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