Descripción: Abstract: Previous comparative genomic studies of genes involved in olfactory behavior in Drosophila focused only on particular gene families such as odorant receptor and/or odorant binding proteins. However, olfactory behavior has a complex genetic architecture that is orchestrated by many interacting genes. In this paper, we present a comparative genomic study of olfactory behavior in Drosophila including an extended set of genes known to affect olfactory behavior. We took advantage of the recent burst of whole genome sequences and the development of powerful statistical tools to analyze genomic data and test evolutionary and functional hypotheses of olfactory genes in the six species of the Drosophila melanogaster species group for which whole genome sequences are available. Our study reveals widespread purifying selection and limited incidence of positive selection on olfactory genes. We show that the pace of evolution of olfactory genes is mostly independent of the life cycle stage, and of the number of life cycle stages, in which they participate in olfaction. However, we detected a relationship between evolutionary rates and the position that the gene products occupy in the olfactory system, genes occupying central positions tend to be more constrained than peripheral genes. Finally, we demonstrate that specialization to one host does not seem to be associated with bursts of adaptive evolution in olfactory genes in D. sechellia and D. erecta, the two specialists species analyzed, but rather different lineages have idiosyncratic evolutionary histories in which both historical and ecological factors have been involved. © the author(s), publisher and licensee Libertas Academica Ltd.

Descripción: The present study first addressed the question of whether developmental time (DT) and viability (VT) vary clinally along latitudinal and altitudinal gradients in Drosophila buzzatii, an autochthonous specialist and the generalist invasive Drosophila melanogaster. Coincident and positive altitudinal clines across species and, direct and inverse latitudinal clines were observed for DT in D. melanogaster and D. buzzatii, respectively. Opposing latitudinal and altitudinal clines were detected for VT only in D. melanogaster. The patterns observed along altitudinal gradients prompted us to investigate whether flies living at lowland and highland environments may respond differentially to thermal treatments consisting of regimes of constant and alternating temperatures. Flies reared at higher mean temperature developed faster than at lower mean temperature in both species. By contrast, the response in VT differed greatly between species. Highland D. melanogaster were more viable than lowland regardless the treatment, whereas, in D. buzzatii, highland flies were more viable than lowland in alternating thermal regimes and the reverse was true in treatments of constant temperature. The results obtained suggest that thermal amplitude may be an important factor that should be considered in investigations of thermal adaptation. © 2008 The Linnean Society of London.

Descripción: Even though substantial progress has been made to elucidate the physiological and environmental factors underpinning differences in body size, little is known about its genetic architecture. Furthermore, all animal species bear a specific relationship between the size of each organ and overall body size, so different body size traits should be investigated as well as their sexual dimorphism that may have an important impact on the evolution of body size. We have surveyed 191 co-isogenic lines of Drosophila melanogaster, each one of them homozygous for a single P-element insertion, and assessed the effects of mutations on different body size traits compared to the P-element-free co-isogenic control. Nearly 60% of the lines showed significant differences with respect to the control for these traits in one or both sexes and almost 35% showed trait- and sex-specific effects. Candidate gene mutations frequently increased body size in males and decreased it in females. Among the 92 genes identified, most are involved in development and/or metabolic processes and their molecular functions principally include protein-binding and nucleic acid-binding activities. Although several genes showed pleiotropic effects in relation to body size, few of them were involved in the expression of all traits in one or both sexes. These genes seem to be important for different aspects related to the general functioning of the organism. In general, our results indicate that the genetic architecture of body size traits involves a large fraction of the genome and is largely sex and trait specific. © 2009 Macmillan Publishers Limited All rights reserved.

Descripción: Insects in general, and Drosophila in particular, are much more capable of surviving anoxia than vertebrates, and the mechanisms involved are of considerable biomedical and ecological interest. Temperature is likely to strongly affect both the rates of damage occurring in anoxia and the recovery processes in normoxia, but as yet there is no information on the effect of this crucial variable on recovery rates from anoxia in any animal. We studied the effects of temperature, and thus indirectly of metabolic flux rates, on survival and recovery times of individual male Drosophila melanogaster following anoxia and O2 reperfusion. Individual flies were reared at 25° and exposed to an anoxic period of 7.5, 25, 42.5 or 60?min at 20, 25 or 30°. Before, during and after anoxic exposure the flies' metabolic rates (MRs), rates of water loss and activity indices were recorded. Temperature strongly affected the MR of the flies, with a Q10 of 2.21. Temperature did not affect the slope of the relationship between time to recovery and duration of anoxic exposure, suggesting that thermal effects on damage and repair rates were similar. However, the intercept of that relationship was significantly lower (i.e. recovery was most rapid) at 25°, which was the rearing temperature. When temperatures during exposure to anoxia and during recovery were switched, recovery times matched those predicted from a model in which the accumulation and clearance of metabolic end-products share a similar dependence on temperature. ©2011. Published by The Company of Biologists Ltd.

Descripción: Odour-guided behaviour is a quantitative trait determined by many genes that are sensitive to gene-environment interactions. Different natural populations are likely to experience different selection pressures on the genetic underpinnings of chemosensory behaviour. However, few studies have reported comparisons of the quantitative genetic basis of olfactory behaviour in geographically distinct populations. We generated isofemale lines of Drosophila melanogaster from six populations in Argentina and measured larval and adult responses to benzaldehyde. There was significant variation within populations for both larval and adult olfactory behaviour and a significant genotype × sex interaction (GSI) for adult olfactory behaviour. However, there is substantial variation in the contribution of GSI to the total phenotypic variance among populations. Estimates of evolvability are orders of magnitude higher for larvae than for adults. Our results suggest that the potential for evolutionary adaptation to the chemosensory environment is greater at the larval feeding stage than at the adult reproductive stage. © 2008 The Authors.

Descripción: Survival of a potentially lethal high temperature stress is a genetically variable thermal adaptation trait in many organisms. Organisms cope with heat stress by basal or induced thermoresistance. Here, we tested quantitative trait loci (QTL) for heat stress survival (HSS) in Drosophila melanogaster, with and without a cyclic heat-hardening pre-treatment, for flies that were reared at low (LD) or high (HD) density. Mapping populations were two panels of recombinant inbred lines (RIL), which were previously constructed from heat stress-selected stocks: RIL-D48 and RIL-SH2, derived from backcrosses to stocks of low and high heat resistance, respectively. HSS increased with heat hardening in both LD and HD flies. In addition, HSS increased consistently with density in non-hardened flies. There was a significant interaction between heat hardening and density effects in RIL-D48. Several QTL were significant for both density and hardening treatments. Many QTL overlapped with thermotolerance QTL identified for other traits in previous studies based on LD cultures only. However, three new QTL were found in HD only (cytological ranges: 12E-16F6; 30A3-34C2; 49C-50C). Previously found thermotolerance QTL were also significant for flies from HD cultures. © 2012. Published by The Company of Biologists Ltd.

Descripción: Understanding the genetic architecture of any quantitative trait requires identifying the genes involved in its expression in different environmental conditions. This goal can be achieved by mutagenesis screens in genetically tractable model organisms such as Drosophila melanogaster. Temperature during ontogenesis is an important environmental factor affecting development and phenotypic variation in holometabolous insects. In spite of the importance of phenotypic plasticity and genotype by environment interaction (GEI) for fitness related traits, its genetic basis has remained elusive. In this context, we analyzed five different adult morphological traits (face width, head width, thorax length, wing size and wing shape) in 42 co-isogenic single P-element insertional lines of Drosophila melanogaster raised at 17°C and 25°C. Our analyses showed that all lines differed from the control for at least one trait in males or females at either temperature. However, no line showed those differences for all traits in both sexes and temperatures simultaneously. In this sense, the most pleiotropic candidate genes were CG34460, Lsd-2 and Spn. Our analyses also revealed extensive genetic variation for all the characters mostly indicated by strong GEIs. Further, our results indicate that GEIs were predominantly explained by changes in ranking order in all cases suggesting that a moderate number of genes are involved in the expression of each character at both temperatures. Most lines displayed a plastic response for at least one trait in either sex. In this regard, P-element insertions affecting plasticity of a large number of traits were associated to the candidate genes Btk29A, CG43340, Drak and jim. Further studies will help to elucidate the relevance of these genes on the morphogenesis of different body structures in natural populations of D. melanogaster. © 2013 Carreira et al.

Descripción: Background. Understanding the genetic architecture of ecologically relevant adaptive traits requires the contribution of developmental and evolutionary biology. The time to reach the age of reproduction is a complex life history trait commonly known as developmental time. In particular, in holometabolous insects that occupy ephemeral habitats, like fruit flies, the impact of developmental time on fitness is further exaggerated. The present work is one of the first systematic studies of the genetic basis of developmental time, in which we also evaluate the impact of environmental variation on the expression of the trait. Results. We analyzed 179 co-isogenic single P[GT1]-element insertion lines of Drosophila melanogaster to identify novel genes affecting developmental time in flies reared at 25°C. Sixty percent of the lines showed a heterochronic phenotype, suggesting that a large number of genes affect this trait. Mutant lines for the genes Merlin and Karl showed the most extreme phenotypes exhibiting a developmental time reduction and increase, respectively, of over 2 days and 4 days relative to the control (a co-isogenic P-element insertion free line). In addition, a subset of 42 lines selected at random from the initial set of 179 lines was screened at 17°C. Interestingly, the gene-by-environment interaction accounted for 52% of total phenotypic variance. Plastic reaction norms were found for a large number of developmental time candidate genes. Conclusion. We identified components of several integrated time-dependent pathways affecting egg-to-adult developmental time in Drosophila. At the same time, we also show that many heterochronic phenotypes may arise from changes in genes involved in several developmental mechanisms that do not explicitly control the timing of specific events. We also demonstrate that many developmental time genes have pleiotropic effects on several adult traits and that the action of most of them is sensitive to temperature during development. Taken together, our results stress the need to take into account the effect of environmental variation and the dynamics of gene interactions on the genetic architecture of this complex life-history trait. © 2008 Mensch et al; licensee BioMed Central Ltd.

Descripción: Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke. © 2010 Dekanty et al.

Descripción: Hypoxia-Inducible Factor (HIF) prolyl hydroxylase domains (PHDs) have been proposed to act as sensors that have an important role in oxygen homeostasis. In the presence of oxygen, they hydroxylate two specific prolyl residues in HIF-α polypeptides, thereby promoting their proteasomal degradation. So far, however, the developmental consequences of the inactivation of PHDs in higher metazoans have not been reported. Here, we describe novel loss-of-function mutants of fatiga, the gene encoding the Drosophila PHD oxygen sensor, which manifest growth defects and lethality. We also report a null mutation in dHIF-α/sima, which is unable to adapt to hypoxia but is fully viable in normoxic conditions. Strikingly, loss-of-function mutations of sima rescued the developmental defects observed in fatiga mutants and enabled survival to adulthood. These results indicate that the main functions of Fatiga in development, including control of cell size, involve the regulation of dHIF/Sima. © 2005 European Molecular Biology Organization.

Descripción: The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of a constitutively expressed HIF-β subunit and an oxygen-regulated HIF-α subunit. We have previously defined a hypoxia-inducible transcriptional response in Drosophila melanogaster that is homologous to the mammalian HIF-dependent response. In Drosophila, the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) are the functional homologues of the mammalian HIF-α and HIF-β subunits, respectively. HIF-α/Sima is regulated by oxygen at several different levels that include protein stability and subcellular localization. We show here for the first time that insulin can activate HIF-dependent transcription, both in Drosophila S2 cells and in living Drosophila embryos. Using a pharmacological approach as well as RNA interference, we determined that the effect of insulin on HIF-dependent transcriptional induction is mediated by PI3K-AKT and TOR pathways. We demonstrate that stimulation of the transcriptional response involves upregulation of Sima protein but not sima mRNA. Finally, we have analyzed in vivo the effect of the activation of the PI3K-AKT pathway on the subcellular localization of Sima protein. Overexpression of dAKT and dPDK1 in normoxic embryos provoked a major increase in Sima nuclear localization, mimicking the effect of a hypoxic treatment. A similar increase in Sima nuclear localization was observed in dPTEN homozygous mutant embryos, confirming that activation of the PI3K-AKT pathway promotes nuclear accumulation of Sima protein. We conclude that regulation of HIF-α/Sima by the PI3K-AKT-TOR pathway is a major conserved mode of regulation of the HIF-dependent transcriptional response in Drosophila.

Descripción: Background: The Hypoxia Inducible Factor (HIF) mediates cellular adaptations to low oxygen. Prolyl-4-hydroxylases are oxygen sensors that hydroxylate the HIF alpha-subunit, promoting its proteasomal degradation in normoxia. Three HIFprolyl hydroxylases, encoded by independent genes, PHD1, PHD2, and PHD3, occur in mammals. PHD2, the longest PHD isoform includes a MYND domain, whose biochemical function is unclear. PHD2 and PHD3 genes are induced in hypoxia to shut down HIF dependent transcription upon reoxygenation, while expression of PHD1 is oxygen-independent. The physiologic significance of the diversity of the PHD oxygen sensors is intriguing. Methodology and Principal Findings: We have analyzed the Drosophila PHD locus, fatiga, which encodes 3 isoforms, FgaA, FgaB and FgaC that are originated through a combination of alternative initiation of transcription and alternative splicing. FgaA includes a MYND domain and is homologous to PHD2, while FgaB and FgaC are shorter isoforms most similar to PHD3. Through a combination of genetic experiments in vivo and molecular analyses in cell culture, we show that fgaB but not fgaA is induced in hypoxia, in a Sima-dependent manner, through a HIF-Responsive Element localized in the first intron of fgaA. The regulatory capacity of FgaB is stronger than that of FgaA, as complete reversion of fga loss-of-function phenotypes is observed upon transgenic expression of the former, and only partial rescue occurs after expression of the latter. Conclusions and Significance: Diversity of PHD isoforms is a conserved feature in evolution. As in mammals, there are hypoxia-inducible and non-inducible Drosophila PHDs, and a fly isoform including a MYND domain co-exists with isoforms lacking this domain. Our results suggest that the isoform devoid of a MYND domain has stronger regulatory capacity than that including this domain.

Descripción: Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. Here, we show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-α homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. We propose that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting. © 2008 Elsevier Inc. All rights reserved.

Descripción: Background: Human β-amyloid, the main component in the neuritic plaques found in patients with Alzheimer's disease, is generated by cleavage of the β-amyloid precursor protein. Beyond the role in pathology, members of this protein family are synaptic proteins and have been associated with synaptogenesis, neuronal plasticity and memory, both in vertebrates and in invertebrates. Consolidation is necessary to convert a short-term labile memory to a long-term and stable form. During consolidation, gene expression and de novo protein synthesis are regulated in order to produce key proteins for the maintenance of plastic changes produced during the acquisition of new information.Results: Here we partially cloned and sequenced the beta-amyloid precursor protein like gene homologue in the crab Chasmagnathus (cappl), showing a 37% of identity with the fruit fly Drosophila melanogaster homologue and 23% with Homo sapiens but with much higher degree of sequence similarity in certain regions. We observed a wide distribution of cappl mRNA in the nervous system as well as in muscle and gills. The protein localized in all tissues analyzed with the exception of muscle. Immunofluorescence revealed localization of cAPPL in associative and sensory brain areas. We studied gene and protein expression during long-term memory consolidation using a well characterized memory model: the context-signal associative memory in this crab species. mRNA levels varied at different time points during long-term memory consolidation and correlated with cAPPL protein levels. Conclusions: cAPPL mRNA and protein is widely distributed in the central nervous system of the crab and the time course of expression suggests a role of cAPPL during long-term memory formation. © 2010 Fustiñana et al; licensee BioMed Central Ltd.

Descripción: Genomes contain the necessary information to ensure that genes are expressed in the right place, at the right time, and with the proper rate. Metazoan developmental genes often possess long stretches of DNA flanking their coding sequences and/or large introns which contain elements that influence gene expression. Most of these regulatory elements are relatively small and can be studied in isolation. For example, transcriptional enhancers, the elements that generate the expression pattern of a gene, have been traditionally studied with reporter constructs in transgenic animals. These studies have provided and will provide invaluable insights into enhancer evolution and function. However, this experimental approach has its limits; often, enhancer elements do not faithfully recapitulate native expression patterns. This fact suggests that additional information in cis-regulatory regions modulates the activity of enhancers and other regulatory elements. Indeed, recent studies have revealed novel functional aspects at the level of whole cis-regulatory regions. First, the discovery of "shadow enhancers." Second, the ubiquitous interactions between cis-regulatory elements. Third, the notion that some cis-regulatory regions may not function in a modular manner. Last, the effect of chromatin conformation on cis-regulatory activity. In this article, I describe these recent findings and discuss open questions in the field. © 2012 Wiley Periodicals, Inc.

Descripción: The spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing foci that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular foci. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing foci termed Smaug 1 foci (S-foci) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny. © 2012 Perez-Pepe et al.