DOI: 10.5061/dryad.hx3ffbgn0
title: The importance of paternal effects in heatwaves

description: As the threat of climate change and associated heatwaves grows, we need to
 understand how natural populations will respond. Inter-generational
 non-genetic inheritance may play a key role in rapid adaptation, but
 whether such mechanisms are truly adaptive and sufficient to protect wild
 populations is unclear. The contribution of paternal effects in particular
 is not fully understood, even though the male reproductive system may be
 highly sensitive to heatwaves. We used the zebrafish Danio rerio to
 investigate the effects of heatwaves on male fertility and assess
 potential adaptive benefits to their offspring in a number of large-scale
 heatwave experiments. Heatwave conditions had negative effects on male
 fertility by reducing gamete quality and fertilisation success, and we
 found indications of an adaptive effect on hatching in offspring produced
 by heatwave-exposed males. Our findings highlight the importance of
 including male and female fertility when determining species ability to
 cope with extreme conditions and suggest that parental effects provide
 limited adaptive benefits.
description: # The importance of paternal effects in heatwaves
 [https://doi.org/10.5061/dryad.hx3ffbgn0](https://doi.org/10.5061/dryad.hx3ffbgn0) ## Description of experiments and files **Experiment 1:** Investigation into whether paternal heatwave exposure and/or elevated temperature on sperm phenotypes and sperm genome integrity, and whether this impacts fertilisation success and offspring performance.  Data files: ``` Note: Any cells in data files containing NA = Not Applicable. Datapoints were either unable to be collected or the variable described does not apply. 1. LP_CASA folder contains all files used for Computer Assisted Semen Analysis (CASA) Variables from output: File names - Identification of individual males and given treatments. For example, file C28 SC34 46A represents Male treatment of 28C, sperm treatment of 34C, and male identification number 46. Field - Represents the 'field' of an image taken every 10 seconds to determine the location of individual sperm and their movement, thus represents a time variable in 10 second intervals #spe. - Sperm ID First - First recorded frame of sperm movement Last - The final recorded frame of sperm movements #points - Number of points tracked for sperm movement between first and final frame Area - Area of sperm head (square micrometers) VCL - Curvilear velocity (micrometers per second) VSL - Linear velocity (micrometers per second) VAP - Average path velocity (micrometers per second) LIN - Linearity, ratio of VSL to VCL (%) STR - Straightness, ratio of VSL to VAP (%) WOB - Wobble, ratio of VAP to VCL (%) ALH - Amplitude of lateral head displacement (micrometers) BCF - Beatcross frequency, frequency at which sperm head crosses trajectory path (Hz) OMS Type: System based classification of sperm movement Type - Variable to determine whether sperm are motile (Static = not motile, all others = motile) DANCE - Movement patterns of sperm Mean DANCE - Mean of DANCE Angularity Index (AI) - Measures angle of sperm movements Mean Angular Velocity (AV) - mean speed of angular movement Mean Displacement Absolute (MDAabs) - Absolute displacement of sperm from starting position Algebraic Mean Displacement Absolute (MDAalg): net diplacement of sperm, accounting for directionality 2. 1_LP_Part1_fertilisation.txt contains all fertilisation data Variables: Male_ID: Identification code for indiviual males Treatment: 28C or 34C treatment Fertilised eggs: Number of fertilised eggs present in a clutch Unfertilized eggs: Number of eggs that were not fertilised in a clutch Total eggs: Total number of eggs in a clutch Percentage: Percentage of eggs in a clutch that are fertilised ID: Observation level identification 3. 1_LP_Part1_Malf.txt contains developmental abnorality data Variables: Male_ID: Identification code for individual males Male treatment: 28C or 34C temperature treatment a male was exposed to Incubation: 28C or 34C temperature treatment half of each clutch was exposed to (clutches were split in half, half going into each treatment) Total: Total number of eggs in a half clutch Deformed: Number of eggs in half clutch that are abnormal Normal: Nubmer of normal eggs in half clutch ID: Individual ID number for each half clutch 4. 1_LP_TUNEL_ASSAYS_FINAL.txt contains data from TUNEL analysis MALE_ID: Identification code for individual male Treatment: 28C or 34C temperature treatment the male was exposed to Healthy: Number of healthy sperm in an ejaculate Damaged: Number of sperm with DNA fragmentation in a sample Total: Total number of sperm in an ejaculate Percentage: Percentage of sperm with DNA damage in an ejaculate Average_percentage: Mean percentage of sperm with DNA damage in an ejaculate Average_Total: Mean total number of sperm in an ejaculate ID: Observation level identification number ``` **Experiment 2:** Repeat of offspring assays from Experiment 1 Data files: ``` Note: Any cells in data files containing NA = Not Applicable. Datapoints were either unable to be collected or the variable described does not apply. 1. 3_MS_2hpf.csv contains fertilisation data (phenotypes measured at 2 hours post fertilisation) Variables: M_ID: Identification code for individual males spawning time: Time range within when spawning occurred total_2hpf: Total number of eggs in a clutch as measured 2 hours post-fertilisation fertilised: Number of fertilised embryos in a clutch fertilised weird: Number of fertilised, but abnormal embryos in a clutch unfertilised: Number of unfertilised eggs in a clutch dead_2hpf: Number of dead eggs in a clutch A eggs: Number of eggs in clutch half A, split for incubation at 28C/34C B eggs: Number of eggs in clutch half B, split for incubation at 28C/34C reset (A/C): Number of egg in clutch half C, split for incubation at 28C/34C produced from a second spawning attempt reset (B/D): Number of egg in clutch half D, split for incubation at 28C/34C produced from a second spawning attempt notes: Extra information regarding spawnings 2. 3_MS_24hpf.csv contains developmental abnormality and survival data Variables: set: Identifier for Experimental Block male: Identification code for individual males petri: Identifier for clutch A or B total: Total number of embryos 24 hours post fertilisation in half clutch dead: Number of dead embryos present in half clutch abnormal: Number of abnormal embryos present in half clutch time: Time when observations occurred photo: Time a photo was taken of dish (x or blank mean no photo) notes: Extra information regarding clutches 3. 3_MS_codes.csv contains all blind code information for parent and offspring treatments. These were incorporated into the datasets within the R code. Variables: M_ID: Identification code for individual males ID: Identification code for individual half clutches maletreat: Temperature treatment males were exposed to (T = 34C, C = 28C) embryotreat: Temperature treatment clutches were exposed to (34C/28C) repeat_spawn: Y/N variable for whether a repeat spawning was used to obtain a clutch ``` **Experiment 3:** We used IVF following heatwave exposure to rule out potential confounding effects of paternal behaviour. We also added a temperature manipulation step during sperm activation for IVF to investigate the potential for environmentally induced heritable effects at the sperm level. Clutches of eggs produced were split into 4 groups, for a fully factorial 34C/28C sperm x embryo treatment.  Data files: ``` Note: Any cells in data files containing NA = Not Applicable. Datapoints were either unable to be collected or the variable described does not apply. 1. 2_AS_IVF_data_errorsRemoved.csv contains data from fertilisation, developmental abnormality and survival data from embryo assays and sperm score data from IVF trials Variables: Block: Experimental block Day: Day males were setup for spawning Male: Identification code for individual males Petri dish: Identification code for portion of clutches based on sperm x embryo treatment combination tot_eggs: Total number of eggs in clutch sample fert: Number of fertilised eggs at 2 hours post fertilisation in clutch sample unfert: Number of unfertilised eggs at 2 hours post fertilisation in clutch sample dead: Number of dead eggs at 2 hours post fertilisation in clutch sample fert_weird: Number of fertilised eggs with abnormalities in clutch sample at 2 hours post fertilisation unfert_weird: Number of unfertilised eggs in clutch sample with abnormalities at 2 hours post fertilisation alive_24: Number of living embryos in clutch sample at 24 hours post fertilisation dead_24: Number of dead embryos in clutch sample at 24 hours post fertilisation normal_24: Number of normal embryos in clutch sample at 24 hours post fertilisation abnormal_24: Number of abnormal embryos in clutch sample at 24 hours post fertilisation abnormal_72: Types of abnormalities identified at 72 hours post fertilisation 2. 2_AS_BlindCodes_embryos.csv contains blind codes for sperm and embryo treatments Variables: Date: Date of experimental setup Block: Experimental Block Day: Day of experimental setup Male: ID number for individual males Sperm: Sperm treatment (28C or 34C) Embryos: Embryo treatment (28C or 34C) Code: Identification code for clutch sample 3. 2_AS_BlindCodes_males.csv contains blind codes for male treatment Block: Experimental block Day: Day of setup Date: Date of setup Male: Identification code for individual males Treatment: Male exposure to 28C/34C temperature treatments Code: Male identification code Ejac: Descriptor of ejaculate size (0-4, where - = 0, ++++ = 4) Comments: Notes on ejaculate extractions Score: Numerical score to match descriptor of ejaculate size 4. 2_AS_BlindCodes_IVF_metadata.csv contains additional information about experimenters Block: Experimental block Day: Day of experimental setup Date: Date of experimental setup Male: Male identification code Sperm 28C: Experimenter who exposed sperm to 28C treatment Sperm 34C: Experimenter who exposed sperm to 34C treatment South: Experimenter location North: Experimenter location ``` **Experiment 4:** We used both male and female fish exposed to heatwaves to test for the relative importance of maternal and paternal effects on offspring resilience to heatwaves during early development via a fully factorial crossing of parents across treatments. Data files: ``` Note: Any cells in data files containing NA = Not Applicable. Datapoints were either unable to be collected or the variable described does not apply. 1. SI_alldata.csv contains all data for fertilisation, developmental abnormalities, survival and early hatching Variables: Date: Date of experimental setup Block: Experimental block F_birthdate: Date of birth of experimental females M_birthdate: Date of birth of experimental males ID_num: Individual identification number for clutches produced and split into halves for different treatments Clutch ID: Numerical identification of clutches before splitting ID: Descriptive individual ID for each split clutch half (B1.1A = Block 1, Clutch 1, Clutch half A) M_temp: Male treatment temperature (C) F_temp: Female treatment temperature (C) Clutch_prod: Binary variable to represent whether a clutch was produced or not (1 = success/0 = failure) Spawn_time: Time eggs collected from spawning Incub_temp: Embryo treatment temperature (C) No_eggs: Total number of eggs in clutch sample Unfert_2h: Number of unfertilised eggs in clutch sample at 2 hours post fertilisation Dead_2h: Number of dead eggs in clutch sample at 2 hours post fertilisation Ab_2h: Number of abnormal eggs in clutch sample at 2 hours post fertilisation Dead_24h: Number of dead eggs in clutch sample at 24 hours post fertilisation Ab_24h: Number of abnormal eggs in clutch sample at 24 hours post fertilisation Dead_48h: Number of dead eggs in clutch sample at 48 hours post fertilisation Ab_48h: Number of abnormal eggs in clutch sample at 48 hours post fertilisation Hatch_48h: Number of hatched eggs in clutch sample at 48 hours post fertilisation Dead_5d: Number of dead offspring in clutch sample 5 days post fertilisation Ab_5d: Number of abnormal offspring in clutch sample 5 days post fertilisation Unhatch_5d: Number of unhatched offspring in clutch sample 5 days post fertilisation Tot_ab_5d: Number of dead offspring in clutch sample 5 days post fertilisation (Ab_5d + Unhatch_5d) ``` **Other:** We performed an experiment to determine the effect of embryo temperature (28 or 34C) 0-2 hpf on embryo survival. Data file: ``` Note: Any cells in data files containing NA = Not Applicable. Datapoints were either unable to be collected or the variable described does not apply. 1. 5_embryo_inc_temp.csv contains data for offspring survival and developmental stage at 24 hours and 48 hours post fertilisation (hpf) to compare relative developmental time between treatments. Staging is based on Kimmel et al., 1995. Variables: ID: Numerical ID to clutches produced from a given treatment tank_ID: ID letter/number for tank fish were housed in incubation_t: Hours post fertilisation clutch incubation temperature treatment began temp: Temperature treatment (C) total_24h: total number of eggs in clutch 24 hpf live_24h: Number of living embryos in clutch 24 hpf dead_24h: Number of dead embryos in clutch 24 hpf sN_24: Number of embryos at 25-somite stage at 24 hpf sA_24: Number of embryos at prim-5 stage at 24 hpf sB_24: Number of embryos at prim-12 stage at 24 hpf total_48h: Total number of embryos in clutch 48 hpf live_48h: Number of living embryos in clutch 48 hpf dead_48h: Number of dead embryos in clutch 48 hpf sB_48h: Number of embryos at prim-12 stage at 48 hpf sD_48h: Number of embryos at prim-20 stage at 48 hpf sF_48h: Number of embryos at prim-25 stage at 48 hpf sH_48h: Number of embryos at high pec stage at 48 hpf hatched: Number of hatched embryos in clutch 48 hpf Stages described above occur in order described below: 1. 25-somite stage 2. prim-5 stage 3. prim-12 stage 4. prim-20 stage 5. prim-25 stage 6. high pec stage ``` ``` Reference: Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. 1995. 'Stages of embryonic development of the zebrafish.' Developmental Dynamics. 203(3): 253-391. ``` ## Code/Software All analyses and visualisations were performed in R (v4.2.0) contained in the file: ``` 1. All_ZFHW_code.R ```