P elements are found in the genomes of nematoceran insects of the genus Anopheles

We report the identification of genomic sequences in various anopheline mosquitoes (family Culicidae: suborder Nematocera: order Diptera) showing homology to the class II, short inverted-terminal-repeat (ITR) transposable element P from Drosophila melanogaster (family Drosophilidae; suborder Brachycera: order Diptera). Anopheles gambiae appears to have at least six distinct P elements. Other anopheline species, including four additional members of the An. gambiae species complex (An. arabiensis, An. merus, An. melas and An. quadriannulatus), Anopheles stephensi (all subgenus Cellia), An. quadrimaculatus (subgenus Anopheles) and Anopheles albimanus (subgenus Nyssorhynchus) also possess P elements similar to those found in An. gambiae. Ten distinct P element types were identified in the genus Anopheles. At least two of the An. gambiae elements appears to be intact and potentially functional. Phylogenetic analysis of the anopheline P elements reveals them to belong to a distinctly different clade from the brachyceran P elements.     

Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti

Glutathione transferases (GSTs) play a central role in the detoxification of xenobiotics such as insecticides and elevated GST expression is an important mechanism of insecticide resistance. In the mosquito, Anopheles gambiae, increased expression of an Epsilon class GST, GSTE2, confers resistance to DDT. We have identified eight GST genes in the dengue vector, Aedes aegypti. Four of these belong to the insect specific GST classes Delta and Epsilon and three are from the more ubiquitously distributed Theta and Sigma classes. The expression levels of the two Epsilon genes, a Theta GST and a previously identified Ae. aegypti GST [Grant and Hammock, 1992. Molecular and General Genetics 234, 169-176] were established for an insecticide susceptible and a resistant strain. We show that the putative ortholog of GSTe2 in Ae. aegypti (AaGSTe2) is over expressed in mosquitoes that are resistant to the insecticides DDT and permethrin. Characterisation of recombinant AaGSTE2-2 confirmed the role of this enzyme in DDT metabolism. In addition, unlike its Anopheles ortholog, AaGSTE2-2 also exhibited glutathione peroxidase activity.     

基于rDNA-ITS2序列的中国按蚊属塞蚊亚属部分种类的系统发育研究(双翅目:蚊科)

应用rDNA-ITS2序列,采用邻接法(NJ)、最大简约法(MP)和最大似然法(ML),以按蚊亚属Anopheles的中华按蚊An.(An.)sinensis和赫坎按蚊An.(An.)hyrcanus为外群,对采自中国的按蚊属Anopheles塞蚊亚属Cellia 21种蚊进行了系统发育分析.结果表明:rDNA-ITS...     

A photomap of the salivary gland chromosomes of Anopheles stephensi liston (Culicidae: Diptera)

A photomap of the banding pattern of the salivary gland chromosomes of Anopheles stephensi Liston, which is first of its kind, has been prepared. The salivary chromosome complement consists of five arms, the shortest of which represents the telocentric X-chromosome, and the remaining four the autosomal arms. A comparison has been made of the banding pattern of this species with other species of the subgenus Cellia.     

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.     

基于mtDNA和rDNA基因序列的中国按蚊属塞蚊亚属种类的系统发育研究（双翅目：蚊科）

【目的】应用mtDNA和rDNA基因特征重建中国按蚊属塞蚊亚属已知种类的系统发育关系, 以阐明亚属内各蚊种的亲缘关系。【方法】对采自中国的按蚊属塞蚊亚属Anopheles (Cellia) 20种蚊的mtDNA-COⅡ和 rDNA-28S-D3序列进行测定和分析, 以按蚊属按蚊亚属Anopheles (Anopheles)的中华按蚊An. (An.) sinensis和赫坎按蚊An. (An.) hyrcanus为外群, 采用COⅡ和D3单基因, 以及“COⅡ+D3”联合数据组以邻接法（NJ）、 最大简约法（MP）、 最大似然法（ML）和贝叶斯法（BI）等重建这些种类的系统发育树。【结果】 mtDNA-COⅡ和rDNA-28S-D3序列的长度范围分别为685 bp和375～410 bp, 在塞蚊亚属蚊种间的遗传距离分别为0.015～0.117和0.003～0.111。各系统树显示外群被合理分开,除在COⅡ树中新塞蚊系为并系外,各系均聚为单系群,新迈蚊系和迈蚊系亲缘关系最近。联合数据组构建的系统合意树显示中国塞蚊亚属各蚊种形成4支,除伪威氏按蚊与多斑按蚊种团未聚为单系群外,其他各种团和复合体成员种均分别聚在一起,各分支的置信值均大于50%。【结论】本研究获得的分子系统发育树清楚地显示了中国按蚊属塞蚊亚属各种类及系之间的系统发育关系, 对其分类和防治研究具有参考价值。     

Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito

Anopheles stephensi is the main urban mosquito vector of malaria in the Indian subcontinent, and belongs to the same subgenus as Anopheles gambiae, the main malaria vector in Africa. Recently the genome and proteome sets of An. gambiae have been described, as well as several protein sequences expressed in its salivary glands, some of which had their expression confirmed by amino terminal sequencing. In this paper, we randomly sequenced a full-length cDNA library of An. stephensi and performed Edman degradation of polyvinylidene difluoride (PVDF)-transferred protein bands from salivary homogenates. Twelve of 13 proteins found by aminoterminal degradation were found among the cDNA clusters of the library. Thirty-three full-length novel cDNA sequences are reported, including a novel secreted galectin; the homologue of anophelin, a thrombin inhibitor; a novel trypsin/chymotrypsin inhibitor; an apyrase; a lipase; and several new members of the D7 protein family. Most of the novel proteins have no known function. Comparison of the putatively secreted and putatively housekeeping proteins of An. stephensi with An. gambiae proteins indicated that the salivary gland proteins are at a faster evolutionary pace. The possible role of these proteins in blood and sugar feeding by the mosquito is discussed. The electronic tables and supplemental material are available at http://www.ncbi.nlm.nih.gov/projects/Mosquito/A_stephensi_sialome/ .     

Cladistic analysis of the subgenus Anopheles (Anopheles) Meigen (Diptera: Culicidae) based on morphological characters

In the present study, we used morphological characters to estimate phylogenetic relationships among members of the subgenus Anopheles Meigen. Phylogenetic analyses were carried out for 36 species of Anopheles (Anopheles). An. (Stethomyia) kompi Edwards, An. (Lophopodomyia) gilesi (Peryassú), Bironella hollandi Taylor, An. (Nyssorhynchus) oswaldoi (Peryassú) and An. (Cellia) maculatus Theobald were employed as outgroups. One hundred one characters of the external morphology of the adult male, adult female, fourth-instar larva, and pupa were scored and analyzed under the parsimony criterion in PAUP. Phylogenetic relationships among the series and several species informal groups of Anopheles (Anopheles) were hypothesized. The results suggest that Anopheles (Anopheles) is monophyletic. Additionally, most species groups included in the analysis were demonstrated to be monophyletic.     

Molecular identification of mosquito species in the Anopheles annularis group in southern Asia

The Anopheles annularis group of subgenus Cellia Theobald (Diptera: Culicidae) includes five currently recognized species in southern Asia: An. annularis Van der Wulp, Anopheles nivipes (Theobald) and Anopheles philippinensis Ludlow, which are widespread in the region, Anopheles pallidus Theobald, which is known in Sri Lanka, India and Myanmar, and Anopheles schueffneri Stanton, which occurs in Java and Sumatra. Identification of the four mainland species based on morphology is problematic. In view of the fact that the three widespread species are variously involved in malaria transmission in different parts of the region, we developed a species-specific polymerase chain reaction assay based on rDNA internal transcribed spacer 2 (ITS2) sequences to facilitate entomological and epidemiological studies of the four species. The method proved to be reliable when tested over a wide geographical area.     

Engorgement response of anopheline mosquitoes to blood fractions and artificial solutions

ABSTRACT. Anopheles stephensi Liston, Anopheles freeborni Aitken, Anopheles gambiae Giles and Anopheles dirus (Peyton & Harrison) fed equally well on whole blood, red blood cells, platelet-rich plasma and platelet-poor plasma. Similar feeding ability on 0.15 M NaCl containing 10^-2 M NaHCO₃ was shown by the first three species, but An. dirus required an addition of albumin. The need for ATP as a phagostimulant could not be demonstrated in any of these species.     

The role of the Aedes aegypti Epsilon glutathione transferases in conferring resistance to DDT and pyrethroid insecticides

The Epsilon glutathione transferase (GST) class in the dengue vector Aedes aegypti consists of eight sequentially arranged genes spanning 53,645 bp on super contig 1.291, which maps to chromosome 2. One Epsilon GST, GSTE2, has previously been implicated in conferring resistance to DDT. The amino acid sequence of GSTE2 in an insecticide susceptible and a DDT resistant strain differs at five residues two of which occur in the putative DDT binding site. Characterization of the respective recombinant enzymes revealed that both variants have comparable DDT dehydrochlorinase activity although the isoform from the resistant strain has higher affinity for the insecticide. GSTe2 and two additional Epsilon GST genes, GSTe5 and GSTe7, are expressed at elevated levels in the resistant population and the recombinant homodimer GSTE5-5 also exhibits low levels of DDT dehydrochlorinase activity. Partial silencing of either GSTe7 or GSTe2 by RNA interference resulted in an increased susceptibility to the pyrethroid, deltamethrin suggesting that these GST enzymes may also play a role in resistance to pyrethroid insecticides.     

Comparative genome analysis of the yellow fever mosquito Aedes aegypti with Drosophila melanogaster and the malaria vector mosquito Anopheles gambiae

An in silico comparative genomics approach was used to identify putative orthologs to genetically mapped genes from the mosquito, Aedes aegypti, in the Drosophila melanogaster and Anopheles gambiae genome databases. Comparative chromosome positions of 73 D. melanogaster orthologs indicated significant deviations from a random distribution across each of the five A. aegypti chromosomal regions, suggesting that some ancestral chromosome elements have been conserved. However, the two genomes also reflect extensive reshuffling within and between chromosomal regions. Comparative chromosome positions of A. gambiae orthologs indicate unequivocally that A. aegypti chromosome regions share extensive homology to the five A. gambiae chromosome arms. Whole-arm or near-whole-arm homology was contradicted with only two genes among the 75 A. aegypti genes for which orthologs to A. gambiae were identified. The two genomes contain large conserved chromosome segments that generally correspond to break/fusion events and a reciprocal translocation with extensive paracentric inversions evident within. Only very tightly linked genes are likely to retain conserved linear orders within chromosome segments. The D. melanogaster and A. gambiae genome databases therefore offer limited potential for comparative positional gene determinations among even closely related dipterans, indicating the necessity for additional genome sequencing projects with other dipteran species.     

Activity and mating competitiveness of γHCH/dieldrin resistant and susceptible male and virgin female Anopheles gambiae and An.stephensi mosquitoes, with assessment of an insecticide-rotation strategy

The effects of gamma HCH/dieldrin resistance genes on flight activity and mating competitiveness were investigated in males from backcrossed strains of Anopheles gambiae Giles and An.stephensi Liston. Activity of males and virgin females of both species, as recorded in an acoustic actograph, occurred mainly at dusk (the E peak). The activity pattern of An.gambiae males was not affected by resistance genes; in mating competition and predator avoidance experiments, however, RR males were less successful than RS males which were less successful than SS males. The activity pattern of An.stephensi differed from An.gambiae in that the E peaks of RR males and females in a gradual dusk regime were out of synchrony with those of SS and RS, the E peaks of RR occurring slightly later. Thus, RR males and females tended to mate assortatively in mate competition experiments. When a sudden dusk regime was substituted for the gradual dusk regime, activity of RR An.stephensi became synchronized with SS and RS activity, but in mating competition experiments RR still tended to mate assortatively. Estimates of male competitiveness, together with previously-obtained estimates of female fitness, were included in population genetics models. Computer simulations showed that the frequency of resistance in populations of An.gambiae and An.stephensi should decrease in the absence of insecticide at a rate comparable with known field reversions.     

What's buzzing? Mosquito genomics and transgenic mosquitoes

Genome projects and associated technologies are now being established for mosquito species that are vectors of human disease. The recent announcement of an award by the National Institute of Allergy and Infectious Diseases (NIAID) to Celera Genomics to sequence the Anopheles gambiae genome will further accelerate the completion of the sequencing of this genome. Completion of the An. gambiae sequence will mean that the genomes of all three organisms involved in the transmission of falciparum malaria--the mosquito, the parasite, and the human--will have been sequenced. This will greatly facilitate the identification of genes and pathways involved in the transmission of malaria. The recent genetic transformation of An. gambiae with the piggyBac transposable element and the transformation of another important malarial vector, Anopheles stephensi using the Minos element, now provide researchers with powerful tools with which to genetically manipulate these medically important vector species. Here we review the recent progress made in the extension of contemporary tools of modern genetics and genomics into these medically important insects.     

Pteridine fluorescence for age determination of Anopheles mosquitoes

The age structure of mosquito populations is of great relevance to understanding the dynamics of disease transmission and in monitoring the success of control operations. Unfortunately, the ovarian dissection methods currently available for determining the age of adult mosquitoes are technically difficult, slow and may be of limited value, because the proportion of diagnostic ovarioles in the ovary declines with age. By means of reversed-phase HPLC this study investigated the malaria vectors Anopheles gambiae and An. stephensi to see if changes in fluorescent pteridine pigments, which have been used in other insects to determine the age of field-caught individuals, may be useful for age determination in mosquitoes. Whole body fluorescence was inversely proportional to age (P < 0.001, r2 > 91%) up to 30 days postemergence, with the regression values: y = 40580-706x for An. gambiae, and y = 52896-681x for An. stephensi. In both species the main pteridines were 6-biopterin, pterin-6-carboxylic acid and an unidentified fluorescent compound. An. gambiae had only 50-70% as much fluorescence as An. stephensi, and fluorescent compounds were relatively more concentrated in the head than in the thorax (ratios 1:0.8 An. gambiae; 1:0.5 An. stephensi). The results of this laboratory study are encouraging. It seems feasible that this simpler and faster technique of fluorescence quantification could yield results of equivalent accuracy to the interpretation of ovarian dissection. A double-blind field trial comparing the accuracy of this technique to marked, released and recaptured mosquitoes is required to test the usefulness of the pteridine method in the field.     

Experimental infection of Anopheles farauti with different species of Plasmodium

Studies were conducted to determine the susceptibility of Anopheles farauti to different species and strains of Plasmodium. Mosquitoes were infected by feeding on animals or cultures infected with different strains of P. vivax, P. falciparum, P. ovale, P. coatneyi, P. gonderi, P. simiovale, P. knowlesi, and P. brasilianum. Infections of P. vivax and P. coatneyi were transmitted via sporozoites from An. farauti to monkeys. Comparative infection studies indicated that An. farauti was less susceptible to infection than An. stephensi, An. gambiae, An. freeborni, and An. dirus with the Salvador I strain of P. vivax, but more susceptible than An. stephensi and An. gambiae to infection with the coindigenous Indonesian XIX strain.     

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC(50) values of 1.5 microM, 3.5 microM, 4 microM, 4.3 microM and 4.8 microM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K(i) of 0.24 microM and 0.14 microM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K(i) = 0.4 microM) whilst GG1 produced mixed type of inhibition. The K(i) and K(i)' for GSH for Tral-1 and GG1 were 0.2 microM and 0.1 microM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.     

In situ hybridization to the Rdl locus on polytene chromosome 3L of Anopheles stephensi

We are interested in generating a Y-autosome translocation of the Resistance to dieldrin (Rdl) locus in the malaria vector mosquito Anopheles stephensi Liston (Diptera: Culicidae), for use in sterile insect release. To ensure stability of the system, a recombination suppressing inversion can also be induced which encompasses the Rdl locus. As a first step, here we report the cloning of fragments of the Rdl gene from both An. stephensi and An. gambiae Giles using degenerate primers in the polymerase chain reaction. These fragments encode the second membrane-spanning region of the gamma-aminobutyric acid receptor and show high levels of both nucleotide and predicted amino acid identity to other Rdl-like receptors. They confirm that, as in all other arthropod species examined, dieldrin resistance in An. stephensi is associated with replacement of alanine302, in this case with a serine. In situ hybridization of the Rdl probe to polytene chromosomes of An. stephensi localizes the gene to the left arm of chromosome 3 (3L) in region 45C. Rdl localization will enable us to identify chromosomal rearrangements encompassing the Rdl locus and help anchor the genome sequence of An. gambiae to the polytene map.     

Studies on the North Korean strain of Plasmodium vivax in Aotus monkeys and different anophelines

Twenty-two Aotus monkeys of different karyotypes were infected with the North Korean strain of Plasmodium vivax. Aotus lemurinus griseimembra animals from Colombia produced higher maximum parasitemias and more readily infected mosquitoes than did Aotus monkeys from Bolivia (K-VI) or Peru (K-V and K-X). Comparative feedings indicated that the most susceptible mosquito species was Anopheles stephensi, followed by An. gambiae, An. dirus, An. freeborni, An. quadrimaculatus, An. culicifacies, and An. maculatus.