DOI: 10.5061/dryad.7sqv9s4z1
title: Supplemental datasets: Opsin gene expression plasticity and spectral sensitivity in male damselflies could mediate female colour morph detection

description: The visual systems of Odonata are characterized by many opsin
 genes, which form the primary light-sensitive photopigments of the eye.
 Female-limited colour polymorphisms are also common in Odonata, with one
 morph typically exhibiting male-like (androchrome) colouration and one or
 two morphs exhibiting female-specific colouration (gynochromes). These
 colour polymorphisms are thought to be maintained by frequency-dependent
 sexual conflict, in which males form search images for certain morphs,
 causing disproportionate mating harassment.  Here, we investigate
 opsin sensitivity and gene expression plasticity in mate-searching males
 of the damselfly Ischnura elegans during adult maturation and across
 populations with different female morph frequencies. We find evidence for
 opsin-specific plasticity in relative and proportional opsin mRNA
 expression, suggesting changes in opsin regulation and visual sensitivity
 during sexual maturation. In particular, expression of the long-wavelength
 sensitive opsin LWF2 changed over development and varied between
 populations with different female morph frequencies. UV-Vis analyses
 indicate that short- and long-wavelength opsins absorb wavelength of light
 between 350 and 650 nm. Assuming opponency between photoreceptors with
 distinct short- and long-wavelength sensitivities, these sensitivities
 suggest male spectral visual discrimination ability of andromorph and
 gynomorph females. Overall, our results suggest that opsin sensitivity and
 expression changes contribute to visual tuning that could impact
 conspecific discrimination.
description: # Data and code for opsin function expression and qPCR analysis for I.
 elegans
 [https://doi.org/10.5061/dryad.7sqv9s4z1](https://doi.org/10.5061/dryad.7sqv9s4z1) We have submitted datasets with measured spectral sensitivity from UV-VIS spectrophotometry for the honey bee (Apis mellifera) short-wavelength sensitive (SW) opsin and for two wild-type damselfly (Ischnura elegans) SW opsins , a negative control I. elegans SW opsin, and 6 I. elegans long-wavelength sensitive (LW) opsins as well as R code to fit spectral sensitivity data to visual pigment templates (dataset 1); newick and fasta files used to generate a phylogeny for Ischnura spp. opsin sequences (dataset 2); dataset with relative expression values and proportion expression for tested opsins in I. elegans and R code used in primary data analysis (dataset 3). ## Description of the data and file structure Dataset 1: Files associated with Fig 2 and Fig S2: A. mellifera SWS opsin functional expression, I. elegans SWS opsin functional expression controls * General information: Files show wavelengths tested in nanometers, measurements for Eluate purification fractions, average, standard deviation (SD), and standard error (SEM). Naming convention for individual replicates in the following format: species tested (Apis mellifera = Amel  or Ischnura elegans = Iel), opsin type (SWb1, SWb2, LWA, LWE1, LWF1 - 4 or Kmut for SWb2 K335E opsin mutant), and Eluate sample number when multiple samples were measured (Ea - Ef). Example: AmelSW-Ea refers to Apis mellifera, opsin SW, Eluate sample a.Files associated with hydrocylamine treatments indicate time points tested in separate columns when there were multiple time points tested (0s, 30s, 60s, 90s, 120s, 150s, or 4m). * AmelSW_opsin_absorbance_values_csv_files  * Amel_SW_Dark_state.csv -  A. mellifera blue SW opsin absorbance spectra in dark adapted state. Columns are Wavelegnth, AmelSW-Ea (Eluate sample-a), AmelSW-Eb (Eluate sample-b), AmelSW-Ec (Eluate sample-c), Average (averages of all eluate samples), SD (standard deviation), SEM (standard error) * Amel_SW+Hydroxylamine.csv -  A. mellifera blue SW opsin absorbance spectra in hydroxylamine treatments across 5 time points. Columns are Wavelegnth, AmelSW+Hydroxy 30s (30 sec hydroxy treatment), AmelSW+Hydroxy 60s (60s treatment), AmelSW+Hydroxy 90s (90s treatment), AmelSW+Hydroxy120s (120s treatment), AmelSW+Hydroxy 4m (4m treatment), Average (average across all treatment times), SD, and SEM. * Amel_SW_Dark_state_lambda_max.Rmd - R-markdown files used in fitting raw data to visual pigment templates for A.mellifera SW opsins in dark-adapted state  and SW opsin + hydroxylamine treatment * Amel_SW+Hydroxylamine(retinal_oxime)_lambda_max.Rmd - R-markdown files used in fitting raw data to visual pigment templates for A.mellifera SW opsins + hydroxylamine treatment * Iel_LW_opsin_absorbance_values_csv_files - * IelLWA2.csv - file with I. elegans LW opsin A2 spectra measurements. Columns are Wavelength, IelLWA2_Ea (Eluate sample-a), IelLWA2_Eb (Eluate sample -b), Average, SD, SEM. * IelLWE1.csv - file with I. elegans LW opsin E1spectra measurements. Columns are Wavelength, IelLWE1_Ea  - IelLWE1_Ef (Individual columns with eluate sample a - f), Average, SD, SEM. * IelLWF1.csv - file with I. elegans LW opsin F1 spectra measurements. Columns are Wavelength, IelLWF1_Ea  - IelLWF1_Eb (Individual columns with eluate sample a - b), Average, SD, SEM. * IelLWF2.csv -file with I. elegans LW opsin F2 spectra measurements. Columns are Wavelength, IelLWF2_Ea  - IelLWF2_Ee (Individual columns with eluate sample a - e), Average, SD, SEM. * IelLWF3.csv -file with I. elegans LW opsin F3 spectra measurements. Columns are Wavelength, IelLWF2_Ea  - IelLWF2_Ef (Individual columns with eluate sample a - f), Average, SD, SEM. * IelLWF4.csv - file with I. elegans LW opsin F4 spectra measurements. Columns are Wavelength, IelLWF4_Ea , Average, SD, SEM. * Iel_LW_Rhodopsin_absorbance_best_fit.Rmd - R-markdown files for fitting of I.elegans LW opsins .csv files to visual pigment template  * Iel_SWb1_opsin_absorbance_values_excel_file - * Diff_Spec_IelSWb1.csv - I. elegans SWb1 opsin data. Columns are Wavelength, Diff spectrum (raw opsin absorbance measures), Average, SD, and SEM. The columns Average, SD, and SEM are not filled in in the.csv files so that they are referenced correctly in the associated R script. * IelSWb1+Hydroxylamine.csv - I. elegans SWb1 opsin data plus Hydroxylamine treatment. Columns are Wavelength, IelSWb1+Hydroxylamine (raw opsin absorbance with hydroxylamine ), Average, SD, and SEM. The columns Average, SD, and SEM are not filled in in the.csv files. These 3 column identifiers are needed to run the associated .Rmd script, and values are computed within the script as needed. Filling these cells with non-numerical values would however interfere with the script and they are therefore left empty. * SWb1_diff_spectrum_lambda_max.Rmd - R-markdown file for fitting of SWb1 opsin sensitivity to visual pigment template for Diff_Spec_IelSWb1.csv. Average, SD, and SEM in the IelSWb1.csv file are calculated as part of this script. * SWb1+Hydroxylamine(retinal_oxime)_lambda_max.Rmd - R-markdown file for fitting of SWb1 opsin sensitivity with hyroxylamine treatment to visual pigment template for IelSWb1+Hydroxylamine.csv Average, SD, and SEM in the IelSWb1+Hydroxylamine.csv file are calculated as part of this script. * Iel_SWb2_K335E - * IelSWb2_K335E_Dark_state.csv - I. elegans SWb2 K335E opsin data. Columns are Wavelength,  IelSWb2_Kmut_Ea -  IelSWb2_Kmut_Eo (Opsin absorbance data for Swb2 K335E eluates a - o), Average, SD, and SEM. * IelSWb2_K335E_Hydroxylamine.csv - I. elegans SWb2 K335E opsin plus hydroxylamine data. Columns are Wavelength,  Iel_SWb2_K335E +Hydroxylamine a_0s -  Iel_SWb2_K335E +Hydroxylamine f_150s (Opsin absorbance data for 0s, 30s, 90s, 120s, and 150s hydroxylamine treatments), Average, SD, and SEM. * SWb2_K335E_dark_state.Rmd - R-markdown file for fitting of SWb2 K335E opsin sensitivity to visual pigment template for IelSWb2_K335E_Dark_state.csv.  * SWb2_K335E+Hydroxylamine(retinal_oxime)_lambda_max.Rmd - R-markdown file for fitting of SWb2 K335E + Hydroxylamine opsin sensitivity data to visual pigment template for IelSWb2_K335E_Hydroxylamine.csv. * Iel_SWb2_opsin_absorbance_values_excel_files - * Diff_Spec_IelSWb2.csv - I. elegans SWb2 opsin data. Columns are Wavelength, Diff spectrum (raw opsin absorbance measures), Average, SD, and SEM. The columns Average, SD, and SEM are not filled in in the.csv files so that they are referenced correctly in the associated R script. * IelSWb2+Hydroxylamine.csv - I. elegans SWb2 opsin data plus Hydroxylamine treatment. Columns are Wavelength, IelSWb2+Hydroxylamine (raw opsin absorbance with hydroxylamine ), Average, SD, and SEM. The columns Average, SD, and SEM are not filled in in the.csv files. These 3 column identifiers are needed to run the associated .Rmd script, and values are computed within the script as needed. Filling these cells with non-numerical values would however interfere with the script and they are therefore left empty. * SWb2_diff_spectrum_lambda_max.Rmd - R-markdown file for fitting of SWb2 opsin sensitivity to visual pigment template for Diff_Spec_IelSWb2.csv. Average, SD, and SEM in the IelSWb2.csv file are calculated as part of this script. * SWb2+Hydroxylamine(retinal_oxime)_lambda_max.Rmd -  R-markdown file for fitting of SWb2 opsin sensitivity with hyroxylamine treatment to visual pigment template for IelSWb2+Hydroxylamine.csv Average, SD, and SEM in the IelSWb2+Hydroxylamine.csv file are calculated as part of this script. - README_spectral_sensitivity.rft - read me file containing general information on how to run R scripts, naming conventions in .csv files, and excel file information * **Additional information:** To run R scripts in each subfolder: * specify own user path to folder * the script detects the .csv files automatically, for publication several related .csv files are grouped in one subfolder. It should be possible to run all .csv files at once, specifying custom “opsin names” in line 80, separated by commas. Else, keep one .csv file in the folder and run them one by one with the script.  Dataset 2: Files associated with Fig S5: I. elegans opsin sequences and  alignment * Ischnura elegans opsin sequence list_final.fasta - file with I. elegans opsin sequences measured in the current study * MAFFT_aligned_opsin_sequences.fasta - file with aligned opsin sequences for I. elegans and I. asiatica used to generate opsin phylogeny * opsin_phylogeny.newick - opsin phylogeny for Ischnura spp. opsins Dataset 3: R code and files for qPCR and population analyses * 20230315_E qpcr data.csv - datafile with relative expression of opsin genes from qPCR data from collected I. elegans. Includes data on Year, male ID, Population, the proportion of each female morph (PropA, PropI, PropO; these columns are na for individuals collected from artificial tanks in Stensoffa), male maturity stage (I_M: T = tenral, I = immature, M = mature), if males were collected singly (S) or in copula (C), female morph type if males were captured in copula (na is entered for males caught not in copula), Opsin, qPCR replicate (Rep), and relative expression (Rel_Exp) * 20230329_propexp.csv -datafile with proportion expression data generated from expression data from qPCR analysis. Categories are the same as above, but with proportion expression for two SW opsins and 5 LW opsins * Ielegans_counts.csv - datafile containing testing locale (Locale), number of females (No.females), number of each female morph collected (No.androchrome, No.infuscans, No.obsoleta), and total females collected (Morph.Total) * Ielegans pop analysis.R - R file for calculation of female morph frequencies for each testing locale associated with Ielegans_counts.csv * Ielegans_qPCR.R - R file for statistical comparisons between maturity stage for relative and proportion opsin expression data and for female morph frequencies for relative expression (associated .csv files: 20230315_E qpcr data.csv and 20230329_propexp.csv) Usage notes:  **CSV Files (`.csv`)** * Can be opened in any text editor (e.g., Notepad, VS Code) * Read and process in R using `read.csv()` or `readr::read_csv()` **R Code (`.R` files)** * Can be opened in any text editor * Recommended IDE: RStudio