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Genomic Organization, Evolution, and Expression of Photoprotein and Opsin Genes in Mnemiopsis Leidyi: A New View of Ctenophore Photocytes

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Genomic Organization, Evolution, and Expression of Photoprotein and Opsin Genes in Mnemiopsis Leidyi: A New View of Ctenophore Photocytes

Christine E Schnitzler et al. BMC Biol.

Abstract

Background: Calcium-activated photoproteins are luciferase variants found in photocyte cells of bioluminescent jellyfish (Phylum Cnidaria) and comb jellies (Phylum Ctenophora). The complete genomic sequence from the ctenophore Mnemiopsis leidyi, a representative of the earliest branch of animals that emit light, provided an opportunity to examine the genome of an organism that uses this class of luciferase for bioluminescence and to look for genes involved in light reception. To determine when photoprotein genes first arose, we examined the genomic sequence from other early-branching taxa. We combined our genomic survey with gene trees, developmental expression patterns, and functional protein assays of photoproteins and opsins to provide a comprehensive view of light production and light reception in Mnemiopsis.

Results: The Mnemiopsis genome has 10 full-length photoprotein genes situated within two genomic clusters with high sequence conservation that are maintained due to strong purifying selection and concerted evolution. Photoprotein-like genes were also identified in the genomes of the non-luminescent sponge Amphimedon queenslandica and the non-luminescent cnidarian Nematostella vectensis, and phylogenomic analysis demonstrated that photoprotein genes arose at the base of all animals. Photoprotein gene expression in Mnemiopsis embryos begins during gastrulation in migrating precursors to photocytes and persists throughout development in the canals where photocytes reside. We identified three putative opsin genes in the Mnemiopsis genome and show that they do not group with well-known bilaterian opsin subfamilies. Interestingly, photoprotein transcripts are co-expressed with two of the putative opsins in developing photocytes. Opsin expression is also seen in the apical sensory organ. We present evidence that one opsin functions as a photopigment in vitro, absorbing light at wavelengths that overlap with peak photoprotein light emission, raising the hypothesis that light production and light reception may be functionally connected in ctenophore photocytes. We also present genomic evidence of a complete ciliary phototransduction cascade in Mnemiopsis.

Conclusions: This study elucidates the genomic organization, evolutionary history, and developmental expression of photoprotein and opsin genes in the ctenophore Mnemiopsis leidyi, introduces a novel dual role for ctenophore photocytes in both bioluminescence and phototransduction, and raises the possibility that light production and light reception are linked in this early-branching non-bilaterian animal.

Figures

Figure 1
Figure 1
Genomic arrangement of Mnemiopsis photoprotein genes based on the current draft genome assembly. There are at least 10 putative full-length photoprotein genes, including seven on scaffold ML0857 and three on ML2154. There are also three partial photoprotein genes, indicated by a 'P'. Photoprotein sequences fall into three sequence groups based on similarity. Sequence groups are color-coded and labeled A, B, or C. Genomic coordinates are indicated below each gene cluster. Scaffold lengths (bp) are shown in black boxes at the end of the scaffold. Scaffolds and genes are not drawn to scale.
Figure 2
Figure 2
Full-length amino acid alignment of functional photoproteins from hydromedusae and ctenophores. EF-hand domains I, III, and IV are indicated by long lines above the alignment, while the 12-residue calcium binding loops within each EF-hand domain are indicated by short lines above the alignment. Calcium-coordinating residues within EF-hand domains (black triangles) and residues in the coelenterazine binding pocket (black circles) are indicated. Residue 6 within the EF-hand I calcium loop is also indicated (black star). Residue numbering is based on the berovin sequence (Ba_berovin). Columns of residues are shaded by similarity group conservation (defined by GeneDoc and the BLOSUM62 matrix) where black shows ≥90%, dark grey shows ≥70% and light grey shows ≥55% similar residues in a column. Species are abbreviated as follows: Mlei = Mnemiopsis leidyi; Ba = Beroe abyssicola; Bi = Bolinopsis infundibulum; Mc = Mitrocoma cellularia; Ol = Obelia longissima; Og = O. geniculata; Cg = Clytia gregarium; Ac = Aequorea coerulescens; Am = A. macrodactyla; Ap = A. parva; Av = A. victoria.
Figure 3
Figure 3
Unrooted maximum likelihood phylogeny of photoprotein and photoprotein-like proteins showing clusters of major groupings. Five major groups were reconstructed: ctenophoran, Nematostella, hydromedusan, and Amphimedon photoproteins, as well as closely related coelenterazine-binding proteins from Renilla (an anthozoan cnidarian). Sarcoplasmic calcium binding proteins (SARCs) and calmodulins (CAMs) from a variety of taxa branch outside of these five major groups. Bayesian methods reconstructed a tree with the same topology. Bootstrap support values greater than 50% are denoted. Bayesian posterior probabilities are shown as colored circles at nodes. Red circles indicate 100% support, blue circles indicate >96% support, and black circles indicate >65% support. Species are abbreviated as follows: Ac = Aequorea coerulescens; Am = Amphimedon macrodactyla; Ap = A. parva; Aque = A. queenslandica; Av = A. victoria; Ba = Beroe abyssicola; Bi = Bolinopsis infundibulum; Bl = Branchiostoma lanceolatum; Co = Capsaspora owczarzaki; Dm = Drosophila melanogaster; Hm = Hydra magnipapillata; Hs = Homo sapiens; Mb = Monosiga brevicollis; Mc = Mitrocoma cellularia; Mlei = Mnemiopsis leidyi; Nd = Nereis diversicolor; Nvec = Nematostella vectensis; Og = Obelia geniculata; Ol = O. longissima; Psp = Penaeus sp.; Rm = Renilla mulleri; Rr = R. reniformis; Sr = Salpingoeca rosetta; Ta = Trichoplax adhaerens.
Figure 4
Figure 4
Evolutionary history of the calcium-regulated photoprotein family. According to currently available sequence data, the family arose at the base of the Metazoa. Filled blue circles indicate that the genes are present and that bioluminescence is observed in these groups. Open blue circles indicate that the genes are present, but that bioluminescence is not observed in these groups. A brown X indicates that photoprotein genes are absent from those groups. A plus sign next to a taxon name indicates that this organism's genome has been fully sequenced.
Figure 5
Figure 5
Maximum likelihood phylogeny of opsin proteins showing clusters of major groupings. Bayesian methods reconstructed a tree with the same topology. Bootstrap support values greater than 50% are denoted. Bayesian posterior probabilities are shown as colored circles at nodes. Red circles indicate 100% support, blue circles indicate >96% support, and black circles indicate >70% support. Gene name abbreviations: 5SR = fifth somatostatin receptor; AR = allatostatin receptor; MAR = muscarinic acetylcholine receptor; MWS = medium-wavelength sensitive; RGR = retinal G protein-coupled receptor; Rh = rhodopsin; SWS = short-wavelength sensitive.
Figure 6
Figure 6
Spectrum of bioluminescent emission for a subset of Mnemiopsis photoproteins representing the three sequence groups (A, B, C) at pH 8.0, 9.0, and 10.0. The color of each curve corresponds to the key shown in the upper right of each graph.
Figure 7
Figure 7
Absorbance spectra of MleiOpsin2 pigment measured in the pre- and post-bleaching difference (inset) conditions. In the latter case, the post-bleaching (light) spectrum was subtracted from the pre-bleaching (dark) spectrum.
Figure 8
Figure 8
In situ hybridization showing Mnemiopsis photoprotein mRNA expression, and Mnemiopsis opsin mRNA expression. (A) Photoprotein expression: Panels A-D and F are lateral views; E is a surface view; and G-L are aboral views. The blastopore is indicated with an asterisk. Tb = tentacle bulb. (B) Opsin expression: Panels A-D and K are lateral views; E, F-J, and L-N are aboral views. The blastopore is indicated with an asterisk. Ao = apical organ. White arrows indicate expression in regions of putative photocytes. Black arrows indicate expression in four putative photoreceptors in apical organ. hpf: hours post-fertilization.
Figure 9
Figure 9
Co-expression of Mnemiopsis photoprotein (MleiPP1) and opsin (MleiOpsin2) mRNA expression. All images are lateral views of a cydippid approximately 18 hpf and a double asterisk denotes the aboral pole of the embryo. (A) Green staining is MleiPP1 mRNA expression via fluorescence in situ hybridization (fluorescein probe). (B) Dark blue staining is MleiOpsin2 mRNA expression via nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (digoxigenin probe). (C) Reflective confocal microscopy of co-fluorescent in situ embryo shown in (A) and (B). Bright blue staining is nuclei via 4'-6-diamidino-2-phenylindole; green staining is MleiPP1 mRNA expression via fluorescence in situ hybridization; red staining is MleiOpsin2 mRNA expression via NBT/BCIP.

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