Evolution at multiple processing levels underlies odor-guided behavior in the genus Drosophila.

Olfaction is a fundamental sense guiding animals to their food. How the olfactory system evolves and influences behavior is still poorly understood. Here, we selected five drosophilid species, including Drosophila melanogaster, inhabiting different ecological niches to compare their olfactory systems at multiple levels. We first identified ecologically relevant natural food odorants from every species and established species-specific odorant preferences. To compare odor coding in sensory neurons, we analyzed the antennal lobe (AL) structure, generated glomerular atlases, and developed GCaMP transgenic lines for all species. Although subsets of glomeruli showed distinct tuning profiles, odorants inducing species-specific preferences were coded generally similarly. Species distantly related or occupying different habitats showed more evident differences in odor coding, and further analysis revealed that changes in olfactory receptor (OR) sequences partially explain these differences. Our results demonstrate that genetic distance in phylogeny and ecological niche occupancy are key determinants in the evolution of ORs, AL structures, odor coding, and behavior. Interestingly, changes in odor coding among species could not be explained by evolutionary changes at a single olfactory processing level but rather are a complex phenomenon based on changes at multiple levels.

Functional olfactory evolution in Drosophila suzukii and the subgenus Sophophora.

Comparative analyses of multiple genomes are used extensively to examine the gains and losses of chemosensory receptors across the genus Drosophila. However, few studies have delved into functional olfactory characteristics. Here we assess olfactory function across 20 species, and identify and describe several similar elements of evolution. We document (a) minor changes in functional ligands based on amino acid substitutions, (b) major changes in olfactory function or perhaps entire receptor replacements, and (c) that only a few receptors are subject to repeated changes, whereas 32 out of 37 OSNs are largely functionally conserved. In addition, we generate a robust model for identifying olfactory function using genomic data and comprehensive ligand-receptor combinations, which includes the prediction of binding pockets. Moreover, this study highlights that functional olfactory evolution does not affect all chemosensory receptors equally, and that ecological, evolutionary, and developmental forces repeatedly affect only a small subset of available receptor proteins.

Mate discrimination among subspecies through a conserved olfactory pathway.

Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster. Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner.

Sobremesa L-type Amino Acid Transporter Expressed in Glia Is Essential for Proper Timing of Development and Brain Growth.

In Drosophila, ecdysone hormone levels determine the timing of larval development. Its production is regulated by the stereotypical rise in prothoracicotropic hormone (PTTH) levels. Additionally, ecdysone levels can also be modulated by nutrition (specifically by amino acids) through their action on Drosophila insulin-like peptides (Dilps). Moreover, in glia, amino-acid-sensitive production of Dilps regulates brain development. In this work, we describe the function of an SLC7 amino acid transporter, Sobremesa (Sbm). Larvae with reduced Sbm levels in glia remain in third instar for an additional 24 hr. These larvae show reduced brain growth with increased body size but do not show reduction in insulin signaling or production. Interestingly, Sbm downregulation in glia leads to reduced Ecdysone production and a surprising delay in the rise of PTTH levels. Our work highlights Sbm as a modulator of both brain development and the timing of larval development via an amino-acid-sensitive and Dilp-independent function of glia.

Olfactory detection of a bacterial short-chain fatty acid acts as an orexigenic signal in Drosophila melanogaster larvae.

Microorganisms inhabiting fermenting fruit produce chemicals that elicit strong behavioral responses in flies. Depending on their ecological niche, individuals confer a positive or a negative valence to a chemical and, accordingly, they trigger either attractive or repulsive behaviors. We studied the case of bacterial short-chain fatty acids (SCFA) that trigger opposite behaviors in adult and larvae of Drosophila melanogaster. We determined that SCFA-attractive responses depend on two larval exclusive chemoreceptors, Or30a and Or94b. Of those SCFA, propionic acid improves larval survival in suboptimal rearing conditions and supports growth. Olfactory detection of propionic acid specifically is sufficient to trigger feeding behaviors, and this effect requires the correct activity of Or30a+ and Or94b+ olfactory sensory neurons. Additionally, we studied the case of the invasive pest Drosophila suzukii that lives on undamaged ripe fruit with less SCFA production. Contrary to D. melanogaster, D. suzukii larvae show reduced attraction towards propionic acid, which does not trigger feeding behavior in this invasive species. Our results demonstrate the relevance of propionic acid as an orexigenic signal in D. melanogaster larvae. Moreover, this study underlines that the changes on ecological niche are accompanied with alterations of olfactory preferences and vital olfactory driven behaviors.

Mmp1 processing of the PDF neuropeptide regulates circadian structural plasticity of pacemaker neurons.

In the Drosophila brain, the neuropeptide PIGMENT DISPERSING FACTOR (PDF) is expressed in the small and large Lateral ventral neurons (LNvs) and regulates circadian locomotor behavior. Interestingly, PDF immunoreactivity at the dorsal terminals changes across the day as synaptic contacts do as a result of a remarkable remodeling of sLNv projections. Despite the relevance of this phenomenon to circuit plasticity and behavior, the underlying mechanisms remain poorly understood. In this work we provide evidence that PDF along with matrix metalloproteinases (Mmp1 and 2) are key in the control of circadian structural remodeling. Adult-specific downregulation of PDF levels per se hampers circadian axonal remodeling, as it does altering Mmp1 or Mmp2 levels within PDF neurons post-developmentally. However, only Mmp1 affects PDF immunoreactivity at the dorsal terminals and exerts a clear effect on overt behavior. In vitro analysis demonstrated that PDF is hydrolyzed by Mmp1, thereby suggesting that Mmp1 could directly terminate its biological activity. These data demonstrate that Mmp1 modulates PDF processing, which leads to daily structural remodeling and circadian behavior.

Circadian pacemaker neurons change synaptic contacts across the day.

Daily cycles of rest and activity are a common example of circadian control of physiology. In Drosophila, rhythmic locomotor cycles rely on the activity of 150-200 neurons grouped in seven clusters [1, 2]. Work from many laboratories points to the small ventral lateral neurons (sLNvs) as essential for circadian control of locomotor rhythmicity [3-7]. sLNv neurons undergo circadian remodeling of their axonal projections, opening the possibility for a circadian control of connectivity of these relevant circadian pacemakers [8]. Here we show that circadian plasticity of the sLNv axonal projections has further implications than mere structural changes. First, we found that the degree of daily structural plasticity exceeds that originally described [8], underscoring that changes in the degree of fasciculation as well as extension or pruning of axonal terminals could be involved. Interestingly, the quantity of active zones changes along the day, lending support to the attractive hypothesis that new synapses are formed while others are dismantled between late night and the following morning. More remarkably, taking full advantage of the GFP reconstitution across synaptic partners (GRASP) technique [9], we showed that, in addition to new synapses being added or removed, sLNv neurons contact different synaptic partners at different times along the day. These results lead us to propose that the circadian network, and in particular the sLNv neurons, orchestrates some of the physiological and behavioral differences between day and night by changing the path through which information travels.

Adult-specific electrical silencing of pacemaker neurons uncouples molecular clock from circadian outputs.

BACKGROUND: Circadian rhythms regulate physiology and behavior through transcriptional feedback loops of clock genes running within specific pacemaker cells. In Drosophila, molecular oscillations in the small ventral lateral neurons (sLNvs) command rhythmic behavior under free-running conditions releasing the neuropeptide PIGMENT DISPERSING FACTOR (PDF) in a circadian fashion. Electrical activity in the sLNvs is also required for behavioral rhythmicity. Yet, how temporal information is transduced into behavior remains unclear. RESULTS: Here we developed a new tool for temporal control of gene expression to obtain adult-restricted electrical silencing of the PDF circuit, which led to reversible behavioral arrhythmicity. Remarkably, PERIOD (PER) oscillations during the silenced phase remained unaltered, indicating that arrhythmicity is a direct consequence of the silenced activity. Accordingly, circadian axonal remodeling and PDF accumulation were severely affected during the silenced phase. CONCLUSIONS: Although electrical activity of the sLNvs is not a clock component, it coordinates circuit outputs leading to rhythmic behavior.

A methyl transferase links the circadian clock to the regulation of alternative splicing.

Circadian rhythms allow organisms to time biological processes to the most appropriate phases of the day-night cycle. Post-transcriptional regulation is emerging as an important component of circadian networks, but the molecular mechanisms linking the circadian clock to the control of RNA processing are largely unknown. Here we show that PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5), which transfers methyl groups to arginine residues present in histones and Sm spliceosomal proteins, links the circadian clock to the control of alternative splicing in plants. Mutations in PRMT5 impair several circadian rhythms in Arabidopsis thaliana and this phenotype is caused, at least in part, by a strong alteration in alternative splicing of the core-clock gene PSEUDO RESPONSE REGULATOR 9 (PRR9). Furthermore, genome-wide studies show that PRMT5 contributes to the regulation of many pre-messenger-RNA splicing events, probably by modulating 5'-splice-site recognition. PRMT5 expression shows daily and circadian oscillations, and this contributes to the mediation of the circadian regulation of expression and alternative splicing of a subset of genes. Circadian rhythms in locomotor activity are also disrupted in dart5-1, a mutant affected in the Drosophila melanogaster PRMT5 homologue, and this is associated with alterations in splicing of the core-clock gene period and several clock-associated genes. Our results demonstrate a key role for PRMT5 in the regulation of alternative splicing and indicate that the interplay between the circadian clock and the regulation of alternative splicing by PRMT5 constitutes a common mechanism that helps organisms to synchronize physiological processes with daily changes in environmental conditions.

[Effects of the early administration of losartan on ventricular remodeling in rabbits with experimental myocardial infarction]. / Efectos de la administración temprana de losartán sobre el remodelamiento ventricular en conejos con infarto de miocardio experimental.

This study was carried out to investigate the effects of early administration of losartan on ventricular remodelling (VR) in rabbits with experimental myocardial infarction (MI). New Zealand rabbits underwent left coronary artery ligation. Four groups were analyzed: sham (G1; n = 13), MI (G2; n = 13), sham + Los (G3; n = 13) and MI + Los (G4; n = 13). Los (12.5 mg/kg/d) was administered 3 hours post-MI during 35 days when the animals were sacrificed. The hearts were isolated and perfused using Langendorff's technique to determine systolic and diastolic pressure-volume (P/V) curves. Hearts were weighed, fixed in formaline, cut from apex to base, and stained with Masson's trichrome and pricrosirius red. The heart weight (HW)/body weight (BW) ratio was used as an index of hypertrophy. Infarct size (IS; %), septum (SeT, mm) and scar thickness (ST, mm) were measured using morphometric analysis. Results were expressed as mean +/- SEM. IS was G2 = 25.38 +/- 5.31; G4 = 21.85 +/- 4.13 (NS). HW/BW was 3.45 +/- 0.16; 3.23 +/- 0.25; 2.87 +/- 0.16; 3.23 +/- 0.18 in G1, G2, G3 and G4, respectively. Los shifted the diastolic P/V relationship to the right in sham and MI (p < 0.05 vs sham), and did not modify the systolic relationship. Scar collagen concentration was lower in G4 (p < 0.05 vs G2). SeT was lower in G3 and G4 (p < 0.05 vs G2). In conclusion, the early administration of Los unfavorably modified post-MI-VR, increasing ventricular dilation and reducing scar collagen concentration and thickness.

Efectos de la administración temprana de losartán sobre el remodelamiento ventricular en conejos con infarto de miocardio experimental. / [Effects of the early administration of losartan on ventricular remodeling in rabbits with experimental myocardial infarction]

This study was carried out to investigate the effects of early administration of losartan on ventricular remodelling (VR) in rabbits with experimental myocardial infarction (MI). New Zealand rabbits underwent left coronary artery ligation. Four groups were analyzed: sham (G1; n = 13), MI (G2; n = 13), sham + Los (G3; n = 13) and MI + Los (G4; n = 13). Los (12.5 mg/kg/d) was administered 3 hours post-MI during 35 days when the animals were sacrificed. The hearts were isolated and perfused using Langendorffs technique to determine systolic and diastolic pressure-volume (P/V) curves. Hearts were weighed, fixed in formaline, cut from apex to base, and stained with Massons trichrome and pricrosirius red. The heart weight (HW)/body weight (BW) ratio was used as an index of hypertrophy. Infarct size (IS;

), septum (SeT, mm) and scar thickness (ST, mm) were measured using morphometric analysis. Results were expressed as mean +/- SEM. IS was G2 = 25.38 +/- 5.31; G4 = 21.85 +/- 4.13 (NS). HW/BW was 3.45 +/- 0.16; 3.23 +/- 0.25; 2.87 +/- 0.16; 3.23 +/- 0.18 in G1, G2, G3 and G4, respectively. Los shifted the diastolic P/V relationship to the right in sham and MI (p < 0.05 vs sham), and did not modify the systolic relationship. Scar collagen concentration was lower in G4 (p < 0.05 vs G2). SeT was lower in G3 and G4 (p < 0.05 vs G2). In conclusion, the early administration of Los unfavorably modified post-MI-VR, increasing ventricular dilation and reducing scar collagen concentration and thickness.