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.

Biological rhythms in the deep-sea hydrothermal mussel Bathymodiolus azoricus.

Biological rhythms are a fundamental property of life. The deep ocean covers 66% of our planet surface and is one of the largest biomes. The deep sea has long been considered as an arrhythmic environment because sunlight is totally absent below 1,000 m depth. In the present study, we have sequenced the temporal transcriptomes of a deep-sea species, the ecosystem-structuring vent mussel Bathymodiolus azoricus. We reveal that tidal cycles predominate in the transcriptome and physiology of mussels fixed directly at hydrothermal vents at 1,688 m depth at the Mid-Atlantic Ridge, whereas daily cycles prevail in mussels sampled after laboratory acclimation. We identify B. azoricus canonical circadian clock genes, and show that oscillations observed in deep-sea mussels could be either a direct response to environmental stimulus, or be driven endogenously by one or more biological clocks. This work generates in situ insights into temporal organisation in a deep-sea organism.

Oyster transcriptome response to Alexandrium exposure is related to saxitoxin load and characterized by disrupted digestion, energy balance, and calcium and sodium signaling.

Harmful Algal Blooms are worldwide occurrences that can cause poisoning in human seafood consumers as well as mortality and sublethal effets in wildlife, propagating economic losses. One of the most widespread toxigenic microalgal taxa is the dinoflagellate Genus Alexandrium, that includes species producing neurotoxins referred to as PST (Paralytic Shellfish Toxins). Blooms cause shellfish harvest restrictions to protect human consumers from accumulated toxins. Large inter-individual variability in toxin load within an exposed bivalve population complicates monitoring of shellfish toxicity for ecology and human health regulation. To decipher the physiological pathways involved in the bivalve response to PST, we explored the whole transcriptome of the digestive gland of the Pacific oyster Crassostrea gigas fed experimentally with a toxic Alexandrium minutum culture. The largest differences in transcript abundance were between oysters with contrasting toxin loads (1098 transcripts), rather than between exposed and non-exposed oysters (16 transcripts), emphasizing the importance of toxin load in oyster response to toxic dinoflagellates. Additionally, penalized regressions, innovative in this field, modeled accurately toxin load based upon only 70 transcripts. Transcriptomic differences between oysters with contrasting PST burdens revealed a limited suite of metabolic pathways affected, including ion channels, neuromuscular communication, and digestion, all of which are interconnected and linked to sodium and calcium exchanges. Carbohydrate metabolism, unconsidered previously in studies of harmful algal effects on shellfish, was also highlighted, suggesting energy challenge in oysters with high toxin loads. Associations between toxin load, genotype, and mRNA levels were revealed that open new doors for genetic studies identifying genetically-based low toxin accumulation.

Influence of temperature on daily locomotor activity in the crab Uca pugilator.

Animals living in the intertidal zone are exposed to prominent temperature changes. To cope with the energetic demands of environmental thermal challenges, ectotherms rely mainly on behavioral responses, which may change depending on the time of the day and seasonally. Here, we analyze how temperature shapes crabs' behavior at 2 different times of the year and show that a transition from constant cold (13.5°C) to constant warm (17.5°C) water temperature leads to increased locomotor activity levels throughout the day in fiddler crabs (Uca pugilator) collected during the summer. In contrast, the same transition in environmental temperature leads to a decrease in the amplitude of the daily locomotor activity rhythm in crabs collected during the winter. In other words, colder temperatures during the cold season favor a more prominent diurnal behavior. We interpret this winter-summer difference in the response of daily locomotor activity to temperature changes within the framework of the circadian thermoenergetics hypothesis, which predicts that a less favorable energetic balance would promote a more diurnal activity pattern. During the winter, when the energetic balance is likely less favorable, crabs would save energy by being more active during the expected high-temperature phase of the day-light phase-and less during the expected low-temperature phase of the day-dark phase. Our results suggest that endogenous rhythms in intertidal ectotherms generate adaptive behavioral programs to cope with thermoregulatory demands of the intertidal habitat.

Role and expression of cry1 in the adductor muscle of the oyster Crassostrea gigas during daily and tidal valve activity rhythms.

Cryptochromes are flavin- and pterin-containing photoreceptors of the cryptochrome/photolyase family. They play critical roles in organisms, among are which light-dependent and light-independent roles in biological rhythms. The present work aimed at describing a cryptochrome gene in the oyster Crassostrea gigas by (i) a characterization and phylogenetic analysis and (ii) by studying its expression in the relationship to rhythmic valve behavior in different entrainment regimes. Cryptochrome expression was focused on the adductor muscle of the oyster, the effector of the valve behavior. The results suggest involvement of Cgcry1 in oyster rhythmicity as a sensor of environmental zeitgebers, associated with circadian rhythms and potentially to tidal activity. The characterized gene belongs to type 1 cryptochrome/insect-type cry. Additionally, Cgcry1 presented a daily oscillation under L:D entrainment, which disappeared in constant darkness. Transcript expression of Cgcry1 also oscillated at tidal frequency under tidal entrainment and in constant darkness. Finally, exposure of tidally entrained oysters to saxitoxin (STX)-producing alga Alexandrium minutum induced a dose effect response in oysters by first altering Cgcry1 expression and then the behavior of oysters with increasing concentrations of toxins. This study initiates the characterization of the molecular clock in the oyster C. gigas and its interactions with environmental zeitgebers.

Identification, Characterization, and Diel Pattern of Expression of Canonical Clock Genes in Nephrops norvegicus (Crustacea: Decapoda) Eyestalk.

The Norway lobster, Nephrops norvegicus, is a burrowing decapod with a rhythmic burrow emergence (24 h) governed by the circadian system. It is an important resource for European fisheries and its behavior deeply affects its availability. The current knowledge of Nephrops circadian biology is phenomenological as it is currently the case for almost all crustaceans. In attempt to elucidate the putative molecular mechanisms underlying circadian gene regulation in Nephrops, we used a transcriptomics approach on cDNA extracted from the eyestalk, a structure playing a crucial role in controlling behavior of decapods. We studied 14 male lobsters under 12-12 light-darkness blue light cycle. We used the Hiseq 2000 Illumina platform to sequence two eyestalk libraries (under light and darkness conditions) obtaining about 90 millions 100-bp paired-end reads. Trinity was used for the de novo reconstruction of transcriptomes; the size at which half of all assembled bases reside in contigs (N50) was equal to 1796 (light) and 2055 (darkness). We found a list of candidate clock genes and focused our attention on canonical ones: timeless, period, clock and bmal1. The cloning of assembled fragments validated Trinity outputs. The putative Nephrops clock genes showed high levels of identity (blastx on NCBI) with known crustacean clock gene homologs such as Eurydice pulchra (period: 47%, timeless: 59%, bmal1: 79%) and Macrobrachium rosenbergii (clock: 100%). We also found a vertebrate-like cryptochrome 2. RT-qPCR showed that only timeless had a robust diel pattern of expression. Our data are in accordance with the current knowledge of the crustacean circadian clock, reinforcing the idea that the molecular clockwork of this group shows some differences with the established model in Drosophila melanogaster.

Genetic and genotoxic impacts in the oyster Crassostrea gigas exposed to the harmful alga Alexandrium minutum.

Genotoxic, genetic and behavioral impacts of the paralytic shellfish toxin (PST)-producing alga Alexandrium minutum on the oyster Crassostrea gigas were assessed using RAPD-PCR, qPCR and valve activity recording. Oysters were exposed to a dose mimicking an algal bloom (≈1600 cells ml(-1)) for 48 h. Results indicate a rapid and sustained behavioral disturbance. Animals remained open but exhibited reduced valve-opening amplitude, correlated to the amount of toxin accumulated in the digestive gland. They also exhibited increased micro-closures. In the gills, gene transcription levels were modified: a transcriptional repression of genes involved in oxidative and mitochondrial metabolism, endogenous clock, immunity and detoxification processes was observed. DNA impacts, both quantitative and qualitative, were observed as well. Indeed, both the PCR product profile and the number of hybridization sites for the RAPD probe OPB7 were modified. These results indicate genotoxic effects and gene repression in C. gigas following behavioral disturbance by A. minutum.

Evidence for a plastic dual circadian rhythm in the oyster Crassostrea gigas.

Although a significant body of literature has been devoted to the chronobiology of aquatic animals, how biological rhythms function in molluscan bivalves has been poorly studied. The first objective of this study was to determine whether an endogenous circadian rhythm does exist in the oyster, Crassostrea gigas. The second objective was to characterize it in terms of robustness. To answer these questions, the valve activity of 15 oysters was continuously recorded for 2 mo in the laboratory under different entrainment and free-running regimes using a high-frequency noninvasive valvometer. The present work demonstrates the presence of a circadian rhythm in the oyster Crassostrea gigas. First, oysters were entrained by 12 L:12 D conditions. Then, free-running conditions (D:D and L:L) indicated that the most frequently observed period ranged from 20 to 28 h, the circadian range. That endogenous circadian rhythm was characterized as weak. Indeed, the period (τ) of the individual animals exhibited high plasticity in D:D and L:L, and the animals immediately followed a 4-h phase advance or delay. Additionally, C. gigas appeared as a dual organism: all oysters were nocturnal at the beginning of the laboratory experiment (January), whereas they were diurnal at the end (March). That shift was progressive. Comparison with a full-year in situ record showed the same behavioral duality as observed in the laboratory: the animals were nocturnal in autumn-winter and diurnal in spring-summer. The significant advantage of a plastic and dual circadian rhythm in terms of adaptability in a highly changing environment is discussed.