UV radiation affects anti-predatory defense traits in Daphnia pulex

This repository contains the raw data and R scripts for “UV radiation affects anti-predatory defense traits in Daphnia pulex” by Eshun-Wilson et al., 2020, Ecology and Evolution, doi:10.1002/ece3.6999. All data is also available on Dryad: https://doi.org/10.5061/dryad.kprr4xh3c

The data folder contains the raw data used for the analyses, and the scripts folder contains the R scripts which were written for the analyses. The fitted models are in the models folder, and all graphics are in the figures folder.

The following R package needs to be installed to run the analysis:

install.packages("brms")

Additionally, all graphics require the tidyverse package collection, especially ggplot2, and patchwork. They can be installed as follows:

install.packages(c("tidyverse", "patchwork"))

1. Data files, definitions of column headers, explanations of indeces used, and units if applicable

Information to data file Data_Experiment_Effectivity of kairomones.csv

Treatment: Three 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.

Mother_ID: Identifier of individual Daphnia pulex females carrying brood of neonates (mothers). Daphnia pulex of a single clonal line, called UNI.

Neonate_ID: Identifier of individual second instar Daphnia pulex juveniles (neonates) per Daphnia pulex female (nested within Mother_ID).

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.

Neckteeth_count: Number of neckteeth counted on individual 2nd instar Daphnia pulex juveniles.

Bump_keel: Pedestal score on individual 2nd instar Daphnia pulex juveniles. 0: no pedestal, 1: small pedestal, 2: large pedestal.

Pedestal_score: Pedestal score recorded on individual 2nd instar Daphnia pulex juveniles. A: no pedestal, B: small pedestal, C: large pedestal.

Induction_score: Neckteeth induction score (in %) recorded on individual 2nd instar Daphnia pulex juveniles.

Further information on methods can be found below in section 2 & 4.

Information to data file Data_Experiment_Direct effect on Daphnia.csv

Clone: Daphnia pulex of two clonal lines were used, called P5 and UNI.

Treatment: Eight 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.

Kairomone: Two levels. +Kairomone: kairomone exposure, -Kairomone: no kairomone exposure.

UVR: Two levels. +UVR: exposure to ultraviolet radiation, -UVR: no exposure to ultraviolet radiation.

Mother_ID: Identifier of individual Daphnia pulex females carrying brood of neonates (mothers).

Sex: Sex of individual Daphnia pulex juveniles (female, male, or NA: not available).

Instar: Instar of individual Daphnia pulex juveniles (instar 1, instar 2, or NA: not available).

Neck_teeth_count: Number of neckteeth counted on individual Daphnia pulex juveniles.

Bump_score: Pedestal score recorded on individual Daphnia pulex juveniles. A: no pedestal, B: small pedestal, C: large pedestal.

Induction: Neckteeth induction score (in %) recorded on individual Daphnia pulex juveniles.

Body_length: Body length (in mm) of individual Daphnia pulex juveniles (NA: not available).

Body_width: Body width (in mm) of individual Daphnia pulex juveniles (NA: not available).

Spina_length: Tail spine (spina) length (in mm) of individual Daphnia pulex juveniles (NA: not available).

Further information on methods can be found below in section 3 & 4.

2. Experiment testing the effect of UVR on kairomone effectivity

Further information to data collection of data file Data_Experiment_Effectivity of kairomones.csv

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 (340-400 nm), medium with kairomones exposed to PAR (400-700 nm), 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).

3. Experiment testing the direct effect of UVR on Daphnia

Further information to data collection of data file Data_Experiment_Direct effect on Daphnia.csv

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).

4. Scoring of morphological defense traits and length measurements

4.1. Neckteeth number, pedestal score, and neckteeth induction score

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.

4.2. Measurements of body length, body width and tail spine (spina) length

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.

5. Statistics and data analyses

Statistical analysis of 2. 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.

Statistical analysis of 3. 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.

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.