Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

Gentile, Carla; Lima, José Bento Pereira; Peixoto, Alexandre A.

doi:10.1590/s0074-02762005000600008

Cited by 49 publications

(26 citation statements)

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“…The comparative Ct method [98] was used to compare gene expression levels. The Aedes aegypti ribosomal protein 49 gene (Rp49) was used as an endogenous control, based on previous data [99]. The primer pairs used for the amplification of cDNA fragments for both conventional and qPCR were: NOX4-art : forward 5-TTG TGT TCG CAC ATC CAA CT-3 and reverse 5-GGT CCA ACG AAA AAT ATC CAA A-3; Rp49 : forward, 5-TGT CGG TGT AAC TGG CAT GT-3 and reverse, 5-TCG GCC AAC AAA AGT ACA CA-3.…”

Section: Methodsmentioning

confidence: 99%

Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase

et al. 2017

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BackgroundNADPH oxidases (NOX) are ROS producing enzymes that perform essential roles in cell physiology, including cell signaling and antimicrobial defense. This gene family is present in most eukaryotes, suggesting a common ancestor. To date, only a limited number of phylogenetic studies of metazoan NOXes have been performed, with few arthropod genes. In arthropods, only NOX5 and DUOX genes have been found and a gene called NOXm was found in mosquitoes but its origin and function has not been examined. In this study, we analyzed the evolution of this gene family in arthropods. A thorough search of genomes and transcriptomes was performed enabling us to browse most branches of arthropod phylogeny.ResultsWe have found that the subfamilies NOX5 and DUOX are present in all arthropod groups. We also show that a NOX gene, closely related to NOX4 and previously found only in mosquitoes (NOXm), can also be found in other taxonomic groups, leading us to rename it as NOX4-art. Although the accessory protein p22-phox, essential for NOX1-4 activation, was not found in any of the arthropods studied, NOX4-art of Aedes aegypti encodes an active protein that produces H2O2. Although NOX4-art has been lost in a number of arthropod lineages, it has all the domains and many signature residues and motifs necessary for ROS production and, when silenced, H2O2 production is considerably diminished in A. aegypti cells.ConclusionsCombining all bioinformatic analyses and laboratory work we have reached interesting conclusions regarding arthropod NOX gene family evolution. NOX5 and DUOX are present in all arthropod lineages but it seems that a NOX2-like gene was lost in the ancestral lineage leading to Ecdysozoa. The NOX4-art gene originated from a NOX4-like ancestor and is functional. Although no p22-phox was observed in arthropods, there was no evidence of neo-functionalization and this gene probably produces H2O2 as in other metazoan NOX4 genes. Although functional and present in the genomes of many species, NOX4-art was lost in a number of arthropod lineages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-017-0940-0) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase

et al. 2017

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“…aegypti dsx gene, an RT-PCR analysis was performed on total RNA extracted from different stages of development using primer pairs spanning the Aeadsx sex-specifically regulated region. The recently isolated Aedes aegypti ( Aearp49 ) homologue of the Drosophila rp49 constitutively expressed gene was tested as the positive control for the RT-PCR analysis in non-saturating conditions [38-42]. Aearp49 was constitutively expressed from the embryonic stage of Aedes until adulthood, as in the fruitfly (Figure 5A).…”

Section: Resultsmentioning

confidence: 99%

Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti

et al. 2011

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BackgroundIn the model system Drosophila melanogaster, doublesex (dsx) is the double-switch gene at the bottom of the somatic sex determination cascade that determines the differentiation of sexually dimorphic traits. Homologues of dsx are functionally conserved in various dipteran species, including the malaria vector Anopheles gambiae. They show a striking conservation of sex-specific regulation, based on alternative splicing, and of the encoded sex-specific proteins, which are transcriptional regulators of downstream terminal genes that influence sexual differentiation of cells, tissues and organs.ResultsIn this work, we report on the molecular characterization of the dsx homologue in the dengue and yellow fever vector Aedes aegypti (Aeadsx). Aeadsx produces sex-specific transcripts by alternative splicing, which encode isoforms with a high degree of identity to Anopheles gambiae and Drosophila melanogaster homologues. Interestingly, Aeadsx produces an additional novel female-specific splicing variant. Genomic comparative analyses between the Aedes and Anopheles dsx genes revealed a partial conservation of the exon organization and extensive divergence in the intron lengths. An expression analysis showed that Aeadsx transcripts were present from early stages of development and that sex-specific regulation starts at least from late larval stages. The analysis of the female-specific untranslated region (UTR) led to the identification of putative regulatory cis-elements potentially involved in the sex-specific splicing regulation. The Aedes dsx sex-specific splicing regulation seems to be more complex with the respect of other dipteran species, suggesting slightly novel evolutionary trajectories for its regulation and hence for the recruitment of upstream splicing regulators.ConclusionsThis study led to uncover the molecular evolution of Aedes aegypti dsx splicing regulation with the respect of the more closely related Culicidae Anopheles gambiae orthologue. In Aedes aegypti, the dsx gene is sex-specifically regulated and encodes two female-specific and one male-specific isoforms, all sharing a doublesex/mab-3 (DM) domain-containing N-terminus and different C-termini. The sex-specific regulation is based on a combination of exon skipping, 5' alternative splice site choice and, most likely, alternative polyadenylation. Interestingly, when the Aeadsx gene is compared to the Anopheles dsx ortholog, there are differences in the in silico predicted default and regulated sex-specific splicing events, which suggests that the upstream regulators either are different or act in a slightly different manner. Furthermore, this study is a premise for the future development of transgenic sexing strains in mosquitoes useful for sterile insect technique (SIT) programs.

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“…cruzii sibling species.The sequences of primer pairs 5'CYCdeg1 + 3'CYCdeg1 and 5'aquaRP1 + 3'aeaquaRP1b are from [39] and [40], respectively. Degenerated primers were used in preliminary amplifications to isolate initial fragments of the cycle and Clock genes.…”

Section: Methodsmentioning

confidence: 99%

Estimation of divergence time between two sibling species of the Anopheles (Kerteszia) cruziicomplex using a multilocus approach

et al. 2010

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BackgroundAnopheles cruzii is the primary human Plasmodium vector in southern and southeastern Brazil. The distribution of this mosquito follows the coast of the Brazilian Atlantic Forest. Previous studies indicated that An. cruzii is a complex of cryptic species.ResultsA multilocus approach using six loci, three circadian clock genes and three encoding ribosomal proteins, was implemented to investigate in more detail the genetic differentiation between the An. cruzii populations from Santa Catarina (southern Brazil) and Bahia States (northeastern Brazil) that represent two sibling species. The analysis revealed very high FST values and fixed differences between the two An. cruzii sibling species in all loci, irrespective of their function. An Isolation with Migration model was fit to the data using the IM program. The results reveal no migration in either direction and allowed a rough estimate of the divergence time between the two sibling species.ConclusionsPopulation genetics analysis of An. cruzii samples from two Brazilian localities using a multilocus approach confirmed that they represent two different sibling species in this complex. The results suggest that the two species have not exchanged migrants since their separation and that they possibly diverged between 1.1 and 3.6 million years ago, a period of intense climatic changes.

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Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

Cited by 49 publications

References 14 publications

Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase

Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase

Genomic organization and splicing evolution of the doublesex gene, a Drosophila regulator of sexual differentiation, in the dengue and yellow fever mosquito Aedes aegypti

Estimation of divergence time between two sibling species of the Anopheles (Kerteszia) cruziicomplex using a multilocus approach

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