Experimental model and subject details
Platynereis dumerilii
All animals were bred and raised in the Marine Facility at the Max F. Perutz Laboratories in accordance with established protocols [30]. Worm cultures are maintained at 18 °C and a 16 h:8 h light:dark cycle. All wildtype animals used in all shadow assays presented here are of the PIN619512 strain background [14]. Platynereis dumerilii worms with an Δ8 base pair deletion in the Go-opsin1 gene were provided by the laboratory of Gaspar Jekely [22]. In order to obtain wildtype siblings for further analyses, these animals were outbred with the PIN619512 inbred line. Resulting heterozygous worms were subsequently incrossed, resulting in offspring with Go-opsin1+/+, Go-opsin1+/− and Go-opsin1−/− genotypes. Worms were genotyped by genomic DNA extraction from a single segment of clipped tail, which was then PCR amplified using standard primer pairs in exon 1 of Go-opsin1 (Additional file 5), given time to regenerate and subsequently used in the shadow response assay. Animals containing the transgene r-opsin1::egfp-f2a-ntr had been generated previously [23]. Platynereis animals used in the shadow reflex assay were at the immature stage of development, and therefore cannot be assessed for sex until later stages when sexual differentiation has occurred.
COS-1 mammalian cells
COS-1 cell lines were maintained at the Institute for Molecular Science, Okazaki, under standard conditions initially established by G. Khorana [31]. The cell line has not undergone authentication, but has been under continual maintenance by the Khorana, Farrens and Tsukamoto laboratories for approximately 30 years.
Method details
Light condition measurements
The behavioural chamber in which the shadow assay took place was a light-tight insulated box (60 cm × 60 cm × 80 cm) placed in a temperature controlled room (18 °C) on 2-cm thick dampening rubber mats to minimise vibrations. White light conditions were installed in this as described previously [16]. LED arrays (Winger Electronics GmbH & Co.KG) of additional monochromatic wavelengths were constructed and installed in the same configuration at the same distance from the sample, which lies 60 cm from the nearest LED (Additional file 2A, B). The spectrum and intensity of emitted light was measured with the ILT950 spectrometer from International Light Technologies Inc. (Peabody, USA) whose detector was placed at the prospective position of the sample. The light intensity was given in irradiance (μW/cm2/s) and was converted to photon flux (photons/cm2/s). We attempted to match the total photon flux between light conditions. However, there is a relative lack of chromatic specificity of commercial green/yellow (500–590 nm) compared to shorter wavelength monochromatic LEDs [32], and so lower intensities were used in this assay to avoid chromatic overlap.
Shadow reflex assay
From 96 h prior to the assay, immature Platynereis dumerilii of comparable sizes (~20 mm in length) were separated into individual wells of six-well dishes (Greiner Bio One International GmbH) and starved of food for the remainder of the assay. At 48 h prior to the assay, the animals were placed in individual hemispherical concave wells (Additional file 1: Movie 1 and Additional file 2B) (diameter = 35 mm, depth = 15 mm) of a custom-made 36-well clear plastic plate and kept under normal culture conditions to allow novel tube formation. Upon introduction to the behavioural chamber (Additional file 2A, B) at ZT5 on the day of assay, the wells were refilled with fresh artificial seawater (ASW) before the assay. The assay was repeated for all six different light conditions (Additional file 2C, D) on each cohort of animals. The order in which the light conditions were presented was randomised for each new cohort of animals. The assay began with a 60-min acclimatisation period of exposure to the given ON light stimulus. After 45 min, each well was spiked with 50 μL of spinach-conditioned ASW to encourage foraging behaviour and make grading of the shadow response more apparent. For the shadow light regime, 12 shadow stimuli, each consisting of 2 s of sudden complete darkness (OFF), were given. These shadows were each separated by 60 s of normal (ON) light conditions to avoid desensitisation to the stimuli (Fig. 1a). Video recording of worm behaviour in both ON and OFF light conditions was facilitated as described previously [16], using an infrared (IR) (λ = 990 nm) LED array (Roschwege GmbH) illuminating the behavioural chamber and an IR high-pass filter restricting the video camera. Video was recorded continuously at 15 frames per second for the duration of the shadow stimuli.
Cirri removal
For the cirri dependence assay, size-matched immature wildtype sibling worms were collected. Half were randomly selected to undergo cirri removal and the other half were not. At 2 days prior to assay onset, all animals (both cut and uncut) were anaesthetised by submersion in an isotonic MgCl2/ASW solution (7.5% w/v in ddH2O mixed 1:1 with ASW). ‘Cut’ animals were placed dorsally on a glass cover slide and their two anal cirri and eight peristomal cirri were surgically removed using fine scalpel blades (Swann Morton Ltd.) and tungsten carbide needles (Fine Science Tools Ltd.) (adapted from [33]). Note that care was taken to avoid causing simultaneous damage to the palps or antennae (Additional file 4). Animals were then transferred to fresh ASW and allowed to recover for 1 h before being placed individually in hemispherical wells alongside their uncut siblings to prepare tubes for the shadow reflex assay. Upon starting the shadow assay 2 days after surgery, ‘cirri-removed’ animals had not begun to regrow their detached cirri (Additional file 4).
Whole mount in situ hybridisation
Expression of Pdu-Go-opsin1 transcripts was localised in adult Platynereis dumerilii using an established whole mount in situ hybridisation protocol [34]. RNA probes were generated by in vitro transcription with T7 RNA Polymerase (New England Biolabs Inc. No. #M0251 L) incorporating digoxigenin-labelled UTPs (Roche Diagnostics GmbH, No. 11277073910). The probe template was generated by a PCR reaction conducted on immature wildtype Platynereis dumerilii cDNA using primers specific to the full coding sequences of the Go-opsin1 gene (Additional file 5). Images of Platynereis heads stained by in situ hybridisation were taken using a Zeiss Axioplan2 light microscope. Overall head images were taken under a 10× and 20× air objective lens and cellular images with a 40× and 63× oil immersion objective.
r-opsin1
+ cell-specific ablation
Transgenic r-opsin1::eGFP-F2A-nitroreductase animals were pre-screened for strong GFP fluorescence, along with the same number of size-matched wildtype animals, and were subjected to mtz (Sigma, catalogue no. M1547) treatment as described previously [24]. Mtz treatment took place for 3 days at a concentration of 12 mM mtz dissolved in 0.2% DMSO in ASW. Following treatment, animals were allowed to recover for 2 days in ASW before being placed in the shadow assay wells. Complete absence of GFP fluorescence was taken as an indicator of a successful ablation procedure. The shadow assay therefore took place 4 days after mtz treatment, well within the 7 day period found to preclude regrowth of the r-opsin1+ cells [24].
In vitro opsin absorption assay
The in vitro opsin absorption assay was conducted as described by Tsukamoto et al. [25]. Briefly, native Platynereis c-opsin2 with the 1D4 tag (ETSQVAPA) on the C-termini was subcloned into the pMT vector. The construct was transiently expressed in mammalian COS-1 cells, incubated with 11-cis-retinal, and proteins were extracted with 1.25% DDM (Dojindo, Kumamoto, Japan), 20 mM HEPES, 140 mM NaCl, 0.25% cholesterol hemisuccinate (Sigma-Aldrich, St. Louis, MO, USA), 25 mM Tris and 10% glycerol at pH 7.0. The extract was mixed with 1D4-agarose overnight, and transferred to Bio-Spin columns (Bio-rad, Hercules, CA, USA). The columns were washed with 0.05% DDM, 2 mM ATP, 1 M NaCl, 3 mM MgCl2, 0.01% cholesterol hemisuccinate, 1 mM Tris and 10% glycerol in PBS, and subsequently washed with 0.05% DDM, 140 mM NaCl, 0.01% cholesterol hemisuccinate, 1 mM Tris, 10% glycerol and 20 mM HEPES at pH 7 (buffer A). The 1D4 tagged c-opsin2 was eluted with buffer A containing 0.45 mg/mL of 1D4 peptide (TETSQVAPA) (TOYOBO, Osaka, Japan). Absorption spectra of purified Platynereis c-opsin2 were recorded with a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). During the spectral measurement, the samples were kept at 10 °C.
Phylogeny
The sequence alignment and tree calculation was performed using CLC Main Workbench, version 7.7.1. The alignment settings were as follows: gap open costs 10, gap extension costs 1, end gap costs as any other, very accurate alignment; settings tree: method UPGMA, protein distance measure: Kimura protein, 1000 bootstraps. The dataset supporting this tree is included in Additional file 6, as well as accession numbers and source organisms of protein sequences used.
Quantification and statistical analysis
Following assay completion, behaviour was analysed manually and individual animals were scored binarily (1 = withdrawal reflex, 0 = no reaction) for each shadow stimulus given. The withdrawal reflex is specifically defined as a shortening of total body length via retraction of the anterior and/or posterior end. If the animal is in the process of undulation associated with swimming or passive ventilation, this reflex can behaviourally appear as a sudden straightening of the body (Additional file 1: Movie 1). Prior to the assay, animals were placed in wells at random by a colleague with genotypes hidden from the behavioural analyser, and were revealed only once scoring had been conducted blindly. Individual SRSRs were then calculated according to the equation below:
$$ Shadow\ Reflex\ Success\ Rate\ (SRSR)=\frac{\# of\ successful\ shadow\ responses}{total\# of\ shadow s\ given} $$
Due to the fact that the number of successful shadow responses is always an integer value, SRSRs are an inherently discrete variable and thus the data takes on a stratified appearance. Statistical analysis and construction of box and whisker plots were conducted using Graphpad Prism 7.03 [35], where boxes represent interquartile range. Individual biological replicates are shown as diamonds in all cases. Datasets are approximately continuous (with 12 potential discrete values), unpaired and similarly distributed. Accordingly, significant differences between groups were calculated using two-tailed Mann–Whitney U tests with a P value threshold of 0.05. The Benjamini–Hochburg correction for multiple testing was applied to these tests in Fig. 1b, where each dataset was tested twice. For Fig. 3b, the lack of statistical significance between groups was calculated by assessment of whether the difference between pseudo-medians, calculated using Hodges–Lehmann non-parametric estimator, exceeds 95% confidence intervals. Under any single condition in every figure, SRSRs were pooled from at least three separate independent experiments. Precise values of n and the statistical parameters used in each figure are provided in the accompanying figure legends.