Drosophila’s lateral posterior neurons (LPNs) belong to a small group of circadian clock neurons that is so far not characterized in detail. Thanks to a new highly specific split‐Gal4 line, here we ...describe LPNs’ morphology in fine detail, their synaptic connections, daily bimodal expression of neuropeptides, and propose a putative role of this cluster in controlling daily activity and sleep patterns. We found that the three LPNs are heterogeneous. Two of the neurons with similar morphology arborize in the superior medial and lateral protocerebrum and most likely promote sleep. One unique, possibly wakefulness‐promoting, neuron with wider arborizations extends from the superior lateral protocerebrum toward the anterior optic tubercle. Both LPN types exhibit manifold connections with the other circadian clock neurons, especially with those that control the flies’ morning and evening activity (M‐ and E‐neurons, respectively). In addition, they form synaptic connections with neurons of the mushroom bodies, the fan‐shaped body, and with many additional still unidentified neurons. We found that both LPN types rhythmically express three neuropeptides, Allostatin A, Allostatin C, and Diuretic Hormone 31 with maxima in the morning and the evening. The three LPN neuropeptides may, furthermore, signal to the insect hormonal center in the pars intercerebralis and contribute to rhythmic modulation of metabolism, feeding, and reproduction. We discuss our findings in the light of anatomical details gained by the recently published hemibrain of a single female fly on the electron microscopic level and of previous functional studies concerning the LPN.
The authors characterized Drosophila’s lateral posterior neurons (LPNs) that belong to the circadian clock neurons. They demonstrate that the LPNs are well connected to the other clock neurons (s‐LNv, LNd, DN2, DN1p) and to putative wake and sleep centers as well as centers that control temperature preference, metabolism, feeding, and oviposition (e.g., the pars intercerebralis (PI), the mushroom body (MB), and the dorsal fan‐shaped body(dFB)). The LPNs get input from the small ventrolateral clock neurons (s‐LNv) and may signal to the downstream neurons and centers via direct synapses and/or via three neuropeptides (AstA, AstC, and DH31), which they express rhythmically. Furthermore, the authors show that the LPNs are heterogeneous (LPN‐Type 1 and LPN‐Type 2) and appear to have opposing roles on activity and sleep.
The fruit fly
Drosophila
is a prime model in circadian research, but still little is known about its circadian regulation of metabolism. Daily rhythmicity in levels of several metabolites has been ...found, but knowledge about hydrophobic metabolites is limited. We here compared metabolite levels including lipids between
period
01
(
per
01
) clock mutants and Canton-S wildtype (WT
CS
) flies in an isogenic and non-isogenic background using LC–MS. In the non-isogenic background, metabolites with differing levels comprised essential amino acids, kynurenines, pterinates, glycero(phospho)lipids, and fatty acid esters. Notably, detectable diacylglycerols (DAG) and acylcarnitines (AC), involved in lipid metabolism, showed lower levels in
per
01
mutants. Most of these differences disappeared in the isogenic background, yet the level differences for AC as well as DAG were consistent for fly bodies. AC levels were dependent on the time of day in WT
CS
in phase with food consumption under LD conditions, while DAGs showed weak daily oscillations. Two short-chain ACs continued to cycle even in constant darkness.
per
01
mutants in LD showed no or very weak diel AC oscillations out of phase with feeding activity. The low levels of DAGs and ACs in
per
01
did not correlate with lower total food consumption, body mass or weight. Clock mutant flies showed higher sensitivity to starvation independent of their background-dependent activity level. Our results suggest that neither feeding, energy storage nor mobilisation is significantly affected in
per
01
mutants, but point towards impaired mitochondrial activity, supported by upregulation of the mitochondrial stress marker 4EBP in the clock mutants.
Significance The moon provides highly reliable time information to organisms. Whereas sunlight is known to set daily animal timing systems, mechanistic insight into the impact of moonlight on such ...systems remains scarce. We establish that the marine bristleworm
times the precise hours of mass spawning by integrating lunar light information into a plastic daily timing system able to run with circadian (∼24 h) or circalunidian (∼24.8 h) periodicity. The correct interpretation of moonlight is mediated by the interplay of two light sensors: a cryptochrome and a melanopsin ortholog provide information on light valence and moonrise time, respectively. Besides its ecological relevance, our work provides a plausible explanation for long-standing observations of light intensity-dependent differences in circadian clock periods.
's dorsal clock neurons (DNs) consist of four clusters (DN
s, DN
s, DN
s, and DN
s) that largely differ in size. While the DN
s and the DN
s encompass only two neurons, the DN
s consist of ∼15 ...neurons, and the DN
s comprise ∼40 neurons per brain hemisphere. In comparison to the well-characterized lateral clock neurons (LNs), the neuroanatomy and function of the DNs are still not clear. Over the past decade, numerous studies have addressed their role in the fly's circadian system, leading to several sometimes divergent results. Nonetheless, these studies agreed that the DNs are important to fine-tune activity under light and temperature cycles and play essential roles in linking the output from the LNs to downstream neurons that control sleep and metabolism. Here, we used the Flybow system, specific split-GAL4 lines,
-Tango, and the recently published fly connectome (called hemibrain) to describe the morphology of the DNs in greater detail, including their synaptic connections to other clock and non-clock neurons. We show that some DN groups are largely heterogenous. While certain DNs are strongly connected with the LNs, others are mainly output neurons that signal to circuits downstream of the clock. Among the latter are mushroom body neurons, central complex neurons, tubercle bulb neurons, neurosecretory cells in the pars intercerebralis, and other still unidentified partners. This heterogeneity of the DNs may explain some of the conflicting results previously found about their functionality. Most importantly, we identify two putative novel communication centers of the clock network: one fiber bundle in the superior lateral protocerebrum running toward the anterior optic tubercle and one fiber hub in the posterior lateral protocerebrum. Both are invaded by several DNs and LNs and might play an instrumental role in the clock network.
Phase response curves (PRCs) for light or temperature stimuli have been shown to be most valuable in understanding how circadian clocks are entrained to daily environmental cycles. Nowadays, PRC ...experiments in which clock neurons are manipulated in a temporally restricted manner by thermogenetic or optogenetic tools are also useful to comprehend clock network properties. Here, we temporally depolarized specific clock neurons of Drosophila melanogaster by activating temperature-sensitive dTrpA1 channels to unravel their role in phase shifting the flies’ activity rhythm. The depolarization of all clock neurons caused a PRC resembling the flies’ light PRC, with strong phase delays in the first half of the subjective night and modest phase advances in its second half. However, the activation of the flies’ pigment-dispersing factor (PDF)-positive morning (M) neurons (s-LNvs) only induced phase advances, and these reached into the subjective day, where the light PRC has its dead zone. This indicates that the M neurons are very potent in accelerating the clock, which is in line with previous observations. In contrast, the evening (E) neurons together with the PDF-positive l-LNvs appear to mediate phase delays. Most interestingly, the molecular clock (Period protein cycling) of the depolarized clock neurons was shifted in parallel to the behavior, and this shift was already visible within the first cycle after the temperature pulse. We identified cAMP response element binding protein B (CREB) as a putative link between membrane depolarization and the molecular clock.
In animals, commensal microbes modulate various physiological functions, including behavior. While microbiota exposure is required for normal behavior in mammals, it is not known how widely this ...dependency is present in other animal species. We proposed the hypothesis that the microbiome has a major influence on the behavior of the vinegar fly (Drosophila melanogaster), a major invertebrate model organism. Several assays were used to test the contribution of the microbiome on some well-characterized behaviors: defensive behavior, sleep, locomotion, and courtship in microbe-bearing, control flies and two generations of germ-free animals. None of the behaviors were largely influenced by the absence of a microbiome, and the small or moderate effects were not generalizable between replicates and/or generations. These results refute the hypothesis, indicating that the Drosophila microbiome does not have a major influence over several behaviors fundamental to the animal's survival and reproduction. The impact of commensal microbes on animal behaviour may not be broadly conserved.
It is assumed that a properly timed circadian clock enhances fitness, but only few studies have truly demonstrated this in animals. We raised each of the three classical
Drosophila period
mutants for ...>50 generations in the laboratory in competition with wildtype flies. The populations were either kept under a conventional 24-h day or under cycles that matched the mutant’s natural cycle, i.e., a 19-h day in the case of
per
s
mutants and a 29-h day for
per
l
mutants. The arrhythmic
per
0
mutants were grown together with wildtype flies under constant light that renders wildtype flies similar arrhythmic as the mutants. In addition, the mutants had to compete with wildtype flies for two summers in two consecutive years under outdoor conditions. We found that wildtype flies quickly outcompeted the mutant flies under the 24-h laboratory day and under outdoor conditions, but
per
l
mutants persisted and even outnumbered the wildtype flies under the 29-h day in the laboratory. In contrast,
per
s
and
per
0
mutants did not win against wildtype flies under the 19-h day and constant light, respectively. Our results demonstrate that wildtype flies have a clear fitness advantage in terms of fertility and offspring survival over the
period
mutants and – as revealed for
per
l
mutants – this advantage appears maximal when the endogenous period resonates with the period of the environment. However, the experiments indicate that
per
l
and
per
s
persist at low frequencies in the population even under the 24-h day. This may be a consequence of a certain mating preference of wildtype and heterozygous females for mutant males and time differences in activity patterns between wildtype and mutants.
Outbreaks of infectious diseases repeatedly affected medieval Europe, leaving behind a large number of dead often inhumed in mass graves. Human remains interred in two burial pits from 14
th
century ...CE Germany exhibited molecular evidence of
Salmonella enterica
Paratyphi C (
S.
Paratyphi C) infection. The pathogen is responsible for paratyphoid fever, which was likely the cause of death for the buried individuals. This finding presented the unique opportunity to conduct a paratyphoid fever association study in a European population. We focused on HLA-DRB1*03:01 that is a known risk allele for enteric fever in present-day South Asians. We generated HLA profiles for 29 medieval
S.
Paratyphi C cases and 24 contemporaneous controls and compared these to a modern German population. The frequency of the risk allele was higher in the medieval cases (29.6%) compared to the contemporaneous controls (13%;
p = 0.189
), albeit not significantly so, possibly because of small sample sizes. Indeed, in comparison with the modern controls (
n = 39,689
; 10.2%;
p = 0.005
) the frequency difference became statistically significant. This comparison also suggested a slight decrease in the allele’s prevalence between the medieval and modern controls. Up to now, this is the first study on the genetic predisposition to
Salmonella
infection in Europeans and the only association analysis on paratyphoid fever C. Functional investigation using computational binding prediction between HLA variants and
S.
Paratyphi and
S.
Typhi peptides supported a reduced recognition capacity of bacterial proteins by DRB1*03:01 relative to other common DRB1 variants. This pattern could potentially explain the disease association. Our results suggest a slightly reduced predisposition to paratyphoid fever in modern Europeans. The causative allele, however, is still common today, which can be explained by a trade-off, as DRB1*03:01 is protective against infectious respiratory diseases such as severe respiratory syndrome (SARS). It is thus possible that the allele also provided resistance to corona-like viruses in the past.
The ability to be synchronized by light-dark cycles is a fundamental property of circadian clocks. Although there are indications that circadian clocks are extremely light-sensitive and that they can ...be set by the low irradiances that occur at dawn and dusk, this has not been shown on the cellular level. Here, we demonstrate that a subset of Drosophila's pacemaker neurons responds to nocturnal dim light. At a nighttime illumination comparable to quarter-moonlight intensity, the flies increase activity levels and shift their typical morning and evening activity peaks into the night. In parallel, clock protein levels are reduced, and clock protein rhythms shift in opposed direction in subsets of the previously identified morning and evening pacemaker cells. No effect was observed on the peripheral clock in the eye. Our results demonstrate that the neurons driving rhythmic behavior are extremely light-sensitive and capable of shifting activity in response to the very low light intensities that regularly occur in nature. This sensitivity may be instrumental in adaptation to different photoperiods, as was proposed by the morning and evening oscillator model of Pittendrigh and Daan. We also show that this adaptation depends on retinal input but is independent of cryptochrome.
Light profoundly affects the circadian clock and the activity levels of animals. Along with the systematic changes in intensity and spectral composition, over the 24-h day, light shows considerable ...irregular fluctuations (noise). Using light as the Zeitgeber for the circadian clock is, therefore, a complex task and this might explain why animals utilize multiple photoreceptors to entrain their circadian clock. The fruit fly
possesses light-sensitive Cryptochrome and seven Rhodopsins that all contribute to light detection. We review the role of Rhodopsins in circadian entrainment, and of direct light-effects on the activity, with a special emphasis on the newly discovered Rhodopsin 7 (Rh7). We present evidence that Rhodopsin 6 in receptor cells 8 of the compound eyes, as well as in the extra retinal Hofbauer-Buchner eyelets, plays a major role in entraining the fly's circadian clock with an appropriate phase-to-light⁻dark cycles. We discuss recent contradictory findings regarding Rhodopsin 7 and report original data that support its role in the compound eyes and in the brain. While Rhodopsin 7 in the brain appears to have a minor role in entrainment, in the compound eyes it seems crucial for fine-tuning light sensitivity to prevent overshooting responses to bright light.
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