Delroisse J et al. (2014), High opsin diversity in a non-visual infaunal b...

ECB-ART-43712

BMC Genomics 2014 Nov 28;15:1035. doi: 10.1186/1471-2164-15-1035.

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High opsin diversity in a non-visual infaunal brittle star.

Delroisse J , Ullrich-Lüter E , Ortega-Martinez O , Dupont S , Arnone MI , Mallefet J , Flammang P .

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BACKGROUND: In metazoans, opsins are photosensitive proteins involved in both vision and non-visual photoreception. Echinoderms have no well-defined eyes but several opsin genes were found in the purple sea urchin (Strongylocentrotus purpuratus) genome. Molecular data are lacking for other echinoderm classes although many species are known to be light sensitive. RESULTS: In this study focused on the European brittle star Amphiura filiformis, we first highlighted a blue-green light sensitivity using a behavioural approach. We then identified 13 new putative opsin genes against eight bona fide opsin genes in the genome of S. purpuratus. Six opsins were included in the rhabdomeric opsin group (r-opsins). In addition, one putative ciliary opsin (c-opsin), showing high similarity with the c-opsin of S. purpuratus (Sp-opsin 1), one Go opsin similar to Sp-opsins 3.1 and 3.2, two basal-branch opsins similar to Sp-opsins 2 and 5, and two neuropsins similar to Sp-opsin 8, were identified. Finally, two sequences from one putative RGR opsin similar to Sp-opsin 7 were also detected. Adult arm transcriptome analysis pinpointed opsin mRNAs corresponding to one r-opsin, one neuropsin and the homologue of Sp-opsin 2. Opsin phylogeny was determined by maximum likelihood and Bayesian analyses. Using antibodies designed against c- and r-opsins from S. purpuratus, we detected putative photoreceptor cells mainly in spines and tube feet of A. filiformis, respectively. The r-opsin expression pattern is similar to the one reported in S. purpuratus with cells labelled at the tip and at the base of the tube feet. In addition, r-opsin positive cells were also identified in the radial nerve of the arm. C-opsins positive cells, expressed in pedicellariae, spines, tube feet and epidermis in S. purpuratus were observed at the level of the spine stroma in the brittle star. CONCLUSION: Light perception in A. filiformis seems to be mediated by opsins (c- and r-) in, at least, spines, tube feet and in the radial nerve cord. Other non-visual opsin types could participate to the light perception process indicating a complex expression pattern of opsins in this infaunal brittle star.

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Genes referenced: LOC100887844 LOC115919910 LOC578116 LOC579470 rhol1 rhol2 tubgcp2
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	Figure 1. Outline of the search pipeline used to identify putative opsin sequences in our datasets (see Methods for details). For reference sequences, see Additional file 2.
	Figure 2. Amino acid similarity (%) between Af-opsins and Sp-opsins (+ Rn Rhodopsin). Similarity measurements were conducted between each Af-opsin and all reference opsins (Sp, Rn) on the basis of a local alignment. Trimming was performed on the local alignment. Each similarity estimation is depending on the length of the local alignment. Values framed in red indicate best similarities.
	Figure 3. Scatter-plots comparing the number of arms observed out of the sediment during the day (specific colour treatments) and the night (means ± S.E.M.). Different treatment conditions (white daylight, no daylight, red/green/blue daylights) are represented in each graph. /*Significant difference between day and night for a particular treatment, nsNo significant difference.
	Figure 4. Deduced amino acid sequences of Amphiura filiformis opsins (names in bold in the figure) aligned with Strongylocentrotus purpuratus opsins and Rattus norvegicus rhodopsin. Alignment is limited to two highly conserved regions including the “DRY-type” tripeptide, the opsin-specific lysine residue, and the “NPxxY(x)6F” pattern. Predicted transmembrane alpha-helices are underlined in red. The lysine residue involved in the Schiff base formation – equivalent to K296 of the R. norvegicus rhodopsin - is highlighted in red in the alignment. The tyrosine residue (Y) in the position equivalent to the glutamate counterion E113 in R. norvegicus rhodopsin, and the DRY-type tripeptide motif (E134/R135/Y136 in R. norvegicus rhodopsin) is highlighted in blue. The pattern “NPxxY(x)6F” (position 302–313 of the R. norvegicus rhodopsin sequence) is highlighted in green. The amino acid triad (in the equivalent position 310–312 in the R. norvegicus rhodopsin) belong to the pattern NPxxY(x)6F. The “NxQ” motif, classically observed in c-opsins is written in red in the alignment and the “HxK” motif, classically observed in r-opsins, in blue. Other amino-acid residues that are highly conserved in the whole opsin family are shown with a grey background. See text and Additional file 3 for more details. Numbers indicated in gray on the left side of each aligned region correspond to the position number of the first amino acid of the considered sequence.
	Figure 5. Phylogenetic tree of metazoan opsins including the new opsins from Amphiura filiformis. Representative bilaterian opsin members cluster into six significantly supported groups in both maximum likelihood and Bayesian analysis. Branch support values are indicated next to the branching points and correspond to posterior probabilities and boostrap proportions (in italics). Branch length scale bar indicate relative amount of amino acid changes (Bayesian analysis). A. filiformis opsins are represented in bold (Af). Other echinoderm opsins were included in the analyses: Strongylocentrotus purpuratus (Sp), Strongylocentrotus droebachiensis (Sd), Paracentrotus lividus (Pl), Hemicentrotus pulcherrimus (Hp), Asterias rubens (Ar).
	Figure 6. Ciliary opsin positive cells in decalcified arms of Amphiura filiformis, detected by Confocal laser scanning microscopy. Double immunolocalisation of c-opsin (red) and acetylated alpha-tubulin (green). A. Partially inverted transmission picture of an arm showing c-opsin positive cells/photoreceptor cells (cop + PRC) in the spines (sp). B. c-opsin proteins seem to be localised in the inner portion of the spines. C. Dense nerve tracts are present at the basis of the spines. D. Nerve tracts partially also run through an internal portion of the spines. Tube feet (tf) show no c-opsin positive cells. E. High magnification reveals connection of the c-opsin positive cells to the nerve tracts (ntr). F. Transmission view of a spine showing the internal c-opsin positive cells and the dark pigment (pig) at the spine base. Scale bars in A-D: 100 μm, E: 20 μm, F: 50 μm.
	Figure 7. Rhabdomeric opsin positive cells in decalcified arms of Amphiura filiformis, detected by confocal laser scanning microscopy. Double immunolocalisation of r-opsin (red) and acetylated alpha-tubulin (green). A. Transmission view of arm (arm) with tube feet (tf) and spines (sp). B. R-opsin positive cells reside in tube feet and radial nerve. C. Innervation of arm showing connection of the radial nerve (radn) to each tube foot nerve ring (tfnr) and spine nerve (spn). D. Tube feet show r-opsin protein (rop+) presence within middle and tip region. R-opsins are also detected within radial nerves. R-opsin positive structures within the radial nerve likely represent axonal/dendritic projections and no stained cell bodies were observed. Scale bars in A-D: 200 μm.
	Figure 8. Rhabdomeric opsin positive cells in the tube feet of Amphiura filiformis, detected by CLSM. Double immunolocalisation of r-opsin (red/hot red) and acetylated alpha-tubulin (green). A. Transmission view of a tube foot (tf) with tip r-opsin positive cells (trop+). B. R-opsin staining shows a cone shaped morphology. C. Cilia (ci) are present along the longitudinal axis of the tube foot. D. Dark areas within the r-opsin positive region indicate location of nuclei. Cilia protrude from the r-opsin positive cells. E. Tube foot proximal r-opsin positive cells (brop+) showing a striated pattern. F. Dense nerve tracts (ntr) but also fine nerve fibers connect to the tube foot nerve ring (tfnr) in the area of proximal r-opsin positive cells. Scale bars in A-D: 10 μm, E-F: 50 μm.

References [+] :

Agca, Neurosensory and neuromuscular organization in tube feet of the sea urchin Strongylocentrotus purpuratus. 2011, Pubmed, Echinobase