniquehas been successfully employed in analyzing the abundance and diversity of viral community in three mosquito samples from Hubei, China. Among 92,304 reads produced through a run with 454 GS FLX system, 39% have high similarities with viral sequences belonging to identified bacterial, fungal, animal, plant and insect viruses, and 0.02% were classed into unidentified viral sequences, demonstrating high abundance and diversity of viruses in mosquitoes. Furthermore, two novel viruses in subfamily Densovirinae and family Dicistroviridae were identified, and six torque tenosus virus1 in family Anelloviridae, three porcine parvoviruses in subfamily Parvovirinae and a Culex tritaeniorhynchus rhabdovirus in Family Rhabdoviridae were preliminarily characterized. The viral metagenomic analysis offered us a deep insight into the viral population of mosquito which played an important role in viral initiative or passive transmission and evolution during the process.     

Controlling malaria transmission with genetically-engineered, Plasmodium-resistant mosquitoes: milestones in a model system

We are developing transgenic mosquitoes resistant to malaria parasites to test the hypothesis that genetically-engineered mosquitoes can be used to block the transmission of the parasites. We are developing and testing many of the necessary methodologies with the avian malaria parasite, Plasmodium gallinaceum, and its laboratory vector, Aedes aegypti, in anticipation of engaging the technical challenges presented by the malaria parasite, P. falciparum, and its major African vector, Anopheles gambiae. Transformation technology will be used to insert into the mosquito a synthetic gene for resistance to P. gallinaceum. The resistance gene will consist of a promoter of a mosquito gene controlling the expression of an effector protein that interferes with parasite development and/or infectivity. Mosquito genes whose promoter sequences are capable of sex- and tissue-specific expression of exogenous coding sequences have been identified, and stable transformation of the mosquito has been developed. We now are developing the expressed effector portion of the synthetic gene that will interfere with the transmission of the parasites. Mouse monoclonal antibodies that recognize the circumsporozoite protein of P. gallinaceum block sporozoite invasion of mosquito salivary glands, as well as abrogate the infectivity of sporozoites to a vertebrate host, the chicken, Gallus gallus, and block sporozoite invasion and development in susceptible cell lines in vitro. Using the genes encoding these antibodies, we propose to clone and express single-chain antibody constructs (scFv) that will serve as the effector portion of the gene that interferes with transmission of P. gallinaceum sporozoites.     

FLP-mediated recombination in the vector mosquito, Aedes aegypti.

The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti. In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos. FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids. In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used. The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid. This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.     

Amblyospora trinus N. sp. (Microsporida: Amblyosporidae) in the Australian mosquito Culex halifaxi (Diptera: Culicidae)

A new species of Amblyospora, a parasite found in wild populations of the predacious Australian mosquito Culex halifaxi, was investigated with light and electron microscopy. This species was found to be heterosporous with two concurrent sporulation sequences in the host larvae, both arising from diplokaryotic meronts and ending with haploid spores. One sequence was dominant and involved meiosis to produce eight thick-walled, broadly oval meiospores in a sporophorous vesicle (SV). The other sequence involved nuclear dissociation to produce lanceolate, thin-walled spores in a subpersistent SV. Horizontal transmission to the mosquito host, by one or both of two distinctly different pathways (one via an intermediate host, the other by cannibalism of infected individuals) and by vertical transmission, are postulated but have not been demonstrated. A new species, Amblyospora trinus, is proposed and its affinities to other heterosporous microsporidia in mosquitoes are discussed.     

Amblyospora trinus N. Sp. (Microsporida: Amblyosporidae) in the Australian Mosquito Culex halifaxi (Diptera: Culicidae)

ABSTRACT. A new species of Amblyospora , a parasite found in wild populations of the predacious Australian mosquito Culex halifaxi , was investigated with light and electron microscopy. This species was found to be heterosporous with two concurrent sporulation sequences in the host larvae, both arising from diplokaryotic meronts and ending with haploid spores. One sequence was dominant and involved meiosis to produce eight thick-walled, broadly oval meiospores in a sporophorous vesicle (SV). The other sequence involved nuclear dissociation to produce lanceolate, thin-walled spores in a subpersistent SV. Horizontal transmission to the mosquito host, by one or both of two distinctly different pathways (one via an intermediate host, the other by cannibalism of infected individuals) and by vertical transmission, are postulated but have not been demonstrated. A new species, Amblyospora trinus , is proposed and its affinities to other heterosporous microsporidia in mosquitoes are discussed.     

Analysis of mosquito bloodmeals by DNA profiling

Abstract. Human specific genetic markers have been used to profile the human DNA found within a mosquito bloodmeal. In this technique, variable numbers of tandem repeat (VNTR) sequences are employed to prime amplification of human DNA in the polymerase chain reaction (PCR) and the radiolabeled products are analysed by high resolution denaturing gel electrophoresis. Matching of DNA profiles allows identification of the individual human host. Bloodmeals of 125 female Anopheles gambiae Giles mosquitoes, caught dead or alive in verandah-trap huts wherein two people had slept overnight protected by intact insecticide-impregnated bednets, were analysed: thirty-five out of thirty-nine profiles generated were identical to those of the sleepers under the nets. Thus the blood-fed mosquitoes found after impregnated nets have been used cannot, in most cases, be explained away by entry of already fed mosquitoes into the huts.     

Using bacteria to express and display anti-Plasmodium molecules in the mosquito midgut

Bacteria capable of colonizing mosquito midguts are attractive vehicles for delivering anti-malaria molecules. We genetically engineered Escherichia coli to display two anti-Plasmodium effector molecules, SM1 and phospholipase-A(2), on their outer membrane. Both molecules significantly inhibited Plasmodium berghei development when engineered bacteria were fed to mosquitoes 24h prior to an infective bloodmeal (SM1=41%, PLA2=23%). Furthermore, prevalence and numbers of engineered bacteria increased dramatically following a bloodmeal. However, E. coli survived poorly in mosquitoes. Therefore, Enterobacter agglomerans was isolated from mosquitoes and selected for midgut survival by multiple passages through mosquitoes. After four passages, E. agglomerans survivorship increased from 2 days to 2 weeks. Since E. agglomerans is non-pathogenic and widespread, it is an excellent candidate for paratransgenic control strategies.     

Mosquito species diversity and abundance in relation to land use in a riceland agroecosystem in Mwea, Kenya.

We conducted an entomological survey to determine the mosquito species diversity and abundance in relation to land use in the Mwea rice scheme, Kenya. Adult mosquitoes were collected by indoor spraying of houses and outdoors by CDC light traps in three villages representing planned (Mbuinjeru) and unplanned (Kiamachiri) rice agroecosystems and a non-irrigated agroecosystem (Murinduko). During the 12-month sampling period, a total of 98,708 mosquitoes belonging to five genera and 25 species were collected. The five most common species collected during this study were Anopheles arabiensis Patton (52.5%), Culex quinquefasciatus Say (36.7%), Anopheles pharoensis Theobald (5.2%), Anopheles coustani Laveran (1.4%), and Anopheles funestus Giles (1.3%). Anopheles arabiensis, Cx quinquefasciatus, and An. pharoensis were more abundant in rice agroecosystems than in the non-irrigated agroecosystem, and in planned than in the unplanned rice agroecosystems. In contrast, An. funestus was more abundant in the non-irrigated agroecosystem. The mosquito species diversity (H) and evenness (E(H)) in the non-irrigated agroecosystem (Shannon diversity Index, H = 1.507, EH = 0.503) was significantly higher than in the rice agroecosystems (H) = 0.968, E(H) = 0.313, unplanned; and H= 1.040, E(H) = 0.367 planned). Results of lag cross correlation analysis revealed a strong relationship between rainfall and the abundance of An. arabiensis, and C. quinquefasciatus in the non-irrigated agroecosystem but not in the rice agroecosystems. It is inferred from the data that different levels of habitat perturbations with regard to rice cultivation have different effects on mosquito diversity and abundance. This provides an understanding of how mosquito diversity is impacted by different habitat management and rice cropping strategies.     

Mosquito (Diptera: Culicidae) grouping based on larval habitat characteristics in high mountain ecosystems of Antioquia,Colombia

Information about mosquito ecology in the high mountain ecosystems of the Neotropical region is sparse. In general, few genera and species have been reported in these ecosystems and there is no information available on habitats and the mosquitoes occupying them. In the present study, specimens collected from NW Colombia in HME were grouped using larval habitat data via an Operational Taxonomic Unit (OTU) determination. A total of 719 mosquitoes was analyzed belonging to 44 OTUs. The analysis considered habitat features and clustered the specimens into six groups from A‐F. Five of these included species from different genera, suggesting common habitat requirements. Group E with four genera, seven subgenera, and six species occupied the highest areas (above 3,000 m), whereas three groups (B, D, F) were detected at lower altitudes (1,960–2,002 m). Bromeliads were the most common larval habitat, with 47% (335/719) of the specimens; five genera, six subgenera, and eight species were identified and classified into 66% (29/44) of the OTUs. This work showed some similarities to the habitat requirements and provides a grouping system that constitutes an important baseline for the classification of mosquito fauna from high mountain ecosystems according to altitude and larval habitat.