Phylogenetic tree inferred from rhodopsin amino acid sequences.

<p>Shown at nodes are bootstrap values from Neighbor-Joining (left), SH-like value of maximum likelihood (medium) and Bayesian analysis (right); only values ≥70%/0.7 at critical nodes are shown. The thickest branches denote bootstrap values of>90%, medium-thick branches values of 70 to 90%, and thin branches values of <70%. The scale bar indicates the substitutions rate per nucleotide. Tree is rooted with <i>Plecoglossus altivelis</i>. The two major types, the proton-pumping (PR) and the sensory type (SR), are indicated on the far right while subgroups within PR are shown next. Pie slices indicate the relative transcript abundances of the two major types (with subtypes within each type combined) in terms of the percentage of reads. Asterisks depict sequences obtained in this study that were either new or matched previously reported sequences (accession numbers indicated).</p

Ratio of expression levels between proton-pump rhodopsin (PR) and sensory rhodopsins (SR1, SR2 and SR3).

<p>LD, cultures grown under light: dark cycle; DD, cultures grown under continuous darkness; error bars, standard deviation. (A) PR/SR1; (B) PR/SR2; (C) PR/SR3. Significant differences between LD and DD groups were marked with “*”.</p

Light-Promoted Rhodopsin Expression and Starvation Survival in the Marine Dinoflagellate <i>Oxyrrhis marina</i>

<div><p>The discovery of microbial rhodopsins in marine proteobacteria changed the dogma that photosynthesis is the only pathway to use the solar energy for biological utilization in the marine environment. Although homologs of these rhodopsins have been identified in dinoflagellates, the diversity of the encoding genes and their physiological roles remain unexplored. As an initial step toward addressing the gap, we conducted high-throughput transcriptome sequencing on <i>Oxyrrhis marina</i> to retrieve rhodopsin transcripts, rapid amplification of cDNA ends to isolate full-length cDNAs of dominant representatives, and quantitative reverse-transcription PCR to investigate their expression under varying conditions. Our phylogenetic analyses showed that <i>O</i>. <i>marina</i> contained both the proton-pumping type (PR) and sensory type (SR) rhodopsins, and the transcriptome data showed that the PR type dominated over the SR type. We compared rhodopsin gene expression for cultures kept under light: dark cycle and continuous darkness in a time course of 24 days without feeding. Although both types of rhodopsin were expressed under the two conditions, the expression levels of PR were much higher than SR, consistent with the transcriptomic data. Furthermore, relative to cultures kept in the dark, rhodopsin expression levels and cell survival rate were both higher in cultures grown in the light. This is the first report of light-dependent promotion of starvation survival and concomitant promotion of PR expression in a eukaryote. While direct evidence needs to come from functional test on rhodopsins <i>in vitro</i> or gene knockout/knockdown experiments, our results suggest that the proton-pumping rhodopsin might be responsible for the light-enhanced survival of <i>O</i>. <i>marina</i>, as previously demonstrated in bacteria.</p></div

Primers used in this study.

<p>Primers used in this study.</p

Expression levels of rhodopsin genes as normalized to <i>cox1</i> cDNA.

<p>LD, cultures grown under light: dark cycle; DD, cultures grown under continuous darkness; error bars, standard deviation. (A) PR; (B) SR1; (C) SR2; (D) SR3. Significant differences between LD and DD groups were marked with “*”.</p

Growth curve (A), cell volume (B), prey 18S rRNA percentage (C), cellular total RNA content (D) of starved <i>O</i>. <i>marina</i>.

<p>RT-qPCR was carried out to quantify the 18S rRNA level of both <i>O</i>. <i>marina</i> and <i>D</i>. <i>tertiolecta</i> in the cDNA libraries from both conditions. Y-axis in (C) indicates the ratio of prey 18S rRNA and <i>O</i>. <i>marina</i> 18S rRNA. LD, cultures grown under light: dark cycle; DD, cultures grown under continuous darkness; error bars, standard deviation. Significant differences between LD and DD groups were marked with “*”. Arrow in the figure denotes time when food was supplied.</p

Expression levels of rhodopsin genes as normalized to 18S rRNA cDNA.

<p>LD, cultures grown under light: dark cycle; DD, cultures grown under continuous darkness; error bars, standard deviation. (A) PR; (B) SR1; (C) SR2; (D) SR3. Significant differences between LD and DD groups were marked with “*”.</p

Taxonomic distribution of 18S rDNA clones retrieved from <i>Can. Pauper.</i>

<p>Taxonomic distribution of 18S rDNA clones retrieved from <i>Can. Pauper.</i></p

Copepod species employed for the test of CEEC primer set^*.

<p>*Adult copepods were used in this experiment, F: female, M: male. The symbol “+” denotes positive result; “−” denotes negative result; * DYB, Daya Bay (22°36.274′N, 114°34.0′E), South China Sea, China; AV, Avery Point (41°18.917′N, 72°3.81′W), Connecticut, USA; PRE, Pearl river estuary (22°7.022′N, 113°52.175′E), South China Sea, China; SYB, Sanya Bay (18°12.794′N, 114°34.0′E), South China Sea, China.</p

Detecting <i>In Situ</i> Copepod Diet Diversity Using Molecular Technique: Development of a Copepod/Symbiotic Ciliate-Excluding Eukaryote-Inclusive PCR Protocol

<div><p>Knowledge of in situ copepod diet diversity is crucial for accurately describing pelagic food web structure but is challenging to achieve due to lack of an easily applicable methodology. To enable analysis with whole copepod-derived DNAs, we developed a copepod-excluding 18S rDNA-based PCR protocol. Although it is effective in depressing amplification of copepod 18S rDNA, its applicability to detect diverse eukaryotes in both mono- and mixed-species has not been demonstrated. Besides, the protocol suffers from the problem that sequences from symbiotic ciliates are overrepresented in the retrieved 18S rDNA libraries. In this study, we designed a blocking primer to make a combined primer set (copepod/symbiotic ciliate-excluding eukaryote-common: CEEC) to depress PCR amplification of symbiotic ciliate sequences while maximizing the range of eukaryotes amplified. We firstly examined the specificity and efficacy of CEEC by PCR-amplifying DNAs from 16 copepod species, 37 representative organisms that are potential prey of copepods and a natural microplankton sample, and then evaluated the efficiency in reconstructing diet composition by detecting the food of both lab-reared and field-collected copepods. Our results showed that the CEEC primer set can successfully amplify 18S rDNA from a wide range of isolated species and mixed-species samples while depressing amplification of that from copepod and targeted symbiotic ciliate, indicating the universality of CEEC in specifically detecting prey of copepods. All the predetermined food offered to copepods in the laboratory were successfully retrieved, suggesting that the CEEC-based protocol can accurately reconstruct the diets of copepods without interference of copepods and their associated ciliates present in the DNA samples. Our initial application to analyzing the food composition of field-collected copepods uncovered diverse prey species, including those currently known, and those that are unsuspected, as copepod prey. While testing is required, this protocol provides a useful strategy for depicting in situ dietary composition of copepods.</p></div