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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.

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.

Monoamines like serotonin, dopamine, and adrenaline/noradrenaline (epinephrine/ norepinephrine) act as neuromodulators that tune the response of the nervous system to the environment with predictable advantages for fitness. For instance, monoamines influence action selection depending on the internal state of the organism, contribute to ‘higher’ cognitive functions like learning and memory formation and modulate fundamental homeostatic needs such as sleep or feeding. Despite their significance and the extensive research in model organisms, the evolutionary origin of the monoaminergic system is uncertain. Here using a phylogenomic approach we study the evolution of the majority of genes involved in the production, modulation, and detection of monoamines. Our analyses suggest that most of the genes of the monoaminergic system originated in the common ancestor of bilaterians. These findings suggest that the monoaminergic synaptic pathway is a bilaterian innovation. We hypothesise that monoaminergic neuromodulation contributed to the diversification and complexification of behaviour and forms found in Bilateria.

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.

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.

Whatever you were doing just before reading this, walking to work, drinking a coffee after lunch or winding down in the evening in your favourite armchair, it is likely you will do it again tomorrow, more or less at the same time. This daily cycle is a reflection of the fact that we live in a 24-hour environment that, through rhythmic changes in light, temperature and social interactions, imposes temporal constraints on our lives. However, we do not simply react to those daily changes; rather, we anticipate them and behave accordingly.

SummaryMany animals use the Earth’s magnetic field (geoMF) for navigation1. The favored mechanism for magnetosensitivity involves a blue-light (BL) activated electron transfer reaction between flavin adenine dinucleotide (FAD) and a chain of tryptophan (Trp) residues within the photoreceptor protein, CRYPTOCHROME (CRY). The spin-state of the resultant radical pair (RP), and hence the concentration of CRY in its active state, is influenced by the geoMF2. The canonical CRY-centric radical pair mechanism (RPM) does not, however, explain many physiological and behavioural observations2–8. Here, using electrophysiology and behavioural analyses, we assay magnetic field (MF) responses at single neuron and organismal level. We show that the 52 C-terminal (CT) amino acids of CRY, which are missing the canonical FAD binding domain and Trp chain, are sufficient to facilitate magnetoreception. We also show that increasing intracellular FAD potentiates both BL-induced and MF-dependent effects on the activity mediated by the CT. Additionally, high levels of FAD alone are sufficient to cause BL neuronal sensitivity and, remarkably, potentiation of this response in the co-presence of a MF. These unexpected results reveal the essential components of a primary magnetoreceptor in flies, providing strong evidence that non-canonical(i.e.,non-CRY-dependent) RPs can elicit MF responses in cells.

Diptera is one of the biggest insect orders and displays a large diversity of visual adaptations. Similarly to other animals, the dipteran visual process is mediated by opsin genes. Although the diversity and function of these genes are well studied in key model species, a comprehensive comparative genomic study across the dipteran phylogeny is missing. Here we mined the genomes of 61 dipteran species, reconstructed the evolutionary affinities of 528 opsin genes, and determined the selective pressure acting in different species. We found that opsins underwent several lineage-specific events, including an independent expansion of Long Wave Sensitive opsins in flies and mosquitoes, and numerous family-specific duplications and losses. Both the Drosophila and the Anopheles complement are derived in comparison with the ancestral dipteran state. Molecular evolutionary studies suggest that gene turnover rate, overall mutation rate, and site-specific selective pressure are higher in Anopheles than in Drosophila. Overall, our findings indicate an extremely variable pattern of opsin evolution in dipterans, showcasing how two similarly aged radiations, Anopheles and Drosophila, are characterized by contrasting dynamics in the evolution of this gene family. These results provide a foundation for future studies on the dipteran visual system.

Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is one of a few neurotrophic factors described in Drosophila melanogaster (DmMANF) but its function is still poorly characterized. In the present study we found that DmMANF is expressed in different clusters of clock neurons. In particular, the PDF-positive large (l-LNv) and small (s-LNv) ventral lateral neurons, the CRYPTOCHROME-positive dorsal lateral neurons (LNd), the group 1 dorsal neurons posterior (DN1p) and different tim-positive cells in the fly’s visual system. Importantly, DmMANF expression in the ventral lateral neurons is not controlled by the clock nor it affects its molecular mechanism. However, silencing DmMANF expression in clock neurons affects the rhythm of locomotor activity in light:dark and constant darkness conditions. Such phenotypes correlate with abnormal morphology of the dorsal projections of the s-LNv and with reduced arborizations of the l-LNv in the medulla of the optic lobe. Additionally, we show that DmMANF is important for normal morphology of the L2 interneurons in the visual system and for the circadian rhythm in the topology of their dendritic tree. Our results indicate that DmMANF is important not only for the development of neurites but also for maintaining circadian plasticity of neurons.

Opsin receptors mediate the visual process in animals and their evolutionary history can provide precious hints on the ecological factors that underpin their diversification. Here we mined the genomes of more than 60 Dipteran species and reconstructed the evolution of their opsin genes in a phylogenetic framework. Our phylogenies indicate that dipterans possess an ancestral set of five core opsins which have undergone several lineage-specific events including an independent expansion of low wavelength opsins in flies and mosquitoes and numerous family specific duplications and losses. Molecular evolutionary studies indicate that gene turnover rate, overall mutation rate, and site-specific selective pressure are higher in Anopheles than in Drosophila; we found signs of positive selection in both lineages, including events possibly associated with their peculiar behaviour. Our findings indicate an extremely variable pattern of opsin evolution in dipterans, showcasing how two similarly aged radiations - Anopheles and Drosophila - can be characterized by contrasting dynamics in the evolution of this gene family.

Aphids display life cycles largely determined by the photoperiod. During the warm long-day seasons, most aphid species reproduce by viviparous parthenogenesis. The shortening of the photoperiod in autumn induces a switch to sexual reproduction. Males and sexual females mate to produce overwintering resistant eggs. In addition to this full life cycle (holocycle), there are anholocyclic lineages that do not respond to changes in photoperiod and reproduce continuously by parthenogenesis. The molecular or hormonal events that trigger the seasonal response (i.e., induction of the sexual phenotypes) are still unknown. Although circadian synthesis of melatonin is known to play a key role in vertebrate photoperiodism, the involvement of the circadian clock and/or of the hormone melatonin in insect seasonal responses is not so well established. Here we show that melatonin levels in the aphid Acyrthosiphon pisum are significantly higher in holocyclic aphids reared under short days than under long days, while no differences were found between anholocyclic aphids under the same conditions. We also found that melatonin is localized in the aphid suboesophageal ganglion (SOG) and in the thoracic ganglionic mass (TGM). In analogy to vertebrates, insect-type arylalkylamine N-acetyltransferases (i-AANATs) are thought to play a key role in melatonin synthesis. We measured the expression of four i-AANAT genes identified in A. pisum and localized two of them in situ in the insect central nervous systems (CNS). Levels of expression of these genes were compatible with the quantities of melatonin observed. Moreover, like melatonin, expression of these genes was found in the SOG and the TGM.

Liang et al. (2017) demonstrate how neuropeptides from two groups of clock cells appear to be responsible for the fly's circadian neurons becoming active at different times of day. By delaying the activity of their clock cell targets, they give rise to morning and evening behavior.

The aim of this study was to investigate the toxic effects of aluminum (Al) on the model organism–Drosophila melanogaster. The study is especially concerned with the effects of aluminum on the fruit fly's development, life span, and circadian rhythm in rest and activity. Flies were exposed to aluminum in concentrations from 40 to 280 mg/kg in rearing media or the flies were raised on control medium. Moreover, the life span of insects exposed to aluminum containing 40, 120, or 240 mg/kg of Al in the medium, only during their larval development, during the whole life cycle and only in their adult life was tested. To check if aluminum and aging cause changes in D. melanogaster behavior, the locomotor activity of flies at different ages was recorded. Results showed that aluminum is toxic in concentrations above 160 mg/kg in the rearing medium. Depending on Al concentration and time of exposure, the life span of the flies was shortened. At intermediate concentrations (120 mg/kg), however, Al had a stimulating effect on males increasing their life span and level of locomotor activity. At higher concentration the aluminum exposure increased or decreased the level of locomotor activity of D. melanogaster depending on age of flies. In addition, in the oldest insects reared on aluminum supplemented media and in mid-aged flies reared on the highest concentration of Al the daily rhythm of activity was disrupted.

The high latitudes inhabited by Antarctic krill Euphausia superba Dana are charac- terised by extreme changes in seasonal conditions, with very low food availability, long periods of darkness and a massive expansion of sea ice in autumn and winter. How krill adapt to these con- ditions has yet to be determined, and it was the purpose of this study to gain more insight by inves- tigating the differences between summer and winter krill populations at the molecular level. To achieve this, we used a cDNA microarray to compare gene expression between female adult krill caught during summer and winter near the Antarctic Peninsula (60° S), and between Peninsula krill and krill caught near South Georgia (54° S) during the same winter. The Peninsula seasonal comparison showed up-regulation in summer krill of genes involved in feeding and digestion, res- piration, motor activity and immunity. Summer krill also showed evidence of increased vitelloge- nesis, with an insulin-related peptide up-regulated and neuroparsin down-regulated. The com- parison between the different overwintering locations showed an up-regulation in the South Georgia krill of genes involved in feeding and digestion, and immunity. There was, however, no differential expression of genes involved in respiration, motor activity or vitellogenesis, suggest- ing that despite the above differences, both populations of overwintering krill were in a quiescent state. This study shows how krill physiology changes seasonally at the molecular level and how these changes are flexible according to the overwintering environment.

1, indicating a role in the initial signalling of the hypertrophic response • Sustained cardiac over-expression of Abra can facilitate deterioration in hypertrophic hearts • Abra homologues has been identified in diverse species, including Drosophila melanogaster (dAbra) • The Drosophila has become a powerful model system for in vivo cardiac research

A change in photoperiod has been implicated in triggering a transition from an active to a quiescent state in Antarctic krill. We examined this process at the molecular level, to identify processes that are affected when passing a photoperiodic threshold. Antarctic krill captured in the austral autumn were divided into two groups and immediately incubated either under a photoperiod of 12 h light:12 h darkness (LD), simulating the natural light cycle, or in continuous darkness (DD), simulating winter. All other conditions were kept identical between incubations. After 7 days of adaptation, krill were sampled every 4 h over a 24 h period and frozen. Total RNA was extracted from the heads and pooled to construct a suppression subtractive hybridisation library. Differentially expressed sequences were identified and annotated into functional categories through database sequence matching. We found a difference in gene expression between LD and DD krill, with LD krill expressing more genes involved in functions such as metabolism, motor activity, protein binding and various other cellular activities. Eleven of these genes were examined further with quantitative polymerase chain reaction analyses, which revealed that expression levels were significantly higher in LD krill. The genes affected by simulated photoperiodic change are consistent with known features of quiescence, such as a slowing of moult rate, a lowering of activity levels and a reduction in metabolic rate. The expression of proteases involved in apolysis, where the old cuticle separates from the epidermis, showed particular sensitivity to photoperiod and point to the mechanism by which moult rate is adjusted seasonally. Our results show that key processes are already responding at the molecular level after just 7 days of exposure to a changed photoperiodic cycle. We propose that krill switch rapidly between active and quiescent states and that the photoperiodic cycle plays a key role in this process.

Antarctic krill (Euphausia superba) is a keystone species in the southern ocean ecosystem where it is the main consumer of phytoplankton and constitutes the main food item of many higher predators. Both food and predators are most abundant at the surface, thus krill hide in the depth of the ocean during the day and migrate to the upper layers at night, to feed at a time when the predatory risk is lowest. Although the functional significance of this diel vertical migration (DVM) is clear and its modulation by environmental factors has been described, the involvement of an endogenous circadian clock in this behaviour is as yet not fully resolved. We have analysed the circadian behaviour of Euphausia superba in a laboratory setting and here we present the first description of locomotor activity rhythms for this species. Our results are in agreement with the hypothesis that the circadian clock plays a key role in DVM. They also suggest that the interplay between food availability, social cues and the light:dark cycle acts as the predominant Zeitgeber for DVM in this species.

The genetic, molecular and anatomical dissection of the circadian clock in Drosophila and other higher organisms relies on the quantification of rhythmic phenotypes. Here, we introduce the methods currently in use in our laboratories for the analysis of fly locomotor activity rhythms. This phenotype provides a relatively simple, automated, efficient, reliable and robust output for the circadian clock. Thus it is not surprising that it is the preferred readout for measuring rhythmicity under a variety of conditions for most fly clock laboratories. The procedure requires at least 10 days of data collection and several days for analysis. In this protocol we advise on fly maintenance and on experimental design when studying the genetics of behavioral traits. We describe the setup for studying locomotor activity rhythms in the fruit fly and we introduce the statistical methods in use in our laboratories for the analysis of periodic data.

The circadian clock percolates through every aspect of behaviour and physiology, and has wide implications for human and animal health. The molecular basis of the Drosophila circadian clock provides a model system that has remarkable similarities to that of mammals. The various cardinal clock molecules in the fly are outlined, and compared to those of their actual and 'functional' homologues in the mammal. We also focus on the evolutionary tinkering of these clock genes and compare and contrast the neuronal basis for behavioural rhythms between the two phyla.

The Drosophila melanogaster circadian clock is generated by interlocked feedback loops, and null mutations in core genes such as period and timeless generate behavioral arrhythmicity in constant darkness. In light-dark cycles, the elevation in locomotor activity that usually anticipates the light on or off signals is severely compromised in these mutants. Light transduction pathways mediated by the rhodopsins and the dedicated circadian blue light photoreceptor cryptochrome are also critical in providing the circadian clock with entraining light signals from the environment. The cry b mutation reduces the light sensitivity of the fly's clock, yet locomotor activity rhythms in constant darkness or light-dark cycles are relatively normal, because the rhodopsins compensate for the lack of cryptochrome function. Remarkably, when we combined a period -null mutation with cry b , circadian rhythmicity in locomotor behavior in light-dark cycles, as measured by a number of different criteria, was restored. This effect was significantly reduced in timeless -null mutant backgrounds. Circadian rhythmicity in constant darkness was not restored, and TIM protein did not exhibit oscillations in level or localize to the nuclei of brain neurons known to be essential for circadian locomotor activity. Therefore, we have uncovered residual rhythmicity in the absence of period gene function that may be mediated by a previously undescribed period -independent role for timeless in the Drosophila circadian pacemaker. Although we do not yet have a molecular correlate for these apparently iconoclastic observations, we provide a systems explanation for these results based on differential sensitivities of subsets of circadian pacemaker neurons to light.

We have isolated the clock gene period (per) from the medfly Ceratitis capitata, one of the most economically important insect pest species. The overall pattern of conserved, non-conserved and functional domains that are observed within dipteran and lepidopteran per orthologues is preserved within the coding sequence. Expression analysis from fly heads revealed a daily oscillation in per mRNA in both light[ratio ]dark cycles and in constant darkness. However PER protein levels from head extracts did not show any significant evidence for cycling in either of these two conditions. When the Ceratitis per transgene under the control of the Drosophila per promoter and 3′UTR was introduced into Drosophila per-null mutant hosts, the transformants revealed a low level of rescue of behavioural rhythmicity. Nevertheless, the behaviour of the rhythmic transformants showed some similarities to that of Ceratitis, suggesting that Ceratitis per carries species-specific information that can evidently affect the Drosophila host's downstream rhythmic behaviour.

Summary Errant polychaete worms in the Orders Eunicida (family Eunicidae) and Phyllodocida (families Nereidae and Polynoidae) exhibit a highly developed biorhythmic capability. The Pacific palolo worms are well known for a precisely timed annual breeding event in which mass spawning occurs at a particular time of day, on one day per year, that day having a fixed relationship to the lunar period. Nereidae and Polynoidae exhibit photoperiodic responses that determine the breeding season by regulation of oocyte growth. Nereis virens shows short-term cycles of foraging activity; automated recording of these patterns has revealed four distinct behaviour rhythm phenotypes: circadian, tidal, lunidian and arrhythmic, the last phenotype being expressed during the photoperiod induced growth diapause. The Eunicids and Phyllodocids are represented in the fossil record by scolecodonts, their fossilised jaws. There was a major radiation of these polychaetes during the Ordovician and the earliest suggested polychaete fo...

Objectives To investigate the emergence of biological rhythms in the first months of life in human infants, by measuring age-related changes in core body temperature during night-time sleep, hormones (cortisol and 6-sulfatoxymelatonin) and the expression of a clock-controlled gene H3f3b in oral epithelial cells. Design Observational longitudinal study. Setting We measured overnight core body temperature, actigraphy, day–night urinary cortisol and 6-sulfatoxymelatonin, as well as circadian gene expression, in infants at home from March 2007 to July 2008 in Leicester. Participants We recruited 35 healthy Caucasian infants who were born at term. They were monitored from 6 to 18 weeks of age. Results At 8 weeks of age the day–night rhythm of cortisol secretion was the first to appear followed by 6-sulfatoxymelatonin 1 week later; at the same time that night-time sleep was established. At 10 weeks, the maximum fall in deep body temperature occurred with the onset of night-time sleep, followed at 11 weeks by the rhythmical expression of the H3f3b gene. Conclusions In human infants, there is a clear sequential pattern for the emergence of diurnal biological rhythms between 6 and 18 weeks of postnatal age, led by the secretion of cortisol and linked with the establishment of consolidated night-time sleep. It is likely that this represents part of a maturation and adaption process as infants gain equilibrium with their external environment after birth.

The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments., Author Summary Low frequency electromagnetic fields (EMFs) are associated with electrical power lines and have been implicated in the development of childhood leukemias. However, the Earth also has a natural EMF that animals can detect and which they use in order to navigate and orient themselves, particularly during migrations. One way they might do this is by using specialised photoreceptors called cryptochromes, which when activated by light, generate changes within the molecule that are susceptible to EMFs. Cryptochromes are important components of animal circadian clocks, the 24 hour timers that determine daily behavioral and physiological cycles. We have studied the circadian behavior of the fruitfly and have observed some novel and robust effects of EMFs on the fly's sleep-wake cycle that are mediated by cryptochrome. By using cryptochrome mutants we find that our results do not support the classic model for how this molecule might respond to EMFs. We also show that mammalian cryptochromes can respond to EMF when placed into transgenic Drosophila, whereas in mammalian clock neurons, they cannot. Consequently, the EMF responsiveness of cryptochrome is determined by its intracellular environment, suggesting that other, unknown molecules that interact with cryptochrome are also very important.

Nest construction is an essential component of the reproductive behavior of many species, and attributes of nests – including their location and structure – have implications for both their functional capacity as incubators for developing offspring, and their attractiveness to potential mates. To maximize reproductive success, nests must therefore be suited to local environmental conditions. Male three-spined sticklebacks (Gasterosteus aculeatus) build nests from collected materials and use an endogenous, glue-like multimeric protein – “spiggin” – as an adhesive. Spiggin is encoded by a multigene family, and differential expression of spiggin genes potentially allows plasticity in nest construction in response to variable environments. Here, we show that the expression of spiggin genes is affected significantly by both the flow regime experienced by a fish and its nesting status. Further, we show the effects of flow on expression patterns are gene-specific. Nest-building fish exhibited consistently higher expression levels of the three genes under investigation (Spg-a,Spg-1, and Spg-2) than non-nesting controls, irrespective of rearing flow treatment. Fish reared under flowing-water conditions showed significantly increased levels of spiggin gene expression compared to those reared in still water, but this effect was far stronger for Spg-a than for Spg-1 or Spg-2. The strong effect of flowing water on Spg-a expression, even among non-nesters, suggests that the increased production of spiggin – or of spiggin rich in the component contributed by Spg-a – may allow more rapid and/or effective nest construction under challenging high flow conditions.

Many higher animals have evolved the ability to use the Earth’s magnetic field, particularly for orientation. Drosophila melanogaster also respond to electromagnetic fields (EMFs), although the reported effects are quite modest. Here we report that negative geotaxis in flies, scored as climbing, is disrupted by a static EMF, and this is mediated by cryptochrome (CRY), the blue-light circadian photoreceptor. CRYs may sense EMFs via formation of radical pairs of electrons requiring photoactivation of flavin adenine dinucleotide (FAD) bound near a triad of Trp residues, but mutation of the terminal Trp in the triad maintains EMF responsiveness in climbing. In contrast, deletion of the CRY C terminus disrupts EMF responses, indicating that it plays an important signalling role. CRY expression in a subset of clock neurons, or the photoreceptors, or the antennae, is sufficient to mediate negative geotaxis and EMF sensitivity. Climbing therefore provides a robust and reliable phenotype for studying EMF responses in Drosophila., The earth’s electromagnetic field has a modest effect on the behaviour of Drosophila melanogaster. Here, Fedele et al. use an assessment of climbing behaviour to describe how the blue-light circadian photoreceptor cryptochrome mediates a negative movement response to gravity in flies.

The negative feedback model for gene regulation of the circadian mechanism is described for the fruitfly,Drosophila melanogaster. The conservation of function of clock molecules is illustrated by comparison with the mammalian circadian system, and the apparent swapping of roles between various canonical clock gene components is highlighted. The role of clock gene duplications and divergence of function is introduced via thetimelessgene. The impressive similarities in clock gene regulation between flies and mammals could suggest that variation between more closely related species within insects might be minimal. However, this is not borne out because the expression of clock molecules in the brain of the giant silk moth,Antheraea pernyi, is not easy to reconcile with the negative feedback roles of theperiodandtimelessgenes. Variation in clock gene sequences between and within fly species is examined and the role of co-evolution between and within clock molecules is described, particularly with reference to adaptive functions of the circadian phenotype.

The ability to anticipate the daily changes occurring in the photic environment, by adjusting their physiology and behavior accordingly, should provide living organisms with a selective advantage. Organisms could thus sample the relevant entraining stimuli at early dawn and/or late dusk. At these times of the day the principal spectral components of sunlight reaching the Earth's surface consist of relatively low levels of irradiance in the blue and red parts of the visible spectrum. Biological circadian systems could entrain to twilight conditions by sampling either one or both of these components. Previous studies, based on the characterization of phase response curves, following brief exposures to relatively high intensity light of varying wavelength, demonstrated the particular sensitivity of the Drosophila circadian system to blue light. In this study, we addressed the capacity of the circadian system of Drosophila to respond to long periods of exposure to red light. Thus flies were initially exposed to 12 h : 12 h LD cycles with full spectrum white light. Following a 1-4 h phase shift, the flies were exposed to LD cycles with red light. Our results suggest that, under these experimental conditions, red light of wavelength between 650-700 nm, can function as an entraining stimulus. Furthermore, analysis of the circadian locomotor activity profiles in visually impaired flies, suggests that in wild type flies locomotor activity is triggered by the circadian clock at key times during the day. Once triggered, the whole cycle (i.e. onset, peak and offset) of locomotor activity occurring both at dawn and dusk can proceed autonomously. However, the occurrence of a lights-off signal (typically at dusk) before the autonomous cessation of locomotor activity, leads to a light-driven termination of such activity. In addition, so1 (eyeless) mutant flies show the presence of a single evening locomotor activity peak during the whole circadian day, suggesting that in wild type flies the morning and evening activity peaks may be under separate control.

The immune response affects learning and memory in insects. Given this and the known fitness costs of both the immune system and learning, does an evolutionary trade-off exist between these two systems? We tested this by measuring the learning ability of 12 bumble-bee ( Bombus terrestris ) colonies in a free-flying paradigm. We then tested their immune response using the zone of inhibition assay. We found a positive relationship between colony learning performance and immune response, that is, fast-learning colonies also show high levels of antimicrobial activity. We conclude that there is no a priori reason to demand an evolutionary relationship between two traits that are linked physiologically.

Parasites can influence different host behaviours including foraging, mate choice and predator avoidance. Several recent papers have shown reduced learning abilities in infected insects. However, it is difficult to separate the effects of the immune response from the direct effects of the parasite. Using a free-flying learning paradigm, this paper shows that learning performance is impaired in bumble-bees ( Bombus terrestris ) that are not infected but whose immune system is stimulated non-pathogenically. This demonstrates that before it is assumed that a parasite has a direct effect on a host's behaviour, the effect of the immune response stimulated by the parasite must first be quantified.

Diapause is a protective response to unfavorable environments that results in a suspension of insect development and is most often associated with the onset of winter. The ls-tim mutation in the Drosophila melanogaster clock gene timeless has spread in Europe over the past 10,000 years, possibly because it enhances diapause. We show that the mutant allele attenuates the photosensitivity of the circadian clock and causes decreased dimerization of the mutant TIMELESS protein isoform to CRYPTOCHROME, the circadian photoreceptor. This interaction results in a more stable TIMELESS product. These findings reveal a molecular link between diapause and circadian photoreception.

The threonine-glycine (Thr-Gly) encoding repeat within the clock gene period of Drosophila melanogaster is polymorphic in length. The two major variants (Thr-Gly)17 and (Thr-Gly)20 are distributed as a highly significant latitudinal cline in Europe and North Africa. Thr-Gly length variation from both wild-caught and transgenic individuals is related to the flies' ability to maintain a circadian period at different temperatures. This phenomenon provides a selective explanation for the geographical distribution of Thr-Gly lengths and gives a rare glimpse of the interplay between molecular polymorphism, behavior, population biology, and natural selection.

Forty years ago, a mutagenesis screening in the fruit fly, Drosophila melanogaster, led to the discovery of period, the first gene to be involved in the endogenous 24-h rhythmicity of an organism. Since then circadian clocks have been identified in fungi, cyanobacteria, plants, and other animals. Although the molecular components are not conserved across the main divisions of life, it appears that in every organism, a common design, based upon a transcription-translation feedback loop (TTL), is in place to regulate endogenous 24 h cycles. The TTL model has informed chronobiology research for the majority of the past 30 years with spectacular results. However, new evidence and the rediscovery of old observations suggest that this model is coming to age. Here, we provide a comprehensive review of the current TTL model in Drosophila highlighting its accomplishments and its limitations. We conclude by offering our personal view on the organization and the evolution of circadian clocks.

The aim of this study was to investigate the toxic effects of aluminum (Al) on the model organism-Drosophila melanogaster. The study is especially concerned with the effects of aluminum on the fruit fly's development, life span, and circadian rhythm in rest and activity. Flies were exposed to aluminum in concentrations from 40 to 280 mg/kg in rearing media or the flies were raised on control medium. Moreover, the life span of insects exposed to aluminum containing 40, 120, or 240 mg/kg of Al in the medium, only during their larval development, during the whole life cycle and only in their adult life was tested. To check if aluminum and aging cause changes in D. melanogaster behavior, the locomotor activity of flies at different ages was recorded. Results showed that aluminum is toxic in concentrations above 160 mg/kg in the rearing medium. Depending on Al concentration and time of exposure, the life span of the flies was shortened. At intermediate concentrations (120 mg/kg), however, Al had a stimulating effect on males increasing their life span and level of locomotor activity. At higher concentration the aluminum exposure increased or decreased the level of locomotor activity of D. melanogaster depending on age of flies. In addition, in the oldest insects reared on aluminum supplemented media and in mid-aged flies reared on the highest concentration of Al the daily rhythm of activity was disrupted.

Forty years ago, a mutagenesis screening in the fruit fly, Drosophila melanogaster, led to the discovery of period, the first gene to be involved in the endogenous 24-h rhythmicity of an organism. Since then circadian clocks have been identified in fungi, cyanobacteria, plants, and other animals. Although the molecular components are not conserved across the main divisions of life, it appears that in every organism, a common design, based upon a transcription–translation feedback loop (TTL), is in place to regulate endogenous 24 h cycles. The TTL model has informed chronobiology research for the majority of the past 30 years with spectacular results. However, new evidence and the rediscovery of old observations suggest that this model is coming to age. Here, we provide a comprehensive review of the current TTL model in Drosophila highlighting its accomplishments and its limitations. We conclude by offering our personal view on the organization and the evolution of circadian clocks.

Circadian rhythms (~24 h) in biochemistry, physiology and behaviour are found in almost all eukaryotes and some bacteria. The elucidation of the molecular components of the 24 h circadian clock in a number of model organisms in recent years has provided an opportunity to assess the adaptive value of variation in clock genes. Laboratory experiments using artificially generated mutants reveal that the circadian period is adaptive in a 24 h world. Natural genetic variation can also be studied, and there are a number of ways in which the signature of natural selection can be detected. These include the study of geographical patterns of genetic variation, which provide a first indication that selection may be at work, and the use of sophisticated statistical neutrality tests, which examine whether the pattern of variation observed is consistent with a selective rather than a neutral (or drift) scenario. Finally, examining the probable selective agents and their differential effects on the circadian phenotype of the natural variants provides the final compelling evidence for selection. We present some examples of how these types of analyses have not only enlightened the evolutionary study of clocks, but have also contributed to a more pragmatic molecular understanding of the function of clock proteins.

The burrow emergence activity of the wild caught ragworm Nereis virens Sars associated with food prospecting was investigated under various photoperiodic (LD) and simulated tidal cycles (STC) using a laboratory based actograph. Just over half (57%) of the animals under LD with STC displayed significant tidal (approximately 12.4 h) and/or lunar-day (approximately 24.8 h) activity patterns. Under constant light (LL) plus a STC, 25% of all animals were tidal, while one animal responded with a circadian (24.2 h) activity rhythm suggestive of cross-modal entrainment where the environmental stimulus of one period entrains rhythmic behavior of a different period. All peaks of activity under a STC, apart from that of the individual cross-modal entrainment case, coincided with the period of tank flooding. Under only LD without a STC, 49% of the animals showed nocturnal (approximately 24 h) activity. When animals were maintained under free-running LL conditions, 15% displayed significant rhythmicity with circatidal and circadian/circalunidian periodicities. Although activity cycles in N. virens at the population level are robust, at the individual level they are particularly labile, suggesting complex biological clock-control with multiple clock output pathways.

The period gene is a key regulator of biological rhythmicity in Drosophila melanogaster. The central part of the gene encodes a dipeptide Thr-Gly repeat that has been implicated in the evolution of both circadian and ultradian rhythms. We have previously observed that length variation in the repeat follows a latitudinal cline in Europe and North Africa, so we have sought to extend this observation to the southern hemisphere. We observe a parallel cline in Australia for one of the two major length variants and find higher levels of some Thr-Gly length variants, particularly at the tropical latitudes, that are extremely rare in Europe. In addition we examined >40 haplotypes from sub-Saharan Africa and find a very different and far more variable profile of Thr-Gly sequences. Statistical analysis of the periodicity and codon content of the repeat from all three continents reveals a possible mechanism that may explain how the repeat initially arose in the ancestors of the D. melanogaster subgroup of species. Our results further reinforce the view that thermal selection may have contributed to shaping the continental patterns of Thr-Gly variability.

The length of the Thr-Gly repeat within the period gene of Drosophilids, coevolves with its immediate flanking region to maintain the temperature compensation of the fly circadian clock. In Drosophila simulans, balancing selection appears to maintain a polymorphism in this region, with three repeat lengths carrying 23, 24 or 25 Thr-Gly pairs, each in complete linkage disequilibrium with a distinctive flanking region amino acid moiety. We wondered whether separating a specific length repeat from its associated flanking haplotype might have functional implications for the circadian clock. We fortuitously discovered a population of flies collected in Kenya, in which a chimeric Thr-Gly haplotype was segregating that carried the (Thr-Gly) 24 repeat, but the flanking region of a (ThrGly) 23 allele. One of the five isofemale lines that carried this ‘mutant’ Thr-Gly sequence showed a dramatically long and temperature-sensitive free-running circadian period. This phenotype was mapped to the X chromosome, close to the D. simulans per gene, but there was also a significant effect of a modifying autosomal locus or loci. It seems remarkable that such a mutant phenotype should be discovered in a screen of chimeric Thr-Gly regions.

The Drosophila simulans per gene is polymorphic for the length of a repeat that encodes a series of Thr-Gly pairs. We have examined the circadian behaviour of flies derived from isofemale lines that carry the major variants, and find some significant differences in the way that the clock responds to temperature challenge, that might relate to the observed frequencies of these alleles in nature. We also observe that circadian thermal behaviour is also predictably influenced by subtle differences in the temperature of the locality from which these flies have been originally collected. There appear to be species-specific differences in the circadian locomotor patterns of D. melanogaster and D. simulans and in the way they may respond to temperature. Using chimeric per transgenes which carry the different species Thr-Gly fragments, we have been able to identify components of the behaviour that are modulated by this region of the PER protein.

Background Circadian rhythms are daily rhythms that control every aspect of our behaviour and physiology in a 24 h fashion. They are common to nearly all living organisms from cyanobacteria to plants and from flies to humans. The underlying molecular mechanisms that control circadian rhythms are well understood and flies are an excellent model to study them as their clock is very similar to ours. Additionally we have high-throughput techniques to analyse the fly’s locomotor activity rhythms, which are equivalent to our sleep/wake cycles. Circadian rhythms are also very sensitive physiological markers of wellbeing and can be easily disturbed by life style changes such as inter continental travel or shift work. Circadian problems can also be the direct or indirect result of brain pathologies as in the case with Huntington’s disease (HD). Aims We are characterising a new fly model of HD that is inducible, tuneable and cell type specific. Our aim is to understand early cellular and physiological processes that lead to neuronal dysfunction by using the circadian clock as a readout. Our work may shed light on several under explored areas. For instance: Is HD pathology directly caused by progressive accumulation of mutated huntingtin (HTT) protein over time or by the ageing cell not being able to cope with the mutant protein? Can we reverse HD pathology by turning off the mutated gene? If so, when is the critical point of no return? Methods Using the molecular toolkit available for flies, we can over-express mutant human HTT protein in the brain targeting the clock neurons. We can then analyse rhythms of locomotor activity, looking for anomalies in period, phase or amount of locomotion. Turning the mutant HTT gene on or off throughout the life span of the fly we can probe the effects of ageing on rhythmic phenotypes. Results Here we show that over expression of mutant human HTT in clock neurons causes arrhythmic behaviour in flies and that this effect is modulated by ageing. Conclusions Using circadian rhythms as a tool and fruit flies as a model we have a powerful experimental system to study the pathology caused by the mutated HD protein. In the future, we will focus on the cellular mechanisms leading to neuronal dysfunction/ neurodegeneration in HD and define the critical point in the progression of the disease before which intervention should be made.

Psychoneuroimmunology studies the increasing number of connections between neurobiology and immunology. We establish Drosophila melanogaster as a tractable model in this field by demonstrating the effects of the immune response on two fundamental behaviours: sleep and memory ability. We used the Geneswitch system to upregulate peptidoglycan receptor protein (PGRP) expression, thereby stimulating the immune system in the absence of infection. Geneswitch was activated by feeding the steroid RU486, to the flies. Importantly, by stimulating the immune system of adult flies in the absence of infection we have avoided the added complications of developmental and disease effects that have confounded other studies. We used an aversive classical conditioning paradigm to quantify memory and measures of activity to infer sleep. Immune stimulated flies exhibited reduced levels of sleep, which could not be explained by a generalised increase in waking activity. The effects on sleep were more pronounced for day compared to night sleep. Immune stimulated flies also showed a reduction in memory abilities. These are important results as they establish Drosophila as a model for immune-neural interactions and provide a possible role for sleep in the interplay between the immune response and memory.

We have developed a modified RNA interference (RNAi) method for generating gene knock-outs in Drosophila melanogaster. We used the sequence of the yellow (y) locus to construct an inverted repeat that will form a double-stranded hairpin structure (y-IR) that is under the control of the upstream activating sequence (UAS) of the yeast transcriptional activator GAL4. Hairpins are extremely difficult to manipulate in Escherichia coli, so our method makes use of a heterologous 330 bp spacer encoding sequences from green fluorescent protein to facilitate the cloning steps. When the UAS–y–IR hairpin is expressed under the control of different promoter–GAL4 fusions, a high frequency of y pigment phenocopies is obtained in adults. Consequently this method for producing gene knock-outs has several advantages over previous methods in that it is applicable to any gene within the fly genome, greatly facilitates cloning of the hairpin, can be used if required with GAL4 drivers to avoid lethality or to induce RNAi in a specific developmental stage and/or tissue, is useful for generating knock-outs of adult phenotypes as reported here and, finally, the system can be manipulated to investigate the trans-acting factors that are involved in the RNAi mechanism.

Entrainment is as fundamental to an organism’s circadian timing as are the molecular mechanisms involved in the functioning of the intracellular clock oscillator. In nature, one of the principle, although not the only, circadian entraining stimulus (Zeitgeber) is provided by the daily light–dark cycles. In animals, the visual processing apparatus alone is inadequate to accomplish the task of transducing circadian photic signals to the clockwork machinery. In fact, it is ever more appreciated by circadian biologists that organisms as divergent as plants and mammals have evolved a wonderfully complex array of partly redundant specializations which can guarantee the precise alignment of biological and environmental time. Research in circadian biology is cruising at such a rate that attempts to review the state of the art can only hope, at best, to provide a snapshot of the speeding cruiser from its wake. This paper will hopefully provide a reasonably sharp portrayal of what is at hand.

R.C. and C.P.K. were supported by grants from the European Community and CRUI-MURST-British Council, R.C. by grants from MURST-progetti nazionali and Ministero per le Politiche Agricole, and E.R. by a David Phillips Fellowship from BBSRC.