DOI: 10.5061/dryad.kprr4xh3c
title: UV radiation affects anti-predatory defense traits in Daphnia pulex

description: In aquatic environments prey perceive predator threats by chemical cues
 called kairomones, which can induce changes in their morphology, life
 histories and behavior. Predator-induced defenses have allowed for prey,
 such as Daphnia pulex, to avert capture by common invertebrate predators,
 such as Chaoborus sp. larvae. However, the influence of additional
 stressors, such as ultraviolet radiation (UVR), on the Daphnia-Chaoborus
 interaction is not settled as UVR may for instance deactivate the
 kairomone. In laboratory experiments, we investigated the combined effect
 of kairomones and UVR at ecologically relevant levels on induced
 morphological defenses of two D. pulex clones. We found that kairomones
 were not deactivated by UVR exposure. Instead, UVR exposure suppressed
 induced morphological defense traits of D. pulex juveniles under predation
 threat by generally decreasing the number of neckteeth and especially by
 decreasing the size of the pedestal beneath the neckteeth. UVR exposure
 also decreased the body length, body width, and tail spine length of
 juveniles, likely additionally increasing the vulnerability to Chaoborus
 predation. Our results suggest potential detrimental effects on fitness
 and survival of D. pulex subject to UVR-stress, with consequences on
 community composition and food web structure in clear and shallow water
 bodies.
description: Details on how the data were collected and analysed are given in
 the methods section of the publication or in the Usage Notes.
 All the data and R scripts are also available on GitHub:
 https://github.com/RaoulWolf/UV-radiation-affects-anti-predatory-defense-traits-in-Daphnia-pulex.  
description: The following information is also given in the
 "README.TXT" file. This repository contains
 the raw data and R scripts for “UV radiation affects anti-predatory
 defense traits in <em>Daphnia pulex</em>” by Eshun-Wilson
 <em>et al.</em>, 2020, Ecology and Evolution. All the data and
 R scripts are also available on GitHub:
 https://github.com/RaoulWolf/UV-radiation-affects-anti-predatory-defense-traits-in-Daphnia-pulex. <strong>1. Data files: definitions of column headers, explanations of indexes used, and units if applicable</strong> Information to data file: “Data_Experiment_Effectivity of kairomones.csv”<br> Treatment: 3 treatment levels, “UVR”= media-filled jars with added kairomone extract exposed to ultraviolet radiation, “PAR”= media-filled jars with added kairomone extract exposed to photosynthetically active radiation, “CONTROL”= media-filled jars without kairomone extract kept in the dark.<br> Mother_ID: identifier of individual Daphnia pulex females carrying brood of neonates (mothers). Daphnia pulex of a single clonal line, called UNI.<br> Neonate_ID: identifier of individual second instar Daphnia pulex juveniles (neonates) per Daphnia pulex female (nested within Mother_ID).<br> Hour: time in hours media-filled jars with added kairomone extract have been exposed to UVR and PAR before Daphnia pulex females carrying brood of neonates have been placed into the jars.<br> Neckteeth_count: number of neckteeth counted on individual 2nd instar Daphnia pulex juveniles.<br> Bump_keel: pedestal score on individual 2nd instar Daphnia pulex juveniles, 0: no pedestal, 1: small pedestal, 2: large pedestal<br> Pedestal_score: pedestal score recorded on individual 2nd instar Daphnia pulex juveniles, A: no pedestal, B: small pedestal, C: large pedestal.<br> Induction_score: neckteeth induction score (in %) recorded on individual 2nd instar Daphnia pulex juveniles.<br> Further information on methods can be found below in section 2 & 4. Information to data file: “Data_Experiment_Direct effect on Daphnia.csv”<br> Clone: Daphnia pulex of two clonal lines were used, called “P5” and “UNI”.<br> Treatment: 8 treatment levels, “P5_Control”= no UVR and no kairomone exposure of clone P5, “P5_Kairomone”= no UVR but kairomone exposure of clone P5, “P5_UVR”= UVR but no kairomone exposure of clone P5, “P5_Both”= UVR and kairomone exposure of clone P5, “UNI_Control”= no UVR and no kairomone exposure of clone UNI, “UNI_Kairomone”= no UVR but kairomone exposure of clone UNI, “UNI_UVR”= UVR but no kairomone exposure of clone UNI, “UNI_Both”= UVR and kairomone exposure of clone UNI.<br> Kairomone: 2 levels, “+Kairomone”= kairomone exposure, “-Kairomone”= no kairomone exposure.<br> UVR: 2 levels, “+UVR”= exposure to ultraviolet radiation, “-UVR”= no exposure to ultraviolet radiation.<br> Mother_ID: identifier of individual Daphnia pulex females carrying brood of neonates (mothers).<br> Sex: sex of individual Daphnia pulex juveniles (female, male, or NA= not available).<br> Instar: instar of individual Daphnia pulex juveniles (instar 1, instar 2, or NA= not available).<br> Neck_teeth_count: number of neckteeth counted on individual Daphnia pulex juveniles.<br> Bump_score: pedestal score recorded on individual Daphnia pulex juveniles, A: no pedestal, B: small pedestal, C: large pedestal.<br> Induction: neckteeth induction score (in %) recorded on individual Daphnia pulex juveniles.<br> Body_length: body length in mm of individual Daphnia pulex juveniles (NA= not available).<br> Body_width: body width in mm of individual Daphnia pulex juveniles (NA= not available).<br> Spina_length: tail spine (spina) length in mm of individual Daphnia pulex juveniles (NA= not available).<br> Further information on methods can be found below in section 3 & 4. Further information to data collection of data file: “Data_Experiment_Effectivity of kairomones.csv”<br> <strong>2. Experiment testing the effect of UVR on Kairomone Effectivity</strong><br> We first conducted an experiment to (i) demonstrate that our kairomone extract effectively induced neckteeth formation as well as to (ii) investigate the hypothesis that UVR may limit neckteeth induction by UVR making the kairomone ineffective. In our study, the integrity of kairomone suspensions was tested for different time intervals with the following three treatments (with 8 replicates/jars per treatment): Medium with kairomones exposed to UVR (340-400nm), medium with kairomones exposed to PAR (400-700nm), and a control with medium that contained no kairomones and was kept in the dark. Kairomone solutions were prepared by adding 60 µL of the kairomone extract into 50 mL glass jars containing 40 mL medium and food algae. The jars containing the kairomone solutions and the control were subjected to the respective treatments for 2, 4, 6, and 8 hours with no daphnids present. After each time period, females of the UNI clone with developing offspring in their brood pouch were placed individually in jars of the different treatment groups (UVR, PAR, and control treatments, respectively), with 2 mother individuals for each group and time interval. Five to ten released offspring juveniles per mother were inspected under the microscope at their 2nd instar to count the number of neckteeth and score the pedestal (see section 4 below). Further information to data collection of data file: “Data_Experiment_Direct effect on Daphnia.csv”<br> <strong>3. Experiment testing the direct effect of UVR on Daphnia</strong><br> In another experiment we tested whether exposure of egg-bearing mothers and offspring to UVR would affect kairomone-induced neckteeth formation in the juveniles. D. pulex clones P5 and UNI were exposed to the following four treatments in a full factorial design: without UVR or kairomone exposure (control), kairomone exposure without UVR exposure, UVR exposure without kairomone exposure, and UVR and kairomone exposure. D. pulex females of both clones carrying the 4th clutch in their brood pouch were used for the experiment (5-7 mothers per treatment). These mother individuals were placed individually in transparent 50 mL open glass jars filled with 40 mL medium and food algae and were exposed to UVR and kairomones depending on treatment. Kairomone treatments were prepared by adding 60 µL of the kairomone extract to the jars. UVR and non-UVR treatment groups were exposed to UVR and PAR light, respectively, in 16:8 hours light:dark cycles. Mother individuals of all treatment groups were transferred daily to freshly prepared kairomone and food suspensions until release of their 4th clutch juveniles. The mothers were removed, and juvenile clutch groups were kept in the same treatments until reaching the 2nd instar. Juveniles were inspected daily alive using a microscope for counting neckteeth, scoring pedestals, and taking photographs of the full body using a computer-aided camera for later length measurements (see section 4 below). <strong>4. Scoring of morphological defense traits and length measurements</strong><br> <em><strong>4.1. Neckteeth number, pedestal score, and neckteeth induction score</strong></em><br> Neckteeth, i.e. small spines at the dorsal head margin, were counted on live individuals of D. pulex juveniles in the 1st and 2nd instar using a microscope (Nikon Eclipse E200) with 100x magnification. At the base of the neckteeth, a pedestal of varying size can develop and was scored in a categorial way with 'A' when absent, 'B' when small, and 'C' when large. Individuals were then photographed at 40x magnification for later length measurements (see below) with a microscope-mounted Nikon camera (DS-5M). From the neckteeth counts and pedestal score, a neckteeth induction score has been calculated: neckteeth were scored 10% each and the pedestal score was translated to A=0%, B=30%, C=50%; the induction score per individual juvenile is the sum of the neckteeth and pedestal scores.<br> 4.2. Measurements of body length, body width and tail spine (spina) length<br> Body length, body width, and tail spine length of D. pulex juveniles were measured from the photographs using ImageJ and a landmark approach. Body length was calculated as the distance between the top of the head and the base of tail spine, body width between the ventral midpoint and dorsal midpoint, and tail spine length between the base and the tip of the tail spine. Technical difficulties caused by image file corruption limited the number of measurements in some treatment groups of instar 2 juveniles. <strong>5. Statistics and data analyses</strong><br> Statistical analysis of the ‘Experiment testing the effect of UVR on kairomone effectivity’ with the associated data file “Data_Experiment_Effectivity of kairomones.csv” can be found in the R script “R_script_Model_kairomone_efficacy.R”.<br> Statistical analysis of the ‘Experiment testing the direct effect of UVR on Daphnia’ with the associated data file “Data_Experiment_Direct effect on Daphnia.csv” can be found in the R scripts “R_script_Model_body_length_body_width_and_spina_length.R”, “R_script_Model_neck_teeth_count_and_bump_score_instar_2.R”, “R_script_Model_neck_teeth_count_and_bump_score_instar_1.R”.<br> The figures of the paper can be reproduced with the R scripts “R_script_figure_1.R”, “R_script_figure_2.R”, “R_script_figure_3.R”, “R_script_figure_4.R”, “R_script_figure_5.R”, “R_script_figure_S1.R”, “R_script_figure_S2.R”, “R_script_figure_S3.R”. The following R package needs to be installed to run the analysis: <pre> install.packages("brms")</pre> Additionally, all graphics require the <code>tidyverse</code> package collection, especially <code>ggplot2</code>, and <code>patchwork</code>. They can be installed as follows: <pre> install.packages(c("tidyverse", "patchwork"))</pre>