Fig 1.
Histological comparison between the retinae of Anomalops katoptron and Carassius auratus.
(A) AFG staining of A. katoptron (left) and C. auratus (right) retinae reveal differences in the organization of the different retinal layers. (B) Quantification of the relative size of the different A. katoptron (blue) and C. auratus (grey) retina layers (photoreceptor layer (PR), outer nuclear layer (ONL) and inner nuclear (INL) layer), and (C) comparison between the ONL/INL ratio between A. katoptron (left) and C. auratus (right). The number of slices analyzed is indicated in parentheses. Statistical significance was evaluated with ANOVA (***p<0.001) (D) Immunohistological analysis of A. katoptron (left) and C. auratus (right) retinae. Cones were visualized using FITC-PNA (FITC conjugated peanut agglutinin) in green, and rods were identified by the expression of rhodopsin (RH) in red detected with mouse anti-rhodopsin antibody and donkey anti-mouse Alexa 549 antibody. Rare incidences of cones could be detected in A. katoptron as shown in the inset. The larger panel from A. katoptron represents the norm.
Fig 2.
Comparison of the amino acid sequence of Rh1 and Rh2 of Anomalops katoptron with bovine rhodopsin.
(A) Amino acid sequence alignment of A. katoptron Rh1, Rh2 and bovine Rh1. Yellow shows the amino acid substitutions; white shows the conserved amino acids; Tm shows the transmembrane region. (B) 2-D plot of the proposed secondary structure based on the crystal structure of bovine Rh1[24]. The amino acid changes in A. katoptron Rh1 are shown in yellow compared to bovine Rh1. (C) Comparison of the relative amounts of retinal mRNA of A. katoptron Rh1 and Rh2 by RNA sequence data. (FPKM, fragments per kilobase million) (D) Example qRT-PCR gels from Rh1, Rh2 and ß actin (internal control) at various dilutions (50 to 59). (E) Relative RNA expression levels of A. katoptron Rh1 and Rh2 from qRT-PCR. The duplicate qRT-PCRs from 5 different retinae mRNAs were analyzed and indicated in parentheses. Statistical significance was evaluated with ANOVA (*p<0.05).
Fig 3.
Phylogenetic analysis of rhodopsin proteins from Anomalps katoptron and other species.
Evolutionary relationship between A. katoptron RH1 anhd RH2 and other species shown as tree diagram. Phylogenetic trees were generated with Neighbor joining. Bootstrap percentages (10,000 bootstrap replicates) are given at the branches. Classifications given on the right are taken from the NCBI Taxonomy database (http://www.ncbi.nlm.nih.gov/taxonomy) [27].
Fig 4.
Characterization of the action spectrum and biophysical properties of Anomalops katoptron RH1 and RH2.
(A) Distribution of mCherry, RH1-mCherry and RH2-mCherry in tsa201 cells. (B) Western blot analysis of tsA201 homogenates expressing mCherry, RH1-mCherry and RH2-mCherry. Protein expression was detected with an antibody against mCherry. (C) Comparison of light-induced GIRK (G protein coupled inward rectifying potassium) currents activated by RH1-Rat, A. katoptron RH1 and A. katoptron RH2 using a 1 s or 10 s light pulse of 490 nm (indicated as blue bar). (D) Wavelength dependence of maximal GIRK current activation induced by RH1-Rat, A. katoptron RH1 and A. katoptron RH2 using a 1 s light pulse of the indicated pseudorandomized wavelength. (E) Light pulse intensity dependence of maximal GIRK current activation induced by RH1-Rat, A. katoptron RH1 and A. katoptron RH2 using a 1 s light pulse of 490 nm with different pseudorandomized light-intensities. (F) Light pulse duration dependence of maximal GIRK current activation induced by A. katoptron RH1 and A. katoptron RH2 using a light pulse of 490 nm with increasing time. (G) Activation and deactivation time constants of GIRK currents induced by RH1-Rat, A. katoptron Rh1 and A. katoptron RH2 using a light pulse of 470 nm for activation with increasing time. The number of cells analyzed is indicated in parentheses. Statistical significance was evaluated with ANOVA (***p<0.001).
Fig 5.
Electroretinogram measurements from Anomalops katoptron and Carassius auratus.
(A) Schematic representation of the experimental set-up to record electroretinograms in fish. Oxygenated (O2) seawater containing 0.01 g/l MS-222 was applied to the fish mouth and gills via a 5 mm plastic tube and a peristaltic pump. Light pulses between 400 nm to 650 nm were applied to the retina with a polychromatic light source (Poly). (recording electrode, Record e-; reference electrode, Ref e; amplifier, amp; computer, PC) (B-C) Comparison of electroretinogram measurements from A. katoptron and C. auratus. (B) Example traces of electroretinograms recorded during a 500 ms, 480 nm light pulse (blue bar) for C. auratus (top) and A. katoptron (bottom). (C) Wavelength dependent electroretinogram activities were plotted against the reciprocal of irradiance for each wavelength for C. auratus (grey) and A. katoptron (blue) using a 500 ms light pulse for indicated wavelengths. Note that the C. auratus retinal responses are probably saturating. The number of retinas analyzed is indicated in parentheses.
Fig 6.
Characterization of the wavelength and intensity dependence on conditioned feeding behavior of Anomalops katoptron.
(A-B) Schematic representation of the behavioral food conditioning experiment (polychromatic light source, Poly). A school of 8 A. katoptron fish were trained to recognize food delivery associated with high intensity red light (100% at 630 nm, 2 mW/mm2, conditioned stimulus). (A) Low intensity blue light (10% at 480 nm) attracted the fish to the feeding area. (B) Low intensity red light (10% at 630 nm) did not attract the fish to the feeding area. (C) Characterization of the wavelength dependence on conditioned feeding behavior of A. katoptron. Wavelength dependent feeding behavior of A. katoptron was measured at 460 nm, 480 nm, 530 nm and 630 nm with 10% light intensities delivered by the polychromatic light source at a given wavelength. (D) Low intensity blue light (10% at 480 nm) but not low intensity red light (10% at 630 nm) activates RH1 mediated GIRK currents. However, high intensity red light (100% at 630 nm) induced RH1 mediated GIRK current, which is about >20% of the current induced by 480 nm (10% intensity). The total number of trials per data point is indicated in parentheses. Statistical significance was evaluated with ANOVA (**p<0.01; ***p<0.001).