Molecular circadian rhythms are robust in marine annelids lacking rhythmic behavior.

The circadian clock controls behavior and metabolism in various organisms. However, the exact timing and strength of rhythmic phenotypes can vary significantly between individuals of the same species. This is highly relevant for rhythmically complex marine environments where organismal rhythmic diversity likely permits the occupation of different microenvironments. When investigating circadian locomotor behavior of Platynereis dumerilii, a model system for marine molecular chronobiology, we found strain-specific, high variability between individual worms. The individual patterns were maintained for several weeks. A diel head transcriptome comparison of behaviorally rhythmic versus arrhythmic wild-type worms showed that 24-h cycling of core circadian clock transcripts is identical between both behavioral phenotypes. While behaviorally arrhythmic worms showed a similar total number of cycling transcripts compared to their behaviorally rhythmic counterparts, the annotation categories of their transcripts, however, differed substantially. Consistent with their locomotor phenotype, behaviorally rhythmic worms exhibit an enrichment of cycling transcripts related to neuronal/behavioral processes. In contrast, behaviorally arrhythmic worms showed significantly increased diel cycling for metabolism- and physiology-related transcripts. The prominent role of the neuropeptide pigment-dispersing factor (PDF) in Drosophila circadian behavior prompted us to test for a possible functional involvement of Platynereis pdf. Differing from its role in Drosophila, loss of pdf impacts overall activity levels but shows only indirect effects on rhythmicity. Our results show that individuals arrhythmic in a given process can show increased rhythmicity in others. Across the Platynereis population, rhythmic phenotypes exist as a continuum, with no distinct "boundaries" between rhythmicity and arrhythmicity. We suggest that such diel rhythm breadth is an important biodiversity resource enabling the species to quickly adapt to heterogeneous or changing marine environments. In times of massive sequencing, our work also emphasizes the importance of time series and functional tests.

Rhythms and Clocks in Marine Organisms.

The regular movements of waves and tides are obvious representations of the oceans' rhythmicity. But the rhythms of marine life span across ecological niches and timescales, including short (in the range of hours) and long (in the range of days and months) periods. These rhythms regulate the physiology and behavior of individuals, as well as their interactions with each other and with the environment. This review highlights examples of rhythmicity in marine animals and algae that represent important groups of marine life across different habitats. The examples cover ecologically highly relevant species and a growing number of laboratory model systems that are used to disentangle key mechanistic principles. The review introduces fundamental concepts of chronobiology, such as the distinction between rhythmic and endogenous oscillator-driven processes. It also addresses the relevance of studying diverse rhythms and oscillators, as well as their interconnection, for making better predictions of how species will respond to environmental perturbations, including climate change. As the review aims to address scientists from the diverse fields of marine biology, ecology, and molecular chronobiology, all of which have their own scientific terms, we provide definitions of key terms throughout the article.

A Cryptochrome adopts distinct moon- and sunlight states and functions as sun- versus moonlight interpreter in monthly oscillator entrainment.

The moon's monthly cycle synchronizes reproduction in countless marine organisms. The mass-spawning bristle worm Platynereis dumerilii uses an endogenous monthly oscillator set by full moon to phase reproduction to specific days. But how do organisms recognize specific moon phases? We uncover that the light receptor L-Cryptochrome (L-Cry) discriminates between different moonlight durations, as well as between sun- and moonlight. A biochemical characterization of purified L-Cry protein, exposed to naturalistic sun- or moonlight, reveals the formation of distinct sun- and moonlight states characterized by different photoreduction- and recovery kinetics of L-Cry's co-factor Flavin Adenine Dinucleotide. In Platynereis, L-Cry's sun- versus moonlight states correlate with distinct subcellular localizations, indicating different signaling. In contrast, r-Opsin1, the most abundant ocular opsin, is not required for monthly oscillator entrainment. Our work reveals a photo-ecological concept for natural light interpretation involving a "valence interpreter" that provides entraining photoreceptor(s) with light source and moon phase information.

Animal behavior is central in shaping the realized diel light niche.

Animal behavior in space and time is structured by the perceived day/night cycle. However, this is modified by the animals' own movement within its habitat, creating a realized diel light niche (RDLN). To understand the RDLN, we investigated the light as experienced by zooplankton undergoing synchronized diel vertical migration (DVM) in an Arctic fjord around the spring equinox. We reveal a highly dampened light cycle with diel changes being about two orders of magnitude smaller compared to the surface or a static depth. The RDLN is further characterized by unique wavelength-specific irradiance cycles. We discuss the relevance of RDLNs for animal adaptations and interactions, as well as implications for circadian clock entrainment in the wild and laboratory.

Seasonal variation in UVA light drives hormonal and behavioural changes in a marine annelid via a ciliary opsin.

The right timing of animal physiology and behaviour ensures the stability of populations and ecosystems. To predict anthropogenic impacts on these timings, more insight is needed into the interplay between environment and molecular timing mechanisms. This is particularly true in marine environments. Using high-resolution, long-term daylight measurements from a habitat of the marine annelid Platynereis dumerilii, we found that temporal changes in ultraviolet A (UVA)/deep violet intensities, more than longer wavelengths, can provide annual time information, which differs from annual changes in the photoperiod. We developed experimental set-ups that resemble natural daylight illumination conditions, and automated, quantifiable behavioural tracking. Experimental reduction of UVA/deep violet light (approximately 370-430 nm) under a long photoperiod (16 h light and 8 h dark) significantly decreased locomotor activities, comparable to the decrease caused by a short photoperiod (8 h light and 16 h dark). In contrast, altering UVA/deep violet light intensities did not cause differences in locomotor levels under a short photoperiod. This modulation of locomotion by UVA/deep violet light under a long photoperiod requires c-opsin1, a UVA/deep violet sensor employing Gi signalling. C-opsin1 also regulates the levels of rate-limiting enzymes for monogenic amine synthesis and of several neurohormones, including pigment-dispersing factor, vasotocin (vasopressin/oxytocin) and neuropeptide Y. Our analyses indicate a complex inteplay between UVA/deep violet light intensities and photoperiod as indicators of annual time.

Rhythms of behavior: are the times changin'?

Animal life is rhythmic. Here we provide an overview of various rhythmic behaviors, connected environmental factors and endogenous mechanisms. We not only cover terrestrial species, but also highlight aquatic environments with typically complex interconnected rhythms. We further address diel, seasonal and potential lunar rhythms of humans. While we cannot be complete, we aim to emphasize three aspects: First, to raise awareness for the all-encompassing presence of behavioral rhythms and their importance in ecology and evolution. Second, to raise awareness how limited our mechanistic understanding is, besides analyses in a small set of model species. Finally, we discuss how anthropogenic effects can affect behavioral rhythmicity and how this might affect ecosystems in the future, as 'For the times they are a-changin''.

Circadian Clock Involvement in Zooplankton Diel Vertical Migration.

Biological clocks are a ubiquitous ancient and adaptive mechanism enabling organisms to anticipate environmental cycles and to regulate behavioral and physiological processes accordingly [1]. Although terrestrial circadian clocks are well understood, knowledge of clocks in marine organisms is still very limited [2-5]. This is particularly true for abundant species displaying large-scale rhythms like diel vertical migration (DVM) that contribute significantly to shaping their respective ecosystems [6]. Here we describe exogenous cycles and endogenous rhythms associated with DVM of the ecologically important and highly abundant planktic copepod Calanus finmarchicus. In the laboratory, C. finmarchicus shows circadian rhythms of DVM, metabolism, and most core circadian clock genes (clock, period1, period2, timeless, cryptochrome2, and clockwork orange). Most of these genes also cycle in animals assessed in the wild, though expression is less rhythmic at depth (50-140 m) relative to shallow-caught animals (0-50 m). Further, peak expressions of clock genes generally occurred at either sunset or sunrise, coinciding with peak migration times. Including one of the first field investigations of clock genes in a marine species [5, 7], this study couples clock gene measurements with laboratory and field data on DVM. While the mechanistic connection remains elusive, our results imply a high degree of causality between clock gene expression and one of the planet's largest daily migrations of biomass. We thus suggest that circadian clocks increase zooplankton fitness by optimizing the temporal trade-off between feeding and predator avoidance, especially when environmental drivers are weak or absent [8].

Metabolic suppression during protracted exposure to hypoxia in the jumbo squid, Dosidicus gigas, living in an oxygen minimum zone.

The jumbo squid, Dosidicus gigas, can survive extended forays into the oxygen minimum zone (OMZ) of the Eastern Pacific Ocean. Previous studies have demonstrated reduced oxygen consumption and a limited anaerobic contribution to ATP production, suggesting the capacity for substantial metabolic suppression during hypoxic exposure. Here, we provide a more complete description of energy metabolism and explore the expression of proteins indicative of transcriptional and translational arrest that may contribute to metabolic suppression. We demonstrate a suppression of total ATP demand under hypoxic conditions (1% oxygen, PO2 =0.8 kPa) in both juveniles (52%) and adults (35%) of the jumbo squid. Oxygen consumption rates are reduced to 20% under hypoxia relative to air-saturated controls. Concentrations of arginine phosphate (Arg-P) and ATP declined initially, reaching a new steady state (~30% of controls) after the first hour of hypoxic exposure. Octopine began accumulating after the first hour of hypoxic exposure, once Arg-P breakdown resulted in sufficient free arginine for substrate. Octopine reached levels near 30 mmol g(-1) after 3.4 h of hypoxic exposure. Succinate did increase through hypoxia but contributed minimally to total ATP production. Glycogenolysis in mantle muscle presumably serves to maintain muscle functionality and balance energetics during hypoxia. We provide evidence that post-translational modifications on histone proteins and translation factors serve as a primary means of energy conservation and that select components of the stress response are altered in hypoxic squids. Reduced ATP consumption under hypoxia serves to maintain ATP levels, prolong fuel store use and minimize the accumulation of acidic intermediates of anaerobic ATP-generating pathways during prolonged diel forays into the OMZ. Metabolic suppression likely limits active, daytime foraging at depth in the core of the OMZ, but confers an energetic advantage over competitors that must remain in warm, oxygenated surface waters. Moreover, the capacity for metabolic suppression provides habitat flexibility as OMZs expand as a result of climate change.