Characterization of Herves-like transposable elements (hATs) in Drosophila species and their evolutionary scenario.

A monophyletic group of Drosophila hAT transposable elements, referred to as Herves-like, was characterized and found to be present in 46% of 57 screened Drosophila species. A remarkable characteristic of these elements is the presence of a long array of minisatellite repeats (MnRs) in both subterminal extremities of the elements. The copy number of these minisatellites was highly variable between and within populations. Twenty-three strains of Drosophila willistoni, covering its geographic distribution, were screened for polymorphism in the copy number of 5' MnRs, showing a variation from 7 to 20 repeat copies. These MnRs are well conserved among Drosophila species and probably function as transposase binding sequences, as provided by short subterminal repeats in other hAT elements. Miniature inverted repeat transposable elements were found in 27% of species carrying Herves-like elements. Phylogenetic analysis showed incongruences between transposable elements and species phylogenies, suggesting that at least four horizontal transfer events have occurred.

Wolbachia from Drosophila incompta: just a hitchhiker shared by Drosophila in the New and Old World?

Wolbachia are intracellular endosymbionts that infect arthropods and filarial nematodes, occasionally causing a wide variety of modifications in host biology, such as male-killing and cytoplasmic incompatibility (CI), amongst others. This study assembled draft genomes for Wolbachia infecting Drosophila incompta, a species that uses flowers as exclusive breeding and feeding sites, in two distinct Brazilian populations. The absence of four genes involved in CI from this genome, together with literature reports of low frequencies of infected flies in wild populations that contain high mitogenome polymorphism, suggests that this bacterium does not induce CI in D. incompta. Phylogenomic analysis placed Wolbachia infecting D. incompta as closely related to the wMel strain which received such name since it was originally detected in Drosophila melanogaster. In addition, phylogenetic analysis using the Wolbachia surface protein gene and five genes used for multilocus sequence typing of Wolbachia found infecting Drosophila and other arthropod species of Old and New World displayed a complex evolutionary scenario involving recent horizontal transfer bursts in all major clades of Wolbachia pipens belonging to the supergroup A in both geographical regions.

Characterization of the complete mitochondrial genome of flower-breeding Drosophila incompta (Diptera, Drosophilidae).

Drosophila incompta belongs to the flavopilosa group of Drosophila, and has a restricted ecology, being adapted to flowers of Cestrum as feeding and oviposition sites. We sequenced, assembled, and characterized the complete mitochondrial genome (mtDNA) of D. incompta. In addition, we performed phylogenomic and polymorphism analyses to assess evolutionary diversification of this species. Our results suggest that this genome is syntenic with the other published mtDNA of Drosophila. This molecule contains 15,641 bp and encompasses two rRNA, 22 tRNA and 13 protein-coding genes. Regarding nucleotide composition, we found a high A-T bias (76.6 %). The recovered phylogenies indicate D. incompta in the virilis-repleta radiation, as sister to the virilis or repleta groups. The most interesting result is the high degree of polymorphism found throughout the D. incompta mitogenome, revealing pronounced intrapopulational variation. Furthermore, intraspecific nucleotide diversity levels varied between different regions of the genome, thus allowing the use of different mitochondrial molecular markers for analysis of population structure of this species.

Effects of Hg(II) exposure on MAPK phosphorylation and antioxidant system in D. melanogaster.

The heavy metal mercury is a known toxin, but while the mechanisms involved in mercury toxicity have been well demonstrated in vertebrates, little is known about toxicological effects of this metal in invertebrates. Here, we present the results of our study investigating the effects associated with exposure of fruit fly Drosophila melanogaster to inorganic mercury (HgCl2 ). We quantify survival and locomotor performance as well as a variety of biochemical parameters including antioxidant status, MAPK phosphorylation and gene expression following mercury treatment. Our results demonstrate that exposure to Hg(II) through diet induced mortality and affected locomotor performance as evaluated by negative geotaxis, in D. melanogaster. We also saw a significant impact on the antioxidant system including an inhibition of acetylcholinesterase (Ache), glutathione S-transferase (GST) and superoxide dismutase (SOD) activities. We found no significant alteration in the levels of mRNA of antioxidant enzymes or NRF-2 transcriptional factor, but did detect a significant up regulation of the HSP83 gene. Mercury exposure also induced the phosphorylation of JNK and ERK, without altering p38(MAPK) and the concentration of these kinases. In parallel, Hg(II) induced PARP cleavage in a 89 kDa fragment, suggesting the triggering of apoptotic cell death in response to the treatment. Taken together, this data clarifies and extends our understanding of the molecular mechanisms mediating Hg(II) toxicity in an invertebrate model.

The copia retrotransposon and horizontal transfer in Drosophila willistoni.

The copia element is a retrotransposon that is hypothesized to have been horizontally transferred from Drosophila melanogaster to some populations of Drosophila willistoni in Florida. Here we have used PCR and Southern blots to screen for sequences similar to copia element in South American populations of D. willistoni, as well as in strains previously shown to be carriers of the element. We have not found the canonical copia element in any of these populations. Unlike the P element, which invaded the D. melanogaster genome from D. willistoni and quickly spread worldwide, the canonical copia element appears to have transferred in the opposite direction and has not spread. This may be explained by differences in the requirements for transposition and in the host control of transposition.

Taxonomic boundaries, phylogenetic relationships and biogeography of the Drosophila willistoni subgroup (Diptera: Drosophilidae).

The Drosophila willistoni subgroup represents a complex with varying taxonomic levels. It encompasses D. willistoni and its five sibling species: D. equinoxialis, D. insularis, D. paulistorum, D. pavlovskiana and D. tropicalis. Of these, D. equinoxialis, D. tropicalis and D. willistoni present differentiation at subspecific level, whereas D. paulistorum represents a superspecies, formed by six semispecies. Despite this taxonomic and evolutionary complexity, many of these semi and subspecific taxa have not yet had their phylogenetic status tested in an explicitly molecular study. Aiming to contribute to the understanding of the evolution of this challenging group, we analyzed nucleotide sequences from two mitochondrial and four nuclear datasets, both individually and simultaneously, through different phylogenetic methods. High levels of incongruence were detected among partitions, especially concerning the mitochondrial sequences. As this incongruence was found to be statistically significant and robust to the use of different models and approaches, and basically restricted to mitochondrial loci, we suggest that it may stem mainly from hybridization-mediated asymmetrical introgression. Despite this, our nuclear data finally led to a phylogenetic hypothesis which further refines several aspects related to the willistoni subgroup phylogeny. In this respect, D. insularis, D. tropicalis, D. willistoni and D. equinoxialis successively branched off from the willistoni subgroup main stem, which recently subdivided to produce D. paulistorum and D. pavlovskiana. As regards the semispecies evolution, we found evidence of a recent diversification, which highly influenced the obtained results due to the associated small levels of genetic differentiation, further worsened by the possibly associated incompletely sorted ancestral polymorphisms and by the possibility of introgression. This study also raises the question of whether these semispecies are monophyletic at all. This reasoning is particularly interesting when one considers that similar levels of reproductive isolation could be attained through infection with different Wolbachia strains.

Harrow: new Drosophila hAT transposons involved in horizontal transfer.

In this study we characterize the transposable elements harrow, which belong to the hAT superfamily of DNA transposons. Searches for harrow sequences were performed in 65 Drosophilidae species, mainly representing Neotropical and cosmopolitan groups from the genus Drosophila. The nucleotide divergence among elements found in these species suggests that harrow sequences could be clustered in a subfamily. The patchy distribution throughout the genus Drosophila and the high similarity presented between all harrow sequences indicate that horizontal transfer could play a major role in the evolution of harrow elements. The results obtained suggest an evolutionary scenario in which harrow would have undergone multiple horizontal transfer events in the Neotropics, involving D. tripuncatata, D. mojavensis (Subgenus Drosophila) and several species of the willistoni and saltans groups (subgenus Sophophora).

Identificação diferencial de Rhodococcus equi e Dietzia maris em bubalinos / Differential identification of Rhodococcus equi and Dietzia maris in buffaloes

Foram analisados 24 isolados bacterianos oriundos de leite e pele de búfalas (Bubalus bubalis), os quais foram previamente identificados como Rhodococcus equi com o auxílio de fenotipia concisa. Testes fenotípicos complementares e ferramentas moleculares foram utilizados com o objetivo de caracterizar esses isolados, bem como diferenciá-los de outros microrganismos intimamente relacionados. Observaram-se três fenótipos distintos, porém a identificação dos isolados foi inconclusiva. Apenas um dos isolados foi comprovado como sendo R. equi com a realização da PCR espécie-específica, sequenciamento e análise dos fragmentos de DNA. Os demais isolados só foram identificados pelo sequenciamento de fragmento do gene que codifica a região 16S do rRNA universal de bactérias, indicando tratar-se de Dietzia maris. O perfil de susceptibilidade aos antimicrobianos revelou maior resistência dos isolados de D. maris para oxacilina (96 por cento) e rifampicina (87 por cento). O isolado de R. equi apresentou resistência à amicacina, oxacilina, penicilina, rifampicina e tetraciclina. Alerta-se para o risco da incorreta identificação dos isolados baseados em testes fenotípicos concisos e para a necessidade de utilização de testes complementares para diferenciação entre R. equi e D. maris.

Twenty-four bacterial isolates from milk and skin of buffalo females (Bubalus bubalis), which previously had been identified as Rhodococcus equi by using a restricted number of phenotypical tests for bacterial characterization, were analyzed. The goal of this study was to perform the characterization of these isolates, as well as the differentiation of other microorganisms closely related by using additional phenotypical tests and molecular tools. Based on the phenotypical results, three different biotypes were obtained. However, the identification of the isolates was inconclusive. Only one of the isolates was confirmed as R. equi by the PCR specifically for this species, as well DNA sequencing and DNA fragment analysis. All the other isolates only could be precisely identified after the DNA sequencing, and they were characterized as Dietzia maris. The sensitivity profile to antimicrobials demonstrated the highest resistance of D. maris to oxacillin and rifampin, 96 percent and 87 percent, respectively. R. equi isolate, presented resistance to amikacin, oxacillin, penicillin, rifampin, and tetracycline. Thus, it is important to alert for the risk of the incorrect identification of the bacterial isolates by using diagnostic analysis based on phenotypical tests in order to differentiate R. equi and D. maris, besides the necessity to use complementary tests for differentiation of these microorganisms.

The mariner transposable element in natural populations of Drosophila simulans.

In cosmopolitan species, geographical variations in copy number and/or level of transposition activity have been observed for several transposable elements (TEs). Environment, history and population structure can contribute to such variation in ways that are difficult to tease apart. For the mariner element, previous studies of the geographic variation of its somatic activity in natural populations of Drosophila simulans have shown contradictory results (latitudinal clines of divergent orientations or no apparent structure). To try and resolve these inconsistencies, we gathered all available data on the mariner somatic activity of worldwide natural populations. This includes previously published results by different groups and also new data. The correlations between the level of activity and several geoclimatic factors were tested. Although no general effect of temperature was found, a relationship with the invasion history was detected. It was also shown that recent invasive populations have a higher level of activity than the putative ancestral ones. Our results strongly suggest that variability of the mariner somatic activity among natural populations of D. simulans is mainly due to populational and historical factors probably related to the recent world colonization of this species. Indeed, this activity is correlated to the main route out of Africa (the Nile route) and the recent colonization of continents such as Australia and South America.

Revisiting horizontal transfer of transposable elements in Drosophila.

Horizontal transfer (HT), defined as the transfer of genetic material between species, is considered to be an essential step in the 'life cycle' of transposable elements. We present a broad overview of suspected cases of HT of transposable elements in Drosophila. Hundred-one putative events of HT have been proposed in Drosophila for 21 different elements (5.0% refer to non-long terminal repeat (LTR) retrotransposons, 42.6% to LTR retrotransposons and 52.4% to DNA transposons). We discuss the methods used to infer HT, their limits and the putative vectors of transposable elements. We outline all the alternative hypotheses and ask how we can be almost certain that phylogenetic inconsistencies are due to HT.

Multiple invasions of Errantivirus in the genus Drosophila.

Aiming to contribute to the knowledge of the evolutionary history of Errantivirus, a phylogenetic analysis of the env gene sequences of Errantivirus gypsy, gtwin, gypsy2, gypsy3, gypsy4 and gypsy6 was carried out in 33 Drosophilidae species. Most sequences were obtained from in silico searches in the Drosophila genomes. The complex evolutionary pattern reported by other authors for the gypsy retroelement was also observed in the present study, including vertical transmission, ancestral polymorphism, stochastic loss and horizontal transfer. Moreover, the elements gypsy2, gypsy3, gypsy4 and gypsy6 were shown to have followed an evolutionary model that is similar to gypsy. Fifteen new possible cases of horizontal transfer were suggested. The infectious potential of these elements may help elucidate the evolutionary scenario described in the present study.

Diphenyl diselenide [(PhSe)2] inhibits Drosophila melanogaster delta-aminolevulinate dehydratase (delta-ALA-D) gene transcription and enzyme activity.

The main objective of the present study was to compare the inhibitory effect of diphenyl diselenide (PhSe)(2) and Pb(2+) on mice and fruit fly delta-Aminolevulinate dehydratase (delta-ALA-D). Optimum pH was quite different for mice (pH 6.5) and flies (pH 8.5). At pH 8.5, the inhibitory potency of (PhSe)(2) was higher for the fruit flies (IC(50) 8.2 micromol/l) than for mice (IC(50) 19.5 micromol/l). Pb(2+) inhibited mice delta-ALA-D at pH 6.5 (IC(50) 6.2 micromol/l) and 8.5 (IC(50) 5.6 micromol/l) with higher potency than the fly enzyme (IC(50) 43.7 micromol/l). delta-ALA-D transcription was reduced by 15% in flies exposed to 0.3 mmol/kg (PhSe)(2), which is similar to the reduction observed in activity measured in the presence of dithiothreitol. The three-dimensional prediction by SWISS-PROT mouse and fly delta-ALA-D revealed differences in the number of hydrogen bonds and turns for the 2 enzymes. Sulfhydryl groups (-SH) that could be oxidized by (PhSe)(2) are conserved in the two sources of enzyme. Distinct responsiveness to pH, (PhSe)(2) and Pb(2+) of these enzymes may be related to subtle differences in tertiary or quaternary structure of mouse and fly delta-ALA-D. Furthermore, mechanism underlying enzyme inhibition after in vivo exposure seems to be different for Drosophila melanogaster and rodent enzymes.

Molecular and antigenic characterization of Brazilian bovine herpesvirus type 1 isolates recovered from the brain of cattle with neurological disease.

Bovine herpesviruses type 1 and 5 (BoHV-1 and BoHV-5) are closely related yet differ markedly in their neuropathogenic potential. BoHV-1 isolates have been associated with respiratory and genital disease whereas BoHV-5 has been consistently isolated from neurological infection. We report the characterization of five Brazilian BoHV-1 isolates associated with neurological disease, an unusual finding. All five viruses were isolated from the brain of cattle presenting neurological disease, yet prominent histological encephalitis was not observed in three cases. The isolated viruses were identified as BoHV-1 by a glycoprotein C gene-based PCR able to differentiate BoHV-1 from BoHV-5. The identity of the isolates was confirmed by nucleotide sequencing of the amplicons and by restriction analysis of PCR products from another gC region. Monoclonal antibody binding and cross-neutralization assays with BoHV-1 and BoHV-5 antisera showed a typical BoHV-1 antigenic profile. Lastly, inoculation of rabbits with these five BoHV-1 isolates did not result in neurological disease, contrasting with fatal meningoencephalitis produced by BoHV-5. Thus, the involvement of BoHV-1 in neurological disease of cattle is more frequent than previously reported, indicating the need for fast and precise means of differentiating it from BoHV-5. Likewise, the potential role of BoHV-1 in neurological infection in cattle should be further investigated.

Evolutionary pattern of the gtwin retrotransposon in the Drosophila melanogaster subgroup.

The gtwin retrotransposon was recently discovered in the Drosophila melanogaster genome and it is evolutionarily closer to gypsy endogenous retrovirus. This study has identified gtwin homologous sequences in the genome of D. simulans, D. sechellia, D. erecta and D. yakuba by performing homology searches against the public genome database of Drosophila species. The phylogenetic analyses of the gtwin env gene sequences of these species have shown some incongruities with the host species phylogeny, suggesting some horizontal transfer events for this retroelement. Moreover, we reported the existence of DNA sequences putatively encoding full-length Env proteins in the genomes of Drosophila species other than D. melanogaster. The results suggest that the gtwin element may be an infectious retrovirus able to invade the genome of new species, supporting the gtwin evolutionary picture shown in this work.

Mobilization of a hobo-related sequence in the genome of Drosophila simulans.

The hobo transposable element can occur under three forms in the Drosophila genome: as a complete element (also called canonical), as internally deleted copies, or as hobo-related sequences (relics). Some evidence indicated that canonical elements and internally deleted copies are recent acquisitions of Drosophila genomes, while the "relics" are old components, normally degenerated and immobile. Here we present the characterization of a hobo-related sequence, found in the genome of a hypermutable strain of D. simulans, which insertion into the white locus raised a de novo white mutation. It is a shorter hobo related element presenting, overall, roughly 18% of divergence at the DNA level from the canonical hobo, with many indels that make clear this element is defective. However, its ITRs and flanking regions are extremely conserved. This is the first hobo "relic" showed to be mobilizable. We suggest, and point up some evidences, toward the idea that this sequence could have been mobilized by the canonical element. The presence of a similar "relic" element in D. sechellia allows us to suggest that these elements have been maintained mobilizable since the time of divergence between these species.

Analyses of P-like transposable element sequences from the genome of Anopheles gambiae.

We have identified 50 P element-homologous sequences in the genome of Anopheles gambiae by performing homology searches against the public genome database of A. gambiae using the canonical P element from Drosophila melanogaster as a query sequence. While most of these sequences belong to P subfamilies previously described from anopheline mosquitoes, at least four new subfamilies were identified. One of these A. gambiae P elements, which we termed AgPLS, was analysed in detail. AgPLS consists of three exons and does not have inverted terminal repeats. This element retains several of the structural features of other P-encoded peptides, such as motifs involved in DNA-protein and protein-protein interaction, and a motif involved in GTP utilization. Strong sequence and structural similarity to functional P elements, a number of nonsynonymous substitutions that is smaller than that of synonymous substitutions and the presence of putative nuclear localization signals suggest that the A. gambiae elements may retain the capacity for transposition or its repression. These sequences seem to be most closely related to P elements described from Musca domestica and Lucilia cuprina, the only P element hosts known outside the family Drosophilidae.