Revealing the profound influence of diapause on gene expression: Insights from the annual transcriptome of the copepod Calanus finmarchicus.

Annual rhythms are observed in living organisms with numerous ecological implications. In the zooplanktonic copepod Calanus finmarchicus, such rhythms are crucial regarding its phenology, body lipid accumulation, and global carbon storage. Climate change drives annual biological rhythms out of phase with the prevailing environmental conditions with yet unknown but potentially catastrophic consequences. However, the molecular dynamics underlying phenology are still poorly described. In a rhythmic analysis of C. finmarchicus annual gene expression, results reveal that more than 90% of the transcriptome shows significant annual rhythms, with abrupt and dramatic upheaval between the active and diapause life cycle states. This work explores the implication of the circadian clock in the annual timing, which may control epigenetic mechanisms to profoundly modulate gene expression in response to calendar time. Results also suggest an increased light sensitivity during diapause that would ensure the photoperiodic entrainment of the endogenous annual clock.

Molecular and behavioural responses of the mussel Mytilus edulis exposed to a marine heatwave.

Marine heatwaves (MHW) threaten marine organisms and tend to increase in frequency and intensity. We exposed the blue mussel Mytilus edulis to a MHW lasting 23 days, including two 10-d periods of thermal intensity increase of +5 °C (20 °C-25 °C) interspersed by 1 day back to 20 °C, followed by a 4-d recovery period. We investigated behaviour responses of mussels and gene expression changes relative to the circadian rhythm (Per), oxidative stress (SOD), cellular apoptosis (CASP3), energy production (ATPs), and general stress response (hsp70). Results showed that the MHW disturbed the valve activity of mussels. Particularly, mussels increased the number of valve micro-closures, showing a stressful state of organisms. Mussels also decreased Per, CASP3, ATPs, and Hsp70 gene expression. Some behavioural and molecular effects persisted after the MHW, suggesting a limited recovery capacity of individuals. This work highlighted the vulnerability of M. edulis to a realistic MHW.

The effects of artificial light at night on behavioral rhythm and related gene expression are wavelength dependent in the oyster Crassostrea gigas.

Artificial light at night (ALAN) constitutes a growing threat to coastal ecosystems by altering natural light cycles, which could impair organisms' biological rhythms, with resulting physiological and ecological consequences. Coastal ecosystems are strongly exposed to ALAN, but its effects on coastal organisms are poorly studied. Besides ALAN's intensity, ALAN's quality exposure may change the impacts on organisms. This study aims to characterize the effects of different ALAN's spectral compositions (monochromatic wavelength lights in red (peak at 626 nm), green (peak at 515 nm), blue (peak at 467 nm), and white (410-680 nm) light) at low and realistic intensity (1 lx) on the oyster Crassostrea gigas daily rhythm. Results reveal that all ALAN's treatments affect the oysters' daily valve activity rhythm in different manners and the overall expression of the 13 studied genes. Eight of these genes are involved in the oyster's circadian clock, 2 are clock-associated genes, and 3 are light perception genes. The blue light has the most important effects on oysters' valve behavior and clock and clock-associated gene expression. Interestingly, red and green lights also show significant impacts on the daily rhythm, while the lowest impacts are shown with the green light. Finally, ALAN white light shows the same impact as the blue one in terms of loss of rhythmic oysters' percentage, but the chronobiological parameters of the remaining rhythmic oysters are less disrupted than when exposed to each of the monochromatic light's treatments alone. We conclude that ALAN's spectral composition does influence its effect on oysters' daily rhythm, which could give clues to limit physiological and ecological impacts on coastal environments.

Is part-night lighting a suitable mitigation strategy to limit Artificial Light at Night effects on the biological rhythm at the behavioral and molecular scales of the oyster Crassostrea gigas?

Artificial Light at Night (ALAN) is a fast-spreading threat to organisms, especially in coastal environments, where night lighting is increasing due to constant anthropization. Considering that ALAN affects a large diversity of coastal organisms, finding efficient solutions to limit these effects is of great importance but poorly investigated. The potential benefit of one strategy, in particular, should be studied since its use is growing: part-night lighting (PNL), which consists in switching off the lights for a few hours during nighttime. The aim of this study is to investigate the positive potential of the PNL strategy on the daily rhythm of the oyster Crassostrea gigas, a key species of coastal areas of ecological and commercial interest. Oysters were exposed to a control condition and three different ALAN modalities. A realistic PNL condition is applied, recreating a strategy of city policy in a coastal city boarding an urbanized bay (Lanton, Arcachon Bay, France). The PNL modality consists in switching off ALAN direct sources (5 lx) for 4 h (23-3 h) during which oysters are in darkness. Then, a PNL + skyglow (PNL + S) modality reproduces the previous one mimicking a skyglow (0.1 lx), an indirect ALAN source, during the direct lighting switch off, to get as close as possible to realistic conditions. Finally, the third ALAN condition mimics full-night direct lighting (FNL). Results revealed that PNL reduces some adverse effects of FNL on the behavioral daily rhythm. But, counterintuitively, PNL + S appears more harmful than FNL for some parameters of the behavioral daily rhythm. PNL + S modality is also the only one that affect oysters' clock and melatonin synthesis gene expression, suggesting physiological consequences. Thus, in realistic conditions, the PNL mitigation strategy might not be beneficial in the presence of skyglow, seeing worse for a coastal organism such as the oysters.

Artificial light at night at environmental intensities disrupts daily rhythm of the oyster Crassostrea gigas.

Artificial Light At Night (ALAN) masks the natural light cycles and thus can disturb the synchronization of organisms' biological rhythms with their environment. Although coastlines are highly exposed to this growing threat, studies concerning the impacts of ALAN on coastal organisms remain scarce. In this study, we investigated the ALAN exposure effects at environmentally realistic intensities (0.1, 1, 10, 25 lx) on the oyster Crassostrea gigas, a sessile bivalve subject to light pollution on shores. We focused on the effects on oyster's daily rhythm at behavioral and molecular levels. Our results showed that ALAN disrupts the oyster's daily rhythm by increasing valve activity and annihilating day / night differences of expression of circadian clock and clock-associated genes. ALAN effects occur starting from 0.1 lx, in the range of artificial skyglow illuminances. We concluded that realistic ALAN exposure affects oysters' biological rhythm, which could lead to severe physiological and ecological consequences.

Annual transcriptome of a key zooplankton species, the copepod Calanus finmarchicus.

The copepod Calanus finmarchicus (Crustacea, Copepoda) is a key zooplanktonic species with a crucial position in the North Atlantic food web and significant contributor to ocean carbon flux. Like many other high latitude animals, it has evolved a programmed arrested development called diapause to cope with long periods of limited food supply, while growth and reproduction are timed to take advantage of seasonal peaks in primary production. However, anthropogenic warming is inducing changes in the expected timing of phytoplankton blooms, suggesting phenological mismatches with negative consequences for the N. Atlantic ecosystem. While diapause mechanisms are mainly studied in terrestrial arthropods, specifically on laboratory model species, such as the fruit fly Drosophila, the molecular investigations of annual rhythms in wild marine species remain fragmentary. Here we performed a rigorous year-long monthly sampling campaign of C. finmarchicus in a Scottish Loch (UK; 56.45°N, 5.18°W) to generate an annual transcriptome. The mRNA of 36 samples (monthly triplicate of 25 individuals) have been deeply sequenced with an average depth of 137 ± 4 million reads (mean ± SE) per sample, aligned to the reference transcriptome, and filtered. We detail the quality assessment of the datasets and provide a high-quality resource for the investigation of wild annual transcriptomic rhythms (35,357 components) in a key diapausing zooplanktonic species.

Widely rhythmic transcriptome in Calanus finmarchicus during the high Arctic summer solstice period.

Solar light/dark cycles and seasonal photoperiods underpin daily and annual rhythms of life on Earth. Yet, the Arctic is characterized by several months of permanent illumination ("midnight sun"). To determine the persistence of 24h rhythms during the midnight sun, we investigated transcriptomic dynamics in the copepod Calanus finmarchicus during the summer solstice period in the Arctic, with the lowest diel oscillation and the highest altitude of the sun's position. Here we reveal that in these extreme photic conditions, a widely rhythmic daily transcriptome exists, showing that very weak solar cues are sufficient to entrain organisms. Furthermore, at extremely high latitudes and under sea-ice, gene oscillations become re-organized to include <24h rhythms. Environmental synchronization may therefore be modulated to include non-photic signals (i.e. tidal cycles). The ability of zooplankton to be synchronized by extremely weak diel and potentially tidal cycles, may confer an adaptive temporal reorganization of biological processes at high latitudes.

Daily transcriptomes of the copepod Calanus finmarchicus during the summer solstice at high Arctic latitudes.

The zooplankter Calanus finmarchicus is a member of the so-called "Calanus Complex", a group of copepods that constitutes a key element of the Arctic polar marine ecosystem, providing a crucial link between primary production and higher trophic levels. Climate change induces the shift of C. finmarchicus to higher latitudes with currently unknown impacts on its endogenous timing. Here we generated a daily transcriptome of C. finmarchicus at two high Arctic stations, during the more extreme time of Midnight Sun, the summer solstice. While the southern station (74.5 °N) was sea ice-free, the northern one (82.5 °N) was sea ice-covered. The mRNAs of the 42 samples have been sequenced with an average of 126 ± 5 million reads (mean ± SE) per sample, and aligned to the reference transcriptome. We detail the quality assessment of the datasets and the complete annotation procedure, providing the possibility to investigate daily gene expression of this ecologically important species at high Arctic latitudes, and to compare gene expression according to latitude and sea ice-coverage.

Evidence for oscillating circadian clock genes in the copepod Calanus finmarchicus during the summer solstice in the high Arctic.

The circadian clock provides a mechanism for anticipating environmental cycles and is synchronized by temporal cues such as daily light/dark cycle or photoperiod. However, the Arctic environment is characterized by several months of Midnight Sun when the sun is continuously above the horizon and where sea ice further attenuates photoperiod. To test if the oscillations of circadian clock genes remain in synchrony with subtle environmental changes, we sampled the copepod Calanus finmarchicus, a key zooplankter in the north Atlantic, to determine in situ daily circadian clock gene expression near the summer solstice at a southern (74.5° N) sea ice-free and a northern (82.5° N) sea ice-covered station. Results revealed significant oscillation of genes at both stations, indicating the persistence of the clock at this time. While copepods from the southern station showed oscillations in the daily range, those from the northern station exhibited an increase in ultradian oscillations. We suggest that in C. finmarchicus, even small daily changes of solar altitude seem to be sufficient to entrain the circadian clock and propose that at very high latitudes, in under-ice ecosystems, tidal cues may be used as an additional entrainment cue.

Bivalve mollusc circadian clock genes can run at tidal frequency.

Marine coastal habitats are complex cyclic environments as a result of sun and moon interactions. In contrast with the well-known circadian orchestration of the terrestrial animal rhythmicity (approx. 24 h), the mechanism responsible for the circatidal rhythm (approx. 12.4 h) remains largely elusive in marine organisms. We revealed in subtidal field conditions that the oyster Crassostrea gigas exhibits tidal rhythmicity of circadian clock genes and clock-associated genes. A free-running (FR) experiment showed an endogenous circatidal rhythm. In parallel, we showed in the field that oysters' valve behaviour exhibited a strong tidal rhythm combined with a daily rhythm. In the FR experiment, all behavioural rhythms were circatidal, and half of them were also circadian. Our results fuel the debate on endogenous circatidal mechanisms. In contrast with the current hypothesis on the existence of an independent tidal clock, we suggest that a single 'circadian/circatidal' clock in bivalves is sufficient to entrain behavioural patterns at tidal and daily frequencies.

Moonlight cycles synchronize oyster behaviour.

Organisms possess endogenous clock mechanisms that are synchronized to external cues and orchestrate biological rhythms. Internal timing confers the advantage of being able to anticipate environmental cycles inherent in life on Earth and to prepare accordingly. Moonlight-entrained rhythms are poorly described, being much less investigated than circadian and circannual rhythms synchronized by sunlight. Yet focus on these lunar rhythms is highly relevant to understanding temporal organization of biological processes. Here, we investigate moonlight cycle effects on valve activity behaviour of the oyster Crassostrea gigas. Our results show that oysters modulate valve behaviour according to both intensity and direction of the lunar illumination cycle. As a consequence, valve opening amplitude is significantly increased at third quarter Moons (decreasing lunar illumination) compared with first quarter Moons (increasing lunar illumination) despite identical lunar illumination, and this indicates that oyster modulation of valve behaviour by moonlight cycles is not a direct response to lunar illumination. We propose that oysters use moonlight cycles to synchronize behaviour and also other physiological and ecological aspects of this benthic mollusc bivalve.

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.

How annual course of photoperiod shapes seasonal behavior of diploid and triploid oysters, Crassostrea gigas.

In this work, we study if ploidy (i.e. number of copies of chromosomes) in the oyster Crassostrea gigas may introduce differences in behavior and in its synchronization by the annual photoperiod. To answer to the question about the effect of the seasonal course of the photoperiod on the behavior of C. gigas according to its ploidy, we quantified valve activity by HFNI valvometry in situ for 1 year in both diploid and triploid oysters. Chronobiological analyses of daily, tidal and lunar rhythms were performed according the annual change of the photoperiod. In parallel, growth and gametogenesis status were measured and spawning events were detected by valvometry. The results showed that triploids had reduced gametogenesis, without spawning events, and approximately three times more growth than diploids. These differences in physiological efforts could explain the result that photoperiod (daylength and/or direction of daylength) differentially drives and modulates seasonal behavior of diploid and triploid oysters. Most differences were observed during long days (spring and summer), where triploids showed longer valve opening duration but lower opening amplitude, stronger daily rhythm and weaker tidal rhythm. During this period, diploids did major gametogenesis and spawning whereas triploids did maximal growth. Differences were also observed in terms of moonlight rhythmicity and neap-spring tidal cycle rhythmicity. We suggest that the seasonal change of photoperiod differentially synchronizes oyster behavior and biological rhythms according to physiological needs based on ploidy.

Trojan Horse Strategy for Non-invasive Interference of Clock Gene in the Oyster Crassostrea gigas.

RNA interference is a powerful method to inhibit specific gene expression. Recently, silencing target genes by feeding has been successfully carried out in nematodes, insects, and small aquatic organisms. A non-invasive feeding-based RNA interference is reported here for the first time in a mollusk bivalve, the pacific oyster Crassostrea gigas. In this Trojan horse strategy, the unicellular alga Heterocapsa triquetra is the food supply used as a vector to feed oysters with Escherichia coli strain HT115 engineered to express the double-stranded RNA targeting gene. To test the efficacy of the method, the Clock gene, a central gene of the circadian clock, was targeted for knockout. Results demonstrated specific and systemic efficiency of the Trojan horse strategy in reducing Clock mRNA abundance. Consequences of Clock disruption were observed in Clock-related genes (Bmal, Tim1, Per, Cry1, Cry2, Rev.-erb, and Ror) and triploid oysters were more sensitive than diploid to the interference. This non-invasive approach shows an involvement of the circadian clock in oyster bioaccumulation of toxins produced by the harmful alga Alexandrium minutum.

Remodeling of the cycling transcriptome of the oyster Crassostrea gigas by the harmful algae Alexandrium minutum.

As a marine organism, the oyster Crassostrea gigas inhabits a complex biotope governed by interactions between the moon and the sun cycles. We used next-generation sequencing to investigate temporal regulation of oysters under light/dark entrainment and the impact of harmful algal exposure. We found that ≈6% of the gills' transcriptome exhibits circadian expression, characterized by a nocturnal and bimodal pattern. Surprisingly, a higher number of ultradian transcripts were also detected under solely circadian entrainment. The results showed that a bloom of Alexandrium minutum generated a remodeling of the bivalve's temporal structure, characterized by a loss of oscillations, a genesis of de novo oscillating transcripts, and a switch in the period of oscillations. These findings provide unprecedented insights into the diurnal landscape of the oyster's transcriptome and pleiotropic remodeling due to toxic algae exposure, revealing the intrinsic plasticity of the cycling transcriptome in oysters.

Molecular Characterization of Voltage-Gated Sodium Channels and Their Relations with Paralytic Shellfish Toxin Bioaccumulation in the Pacific Oyster Crassostrea gigas.

Paralytic shellfish toxins (PST) bind to voltage-gated sodium channels (Nav) and block conduction of action potential in excitable cells. This study aimed to (i) characterize Nav sequences in Crassostrea gigas and (ii) investigate a putative relation between Nav and PST-bioaccumulation in oysters. The phylogenetic analysis highlighted two types of Nav in C. gigas: a Nav1 (CgNav1) and a Nav2 (CgNav2) with sequence properties of sodium-selective and sodium/calcium-selective channels, respectively. Three alternative splice transcripts of CgNav1 named A, B and C, were characterized. The expression of CgNav1, analyzed by in situ hybridization, is specific to nervous cells and to structures corresponding to neuromuscular junctions. Real-time PCR analyses showed a strong expression of CgNav1A in the striated muscle while CgNav1B is mainly expressed in visceral ganglia. CgNav1C expression is ubiquitous. The PST binding site (domain II) of CgNav1 variants possess an amino acid Q that could potentially confer a partial saxitoxin (STX)-resistance to the channel. The CgNav1 genotype or alternative splicing would not be the key point determining PST bioaccumulation level in oysters.

Vaccination with metastasis-related tumor associated antigen TPD52 and CpG/ODN induces protective tumor immunity.

Tumor protein D52 (TPD52) is involved in transformation and metastasis and has been shown to be over-expressed in tumor cells compared to normal cells and tissues. Murine TPD52 (mD52) shares 86% protein identity with the human TPD52 orthologue (hD52). To study TPD52 protein as a target for active vaccination recombinant, mD52 was administered as a protein-based vaccine. Naïve mice were immunized with either mD52 protein and CpG/ODN as a molecular adjuvant or CpG/ODN alone. Two weeks following the final immunization, mice were challenged s.c. with syngeneic tumor cells that over-express mD52. Two distinct murine tumor cell lines were used for challenge in this model, mKSA and 3T3.mD52. Half of the mice immunized with mD52 and CpG/ODN rejected or delayed onset of mKSA s.c. tumor cell growth, and 40% of mice challenged with 3T3.mD52 rejected s.c. tumor growth, as well as the formation of spontaneous lethal lung metastases. Mice immunized with mD52 and CpG/ODN generated detectable mD52-specific IgG antibody responses indicating that mD52 protein vaccination induced an adaptive immune response. In addition, mice that rejected tumor challenge generated tumor-specific cytotoxic T lymphocytes' responses. Importantly, microscopic and gross evaluation of organs from mD52 immunized mice revealed no evidence of autoimmunity as assessed by absence of T cell infiltration and absence of microscopic pathology. Together, these data demonstrate that mD52 vaccination induces an immune response that is capable of rejecting tumors that over-express mD52 without the induction of harmful autoimmunity.

Induction of tumorigenesis and metastasis by the murine orthologue of tumor protein D52.

Expression studies have consistently identified tumor protein D52 (TPD52) overexpression in tumor cells. Murine TPD52 (mD52) shares 86% identity with the human orthologue. To study a possible role for TPD52 in transformation, 3T3 fibroblasts were transfected with the full-length cDNA for mD52. Expression of mD52 was confirmed by reverse transcription-PCR (RT-PCR), real-time PCR, and Western blot analysis compared with 3T3 and vector-transfected 3T3 (3T3.V), and the resultant cell line was designated 3T3.mD52. At 4 weeks, 3T3.mD52 gained a 2-fold increase in growth rate, lost contact inhibition, and exhibited a marked phenotype change. Further characterization revealed an acquired ability for anchorage-independent cell growth. To determine whether 3T3.mD52 had become tumorigenic, naïve, healthy, immunocompetent syngeneic mice were inoculated subcutaneously with varying cell doses. Tumors measuring >1 cm(2) were detected 60 days postinoculation with 3T3.mD52, and a 50% subcutaneous tumor incidence was obtained with as few as 5 x 10(5) 3T3.mD52 cells. Remarkably, when lungs from 3T3.mD52 tumor-bearing mice were analyzed, numerous tumor nodules were observed, ranging from nodules less than 10 to nodules too numerous to count (inoculation with 1 x 10(5) and 5 x 10(6) cells, respectively). Further support for the metastatic capacity of 3T3.mD52 was the demonstration that transforming growth factor (TGF)-betaR1 (receptor) expression decreased and TGF-beta1 secretion increased in 3T3.mD52 compared with 3T3 controls. cDNA microarray analysis showed a gene expression pattern that further supported mD52-induced transformation and metastasis. Together, these data suggest that mD52 expression in 3T3 cells initiated cellular transformation, tumorigenesis, and progression to metastasis.