Quantitative genetic analysis of attractiveness of yeast products to Drosophila.

An attractive perfume is a complex mixture of compounds, some of which may be unpleasant on their own. This is also true for the volatile combinations from yeast fermentation products in vineyards and orchards when assessed by Drosophila. Here we used crosses between a yeast strain with an attractive fermentation profile and another strain with a repulsive one and tested flies responses using a T-maze. QTL analysis reveals allelic variation in four yeast genes, PTC6, SAT4, YFL040W, and ARI1, that modulated expression levels of volatile compounds (assessed by GC-MS) and in different combinations, generated various levels of attractiveness. The parent strain that is more attractive to Drosophila has repulsive alleles at two of the loci while the least attractive parent has attractive alleles. Behavioral assays using artificial mixtures mimicking the composition of odors from fermentation validated the results of GC-MS and QTL mapping, thereby directly connecting genetic variation in yeast to attractiveness in flies. This study can be used as a basis for dissecting the combination of olfactory receptors that mediate the attractiveness/repulsion of flies to yeast volatiles and may also serve as a model for testing the attractiveness of pest species such as Drosophila suzukii to their host fruit.

Correction: The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).

[This corrects the article DOI: 10.1371/journal.pgen.1011011.].

The circadian clock gene bmal1 is necessary for co-ordinated circatidal rhythms in the marine isopod Eurydice pulchra (Leach).

Circadian clocks in terrestrial animals are encoded by molecular feedback loops involving the negative regulators PERIOD, TIMELESS or CRYPTOCHROME2 and positive transcription factors CLOCK and BMAL1/CYCLE. The molecular basis of circatidal (~12.4 hour) or other lunar-mediated cycles (~15 day, ~29 day), widely expressed in coastal organisms, is unknown. Disrupting circadian clockworks does not appear to affect lunar-based rhythms in several organisms that inhabit the shoreline suggesting a molecular independence of the two cycles. Nevertheless, pharmacological inhibition of casein kinase 1 (CK1) that targets PERIOD stability in mammals and flies, affects both circadian and circatidal phenotypes in Eurydice pulchra (Ep), the speckled sea-louse. Here we show that these drug inhibitors of CK1 also affect the phosphorylation of EpCLK and EpBMAL1 and disrupt EpCLK-BMAL1-mediated transcription in Drosophila S2 cells, revealing a potential link between these two positive circadian regulators and circatidal behaviour. We therefore performed dsRNAi knockdown of Epbmal1 as well as the major negative regulator in Eurydice, Epcry2 in animals taken from the wild. Epcry2 and Epbmal1 knockdown disrupted Eurydice's circadian phenotypes of chromatophore dispersion, tim mRNA cycling and the circadian modulation of circatidal swimming, as expected. However, circatidal behaviour was particularly sensitive to Epbmal1 knockdown with consistent effects on the power, amplitude and rhythmicity of the circatidal swimming cycle. Thus, three Eurydice negative circadian regulators, EpCRY2, in addition to EpPER and EpTIM (from a previous study), do not appear to be required for the expression of robust circatidal behaviour, in contrast to the positive regulator EpBMAL1. We suggest a neurogenetic model whereby the positive circadian regulators EpBMAL1-CLK are shared between circadian and circatidal mechanisms in Eurydice but circatidal rhythms require a novel, as yet unknown negative regulator.

Bypassing mitochondrial defects rescues Huntington's phenotypes in Drosophila.

Huntington's disease (HD) is a fatal neurodegenerative disease with limited treatment options. Human and animal studies have suggested that metabolic and mitochondrial dysfunctions contribute to HD pathogenesis. Here, we use high-resolution respirometry to uncover defective mitochondrial oxidative phosphorylation and electron transfer capacity when a mutant huntingtin fragment is targeted to neurons or muscles in Drosophila and find that enhancing mitochondrial function can ameliorate these defects. In particular, we find that co-expression of parkin, an E3 ubiquitin ligase critical for mitochondrial dynamics and homeostasis, produces significant enhancement of mitochondrial respiration when expressed either in neurons or muscles, resulting in significant rescue of neurodegeneration, viability and longevity in HD model flies. Targeting mutant HTT to muscles results in larger mitochondria and higher mitochondrial mass, while co-expression of parkin increases mitochondrial fission and decreases mass. Furthermore, directly addressing HD-mediated defects in the fly's mitochondrial electron transport system, by rerouting electrons to either bypass mitochondrial complex I or complexes III-IV, significantly increases mitochondrial respiration and results in a striking rescue of all phenotypes arising from neuronal mutant huntingtin expression. These observations suggest that bypassing impaired mitochondrial respiratory complexes in HD may have therapeutic potential for the treatment of this devastating disorder.

Essential elements of radical pair magnetosensitivity in Drosophila.

Many animals use Earth's magnetic field (also known as the geomagnetic field) for navigation1. The favoured mechanism for magnetosensitivity involves a blue-light-activated electron-transfer reaction between flavin adenine dinucleotide (FAD) and a chain of tryptophan residues within the photoreceptor protein CRYPTOCHROME (CRY). The spin-state of the resultant radical pair, and therefore the concentration of CRY in its active state, is influenced by the geomagnetic field2. However, the canonical CRY-centric radical-pair mechanism does not explain many physiological and behavioural observations2-8. Here, using electrophysiology and behavioural analyses, we assay magnetic-field responses at the single-neuron and organismal levels. We show that the 52 C-terminal amino acid residues of Drosophila melanogaster CRY, lacking the canonical FAD-binding domain and tryptophan chain, are sufficient to facilitate magnetoreception. We also show that increasing intracellular FAD potentiates both blue-light-induced and magnetic-field-dependent effects on the activity mediated by the C terminus. High levels of FAD alone are sufficient to cause blue-light neuronal sensitivity and, notably, the potentiation of this response in the co-presence of a magnetic field. These results reveal the essential components of a primary magnetoreceptor in flies, providing strong evidence that non-canonical (that is, non-CRY-dependent) radical pairs can elicit magnetic-field responses in cells.

Genetic analysis of cryptochrome in insect magnetosensitivity.

The earth's magnetic field plays an important role in the spectacular migrations and navigational abilities of many higher animals, particularly birds. However, these organisms are not amenable to genetic analysis, unlike the model fruitfly, Drosophila melanogaster, which can respond to magnetic fields under laboratory conditions. We therefore review the field of insect magnetosensitivity focusing on the role of the Cryptochromes (CRYs) that were first identified in Arabidopsis and Drosophila as key molecular components of circadian photo-entrainment pathways. Physico-chemical studies suggest that photo-activation of flavin adenine dinucleotide (FAD) bound to CRY generates a FADo- Trpo+ radical pair as electrons skip along a chain of specific Trp residues and that the quantum spin chemistry of these radicals is sensitive to magnetic fields. The manipulation of CRY in several insect species has been performed using gene editing, replacement/rescue and knockdown methods. The effects of these various mutations on magnetosensitivity have revealed a number of surprises that are discussed in the light of recent developments from both in vivo and in vitro studies.

Diurnal Differences in Intracellular Replication Within Splenic Macrophages Correlates With the Outcome of Pneumococcal Infection.

Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-α concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection.

Visualization of Mutant Aggregates from Clock Neurons by Agarose Gel Electrophoresis (AGERA) in Drosophila melanogaster.

The clock neurons of the fruit fly Drosophila melanogaster have become a useful model for expressing misfolded protein aggregates that accumulate in several human neurodegenerative diseases. One advantage of such an approach is that the behavioral effects can be readily quantified on circadian locomotor rhythms, sleep or activity levels via automated, highly reliable and objective procedures. Therefore, a rapid assay is required to visualize whether these neurons develop aggregates. Here we describe a modified immunoblot method, agarose gel electrophoresis (AGERA) that has been optimized for resolving aggregates from fly clock neurons.

Methods for Delivery of dsRNAi Against Canonical Clock Genes and Immunocytodetection of Clock Proteins in Crustacea.

The use of nonclassical model organisms for biological rhythm research has become popular in the last two decades. Here we describe techniques for delivery of dsRNAi molecules to knock down clock gene transcripts in a small intertidal crustacean, Eurydice pulchra, as well as our method for immunodetection of clock proteins in the brain. These methods can be generalized for gene knockdown in any small crustacean or arthropod in which mutagenesis by other methods is neither practical nor possible.

Photoperiod-Dependent Expression of MicroRNA in Drosophila.

Like many other insects in temperate regions, Drosophila melanogaster exploits the photoperiod shortening that occurs during the autumn as an important cue to trigger a seasonal response. Flies survive the winter by entering a state of reproductive arrest (diapause), which drives the relocation of resources from reproduction to survival. Here, we profiled the expression of microRNA (miRNA) in long and short photoperiods and identified seven differentially expressed miRNAs (dme-mir-2b, dme-mir-11, dme-mir-34, dme-mir-274, dme-mir-184, dme-mir-184*, and dme-mir-285). Misexpression of dme-mir-2b, dme-mir-184, and dme-mir-274 in pigment-dispersing, factor-expressing neurons largely disrupted the normal photoperiodic response, suggesting that these miRNAs play functional roles in photoperiodic timing. We also analyzed the targets of photoperiodic miRNA by both computational predication and by Argonaute-1-mediated immunoprecipitation of long- and short-day RNA samples. Together with global transcriptome profiling, our results expand existing data on other Drosophila species, identifying genes and pathways that are differentially regulated in different photoperiods and reproductive status. Our data suggest that post-transcriptional regulation by miRNA is an important facet of photoperiodic timing.

A latitudinal cline in a courtship song character of Drosophila melanogaster.

The courtship song of male Drosophila melanogaster is generated by wing vibration and contains an interpulse interval (IPI) which is species-specific and usually falls in the mean range of 30-40 ms. The IPI is extremely temperature-sensitive, so we wondered whether flies collected along the eastern coast of Australia between latitudes 16.9°S and 42.9°S might have adapted to the different thermal conditions and show differences in mean IPI. We observe a significant correlation between IPI and latitude in addition to the well-known association between latitude and body size (Bergmannn's Rule). However, somewhat surprisingly we could not detect a significant association between body size and IPI. We also examined flies collected from the North and South-facing slopes of 'Evolution Canyon' in Israel and observed differences in IPI that support the view that thermal adaptation can shape this important song character. We also examined the songs of flies from Kenya and observed no correlation between altitude of collection and IPI. In all three experiments, body size did not correlate with IPI. A global analysis of all three sets of populations on three continents revealed a strong association between IPI and latitude. We speculate that IPI is shaped by thermal and sexual selection whereas body size is also shaped by natural selection.

A novel role for kynurenine 3-monooxygenase in mitochondrial dynamics.

The enzyme kynurenine 3-monooxygenase (KMO) operates at a critical branch-point in the kynurenine pathway (KP), the major route of tryptophan metabolism. As the KP has been implicated in the pathogenesis of several human diseases, KMO and other enzymes that control metabolic flux through the pathway are potential therapeutic targets for these disorders. While KMO is localized to the outer mitochondrial membrane in eukaryotic organisms, no mitochondrial role for KMO has been described. In this study, KMO deficient Drosophila melanogaster were investigated for mitochondrial phenotypes in vitro and in vivo. We find that a loss of function allele or RNAi knockdown of the Drosophila KMO ortholog (cinnabar) causes a range of morphological and functional alterations to mitochondria, which are independent of changes to levels of KP metabolites. Notably, cinnabar genetically interacts with the Parkinson's disease associated genes Pink1 and parkin, as well as the mitochondrial fission gene Drp1, implicating KMO in mitochondrial dynamics and mitophagy, mechanisms which govern the maintenance of a healthy mitochondrial network. Overexpression of human KMO in mammalian cells finds that KMO plays a role in the post-translational regulation of DRP1. These findings reveal a novel mitochondrial role for KMO, independent from its enzymatic role in the kynurenine pathway.

Rab8 Promotes Mutant HTT Aggregation, Reduces Neurodegeneration, and Ameliorates Behavioural Alterations in a Drosophila Model of Huntington's Disease.

BACKGROUND: Altered cellular vesicle trafficking has been linked to the pathogenesis of Huntington's disease (HD), a fatal, inherited neurodegenerative disorder caused by mutation of the huntingtin (HTT) protein. The Rab GTPase family of proteins plays a key role in regulation of vesicle trafficking, with distinct Rabs helping specify membrane identity and mediating cellular processes including budding, motility and tethering of vesicles to their targets. In recent years several Rab GTPases-notably, Rab5 and Rab11-have been linked to the pathogenesis of neurodegenerative disorders, including HD. OBJECTIVE: We investigated whether Rab8, which regulates post-Golgi vesicle trafficking, is able to improve HD-relevant phenotypes in a well-characterised model. METHODS: We overexpressed Rab8 in a Drosophila model of HD testing cellular, behavioural, and molecular phenotypes. RESULTS: We found that Rab8 overexpression ameliorated several disease-related phenotypes in fruit flies expressing a mutant HTT fragment throughout the nervous system, including neurodegeneration of photoreceptor neurons, reduced eclosion of the adult fly from the pupal case and shortened lifespan. Rab8 overexpression also normalised aberrant circadian locomotor behaviour in flies expressing mutant HTT in a specific population of neurons that regulate the circadian clock. Intriguingly, expression of Rab8 increased the accumulation of SDS-insoluble aggregated species of mutant HTT. CONCLUSION: Collectively, our findings demonstrate that increased Rab8 levels protect against mutant HTT toxicity and potentiate its aggregation, likely reducing the accumulation of downstream toxic soluble species.

Norpa Signalling and the Seasonal Circadian Locomotor Phenotype in Drosophila.

In this paper, we review the role of the norpA-encoded phospholipase C in light and thermal entrainment of the circadian clock in Drosophila melanogaster. We extend our discussion to the role of norpA in the thermo-sensitive splicing of the per 3' UTR, which has significant implications for seasonal adaptations of circadian behaviour. We use the norpA mutant-generated enhancement of per splicing and the corresponding advance that it produces in the morning (M) and evening (E) locomotor component to dissect out the neurons that are contributing to this norpA phenotype using GAL4/UAS. We initially confirmed, by immunocytochemistry and in situ hybridisation in adult brains, that norpA expression is mostly concentrated in the eyes, but we were unable to unequivocally reveal norpA expression in the canonical clock cells using these methods. In larval brains, we did see some evidence for co-expression of NORPA with PDF in clock neurons. Nevertheless, downregulation of norpA in clock neurons did generate behavioural advances in adults, with the eyes playing a significant role in the norpA seasonal phenotype at high temperatures, whereas the more dorsally located CRYPTOCHROME-positive clock neurons are the likely candidates for generating the norpA behavioural effects in the cold. We further show that knockdown of the related plc21C encoded phospholipase in clock neurons does not alter per splicing nor generate any of the behavioural advances seen with norpA. Our results with downregulating norpA and plc21C implicate the rhodopsins Rh2/Rh3/Rh4 in the eyes as mediating per 3' UTR splicing at higher temperatures and indicate that the CRY-positive LNds, also known as 'evening' cells are likely mediating the low-temperature seasonal effects on behaviour via altering per 3'UTR splicing.

Disrupted Glutamate Signaling in Drosophila Generates Locomotor Rhythms in Constant Light.

We have used the Cambridge Protein Trap resource (CPTI) to screen for flies whose locomotor rhythms are rhythmic in constant light (LL) as a means of identifying circadian photoreception genes. From the screen of ∼150 CPTI lines, we obtained seven hits, two of which targeted the glutamate pathway, Got1 (Glutamate oxaloacetate transaminase 1) and Gs2 (Glutamine synthetase 2). We focused on these by employing available mutants and observed that variants of these genes also showed high levels of LL rhythmicity compared with controls. It was also clear that the genetic background was important with a strong interaction observed with the common and naturally occurring timeless (tim) polymorphisms, ls-tim and s-tim. The less circadian photosensitive ls-tim allele generated high levels of LL rhythmicity in combination with Got1 or Gs2, even though ls-tim and s-tim alleles do not, by themselves, generate the LL phenotype. The use of dsRNAi for both genes as well as for Gad (Glutamic acid decarboxylase) and the metabotropic glutamate receptor DmGluRA driven by clock gene promoters also revealed high levels of LL rhythmicity compared to controls. It is clear that the glutamate pathway is heavily implicated in circadian photoreception. TIM levels in Got1 and Gs2 mutants cycled and were more abundant than in controls under LL. Got1 but not Gs2 mutants showed diminished phase shifts to 10 min light pulses. Neurogenetic dissection of the LL rhythmic phenotype using the gal4/gal80 UAS bipartite system suggested that the more dorsal CRY-negative clock neurons, DNs and LNds were responsible for the LL phenotype. Immunocytochemistry using the CPTI YFP tagged insertions for the two genes revealed that the DN1s but not the DN2 and DN3s expressed Got1 and Gs2, but expression was also observed in the lateral neurons, the LNds and s-LNvs. Expression of both genes was also found in neuroglia. However, downregulation of glial Gs2 and Got1 using repo-gal4 did not generate high levels of LL rhythmicity, so it is unlikely that this phenotype is mediated by glial expression. Our results suggest a model whereby the DN1s and possibly CRY-negative LNds use glutamate signaling to supress the pacemaker s-LNvs in LL.

A Computational Error and Restricted Use of Time-series Analyses Underlie the Failure to Replicate period-Dependent Song Rhythms in Drosophila.

From 1980 to 1991, Kyriacou, Hall, and collaborators (K&H) reported that the Drosophila melanogaster courtship song has a 1-min cycle in the length of mean interpulse intervals (IPIs) that is modulated by circadian rhythm period mutations. In 2014, Stern failed to replicate these results using a fully automated method for detecting song pulses. Manual annotation of Stern's song records exposed a ~50% error rate in detection of IPIs, but the corrected data revealed period-dependent IPI cycles using a variety of statistical methods. In 2017, Stern et al. dismissed the sine/cosine method originally used by K&H to detect significant cycles, claiming that randomized songs showed as many significant values as real data using cosinor analysis. We first identify a simple mathematical error in Stern et al.'s cosinor implementation that invalidates their critique of the method. Stern et al. also concluded that although the manually corrected wild-type and perL mutant songs show similar periods to those observed by K&H, each song is usually not significantly rhythmic by the Lomb-Scargle (L-S) periodogram, so any genotypic effect simply reflects "noise." Here, we observe that L-S is extremely conservative compared with 3 other time-series analyses in assessing the significance of rhythmicity, both for conventional locomotor activity data collected in equally spaced time bins and for unequally spaced song records. Using randomization of locomotor and song data to generate confidence limits for L-S instead of the theoretically derived values, we find that L-S is now consistent with the other methods in determining significant rhythmicity in locomotor and song records and that it confirms period-dependent song cycles. We conclude that Stern and colleagues' failure to identify song cycles stems from the limitations of automated methods in accurately reflecting song parameters, combined with the use of an overly stringent method to discriminate rhythmicity in courtship songs.

Heme binding to human CLOCK affects interactions with the E-box.

The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.