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.

Apoptosis stain

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.

Behavioral Assay

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).

Closed Respirometry

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.

Ejection Fraction

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.

Heart Rate

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).

Quantitative-PCR

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).

Vasculature Staining

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.