BackgroundCircadian rhythms in behavior and physiology are the observable phenotypes from cycles in expression of, interactions between, and degradation of the underlying molecular components. In bilaterian animals, the core molecular components include Timeless-Timeout, photoreceptive cryptochromes, and several members of the basic-loop-helix-Per-ARNT-Sim (bHLH-PAS) family. While many of core circadian genes are conserved throughout the Bilateria, their specific roles vary among species. Here, we identify and experimentally study the rhythmic gene expression of conserved circadian clock members in a sea anemone in order to characterize this gene network in a member of the phylum Cnidaria and to infer critical components of the clockwork used in the last common ancestor of cnidarians and bilaterians.Methodology/Principal FindingsWe identified homologs of circadian regulatory genes in the sea anemone Nematostella vectensis, including a gene most similar to Timeout, three cryptochromes, and several key bHLH-PAS transcription factors. We then maintained N. vectensis either in complete darkness or in a 12 hour light: 12 hour dark cycle in three different light treatments (blue only, full spectrum, blue-depleted). Gene expression varied in response to light cycle and light treatment, with a particularly strong pattern observed for NvClock. The cryptochromes more closely related to the light-sensitive clade of cryptochromes were upregulated in light treatments that included blue wavelengths. With co-immunoprecipitation, we determined that heterodimerization between CLOCK and CYCLE is conserved within N. vectensis. Additionally, we identified E-box motifs, DNA sequences recognized by the CLOCK:CYCLE heterodimer, upstream of genes showing rhythmic expression.Conclusions/SignificanceThis study reveals conserved molecular and functional components of the circadian clock that were in place at the divergence of the Cnidaria and Bilateria, suggesting the animal circadian clockwork is more ancient than previous data suggest. Characterizing circadian regulation in a cnidarian provides insight into the early origins of animal circadian rhythms and molecular regulation of environmentally cued behaviors.

We assessed the microbial diversity and microenvironmental niche characteristics in the didemnid ascidian Lissoclinum patella using 16S rRNA gene sequencing, microsensor and imaging techniques. L. patella harbors three distinct microbial communities spatially separated by few millimeters of tunic tissue: (i) a biofilm on its upper surface exposed to high irradiance and O 2 levels, (ii) a cloacal cavity dominated by the prochlorophyte Prochloron spp. characterized by strong depletion of visible light and a dynamic chemical microenvironment ranging from hyperoxia in light to anoxia in darkness and (iii) a biofilm covering the underside of the animal, where light is depleted of visible wavelengths and enriched in near-infrared radiation (NIR). Variable chlorophyll fluorescence imaging demonstrated photosynthetic activity, and hyperspectral imaging revealed a diversity of photopigments in all microhabitats. Amplicon sequencing revealed the dominance of cyanobacteria in all three layers. Sequences representing the chlorophyll d containing cyanobacterium Acaryochloris marina and anoxygenic phototrophs were abundant on the underside of the ascidian in shallow waters but declined in deeper waters. This depth dependency was supported by a negative correlation between A. marina abundance and collection depth, explained by the increased attenuation of NIR as a function of water depth. The combination of microenvironmental analysis and fine-scale sampling techniques used in this investigation gives valuable first insights into the distribution, abundance and diversity of bacterial communities associated with tropical ascidians. In particular, we show that microenvironments and microbial diversity can vary significantly over scales of a few millimeters in such habitats; which is information easily lost by bulk sampling.

Enzymes in the cytochrome P450 1 family oxidize many common environmental toxicants. We identified a new CYP1, termed CYP1D1, in zebrafish. Phylogenetically, CYP1D1 is paralogous to CYP1A and the two share 45% amino acid identity and similar gene structure. In adult zebrafish, CYP1D1 is most highly expressed in liver and is relatively highly expressed in brain. CYP1D1 transcript levels were higher at 9 hours post-fertilization than at later developmental times. Treatment of zebrafish with potent aryl hydrocarbon receptor (AHR) agonists (3,3′,4,4′,5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin) did not induce CYP1D1 transcript expression. Morpholino oligonucleotide knockdown of AHR2, which mediates induction of other CYP1s, did not affect CYP1D1 expression. Zebrafish CYP1D1 heterologously expressed in yeast exhibited ethoxyresorufin-and methoxyresorufin-O-dealkylase activities. Antibodies against a CYP1D1 peptide specifically detected a single electrophoretically-resolved protein band in zebrafish liver microsomes, distinct from CYP1A. CYP1D1 in zebrafish is a CYP1A-like gene that could have metabolic functions targeting endogenous compounds.

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.

The discovery of the cyanobacterium Prochloron was the first finding of a bacterial oxyphototroph with chlorophyll (Chl) b, in addition to Chl a. It was first described as Prochloron didemni but a number of clades have since been described. Prochloron is a conspicuously large (7–25 μm) unicellular cyanobacterium living in a symbiotic relationship, primarily with (sub-) tropical didemnid ascidians; it has resisted numerous cultivation attempts and appears truly obligatory symbiotic. Recently, a Prochloron draft genome was published, revealing no lack of metabolic genes that could explain the apparent inability to reproduce and sustain photosynthesis in a free-living stage. Possibly, the unsuccessful cultivation is partly due to a lack of knowledge about the microenvironmental conditions and ecophysiology of Prochloron in its natural habitat. We used microsensors, variable chlorophyll fluorescence imaging and imaging of O2 and pH to obtain a detailed insight to the microenvironmental ecology and photobiology of Prochloron in hospite in the didemnid ascidian Lissoclinum patella. The microenvironment within ascidians is characterized by steep gradients of light and chemical parameters that change rapidly with varying irradiances. The interior zone of the ascidians harboring Prochloron thus became anoxic and acidic within a few minutes of darkness, while the same zone exhibited O2 super-saturation and strongly alkaline pH after a few minutes of illumination. Photosynthesis showed lack of photoinhibition even at high irradiances equivalent to full sunlight, and photosynthesis recovered rapidly after periods of anoxia. We discuss these new insights on the ecological niche of Prochloron and possible interactions with its host and other microbes in light of its recently published genome and a recent study of the overall microbial diversity and metagenome of L. patella.