UV Light Greatly Facilitates Visually Guided Prey Capture in Larval Zebrafish

(A) Schematic representation of visual prey capture by larval zebrafish.

(B) Setup for filming paramecia. A filter wheel equipped with UV and yellow bandpass filters was positioned in front of the charge-coupled device (CCD) camera to image paramecia in a naturalistic tank in the sun.

(C) Peak-normalized spectra for the UV and yellow channels (thick lines; STAR Methods) superimposed on the zebrafish’s four opsin absorption spectra (shadings). The spectral overlap between the UV and yellow channels with each opsin is indicated (thin lines). Abs., absorption; Tr., transmittance.

(D) Example frames from the yellow and UV channels taken consecutively from the same position.

(E) Zoom in from (D), with line profiles extracted as indicated. Arrowheads highlight paramecia visible in the UV channel. See also Video S1.

(F) Schematic of behavioral setup. Individual larval zebrafish (7–8 dpf) in the presence of free-swimming paramecia were head-mounted and filmed from above, with infrared illumination from below.

(G) Top illumination was provided by intensity-matched UV (374 ± 15 nm) or yellow (507 ± 10 nm) LEDs, which mainly activated UV/blue and red/green opsins, respectively, as indicated.

(H) Top: zebrafish consistently responded more readily to passing paramecia with full prey-capture bouts (eye convergence + tail flicks, each event indicated with a marker) during UV-illumination periods. See also Video S2. Individual trials (left) and summary statistics (right). This difference was abolished when UV cones were ablated (bottom). Mann-Whitney U test, UV versus yellow light in wild-type (WT) fish: p < 0.01; WT versus UV killing under UV light: p < 0.001; UV versus yellow light in UV killing fish: p > 0.05; n = 12 each for WT and UV cone ablation.

The Detector Hardware for UV Vision in Larval Zebrafish

(A–D) UV cone density projected into sinusoidal map of visual space when eyes are in resting position for initial prey detection (A) and once converged for prey localization following detection (C). A 100-μm paramecium is too small for reliable detection at ∼3 mm distance and can therefore only be seen by a single UV cone at a time (B). Even at ∼1 mm strike distance, it covers at most a handful of UV cones per eye (D). 3D schematics (A and C) illustrate approximate visual space surveyed by the two SZs. Scale bars, UV cones/°. See also Figures S1A–S1D.

(E) Sagittal section across the eye with outer segments (OSs) stained by BODIPY (magenta) and UV cones expressing GFP (green, Tg(opn1sw1:GFP)) in an 8 dpf larva.

(F) Higher magnification sections from (E). Note that BODIPY stains the OSs of all photoreceptors, as well as the spot-like pocket of mitochondria immediately below the OS (Figures S1E–S1G). Note also that region-specific OS enlargements are restricted to UV cones.

(G) Mean and 95% confidence intervals of UV cone OS lengths across the eye. V, ventral; SZ, strike zone; D, dorsal; N, nasal. Open-source 3D fish model created by M.Y. Zimmermann.

Imaging Cone Calcium in the Live Eye

(A) Confocal images of synaptically targeted GCaMP6f (green, Tg(opn1sw1:SyGCaMP6f)) in UV cones (magenta, Tg(opn1sw1:nfsBmCherry)).

(B) Mean and single trial dorsal and SZ single cone 2-photon calcium responses to varying duration light- (6 × 10⁵ photon/s/μm²) and dark-steps (0 photon/s/μm²) from a constant UV background (2.4 × 10⁴ photon/s/μm²).

(C) Mean calcium responses to the same stimulus as in (B) from ventral, nasal, dorsal, and SZ cones (V, N, D, and SZ; n = 9, 21, 23, and 29, respectively). Shadings represent ±1 SD. Left panel shows an enlargement of the response to the 20-ms light step.

(D) Mean and 95% confidence intervals of peak amplitudes from (C).

(E) Enlargement from (D). All responses except nasal and ventral 20-ms dark-flash conditions were significantly different from zero (Mann-Whitney U test). Within-condition pairwise comparisons across for SZ versus the other three zones are indicated with asterisks (^∗p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001, respectively; p value adjustment, Tukey method for comparing a family of four estimates).

(F) Light and dark responses from (C) and (D) plotted against each other for equivalent stimulus durations, with 95% confidence intervals indicated.

(G and H) Mean calcium responses to increasing-amplitude 5-ms light flashes from darkness, as indicated (G), and quantification (mean and 95% confidence intervals) with Hill functions fitted (H).

(I) Quantification of calcium responses as in (G) and (H) following horizontal cell (HC) blockage using CNQX. For better comparison, curves from (H) are added as faint dashed lines. n = 51, 29, 46, and 17 for SZ, D, N, and V, respectively, for control and n = 51, 32, 46, and 19 after HC block.

Temporal Tuning of UV Cones

(A) Mean ± 1 SD responses to a 200-ms flash of light (6 × 10⁵ photon/s/μm²) from darkness (0 photon/s/μm²).

(B) Box and violin plots of recovery time constants from (G). n = 29, 29, 23, and 13 for SZ, D, N, and V, respectively.

(C and D) As in (A) and (B), but for an equivalent contrast dark flash. n = 27, 24, 19, and 13 for SZ, D, N, and V, respectively. ANOVA test ^∗p < 0.02, ^∗∗∗p < 0.0001 (H and J). n.s., not significant.

(E) Mean ± 1 SD (shadings) calcium responses to a 5-ms light flash from darkness before (shades of purple) and after HC blockage using CNQX (green).

(F) Quantification of recovery time constant after a 5-ms UV flash at 10⁴ photons/cone. n = 51, 29, 46, and 17 for SZ, D, N and V, respectively for the control condition and n = 51, 32, 46, and 19 after HC block. ANOVA test ^∗∗p < 0.01, ^∗∗∗p < 0.001. n.s., not significant.

A Model of UV Cone Activation by a Small Moving Target

(A) Model setup. Monocular UV cone distribution across the visual field (gray dots) with model bipolar cell (BC) array superimposed (filled circles) and target paths black line. The SZ was centered in the upper left quadrant, corresponding to the upper frontal visual field.

(B) Number of cones touched by a moving 2° plotted as a trace over time, with histogram to the right. Top: UV cones. Bottom: any-type cone.

(C) Time trace of model cone activation of four example cones, taken from representative regions across the array. Responses below and above zero correspond to activation in response to a 2° bright and 5° dark target, respectively.

(D) Maximal activation levels of each cone over the full path for a 2° bright target, normalized to peak activation across the entire array.

(E) Activation of BCs driven by UV cones in (D).

(F and G) As in (D) and (E) but for a 5° dark target.

Calcium Baseline Predicts Dark-Light Responses

(A and B) Whole-eye sagittal view of UV cone SyGCaMP6f in live Tg(opn1sw1:SyGCaMP6f) zebrafish under 3 × 10⁵ photon/s/μm² UV background light (A) and after immunostaining against SyGCaMP6f using anti-GFP antibody (B).

(C) Mean and 95% confidence intervals of the difference between live SyGCaMP6f signal per cone as in (A) and fixed signal as in (B), with red lines indicating regions that were significantly different from zero.

(D) Example mean and individual trial single cone response to 0 photon/s/μm² dark and 6 × 10⁵ photon/s/μm² light steps from a constant brightness UV 3 × 10⁵ photon/s/μm² without and with spectrally broad background light. After five repeats, a 1.5 × 10⁷ photon/s/μm² UV light step was presented to drive calcium to a minimum (right).

(E) Mean and 95% confidence interval of calcium baseline relative to the full dynamic range as indicated, with single datapoints in the back.

(F) Mean ± 1 SD calcium responses to light and dark contrasts with naturalistic RGB background light across all UV cones in specified regions. Traces were shifted and scaled to align the baseline and peak dark response.

(G) Mean and 95% confidence intervals of dark-light index (DLi) with single datapoints in the back.

Tuning of Phototransduction Cascade Elevates SZ Baseline

(A) UV cone RNA sequencing (RNA-seq) workflow. Retinas from 7 dpf zebrafish Tg(opn1sw1:GFP) were dissected and separated into SZ and non-SZ. After cell dissociation, UV cones were FACS sorted and immediately flash frozen. Samples were then subjected to library preparation for next-generation sequencing.

(B and C) All detected genes in UV cones ranked by expression label, with phototransduction genes highlighted (B), and zoom in to the top 200 genes (C). The two most highly expressed genes are both non-protein-coding genes; therefore, UV opsin is the highest expressed protein-coding gene.

(D) Mean gene expression ratio between SZ and non-SZ batches, with phototransduction genes highlighted.

(E) As in (D), but normalized to UV-opsin expression level in each batch and zoomed in to high expression phototransduction targets. Green and gray markers denote activators and repressors of the photo-response, respectively. Error bars represent SEM.

(F) Schematic of phototransduction based on Yau and Hardie (2009), with activators and repressors denoted in green and gray, respectively.

(G) Simulated current response of SZ and non-SZ UV cones to 100% dark and light contrasts from a 50% contrast background based on Invergo et al. (2014). Non-SZ was based on default model parameters, while SZ uses relatively scaled parameters according to gene expression ratios as in (E).

(H) Effects of expression changes of individual phototransduction components compared to non-SZ.

(I) Mean calcium responses to a flash of light from darkness in SZ, nasal, and dorsal UV cones from Figure 4E.

(J) Output of full phototransduction model to an equivalent stimulus between SZ and non-SZ batches.

(K) Full model output to a series of increasing amplitude 5-ms light flashes from darkness for SZ and non-SZ batches.

(L and M) Stimulus-response data from SZ and average of non-SZ data (N+D+V) from Figure 3H (L) and corresponding quantification of the phototransduction model output (K) (M).

Synaptic Release Accentuates Functional Differences between UV Cones

(A and B) Schematic of HC dendrites at photoreceptor synaptic invaginations. SFiGluSnFR expression in HC dendrites is well positioned to detect glutamate release from ribbon synapses (bar structure) at single terminals of any cone type. UV cones are identified by co-expression of mCherry as before. OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer.

(C) In vivo two-photon image of SFiGluSnFR in HCs and nfsBmCherry in UV cones.

(D) Scan field for SFiGluSnFR recordings. Individual HC dendritic bundles at single cone terminals are readily visible. ROIs 3 and 7 are associated with UV cones as seen by overlap with the mCherry signal. A map of pixel-to-pixel correlation over time (Franke et al., 2017) highlights localized activity at each cone terminal.

(E) Partial example trace of mean and individual trial glutamate responses of ROIs from (D) to a tetrachromatic binary noise stimulus (STAR Methods). UV cone responses highlighted in magenta.

(F) Correlation of glutamate responses across pairs of ROIs. ROIs 3 and 7 are highly correlated only to each other. Color code is based on each ROI’s preferred response as in (G).

(G) Linear filters (“kernels”) recovered by reverse correlation of each ROI’s response to the noise tetrachromatic stimulus (E). R, G, B, and U denote red, green, blue, and UV light, respectively. UV cones are highlighted by asterisks.

(H) Partial example trace of mean calcium (SyGCaMP6f) and glutamate (SFiGluSnFR) responses of SZ and dorsal UV cones to the tetrachromatic noise stimulus. Background shading indicates UV light and dark stimulus periods. Arrowheads highlight enhanced glutamate response transients from SZ relative to dorsal UV cones.

(I) Signal-to-noise ratio in the Fourier domain and resulting information rate in calcium responses across UV cones from different regions.

(J) As in (I), computed for glutamate responses. n = 35, 20, 28, and 18 for calcium in SZ, D, N, and V, respectively, and 51, 20, 22, and 18 for glutamate SZ, D, N, and V, respectively.