DOI: 10.5061/dryad.280gb5mtf
title: Multiple rod layers increase the speed and sensitivity of vision in nocturnal reef fishes

description: Most vertebrates have one layer of the dim-light active rod
 photoreceptors. However, multiple rod layers, known as a multibank retina,
 can be found in over 100 species of fish, including several deep-sea
 species and one family of nocturnally active reef fish, the Holocentridae.
 Although seemingly associated with increased photon catch, the function of
 multibank retinas remained unknown. We used an integrative approach,
 combining histology, electrophysiology, and amino acid sequence analysis,
 applied to three species of nocturnal reef fishes, two holocentrids with a
 multibank retina (Neoniphon sammara and Myripristis violacea) and an
 apogonid with a single rod bank (Ostorhinchus compressus), to determine
 the sensory advantage of multiple rod layers. Our results showed that fish
 with multibank retinas have both faster vision and enhanced sensitivity to
 bright and dim light intensities. Faster vision was indicated by higher
 flicker fusion frequencies during temporal resolution electroretinography
 as well as faster retinal release kinetics estimated from their rhodopsin
 proteins. Enhanced sensitivity was demonstrated by broadened
 intensity-response curves derived from luminous sensitivity
 electroretinography. Overall, our findings provide the first functional
 evidence for enhanced dim-light sensitivity using a multibank retina while
 also suggesting novel roles for the adaptation in enhancing bright-light
 sensitivity and the speed of vision.
description: Data are derived from electroretinograms recorded from individual
 nocturnal coral reef fish as described in the associated
 preprint.  Data are either normalised to the maximal
 response (Vmax) or to both the maximal response and eye size.
 Eye diameter (in millimetres) is given in an additional
 file.
description: Files are given in tsv format. No proprietary software is
 required to open the files or manipulate the data.
description: This README.md file is for he dataset "Multiple rod layers increase
 the speed and sensitivity of vision in nocturnal reef fishes" The
 associated preprint can be accessed at:
 https://www.biorxiv.org/content/10.1101/2022.11.27.518067v1 Dataset
 Contributors: Lily Fogg, Wen-Sung Chung, N. Justin Marshall, Fabio
 Cortesi, Fanny de Busserolles Date Created: 2023.02.09 Date Updated:
 2023.10.03 License: Use of these data is covered by the CC0 license
 Contact Information Name: Affiliations: Queensland Brain Institute
 University: University of Queensland, Brisbane, Australia ORCID ID:
 0000-0002-5378-8781 Email: lily.fogg@uqconnect.edu.au Alternate Email:
 lily.fogg@unibas.ch Address: e-mail preferred *** ### Additional dataset
 metadata #### Format All data files are given as tsv or csv files (file
 type is indicated in the description of individual data files below). All
 files can be opened in Microsoft Excel or an alternative non-proprietary
 software. No unpacking of the data is required. Note that any blank fields
 have been replaced with NA. #### Animal collection and ethics Adult fish
 were collected from the Great Barrier Reef around Lizard Island, Australia
 or sourced from a supplier, Cairns Marine, which also collects from the
 northern Great Barrier Reef. All collections and procedures were conducted
 under a Great Barrier Reef Marine Park Permit (G17/38160.1), a Queensland
 General Fisheries Permit (180731), and a University of Queensland’s Animal
 Ethics Permit (QBI 304/16). Following euthanasia, all animals were
 photographed with a scale reference to quantify body length and eye
 diameter. Eyes were dissected and the eye cup preserved in RNAlater or
 paraformaldehyde [PFA; 4% (w/v) PFA in 0.01M phosphate-buffered saline
 (PBS), pH 7.4] depending on the analyses. *** ### Overview of data These
 data files are derived from electroretinography (ERG) recordings conducted
 on individual nocturnal coral reef fish. Three species were assessed:
 Ostorhinchus compressus (referred to as cardinalfish in Dryad data files),
 Neoniphon sammara (squirrelfish) and Myripristis violacea (soldierfish).
 They were collected as described below. Further details of data collection
 can also be found in the associated preprint
 (https://www.biorxiv.org/content/10.1101/2022.11.27.518067v1). Eye
 diameter for each individual is also given in a separate file in this
 dataset (in millimetres). *** ### Description of individual data files
 #### Name: EyeDiameter\_AllSpecies.tsv Size: 250 bytes Description of
 data: this file contains eye diameter (in millimetres) of each individual
 fish used in electroretinography recordings. Species and replicates:
 Measurements were obtained for Ostorhinchus compressus (a cardinalfish; n
 = 10 individuals), Myripristis violacea (a soldierfish; n = 8 individuals)
 and Neoniphon sammara (a squirrelfish; n = 9 individuals). Description of
 methods used to generate data: Eye diameter was measured in millimetres
 from photographs using the image processing software, Fiji. #### Name:
 TemporalResolutionERG\_AllSpecies.tsv Size: 880 bytes Description of data:
 this file contains results from temporal resolution experiments. Data are
 provided as maximal flicker fusion frequency (FFF) values in hertz (Hz).
 Species and replicates: Measurements were obtained for Ostorhinchus
 compressus, Myripristis violacea and Neoniphon sammara (all species: n = 3
 and 5 individuals each for day and night recordings, respectively, for
 both dim and bright stimuli). Description of methods used to generate
 data: Corneal ERG recordings were conducted in vivo on whole, intact eyes
 to assess visual function. Fish were acclimatised to the recording chamber
 for 30 min, anaesthetised with 0.2 mL clove oil/litre seawater,
 immobilised with an intramuscular injection of 8.5 mg/kg gallamine
 triethiodide and ventilated with oxygenated seawater. After ≥40 min of
 dark adaptation, light stimuli were delivered to the eye using a
 custom-built, calibrated, broad-spectrum light source controlled via a
 PowerLab 4/26 DAQ module. Visual responses were detected through silver
 wire electrodes placed on the surface of the eye, amplified via a DP-103
 amplifier and acquired in LabChart 8 (v8.1.16). The system was grounded to
 the water of the recording chamber. Recordings were conducted at 28 ± 1˚C
 at both day and night to control for any effects of temperature and
 circadian rhythm, respectively. Recordings were performed at the Lizard
 Island Research Station (LIRS) or the Queensland Brain Institute (QBI).
 The temporal resolution of vision was assessed using flicker fusion
 frequency (FFF) ERGs. FFF is the point at which evenly spaced light pulses
 can no longer be distinguished as separate. Dark-adapted FFF ERGs were
 recorded by increasing the frequency of white light stimuli of constant
 intensity from 5 Hz to 95 Hz at increments of 5 Hz. Light pulses were 10
 ms in duration and were repeated 30 times. Recordings were conducted for
 bright (384 lux) and dim (4 lux) stimuli. The FFF threshold was determined
 either through visual inspection (at lower frequencies, &lt;65 Hz) or
 by using the power spectrum to differentiate the signal and noise (at
 higher frequencies, ≥65 Hz). #### Name:
 LuminousSensitivityERGData\_AllSpeciesCombined\_Revised.csv Size: 14 KB
 Description of data: this file contains results from absolute sensitivity
 experiments. Data labelled as "Normalised to Vmax" are provided
 as a percentage of the maximal response recorded for each individual per
 stimulus intensity step (given in lux). Data labelled as "Eye size
 Normalised" are provided as the percentage of the maximal response
 divided by the eye diamter for each individual per stimulus intensity
 step. Species and replicates: Measurements were obtained for Ostorhinchus
 compressus (n = 5 individuals each for day and night recordings, labelled
 as Fish 1, Fish 2, etc.), Myripristis violacea (n = 4 individuals each for
 day and night recordings) and Neoniphon sammara (n = 4 and 5 individuals
 for day and night recordings, respectively). Description of methods used
 to generate data: Corneal ERG recordings were conducted in vivo on whole,
 intact eyes to assess visual function. Fish were acclimatised to the
 recording chamber for 30 min, anaesthetised with 0.2 mL clove oil/litre
 seawater, immobilised with an intramuscular injection of 8.5 mg/kg
 gallamine triethiodide and ventilated with oxygenated seawater. After ≥40
 min of dark adaptation, light stimuli were delivered to the eye using a
 custom-built, calibrated, broad-spectrum light source controlled via a
 PowerLab 4/26 DAQ module. Visual responses were detected through silver
 wire electrodes placed on the surface of the eye, amplified via a DP-103
 amplifier and acquired in LabChart 8 v8.1.16. The system was grounded to
 the water of the recording chamber. Recordings were conducted at 28 ± 1˚C
 at both day and night to control for any effects of temperature and
 circadian rhythm, respectively. Recordings were performed at the Lizard
 Island Research Station (LIRS) or the Queensland Brain Institute (QBI).
 The absolute (luminous) sensitivity of vision was determined using V/logI
 curves, which plot the normalised amplitude of the response (V) against
 the log of the intensity (I). These ERGs were recorded by increasing the
 intensity of a white light from 2.4x10-8 to 240,000 lux [i.e., -7.6 to 5.4
 log10(lux)] in 0.3-0.6 log unit steps. Light stimuli were 100 ms pulses
 presented at 0.1 – 0.4 Hz and were repeated ten times for each intensity.
 The mean response amplitudes were normalised to the maximal response
 (Vmax) and plotted against stimulus intensity to obtain the V/logI curve
 (labelled as "Normalised to Vmax" in Dryad data files). The area
 under the curve (AUC) was calculated as a proxy for the magnitude and
 breadth of the visual responses. AUC was calculated for either all
 intensities, dim intensities (&lt;0.002 lux) or bright intensities
 (&gt;10 lux) for each species. To isolate the effect of the multibank
 retina, the Vmax-normalised responses were also normalised to eye size (to
 obtain responses per unit of retina) and analysed again as described above
 (labelled as "Eye Size Normalised" in Dryad data files). ####
 Name:
 LuminousSensitivityERGData\_CardinalfishLocationComparison\_Revised.csv
 Size: 2 KB Description of data: Data are provided as a percentage of the
 maximal response recorded for each individual per stimulus intensity step
 (given in lux). Species and replicates: Measurements were obtained from
 Ostorhinchus compressus in two recording locations: the Queensland Brain
 Insitute (QBI) in Brisbane, Australia (n = 5 individuals) and the Lizard
 Island Research Station (LIRS) on the northern Great Barrier Reef,
 Australia (n = 2 individuals). All recordings were conducted during the
 day.Description of methods used to generate data: These data were
 collected in the same way as described above for the file
 "LuminousSensitivityERG\_AllSpecies.tsv". However, recordings
 were taken at both Lizard Island Research Station, Australia (field) and
 Queensland Brain Institute, Australia (lab) to compare results between the
 recording locations. *** #### Explanation of Update on 2023.10.03
 Originally the b wave amplitudes in the luminous sensitivity ERG files
 were calculated from the isoelectric line. During the peer review process,
 a reviewer pointed out that the b wave amplitude would be affected by a
 deepening of the a wave at higher light intensities. Thus, the reviewer
 suggested that it would be more correct to calculate the b wave amplitude
 from the base of the a wave instead of the isoelectric line. This
 suggestion was also consistent with previously published analysis
 approaches and so was applied to the dataset. This change in analysis
 approach is incorporated into the updated luminous sensitivity ERG files.