The Neuronal Circuit of the Dorsal Circadian Clock Neurons in Drosophila melanogaster.

Drosophila's dorsal clock neurons (DNs) consist of four clusters (DN1as, DN1ps, DN2s, and DN3s) that largely differ in size. While the DN1as and the DN2s encompass only two neurons, the DN1ps consist of ∼15 neurons, and the DN3s 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, trans-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.

The lateral posterior clock neurons of Drosophila melanogaster express three neuropeptides and have multiple connections within the circadian clock network and beyond.

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.

Highly contiguous assemblies of 101 drosophilid genomes.

Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

Flies as models for circadian clock adaptation to environmental challenges.

Life on earth is assumed to have developed in tropical regions that are characterized by regular 24 hr cycles in irradiance and temperature that remain the same throughout the seasons. All organisms developed circadian clocks that predict these environmental cycles and prepare the organisms in advance for them. A central question in chronobiology is how endogenous clocks changed in order to anticipate very different cyclical environmental conditions such as extremely short and long photoperiods existing close to the poles. Flies of the family Drosophilidae can be found all over the world-from the tropics to subarctic regions-making them unprecedented models for studying the evolutionary processes that underlie the adaptation of circadian clocks to different latitudes. This review summarizes our current understanding of these processes. We discuss evolutionary changes in the clock genes and in the clock network in the brain of different Drosophilids that may have caused behavioural adaptations to high latitudes.

Life at High Latitudes Does Not Require Circadian Behavioral Rhythmicity under Constant Darkness.

Nearly all organisms evolved endogenous self-sustained timekeeping mechanisms to track and anticipate cyclic changes in the environment. Circadian clocks, with a periodicity of about 24 h, allow animals to adapt to day-night cycles. Biological clocks are highly adaptive, but strong behavioral rhythms might be a disadvantage for adaptation to weakly rhythmic environments such as polar areas [1, 2]. Several high-latitude species, including Drosophila species, were found to be highly arrhythmic under constant conditions [3-6]. Furthermore, Drosophila species from subarctic regions can extend evening activity until dusk under long days. These traits depend on the clock network neurochemistry, and we previously proposed that high-latitude Drosophila species evolved specific clock adaptations to colonize polar regions [5, 7, 8]. We broadened our analysis to 3 species of the Chymomyza genus, which diverged circa 5 million years before the Drosophila radiation [9] and colonized both low and high latitudes [10, 11]. C. costata, pararufithorax, and procnemis, independently of their latitude of origin, possess the clock neuronal network of low-latitude Drosophila species, and their locomotor activity does not track dusk under long photoperiods. Nevertheless, the high-latitude C. costata becomes arrhythmic under constant darkness (DD), whereas the two low-latitude species remain rhythmic. Different mechanisms are behind the arrhythmicity in DD of C. costata and the high-latitude Drosophila ezoana, suggesting that the ability to maintain behavioral rhythms has been lost more than once during drosophilids' evolution and that it might indeed be an evolutionary adaptation for life at high latitudes.

Closely Related Fruit Fly Species Living at Different Latitudes Diverge in Their Circadian Clock Anatomy and Rhythmic Behavior.

Recently, we reported differences in the expression pattern of the blue light-sensitive flavoprotein cryptochrome (CRY) and the neuropeptide pigment-dispersing factor (PDF) in the neuronal clock network of high-latitude Drosophila species, belonging to the Drosophila subgenus ( virilis-repleta radiation), compared with cosmopolitan D. melanogaster flies, belonging to the Sophophora subgenus. Alterations in rhythmic patterns of activity due to these differences might have adaptive significance for colonizing high-latitude habitats and, hence, adjusting to long photoperiods. Here, we show that these differing CRY/PDF expression patterns are only present in those species of the virilis-repleta radiation that colonized high latitudes. The cosmopolitan species D. mercatorum and D. hydei have a D. melanogaster-like clock network and behavior despite belonging to the virilis-repleta radiation. Similarly, 2 species of the holotropical Zaprionus genus, more closely related to the Drosophila subgenus than to the Sophophora subgenus, retain a D. melanogaster-like clock network and rhythmic behavior. We therefore suggest that the D. melanogaster-like clock network is the "ancestral fly clock phenotype" and that alterations in the CRY/PDF clock neurochemistry have allowed some species of the virilis-repleta radiation to colonize high-latitude environments.

Author Correction: The characterization of the circadian clock in the olive fly Bactrocera oleae (Diptera: Tephritidae) reveals a Drosophila-like organization.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

The characterization of the circadian clock in the olive fly Bactrocera oleae (Diptera: Tephritidae) reveals a Drosophila-like organization.

The olive fruit fly, Bactrocera oleae, is the single most important pest for the majority of olive plantations. Oxitec's self-limiting olive fly technology (OX3097D-Bol) offers an alternative management approach to this insect pest. Because of previously reported asynchrony in the mating time of wild and laboratory strains, we have characterized the olive fly circadian clock applying molecular, evolutionary, anatomical and behavioural approaches. Here we demonstrate that the olive fly clock relies on a Drosophila melanogaster-like organization and that OX3097D-Bol carries a functional clock similar to wild-type strains, confirming its suitability for operational use.