Characterization of pre-diapause phase in the northern Drosophila species D. ezoana.

Drosophila ezoana is a virilis group Drosophila species inhabiting northern latitudes. The flies enter adult reproductive diapause to survive winter upon exposure to short photoperiod conditions (short-day) over several consecutive days. Insect pre-diapause phase - the duration between the beginning of exposure to short days and expression of diapause is thought to be comprised of two distinct phases - (a) photoperiodic time measurement that detects short-days, followed by (b) physiological events leading to the expression of diapause phenotype. A short-day dependent segment of the pre-diapause phase thus approximates the process of photoperiodic time measurement. Continuous darkness has been found to be a neutral condition with respect to diapause regulation in many insect species. The effect of variable number of short-days followed by continuous darkness on diapause incidence thus allows identification of short-day dependent segment of pre-diapause phase thereby mapping the process of photo-periodic time measurement. Although, few weeks of exposure to short-days in adult stage is known to be sufficient for the expression of diapause in D. ezoana, the number of short days required for the completion of photo-periodic time measurement has never been systematically analysed. Our experiments show that continuous darkness is a neutral condition for diapause regulation also in D. ezoana. We utilized the neutral nature of continuous darkness to map the process of photoperiodic time measurement in the D. ezoana strain 124OJ8 which showed that integration of short-day photic cues over the first 10 days of pre-diapause phase is essential for diapause induction.

A clock for all seasons.

Circadian clocks play an essential role in adapting locomotor activity as well as physiological, and metabolic rhythms of organisms to the day-night cycles on Earth during the four seasons. In addition, they can serve as a time reference for measuring day length and adapt organisms in advance to annual changes in the environment, which can be particularly pronounced at higher latitudes. The physiological responses of organisms to day length are also known as photoperiodism. This special issue of the Journal of Comparative Physiology A aims to account for diurnal and photoperiodic adaptations by presenting a collection of ten review articles, five original research articles, and three perspective pieces. The contributions include historical accounts, circadian and photoperiodic clock models, epigenetic, molecular, and neuronal mechanisms of seasonal adaptations, latitudinal differences in photoperiodic responses and studies in the wild that address the challenges of global change.

Erwin Bünning and Wolfgang Engelmann: establishing the involvement of the circadian clock in photoperiodism.

In 1936, Erwin Bünning published his groundbreaking work that the endogenous clock is used to measure day length for initiating photoperiodic responses. His publication triggered years of controversial debate until it ultimately became the basic axiom of rhythm research and the theoretical pillar of chronobiology. Bünning's thesis is frequently quoted in the articles in this special issue on the subject of "A clock for all seasons". However, nowadays only few people know in detail about Bünning's experiments and almost nobody knows about the contribution of his former doctoral student, Wolfgang Engelmann, to his theory because most work on this topic is published in German. The aim of this review is to give an overview of the most important experiments at that time, including Wolfgang Engelmann's doctoral thesis, in which he demonstrated the importance of the circadian clock for photoperiodic flower induction in the Flaming Katy, Kalanchoë blossfeldiana, but not in the Red Morning Glory, Ipomoea coccinea.

Photoreceptors for immediate effects of light on circadian behavior.

Animals need to sharpen their behavioral output in order to adapt to a variable environment. Hereby, light is one of the most pivotal environmental signals and thus behavioral plasticity in response to light can be observed in diurnal animals, including humans. Furthermore, light is the main entraining signal of the clock, yet immediate effects of light enhance or overwrite circadian output and thereby mask circadian behavior. In Drosophila, such masking effects are most evident as a lights-on response in two behavioral rhythms - the emergence of the adult insect from the pupa, called eclosion, and the diurnal rhythm of locomotor activity. Here, we show that the immediate effect of light on eclosion depends solely on R8 photoreceptors of the eyes. In contrast, the increase in activity by light at night is triggered by different cells and organs that seem to compensate for the loss of each other, potentially to ensure behavioral plasticity.

Neuropeptidergic regulation of insect diapause by the circadian clock.

Diapause is an endocrine-mediated strategy used by insects to survive seasons of adverse environmental conditions. Insects living in temperate zones are regularly exposed to such conditions in the form of winter. To survive winter, they must prepare for it long before it arrives. A reliable indicator of impending winter is the shortening of day length. To measure day length, insects need their circadian clock as internal time reference. In this article, I provide an overview of the current state of knowledge on the neuropeptides that link the clock to the diapause inducing hormonal brain centers.

One hundred years of excellence: the top one hundred authors of the Journal of Comparative Physiology A.

The Journal of Comparative Physiology A is the premier peer-reviewed scientific journal in comparative physiology, in particular sensory physiology, neurophysiology, and neuroethology. Founded in 1924 by Karl von Frisch and Alfred Kühn, it celebrates its 100th anniversary in 2024. During these 100 years, many of the landmark achievements in these disciplines were published in this journal. To commemorate these accomplishments, we have compiled a list of the Top 100 Authors over these 100 years, representing approximately 1% of all its authors. To select these individuals, three performance criteria were applied: number of publications, total number of citations attracted by these articles, and mean citation rate of the papers published by each author. The resulting list of the Top 100 Authors provides a fascinating insight into the history of the disciplines covered by the Journal of Comparative Physiology A and into the academic careers of many of their leading representatives.

Ingeborg Beling and the time memory in honeybees: almost one hundred years of research.

Bees are known for their ability to forage with high efficiency. One of their strategies to avoid unproductive foraging is to be at the food source at the right time of the day. Approximately one hundred years ago, researchers discovered that honeybees have a remarkable time memory, which they use for optimizing foraging. Ingeborg Beling was the first to examine this time memory experimentally. In her doctoral thesis, completed under the mentorship of Karl von Frisch in 1929, she systematically examined the capability of honeybees to remember specific times of the day at which they had been trained to appear at a feeding station. Beling was a pioneer in chronobiology, as she described the basic characteristics of the circadian clock on which the honeybee's time memory is based. Unfortunately, after a few years of extremely productive research, she ended her scientific career, probably due to family reasons or political pressure to reduce the number of women in the workforce. Here, we present a biographical sketch of Ingeborg Beling and review her research on the time memory of honeybees. Furthermore, we discuss the significance of her work, considering what is known about time memory today - nearly 100 years after she conducted her experiments.

Optimized design and in vivo application of optogenetically functionalized Drosophila dopamine receptors.

Neuromodulatory signaling via G protein-coupled receptors (GPCRs) plays a pivotal role in regulating neural network function and animal behavior. The recent development of optogenetic tools to induce G protein-mediated signaling provides the promise of acute and cell type-specific manipulation of neuromodulatory signals. However, designing and deploying optogenetically functionalized GPCRs (optoXRs) with accurate specificity and activity to mimic endogenous signaling in vivo remains challenging. Here we optimize the design of optoXRs by considering evolutionary conserved GPCR-G protein interactions and demonstrate the feasibility of this approach using two Drosophila Dopamine receptors (optoDopRs). These optoDopRs exhibit high signaling specificity and light sensitivity in vitro. In vivo, we show receptor and cell type-specific effects of dopaminergic signaling in various behaviors, including the ability of optoDopRs to rescue the loss of the endogenous receptors. This work demonstrates that optoXRs can enable optical control of neuromodulatory receptor-specific signaling in functional and behavioral studies.

David S. Saunders: man of insects and photoperiodism (1935-2023).

David S. Saunders was an outstanding scientist, who devoted his life to his family and to insects. He has made many fundamental contributions to our understanding of how insects reproduce and adapt their reproduction and development to the seasonal changes on our planet. Most importantly, he was a pioneer in demonstrating the role of the circadian clock in insect photoperiodic time measurement, first in the jewel wasp Nasonia vitripennis, and later in varies species of flies. His books on biological rhythms and insect clocks are important undergraduate, graduate and research reference literature. David was also a brilliant teacher and mentor and played a major role in establishing and teaching a series of successful Erasmus-funded Chronobiology Summer Schools in Europe. He leaves behind a legacy, both professionally and personally.

The circadian and photoperiodic clock of the pea aphid.

The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species that exhibits a remarkable annual life cycle, which is tightly coupled to the seasonal changes in day length. During spring and summer, characterised by longer days, aphid populations consist exclusively of viviparous females that reproduce parthenogenetically. When autumn comes and the days shorten, aphids switch their reproductive mode and generate males and oviparous sexual females, which mate and produce cold-resistant eggs that overwinter and survive the unfavourable season. While the photoperiodic responses have been well described, the nature of the timing mechanisms which underlie day length discrimination are still not completely understood. Experiments from the 1960's suggested that aphids rely on an 'hourglass' clock measuring the elapsed time during the dark night by accumulating a biochemical factor, which reaches a critical threshold at a certain night length and triggers the switch in reproduction mode. However, the photoperiodic responses of aphids can also be attributed to a strongly dampened circadian clock. Recent studies have uncovered the molecular components and the location of the circadian clock in the brain of the pea aphid and revealed that it is well connected to the neurohormonal system controlling aphid reproduction. We provide an overview of the putative mechanisms of photoperiodic control in aphids, from the photoreceptors involved in this process to the circadian clock and the neuroendocrine system.

The circadian clock is required for rhythmic lipid transport in Drosophila in interaction with diet and photic condition.

Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality.

Characterization of clock-related proteins and neuropeptides in Drosophila littoralis and their putative role in diapause.

Insects from high latitudes spend the winter in a state of overwintering diapause, which is characterized by arrested reproduction, reduced food intake and metabolism, and increased life span. The main trigger to enter diapause is the decreasing day length in summer-autumn. It is thus assumed that the circadian clock acts as an internal sensor for measuring photoperiod and orchestrates appropriate seasonal changes in physiology and metabolism through various neurohormones. However, little is known about the neuronal organization of the circadian clock network and the neurosecretory system that controls diapause in high-latitude insects. We addressed this here by mapping the expression of clock proteins and neuropeptides/neurohormones in the high-latitude fly Drosophila littoralis. We found that the principal organization of both systems is similar to that in Drosophila melanogaster, but with some striking differences in neuropeptide expression levels and patterns. The small ventrolateral clock neurons that express pigment-dispersing factor (PDF) and short neuropeptide F (sNPF) and are most important for robust circadian rhythmicity in D. melanogaster virtually lack PDF and sNPF expression in D. littoralis. In contrast, dorsolateral clock neurons that express ion transport peptide in D. melanogaster additionally express allatostatin-C and appear suited to transfer day-length information to the neurosecretory system of D. littoralis. The lateral neurosecretory cells of D. littoralis contain more neuropeptides than D. melanogaster. Among them, the cells that coexpress corazonin, PDF, and diuretic hormone 44 appear most suited to control diapause. Our work sets the stage to investigate the roles of these diverse neuropeptides in regulating insect diapause.

Drosophila ezoana uses morning and evening oscillators to adjust its rhythmic activity to different daylengths but only the morning oscillator to measure night length for photoperiodic responses.

Animals living at high latitudes are exposed to prominent seasonal changes to which they need to adapt to survive. By applying Zeitgeber cycles of different periods and photoperiods we show here that high-latitude D. ezoana flies possess evening oscillators and highly damped morning oscillators that help them adapting their activity rhythms to long photoperiods. In addition, the damped morning oscillators are involved in timing diapause. The flies measure night length and use external coincidence for timing diapause. We discuss the clock protein TIMELESS (d-TIM) as the molecular correlate and the small ventrolateral clock neurons (s-LNvs) as the anatomical correlates of the components measuring night length.

Pigment-dispersing factor is present in circadian clock neurons of pea aphids and may mediate photoperiodic signalling to insulin-producing cells.

The neuropeptide pigment-dispersing factor (PDF) plays a pivotal role in the circadian clock of most Ecdysozoa and is additionally involved in the timing of seasonal responses of several photoperiodic species. The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species with an annual life cycle tightly coupled to the seasonal changes in day length. Nevertheless, PDF could not be identified in A. pisum so far. In the present study, we identified a PDF-coding gene that has undergone significant changes in the otherwise highly conserved insect C-terminal amino acid sequence. A newly generated aphid-specific PDF antibody stained four neurons in each hemisphere of the aphid brain that co-express the clock protein Period and have projections to the pars lateralis that are highly plastic and change their appearance in a daily and seasonal manner, resembling those of the fruit fly PDF neurons. Most intriguingly, the PDF terminals overlap with dendrites of the insulin-like peptide (ILP) positive neurosecretory cells in the pars intercerebralis and with putative terminals of Cryptochrome (CRY) positive clock neurons. Since ILP has been previously shown to be crucial for seasonal adaptations and CRY might serve as a circadian photoreceptor vital for measuring day length, our results suggest that PDF plays a critical role in aphid seasonal timing.

Biological timing: Linking the circadian clock to the season.

The circadian clock is thought to provide the internal time reference for measuring day length, allowing organisms to prepare in advance for the coming winter and summer. A new study sheds light on the neural link between the circadian clock and seasonal timing.

Contact chemoreception, magnetic maps, thermoregulation by a superorganism, and, thanks to Einstein, an all-time record: the Editors' and Readers' Choice Awards 2023.

During the 99 years of its history, the Journal of Comparative Physiology A has published many of the most influential papers in comparative physiology and related disciplines. To celebrate this achievement of the journal's authors, annual Editors' Choice Awards and Readers' Choice Awards are presented. The winners of the 2023 Editors' Choice Awards are 'Contact chemoreception in multi­modal sensing of prey by Octopus' by Buresch et al. (J Comp Physiol A 208:435-442, 2022) in the Original Paper category; and 'Magnetic maps in animal navigation' by Lohmann et al. (J Comp Physiol A 208:41-67, 2022) in the Review/Review-History Article category. The winners of the 2023 Readers' Choice Awards are 'Coping with the cold and fighting the heat: thermal homeostasis of a superorganism, the honeybee colony' by Stabentheiner et al. (J Comp Physiol A 207:337-351; 2021) in the Original Paper category; and 'Einstein, von Frisch and the honeybee: a historical letter comes to light' by Dyer et al. (J Comp Physiol A 207:449-456, 2021) in the Review/Review-History category.

Contribution of cryptochromes and photolyases for insect life under sunlight.

The cryptochrome/photolyase (CRY/PL) family is essential for life under sunlight because photolyases repair UV-damaged DNA and cryptochromes are normally part of the circadian clock that controls the activity-sleep cycle within the 24-h day. In this study, we aim to understand how the lineage and habitat of an insect affects its CRY/PL composition. To this end, we searched the large number of annotated protein sequences of 340 insect species already available in databases for CRY/PLs. Using phylogenetic tree and motif analyses, we identified four frequent CRY/PLs in insects: the photolyases 6-4 PL and CPDII PL, as well as the mammalian-type cryptochrome (MCRY) and Drosophila-type cryptochrome (DCRY). Assignment of CRY/PLs to the corresponding insects confirmed that light-exposed insects tend to have more CRY/PLs than insects with little light exposure. Nevertheless, even insects with greatly reduced CRY/PLs still possess MCRY, which can be regarded as the major insect cryptochrome. Only flies of the genus Schizophora, which includes Drosophila melanogaster, lost MCRY. Moreover, we found that MCRY and CPDII PL as well as DCRY and 6-4 PL occur very frequently together, suggesting an interaction between the two pairs.

The Gain and Loss of Cryptochrome/Photolyase Family Members during Evolution.

The cryptochrome/photolyase (CRY/PL) family represents an ancient group of proteins fulfilling two fundamental functions. While photolyases repair UV-induced DNA damages, cryptochromes mainly influence the circadian clock. In this study, we took advantage of the large number of already sequenced and annotated genes available in databases and systematically searched for the protein sequences of CRY/PL family members in all taxonomic groups primarily focusing on metazoans and limiting the number of species per taxonomic order to five. Using BLASTP searches and subsequent phylogenetic tree and motif analyses, we identified five distinct photolyases (CPDI, CPDII, CPDIII, 6-4 photolyase, and the plant photolyase PPL) and six cryptochrome subfamilies (DASH-CRY, mammalian-type MCRY, Drosophila-type DCRY, cnidarian-specific ACRY, plant-specific PCRY, and the putative magnetoreceptor CRY4. Manually assigning the CRY/PL subfamilies to the species studied, we have noted that over evolutionary history, an initial increase of various CRY/PL subfamilies was followed by a decrease and specialization. Thus, in more primitive organisms (e.g., bacteria, archaea, simple eukaryotes, and in basal metazoans), we find relatively few CRY/PL members. As species become more evolved (e.g., cnidarians, mollusks, echinoderms, etc.), the CRY/PL repertoire also increases, whereas it appears to decrease again in more recent organisms (humans, fruit flies, etc.). Moreover, our study indicates that all cryptochromes, although largely active in the circadian clock, arose independently from different photolyases, explaining their different modes of action.

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.

Two light sensors decode moonlight versus sunlight to adjust a plastic circadian/circalunidian clock to moon phase.

Many species synchronize their physiology and behavior to specific hours. It is commonly assumed that sunlight acts as the main entrainment signal for ∼24-h clocks. However, the moon provides similarly regular time information. Consistently, a growing number of studies have reported correlations between diel behavior and lunidian cycles. Yet, mechanistic insight into the possible influences of the moon on ∼24-h timers remains scarce. We have explored the marine bristleworm Platynereis dumerilii to investigate the role of moonlight in the timing of daily behavior. We uncover that moonlight, besides its role in monthly timing, also schedules the exact hour of nocturnal swarming onset to the nights' darkest times. Our work reveals that extended moonlight impacts on a plastic clock that exhibits <24 h (moonlit) or >24 h (no moon) periodicity. Abundance, light sensitivity, and genetic requirement indicate that the Platynereis light receptor molecule r-Opsin1 serves as a receptor that senses moonrise, whereas the cryptochrome protein L-Cry is required to discriminate the proper valence of nocturnal light as either moonlight or sunlight. Comparative experiments in Drosophila suggest that cryptochrome's principle requirement for light valence interpretation is conserved. Its exact biochemical properties differ, however, between species with dissimilar timing ecology. Our work advances the molecular understanding of lunar impact on fundamental rhythmic processes, including those of marine mass spawners endangered by anthropogenic change.