DOI: 10.5061/dryad.c2fqz61m8
title: Data from: Elevated temperature during rearing diminishes swimming and disturbs the metabolism of yellow perch larvae

description: Temperate waters, such as the Great Lakes, are predicted to increase by
 1°C every decade. Poikilothermic fish thermoregulate behaviourally, moving
 to more suitable thermal environments. Embryos are incapable of locomotion
 and may be exposed to non-optimal temperatures during development.
 Increased temperature alters the normal development of the yellow perch
 (Perca flavescens); however, whether altered incubation temperature
 influences the development of metabolism and function in these fish
 remains unknown. We hypothesized that increased embryonic incubation
 temperature would disturb cardiac and metabolic function and the behaviour
 of yellow perch larvae. We reared yellow perch embryos at 12°C, 15°C, or
 18°C until hatching; after hatching, the temperature was raised to a
 common garden 18°C, their preferred post-hatch temperature. We assessed
 exploratory behaviour, metabolism (oxygen consumption), and cardiac
 performance throughout early development. At hatch, 12°C fish exhibited
 the greatest swimming activity, with 18°C fish consuming the least oxygen
 and possibly experiencing mitochondrial dysfunction. Cardiac development
 was more advanced at hatch in 18°C fish. Yet, warmer incubated fish had
 diminished movement and increased oxygen consumption at 20 days
 post-hatch, demonstrating long-term disruptions of increased temperature
 in the embryonic environment. Overall, elevations in rearing temperature
 may cause metabolic dysfunction and behavioural alterations, potentially
 impacting the survival of yellow perch.
description: <strong>Apoptosis stain</strong>
 The acridine orange stain protocol for apoptotic cell death was
 adapted from Wong et al. (2021). At hatch, 20 yellow perch larvae were
 added to each well on a 6-well plate along with E2 fish water. After quick
 removal of fish water from each well, 3mL of fresh E2 at the appropriate
 incubation temperature or acridine orange solution (5 µg/ml in E2;
 Acridine orange hemi (zinc chloride) salt; Sigma-Aldrich; Cat No. A6014)
 was added for the control and stained groups, respectively. The plate was
 immediately covered and moved into a dark incubator at the respective
 temperature for 60 min. Post-incubation, embryos were washed with E2 at
 incubation temperatures. To measure whole-body fluorescence at hatch,
 stained and control fish were anesthetized with 0.016% MS-222 (Sigma
 Aldrich, Canada) in E2, and individual fish were placed into each well of
 a 96-well plate. The plate was read using the Synergy 2 Multi-Mode plate
 reader (BioTek Instruments, Winooski, United States) and Gen 5 software
 (BioTek Instruments, Winooski, United States) set to an excitation of
 485/20 λ and emission of 528/20 λ. Whole body fluorescence was measured in
 72, 71, and 69 stained larvae for 12°C, 15°C, and 18°C treatment groups,
 respectively. 18 control larvae and 6 blanks (E2) were also run on each
 plate along with the stained larvae of each treatment. The fluorescence in
 each group of unstained larvae was averaged and subtracted from the
 fluorescence of stained larvae. Mean fluorescence was then determined for
 each of the temperature treatments. Live-stained and control fish were
 imaged with the X-cite series 120Q fluorescence lamp and the Zeiss
 Discovery V8 stereoscope while using the Zeiss ZEN Pro 2012 imaging
 software. Fish were positioned laterally on agar using a transfer pipette,
 and images focusing on the heart were captured. Using Image J (Fiji;
 v1.54f), images were analyzed, consisting of manually outlining the fish
 heart with the freehand ROI tool. Parameters were set to include area,
 integrated density, and mean grey value.
 <strong>Behavioral Assay</strong>
 Yellow perch larvae were assessed for exploratory behaviour using
 a general swimming assay, tracking movement immediately after introduction
 to a novel environment. Individual fish were hatched shortly after hatch,
 and at 1, 2, 3, 4, 5, and 10 DPH were transferred into a 24-well clear
 plate in 2 mL/well E2 media (1 individual per well). For 20 DPH, a 12-well
 clear plate with 5 mL/well E2 media was used to account for the change in
 overall growth between 10 and 20 DPH (Fraz et al., 2024). Plates were
 moved to the Daniovision observation chamber (Noldus, Wageningen,
 Netherlands) and recorded for 20 min under natural light. The total
 distance travelled and the maximum velocity were determined using the
 Ethovision XT software (version 15.0.1416; Noldus, Wageningen,
 Netherlands). Individuals who did not move were removed from further
 statistical analysis (total n=44-48).
 <strong>Closed Respirometry</strong>
 Closed respirometry was conducted to determine the oxygen uptake
 rates as a measure of metabolic rates in the animal. This was measured at
 the respective incubation temperatures of fish at hatch, 5 DPH, 10 DPH,
 and 20 DPH, from each treatment group (12°C, 15°C, and 18°C). Additional
 closed respirometry trials were run at hatch at respective incubation
 temperatures and at 25°C, the temperature at which the Agilent Seahorse
 mitochondrial respiration data were collected, to confirm animal
 viability. The replicates for oxygen consumption analyses ranged from
 12-18 per group. Four oxygen respiration vials (volume: 4mL;
 diameter/height: 15mm/48mm) with integrated oxygen sensors were connected
 to the Pyro Science FireSting-O2 (4 Channels; Aachen, Germany), containing
 data logging software. One respirometer vial in every trial contained only
 E2 water to account for background microbial respiration. Respirometers
 were submerged in an insulated and temperature-regulated water bath, with
 a circulating pump to ensure mixing. Oxygen saturation values were
 collected from individual vials every 5s for 1h. Immediately after, the
 pool of larvae in each vial was weighed. Oxygen saturation values were
 analyzed in R (version 4.3.0; Vienna, Austria) and RStudio (version
 2023.06.0+421; Boston, United States) using the ‘Respirometry’
 package. <strong>Ejection
 Fraction</strong> Ejection fraction was estimated
 from the videos, using the image analysis protocol by Perrichon et al.
 (2017). Suitable clips were chosen, videos were examined frame by frame,
 and screenshots were captured at the precise moment of end-diastole
 (maximum ventricle volume) and end-systole (minimum ventricle volume) for
 5 continuous cardiac cycles. Using ImageJ (Fiji; v1.54f), the perimeter of
 the cardiac ventricle was traced with the freehand ROI tool, and
 parameters were set to include the best-fitted ellipse within the selected
 area. From the best-fitted ellipse, measurements of the major (a) and
 minor (b) axes were estimated by ImageJ and input into the volume of a
 prolate spheroid equation to quantify ventricle volume. Once diastolic and
 systolic volumes were calculated for each fish at hatch, the difference
 between the two measures was considered the stroke volume. However, to
 account for differences in zoom magnification (no standardized scale was
 used), ejection fraction was calculated and expressed as relative changes
 in volume (percent difference) per cycle. This was repeated for each of
 the 5 cycles of diastolic and systolic events per larva, and the ejection
 fraction was averaged. <strong>Heart
 Rate</strong> Live perch embryos from each
 treatment were transferred into Petri dishes containing E2 media at the
 onset of heartbeat and eye pigmentation. Temperature was maintained at the
 appropriate incubation temperature using a temperature-regulated water
 bath. Videos of the heart were approximately 30s and recorded on an Axio
 Zoom V16 microscope (Carl Zeiss, Oberkochen, Germany) connected to a Canon
 EOS Rebel T1i camera using AxioVision software (Rel. 4.8; Carl Zeiss,
 Oberkochen, Germany). At hatch, videos were recorded at their incubation
 temperature and after an acute increase to 18°C (rate increased 3°C/30
 min). Yellow perch at 20 DPH were recorded at 18°C only because they were
 all at the same common garden temperature; individual larvae were
 immobilized using mild anesthesia for 2 minutes before recording. Using
 VLC media player (version 3.0; Paris, France), videos were slowed to
 0.25-0.5x and trimmed (20-30s). Heartbeats were visually counted a total
 of 3 times per video, with the observer blind to the treatment and
 converted to beats per minute (bpm). At hatch (n=37-48), fish were
 recorded at their respective incubation temperatures and then raised
 acutely to 18°C, and heart rate was recorded for the 12 and 15°C groups
 again (n=44-45). Heart rate for each group at 20 DPH was recorded at 18°C
 (n=44-45).
 <strong>Quantitative-PCR</strong>
 Quantification of transcript abundance was carried out as
 described by Thompson and Vijayan (2020). A total of 8 samples containing
 12-15 pooled fish had RNA extracted using TRIzol reagent per manufacturer
 instructions (Thermo-Fisher Scientific, Waltham, United States; Cat No.
 15596018) with the addition of glycogen to enhance precipitation (RNA
 grade; Thermo-Fisher Scientific, Waltham, United States; Cat No. R0551). A
 total of 1μg of RNA was used to create cDNA using a QuantiTect Reverse
 Transcription Kit (QIAGEN, Hilden, Germany; Cat No. 205311). Before qPCR,
 testing was conducted for optimal annealing temperature via a primer PCR
 gradient (Taq DNA Polymerase; Thermo-Fisher Scientific, Waltham, United
 States; Cat No. 10342053). The optimal annealing temperature was 60°C for
 all primers. Our primers were for the genes vascular endothelial growth
 factor- A (VEGFA), myosin heavy chain (MyHC), and NKX2-homeobox 5
 (NKX2.5), and were confirmed via sequencing after amplification in yellow
 perch samples. qPCR was performed with SYBR Green supermix (Bio-Rad,
 Hercules, United States; Cat No. 1725270) using a CFX96 Real-Time System
 C1000 Touch thermal cycler (Bio-Rad, Hercules, United States). The raw Cq
 (cycle of quantification) values were obtained using the CFX Manager
 Software (Bio-Rad, Hercules, United States; version 3.1).
 <strong>Vasculature Staining</strong>
 The alkaline phosphatase staining protocol was modified from a
 protocol for zebrafish by Eliceiri et al. (2011) for use with yellow
 perch. Briefly, dechorionated embryos and larvae were washed in 500 µL of
 developing buffer (0.1 M Tris-HCl, pH 9.5; 0.1 M NaCl; and 0.05 M MgCl2)
 twice for 10 minutes each. Following this, the samples were incubated with
 100 µL of 1:1, 1-step NBT/BCIP substrate solution (Nitro blue
 tetrazolium/5-Bromo-4-chloro-3-indoyl phosphate; Thermo-Fisher Scientific;
 Cat No. 34042) and development buffer for 1h. Following this samples were
 washed and fixed with 10% neutral buffered formalin for 30 min. Finally,
 samples were washed with developing buffer nd stored at 4°C in the dark.
 The yellow perch samples were imaged either the same day as staining or
 the following day in a dimly lit room to ensure minimal fading. Imaging
 was performed using a Zeiss Discovery V8 stereoscope and Zeiss ZEN Pro
 2012 imaging software. Images were of lateral and dorsal views of the yolk
 sac, brain, and muscle of the yellow perch. The presence of vasculature at
 these regions was determined by examining images for various markers of
 stained vasculature based on those observed in zebrafish (Ulrich et al.,
 2011). A total of 8 embryos or larvae were stained at each developmental
 stage for each incubation temperature. Vasculature staining was assessed
 as the presence/absence of vessels and was analyzed using a simple
 logistic regression model.
description: # Data from: Elevated temperature during rearing diminishes swimming and
 disturbs the metabolism of yellow perch larvae
 [https://doi.org/10.5061/dryad.c2fqz61m8](https://doi.org/10.5061/dryad.c2fqz61m8) ## Description of the data and file structure **GENERAL INFORMATION** This dataset is divided into folders according to the experiments from which the data was collected. We characterized cardiac development by assessing ejection fraction at hatch and heart rate at hatch and 20 days post-hatch (DPH). Whole-animal metabolic rates were measured at hatch, 5, 10, and 20 DPH. To probe into possible changes in mitochondrial respiration, we estimated basal metabolic rate, ATP production, proton leak, and non-mitochondrial respiration. To gauge locomotory function in larvae, we performed general swimming assessments from hatch until 5 DPH, and at 10 and 20 DPH. At hatch, transcript abundance was determined for vascular endothelial growth factor- A (vegfa), myosin heavy chain (myhc), and NKX2-homeobox 5 (nkx2.5). Apoptotic cell death (hatch) and the vasculature of the circulatory system (pre-hatch and at hatch) were determined in whole animals. **DATA & FILE OVERVIEW** 1\. File List ├── Apoptosis/ ├── Behaviour/ ├── Closed Respirometry/ ├── Ejection Fraction/ ├── Heart Rate/ ├── Quantitative-PCR/ └── Vasculature/ ### Files and variables #### File: Apoptosis.zip **Description:** └── Apoptosis/ ``` ├── Heart/ └── Raw Images/ ├── 12C Hatch/ ├── 15C Hatch/ └── 18C_Hatch/ ├── 12C Apoptosis Heart Data.xlsx ├── 15C Apoptosis Heart Data.xlsx ├── 18C Apoptosis Heart Data.xlsx └── Apoptosis Image Results.xlsx └── Whole-body/ ├── 12C Fluorescence plate.xlsx ├── 15C Fluorescence plate.xlsx ├── 18C Fluorescence plate.xlsx └── Whole-body Fluorescence Results.xlsx ``` File-Specific Information for: Raw Images/ This folder has image files (.czi and .jpg) that were used for the measurements of total relative fluorescence following acridine orange staining in the area of the heart in yellow perch larvae. Column Headers: In the "Apoptosis Image Results.xlsx" and "Whole-body Fluorescence Results.xlsx" files, temp: the treatment temperature (°C) of the yellow perch larvae, fluorescence: the measured fluorescence (relative fluorescence units). #### File: Behaviour.zip **Description:**  └── Behaviour/ ``` ├── Raw Media Files/ ├── Hatch/ ├── 12C/ ├── 15C/ └── 18C/ ├── 1 DPH/ ├── 12C/ ├── 15C/ └── 18C/ ├── 2 DPH/ ├── 12C/ ├── 15C/ └── 18C/ ├── 3 DPH/ ├── 12C/ ├── 15C/ └── 18C/ ├── 4 DPH/ ├── 12C/ ├── 15C/ └── 18C/ ├── 5 DPH/ ├── 12C/ ├── 15C/ └── 18C/ ├── 10 DPH/ ├── 12C/ ├── 15C/ └── 18C/ └── 20 DPH/ ├── 12C/ ├── 15C/ └── 18C/ ├── General Swimming-Total Distance Data.xlsx └── General Swimming-Maximum Velocity Data.xlsx ``` File-Specific Information for: Raw Media Files/ This folder has subfolders "Hatch", "1 DPH", "2 DPH", "3 DPH", "4 DPH", "5 DPH", "10 DPH", "20 DPH"- corresponding to days post-hatch at which the yellow perch samples were run. 12C, 15C, and 18C treatments were run at each time point and are found within these folders as subfolders. These contain the raw media files (.mpg) that were analyzed using the Ethovision XT software to determine total distance travelled and maximum velocity. Abbreviations used for Naming Folders/Files: DPH: Days post-hatch Column Headers: In the "General Swimming-Total Distance Data.xlsx" and "General Swimming-Maximum Velocity Data.xlsx" files, headers indicate the treatment temperature and DPH of the total distance and maximum velocity of yellow perch larvae measured in behavioural trials. #### File: Closed_Respirometry.zip **Description:** . └── Closed Respirometry/ ``` ├── Acute Exposure/ ├── Blue/ ├── Pink/ └── Seahorse Respirometry Results.xlsx ├── Incubation Temperatures/ ├── Hatch/ ├── 12C/ ├── 15C/ └── 18C/ ├── 5 DPH/ ├── 12C/ ├── 15C/ └── 18C/ ├── 10 DPH/ ├── 12C/ ├── 15C/ └── 18C/ ├── 20 DPH/ ├── 12C/ ├── 15C/ └── 18C/ └── Respirometry Results.xlsx └── Metabolic Rate Script.R ``` File-Specific Information for: Acute Exposure/ This folder has subfolders "Blue" and "Pink," which were used to differentiate the 2 respirometry setups that were run simultaneously to confirm animal viability from the seahorse experiments. Blue and Pink contain the raw respirometry output files from Pyro Science Firesting software. File-Specific Information for: Incubation Temperature/ This folder has subfolders "Hatch", "5 DPH", "10 DPH", "20 DPH"- corresponding to days post-hatch at which the yellow perch samples were run. 12C, 15C, and 18C treatments were run at each time point and are found within these folders as subfolders. Abbreviations used for Naming Folders/Files: SH: Seahorse; R1: Replicate 1; R2: Replicate 2; DPH: Days post-hatch Column Headers: In the "Seahorse Respirometry Results.xlsx" file, basal: the treatment temperature (°C) of the yellow perch larvae, ran: the water temperature at which the larvae were run respirometry at (either ambient or room temperature), oxygen: the oxygen consumption rate (µmol/hr/g) of the larvae. In the "Respirometry Results.xlsx" file, temp: the treatment temperature (°C) of the yellow perch larvae, dph: the days-post hatch age group of the larvae, oxygen: the oxygen consumption rate (µmol/hr/g) of the larvae. #### File: Ejection_Fraction.zip **Description:**  └── Ejection Fraction/ ``` ├── 12C Hatch, t12/ ├── 12C Hatch, t18/ ├── 15C Hatch, t15/ ├── 15C Hatch, t18/ ├── 18C Hatch/ ├── 12C Hatch, t12 Data.xlsx ├── 12C Hatch, t18 Data.xlsx ├── 15C Hatch, t15 Data.xlsx ├── 15C Hatch, t18 Data.xlsx ├── 18C Hatch Data.xlsx └── Ejection Fraction Results.xlsx ``` File-Specific Information for: Ejection Fraction/ The subfolders contain the screenshots for 5 continuous cardiac cycles that were used in the quantification of ventricular volume. The subfolders for each treatment contain labelled folders that correspond to the heart rate videos the images (.png) were captured. Along with the images is a "Results.csv" file that contains the major and minor axes measurements used to determine ventricle volume. Abbreviations used for Naming Folders/Files: t12: trials were run at 12C water temperature; t15: trials were run at 15C; t18: trials were run at 18C. DPH: Days post-hatch. Column Headers: In the "Ejection Fraction Results.xlsx" file, basal: the treatment temperature (°C) of the yellow perch larvae, ran: the water temperature at which the video recordings of larvae were collected, ejection_fraction: the ejection fraction of the larvae. #### File: Heart_Rate.zip **Description:**  └── Heart Rate/ ``` ├── Hatch/ ├── 12C Hatch, t12/ ├── 12C Hatch, t18/ ├── 15C Hatch, t15/ ├── 15C Hatch, t18/ ├── 18C Hatch/ ├── 12C Hatch, t12 Data.xlsx ├── 12C Hatch, t18 Data.xlsx ├── 15C Hatch, t15 Data.xlsx ├── 15C Hatch, t18 Data.xlsx └── 18C Hatch Data.xlsx ├── 20 DPH/ ├── 12C/ ├── 15C/ ├── 18C/ ├── 12C 20DPH Data/ ├── 15C 20DPH Data/ └── 18C 20DPH Data/ └── Heart Rate Results.xlsx ``` File-Specific Information for: Heart Rate/ The subfolders contain videos (.MOV) of the heart beating that were used to determine the heart rate (beats/min) of each yellow perch larva. Abbreviations used for Naming Folders/Files: t12: trials were run at 12C water temperature; t15: trials were run at 15C; t18: trials were run at 18C. DPH: Days post-hatch. Column Headers: In the "Heart Rate Results.xlsx" file, treatment: the treatment temperature (°C) of the yellow perch larvae, ran: the water temperature at which the video recordings of larvae were collected, dph: the days-post hatch age group of the larvae, heart_rate: the heart rate (beats per minute) of the larvae. #### File: Quantitative-PCR.zip **Description:**  └── Quantitative-PCR/ ``` ├── YPeff.csv └── YPgene.csv ``` Abbreviations used for Naming Folders/Files: YPeff: quantitative-PCR efficiencies for yellow perch; YPgene: raw cycle of quantification (Cq) values obtained from CFX Manager Software. Column Headers: In the "YPeff.csv" file, gene: the genes that were measured using quantitative-PCR, efficiency: quantitative-PCR efficiencies for yellow perch. In the "YPgene.csv" file, sample: the same letter denotes replicates of the same sample, tr: the treatment temperature (°C) of the yellow perch larvae, myhc1: myosin heavy chain-1 gene, vegfa: vascular endothelial growth factor -A gene, slc5a5: solute carrier family 5 member 5 gene, tpo5: thyroid peroxidase gene, nkx25: NKX2- homeobox 5 gene. Listed under each gene are the raw cycle of quantification (Cq) values obtained from CFX Manager Software. #### File: Vasculature.zip **Description:**  └── Vasculature/ ``` ├── Hatch/ ├── 12C/ ├── 15C/ ├── 18C/ └── Hatch Data.xlsx ├── Eye Pigmentation/ ├── 12C/ ├── 15C/ ├── 18C/ └── Eye Pigmentation Data.xlsx ├── Onset of Heartbeat/ ├── 12C/ ├── 15C/ ├── 18C/ └── Onset of Heartbeat Data.xlsx └── Vasculature Staining Results.xlsx ``` File-Specific Information for: Vasculature/ This folder has subfolders "Hatch", "Eye Pigmentation", "Onset of Heartbeat"- corresponding to the stages at which the yellow perch samples were stained for vasculature visualization. 12C, 15C, and 18C treatments were stained at each stage and are found within these folders as subfolders. These contain the raw images (.jpg) that were used to evaluate the presence/absence of vasculature in the heart, brain, and muscle of yellow perch embryos/larvae. Abbreviations used for Naming Folders/Files: T1: Timepoint 1- Onset of Heartbeat stage; T2: Timepoint 2- Eye Pigmentation stage; T3: Timepoint 3- Hatch stage. R3: Ribbon 3; R4: Ribbon 4; R6: Ribbon 6; R8: Ribbon 8- the ribbon number from which the samples were collected. P1, P2, and P3 correspond to the vial number from which the samples were taken for staining. There are multiple image files per fish. The images are named with the use of a common first numeric in the name (1 to 3/4), and multiple images of one animal are named with the addition of .1, .2, .3, .4, etc., to the first common numeric. For example, images named as 1.1, 1.2, 1,3, 1.4 correspond to 4 images of fish 1. The naming system applies to all the images in the folders in this directory. Column Headers: In the "Vasculature Staining Results.xlsx" file, temp: the treatment temperature (°C) of the yellow perch larvae, stage: the stages at which the yellow perch samples were stained for vasculature visualization, heart: presence or absence of vasculature within the heart (yes or no), brain: presence or absence of vasculature within the brain (yes or no), muscle: presence or absence of vasculature within the muscle (yes or no).