Critical windows in embryonic development: Shifting incubation temperatures alter heart rate and oxygen consumption of Lake Whitefish (Coregonus clupeaformis) embryos and hatchlings

Comparative Biochemistry and Physiology, Part A 179 (2015) 71–80

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Critical windows in embryonic development: Shifting incubation temperatures alter heart rate and oxygen consumption of Lake Whitefish (Coregonus clupeaformis) embryos and hatchlings J. Eme a,⁎, C.A. Mueller a, R.G. Manzon b, C.M. Somers b, D.R. Boreham c,d,e, J.Y. Wilson a a

Department of Biology, McMaster University, 1280 Main St. West, Hamilton, ON L8S 4K1, Canada Department of Biology, University of Regina, 3737 Wascana Parkway, Regina, SK S4S 0A2, Canada c Medical Sciences, Northern Ontario School of Medicine, Laurentian University, 935 Ramsey Lake Road, Sudbury, ON P3E 2C6, Canada d Bruce Power, Tiverton, ON, Canada e Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, ON, Canada b

a r t i c l e

i n f o

Article history: Received 15 July 2014 Received in revised form 3 September 2014 Accepted 9 September 2014 Available online 16 September 2014

a b s t r a c t Critical windows are periods of developmental susceptibility when the phenotype of an embryonic, juvenile or adult animal may be vulnerable to environmental fluctuations. Temperature has pervasive effects on poikilotherm physiology, and embryos are especially vulnerable to temperature shifts. To identify critical windows, we incubated whitefish embryos at control temperatures of 2 °C, 5 °C, or 8 °C, and shifted treatments among temperatures at the end of gastrulation or organogenesis. Heart rate (fH) and oxygen consumption (VO2 ) were measured across embryonic development, and VO2 was measured in 1-day old hatchlings. Thermal shifts, up or down, from initial incubation temperatures caused persistent changes in fH and VO2 compared to control embryos measured at the same temperature (2 °C, 5 °C, or 8 °C). Most prominently, when embryos were measured at organogenesis, shifting incubation temperature after gastrulation significantly lowered VO2 or fH. Incubation at 2 °C or 5 °C through gastrulation significantly lowered VO2 (42% decrease) and fH (20% decrease) at 8 °C, incubation at 2 °C significantly lowered VO2 (40% decrease) and fH (30% decrease) at 5 °C, and incubation at 5 °C and 8 °C significantly lowered VO2 at 2 °C (27% decrease). Through the latter half of development, VO2 and fH in embryos were not different from control values for thermally shifted treatments. However, in hatchlings measured at 2 °C, VO2 was higher in groups incubated at 5 °C or 8 °C through organogenesis, compared to 2 °C controls (43 or 65% increase, respectively). Collectively, these data suggest that embryonic development through organogenesis represents a critical window of embryonic and hatchling phenotypic plasticity. This study presents an experimental design that identified thermally sensitive periods for fish embryos. Crown Copyright © 2014 Published by Elsevier Inc. All rights reserved. 



Keywords: Critical windows Development Embryo Fish Heart rate Oxygen consumption Developmental plasticity Temperature















1. Introduction Embryos of egg-laying vertebrates are susceptible to fluctuations in abiotic environmental conditions, including temperature, water availability, salinity, and respiratory gas levels (Booth, 1998; Dzialowski et al., 2002; Du and Shine, 2008; Mueller et al., 2011a,b; Eme et al., 2013; Anderson and Podrabsky, 2014; Hopkins et al., 2014). Fluctuations in environmental factors can permanently or transiently alter embryonic growth, metabolism, and survival through phenotypic plasticity (Hodin, 2000; Garland and Kelly, 2006; Burggren et al., 2014). Developmental phenotypic plasticity may occur through changes in physiology,

⁎ Corresponding author. Tel.: +1 905 525 9140x20075; fax: +1 905 522 6066. E-mail addresses: [email protected] (J. Eme), [email protected] (C.A. Mueller), [email protected] (R.G. Manzon), [email protected] (C.M. Somers), [email protected] (D.R. Boreham), [email protected] (J.Y. Wilson).

http://dx.doi.org/10.1016/j.cbpa.2014.09.005 1095-6433/Crown Copyright © 2014 Published by Elsevier Inc. All rights reserved.

morphology and/or biochemistry, enabling the organism to adjust and respond to the environment (West-Eberhard, 2003). These responses may result in beneficial physiological variation, deleterious phenotypes, or self-repair to a normal phenotype during the embryonic period or following hatching. Typically, studies investigate whether chronic changes in abiotic factors throughout embryonic development alter phenotypic characteristics (Eme et al., 2013; Johnston et al., 2013; Anderson and Podrabsky, 2014), but identification of critical windows of susceptibility to environmental fluctuation has received added emphasis (Burggren and Wartburton, 1994; Macqueen et al., 2008; Burggren and Reyna, 2011; Burggren et al., 2014). In this paradigm, it is recognized that there may be periods of increased or decreased sensitivity to environmental fluctuations during embryonic development. Exposure to fluctuations during only these ‘critical windows’ of susceptibility may be all that is required to alter the embryonic, juvenile or adult phenotype, rather than chronic exposure throughout the entire embryonic period. Environmental perturbations during embryonic critical windows may

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transiently or permanently alter phenotypes during the embryonic period, as well as possibly juvenile or adult phenotypes. Temperature has been shown to have lasting, potentially transgenerational impacts on the muscle type, thermal acclimation, growth, gene expression, and metabolic enzyme activity of fishes (Johnston et al., 2009; Finstad and Jonsson, 2012; Salinas and Munch, 2012; Scott and Johnston, 2012; Schnurr et al., 2014), and critical windows of thermal exposure may exist prior to the eyed stage in fish embryonic development (Macqueen et al., 2008). Studies have not only focused on model organisms, such as zebrafish (Danio rerio), but also included cold-water species of economic concern, such as Atlantic salmon (Salmo salar) (Finstad and Jonsson, 2012; Scott and Johnston, 2012). Atlantic salmon embryos raised through to a larval feeding stage in heated water, 4.6 °C above ambient (2.6 °C or 7.2 °C), displayed higher maximum growth rates compared to control fish when both groups were raised after feeding at common temperatures (Finstad and Jonsson, 2012). Zebrafish raised from hatching to adulthood at 27 °C, but incubated as embryos at 22 °C, 27 °C (control), or 32 °C, showed increased thermal sensitivity to exercise performance at temperatures different than respective embryonic incubation temperatures, and both high and low temperature incubation groups displayed better exercise performance than control fish at 16 °C (Scott and Johnston, 2012). While it is clear that embryonic temperature can have long-term effects on fish physiology, it is less clear how temperature affects the metabolic or cardiac phenotype of embryos or hatchlings during discrete periods of early or late development. However, the thermal environment in embryogenesis up to the eyed embryo stage can persistently alter the muscle phenotype of Atlantic salmon (Macqueen et al., 2008), and development to the eyed stage may be a period of metabolic or cardiac phenotypic plasticity. Vertebrate embryogenesis and development progress through a series of conserved morphological and physiological milestones prior to hatching, including fertilization, cellular differentiation and formation of the blastoderm, development of neural folds, gastrulation, organogenesis, and growth (Carlson, 1988). Gastrulation is a timepoint where progenitor cells for organ systems including the heart have begun to differentiate and is an early developmental milestone with morphological and potentially physiological significance (Stainier et al., 1993; Stainier, 2001; Jensen et al., 2013). At the end of primordial organogenesis, two distinct morphological markers are present for many developing embryos, the presence of a beating heart tube and the formation of eyes (Price, 1934a,b; Ferguson, 1985; Burggren and Fritsche, 1997). Therefore, the end of primordial organogenesis (eyed stage) is a timepoint where the first functional organ system, the cardiovascular system, is present and beginning to rapidly take shape, and the establishment of a majority of the other organ systems has occurred. The present study is the first to permanently shift fish embryonic thermal incubation and measure metabolism and heart rate. The purpose of this study was to determine if exposure to permanent shifts in incubation temperature following the end of gastrulation and/or primordial organogenesis altered the oxygen consumption and/ or heart rate of Lake Whitefish (Coregonus clupeaformis) embryos or hatchlings. The North American Lake Whitefish is a commercially fished, cold-water species found in all the Great Lakes and throughout Canada and the northern United States. This species lays eggs in late autumn/early winter, which develop at 0.5 °C–8 °C for 60–180 days through to spring (Price, 1940; Brooke, 1975). We used an experimental design where embryos were incubated immediately following fertilization at three constant control temperatures across the normal developmental thermal regime for Lake Whitefish, 2 °C, 5 °C, or 8 °C, or were shifted into another of the three temperatures. Shifted treatments were exposed to a temperature shift at the end of gastrulation or primordial organogenesis, and remained in the new temperature for the remainder of embryonic development. We hypothesized that the formation of the three germ layers (gastrulation) or the initial formation of major organs (primordial organogenesis) represents critical windows

for embryonic or hatchling physiology. Therefore, shifts in thermal incubation temperature of Lake Whitefish embryos would result in persistent alteration of heart rate and oxygen consumption at subsequent embryonic or hatchling stages. 2. Materials and methods 2.1. Embryo acquisition and incubation Male (N = 30; 1.39 ± 0.04 kg, 49.6 ± 0.4 cm fork length) and female (N = 18; 1.51 ± 0.07 kg, 50.9 ± 0.7 cm fork length; ±SEM) Lake Whitefish were caught by gill net near the Fishing Islands of Lake Huron adjacent to the town of South Bruce Peninsula, Ontario, Canada on November 21, 2013 (44°42′37.74″N 81°18′38.94″W; lake water surface temperature = 7.0 °C; Ontario Ministry of Natural Resources Permit UGLMU2013-08.). Eggs (N = 120,000; ~ 3 mm diameter) and milt were stripped, pooled, and fertilized dry for 4 min. Fertilized eggs were subsequently put into fresh lake water at a 50:50 ratio by volume, exposed to 0.5% iodine for 30 min, and then thoroughly rinsed three times with lake water. Eggs were transported to the laboratory in a 50:50 ratio of eggs:fresh lake water on ice. Eggs were distributed into McDonald Bell hatching jars at 5 °C at a density of 4500–5500 per liter of dechlorinated city water, which was recirculated, filtered and UV sterilized within a custom-built temperature controlled incubator (Mitz et al., 2014). On 1 day post fertilization (dpf), a subset of eggs (n ~ 7800 total) was distributed into 150 sterile polystyrene petri dishes (100 mm × 20 mm) at a density of 52.4 ± 0.5 (±SEM) per dish, and each dish contained 75– 80 ml of dechlorinated city water (Mitz et al., 2014). Eggs were removed from hatching jars and distributed evenly into petri dishes (on ice) among 15 temperature treatments with 10 replicate dishes per treatment (Table 1). Each temperature treatment began incubation at 1 dpf at 2 °C (~ 2500 eggs), 5 °C (~ 2500 eggs) or 8 °C (~ 2500 eggs) in one of three incubators. As embryos developed, each treatment was monitored for one of two developmental timepoints: (1) the end of gastrulation and (2) the end of primordial organogenesis according to Price (1940) and Brooke (1975). Monitoring consisted of visually inspecting and photographing a subset of each treatment (while in petri dishes on ice, 2–4 times per week) using a CANON SL6 camera (Tokyo, Japan), attached to a Zeiss AXIO Zoom V16 microscope (Carl Zeiss AG; Oberkochen, Germany) at 10 × magnification, attached to a computer running Axio Vision 4.8.2 (Carl Zeiss AG). Photographic records during incubation allowed for precise timing of temperature shifts. At each of the two developmental timepoints, embryos were either switched to another constant temperature or remained at the same constant temperature (controls). For those treatments that underwent a ΔT of 3 °C (2 °C to 5 °C, 5 °C to 2 °C, 5 °C to 8 °C, or 8 °C to 5 °C), embryos were directly moved to their new constant temperature. For those treatments that underwent a ΔT of 6 °C (2 °C to 8 °C or 8 °C to 2 °C), embryos were first moved to 5 °C for 4–6 h and then subsequently to 2 °C or 8 °C. Embryos in all replicate dishes were visually monitored for mortality (petri dishes on ice), dead embryos were removed, and 70% of the water was changed 1–2 times per week with clean dechlorinated water of identical temperature. A HOBO® data logger (±0.2 °C; TidbiT v2 Water Temperature Data Logger UTBI001; Onset Computer Corporation, Bourne, MA) recorded temperature every 5 min in 100 ml of water in a glass beaker adjacent to dishes within each incubator, and weekly average temperatures were used to generate a grand mean for each constant temperature: 1.9 ± 0.1 °C, 5.1 ± 0.1 °C, and 8.0 ± 0.1 °C (±SEM). Beginning at 50 dpf, each treatment was inspected daily for hatchlings. Hatchlings were removed from the dish placed into separate dishes for each replicate with water of identical temperature, and VO2 was measured at ~ 24 h post-hatch as described below. Oxygen consumption rate (VO2 ) and heart rate (fH) were measured for each treatment at the constant temperature at which embryos were currently being incubated, regardless of previous or subsequent 



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Table 1 

Critical window experimental design, temperatures, days post-fertilization (dpf), and Price stages for fH or VO2 measurements. White represents incubation conditions and measurements made at 2 °C, light gray represents 5 °C, and dark gray represents 8 °C.

Experimental group

Temperature (dpf) end of gastrulation price stage 128

Temperature (dpf) end of primordial organogenesis price stage 320

Temperature (dpf) intermittent fin flutter Price stage 680

Temperature (dpf) constant fin flutter price stage 776

Temperature (dpf) pre–hatch and hatchling price stage 800

2oC 5 → 2 → 2oC 8 → 2 → 2oC 5 → 5 → 2oC 8 → 8 → 2oC 5oC 2 → 5 → 5oC 8 → 5 → 5oC 2 → 2 → 5oC 8 → 8 → 5oC 8oC 2 → 8 → 8oC 5 → 8 → 8oC 2 → 2 → 8oC 5 → 5 → 8oC

2oC (23) 5oC (14) 8oC (8) 5oC (14) 8oC (8) 5oC (14) 2oC (23) 8oC (8) 2oC (23) 8oC (8) 8oC (8) 2oC (23) 5oC (14) 2oC (23) 5oC (14)

2oC (38–39) 2oC (30) 2oC (24–26) 5oC (24–25) 8oC (17–18) 5oC (24–25) 5oC (33–34) 5oC (21–22) 2oC (38–39) 8oC (17–18) 8oC (17–18) 8oC (29–30) 8oC (20–21) 2oC (38–39) 5oC (24–25)

2oC (71–72) 2oC (67) 2oC (64–65) 2oC (55) 2oC (43) 5oC (46) 5oC (53) 5oC (40–42) 5oC (61) 5oC (35) 8oC (32–33) 8oC (45) 8oC (36–37) 8oC (52) 8oC (37)

2oC (88) 2oC (84) 2oC (81) 2oC (70) 2oC (60) 5oC (55–56) 5oC (63) 5oC (51) 5oC (71) 5oC (45) 8oC (36–37) 8oC (50) 8oC (42) 8oC (58) 8oC (43)

2oC (151–152) 2oC (147–148) 2oC (145) 2oC (147–148) 2oC (130–131) 5oC (95–96) 5oC (102–103) 5oC (90) 5oC (102–103) 5oC (86–87) 8oC (62) 8oC (75) 8oC (66–67) 8oC (82) 8oC (68–69)

Each treatment consisted of 10 replicate petri dishes held at constant temperatures, from which N = 6 embryos or hatchlings from replicates within each treatment were measured at 

each of the timepoints — gastrulation, organogenesis, intermittent fin flutter, constant fin flutter, pre-hatch, and hatchling. At the gastrulation timepoint, only VO2 was measured. Embryos in each replicate were shifted from one constant temperature to another at either the end of gastrulation or the end of primordial organogenesis, and embryos or hatchlings remained at 

the shifted temperature for the duration of development until measurement. The sample sizes used in statistical analyses for each timepoint, by temperature, were: 1. Gastrulation. VO2 only. N = 30, each treatment; 2. Primordial organogenesis. N = 18, at control 2, 5, or 8 °C groups, and N = 6, other treatments; 3. Intermittent fin flutter. N = 6, each treatment; 4. Con

stant fin flutter. N = 6, each treatment; 5. Pre-hatch embryo. N = 6, each treatment; 6. Hatchlings (1 day post-hatch). VO2 only. N = 6, each treatment.

temperature shifts, such that embryos in 2 °C water were measured at 2 °C, embryos in 5 °C water were measured at 5 °C, and embryos in 8 °C water were measured at 8 °C. We provide for reference the ‘Price stages’ for each developmental timepoint used for fH or VO2 measurements (Price, 1934a,b, 1935). Price stages 128 and 320 corresponded to the end of gastrulation and the end of primordial organogenesis, respectively. Price stages 680 and 776 corresponded to the intermittent and constant fin flutter stages, respectively. Price stage 800 corresponded to ~ 25% of total embryos hatched for each treatment and is therefore described as a ‘pre-hatch stage’. Pre-hatch stage embryos were within the chorion for both fH and VO2 measurements, i.e. no hatched embryos were used in these measurements. VO2 only was measured at the end of gastrulation (Price stage 128; Table 1) prior to the first critical window temperature switch. VO2 and fH were each measured at the end of primordial organogenesis (Price stage 320) prior to the second critical window temperature switch, as well as at later embryonic stages (Price stages 680, 776, 800; Table 1). 







2.2. Heart rate fH measurements were completed in a custom made, water-jacketed acrylic observation chamber. The observation chamber measured 5 cm3 and was approximately half filled with ~60 ml of clean dechlorinated water at the experimental temperature. Water temperature was monitored with a Fibox 3 thermocouple (PreSens Precision Sensing GmbH, Regensburg, Germany), and temperature for each trial recorded using OxyView software (PST3v6.02, PreSens). The average temperature for each trial was ± 0.1 °C of the desired experimental temperature. Water from a recirculating chiller (Thermo Scientific® HAKE A 25; Waltham, MA, USA) was pumped into the jacketed chamber (15 cm × 10 cm × 10 cm) at 15 l·min− 1. Recirculating water 0.6–0.8 °C below the experimental temperature allowed for the observation chamber temperature to equilibrate at the appropriate experimental temperature in ~ 30 min. Embryos were removed from their treatments, placed individually into the observation chamber and allowed to rest for 15– 30 min, after which fH (beats·min− 1) was estimated in triplicate by visual counting for 1 min, with N1 min between each measurement. Visual counting was done on a 76 cm television monitor (Samsung, Seoul, South Korea) attached to the AXIO Zoom V16 microscope (Carl

AG). After the triplicate visual counting, fH was recorded at 30 fps for 1–2 min at a resolution of 640 × 480 using the CANON SL6 camera. Mean fH was calculated as the average of the triplicate measurements and the video recording measurement. 2.3. Oxygen consumption 

VO2 of embryos and hatchlings was determined from the decrease in PO2 in a closed respiratory system using separate embryos from the fH experiments. Custom-built, water-filled respiratory chambers were created from borosilicate glass vials with screw top caps. A 5 mm O2 sensor spot (Loligo Systems, Tjele, Denmark) was placed onto the flat bottom of the chamber with silicone glue (555-588, RS Components Ltd, Northants, UK). The final volume of the chambers with the sensor spot, determined by the mass of the empty chambers versus chambers filled with water, ranged from 300 to 325 μl for small chambers used for embryos, and 400 to 470 μl for large chambers used for hatchlings. For each VO2 trial, six chambers were filled with clean dechlorinated water within a larger reservoir at the relevant temperature. Embryos and hatchlings were removed from their treatments placed individually in the chambers, and the caps closed under the water within the reservoir. The chambers were held cap side down (oxygen sensor spot up) in tubing, which was housed in a weighted plastic tube holder within the water-bath of a recirculating chiller set at the relevant temperature (HAKE A 25). The sensor spot was read with a bare tip fiber optic cable (PreSens) connected to either an Oxy-4 mini oxygen meter (PreSens) or a Fibox 3 oxygen transmitter (PreSens), with a computer running either Oxy4 (v2, PreSens) or OxyView (PST3v6.02, PreSens) software. Water temperature was recorded, as above for fH, and average temperature was ±0.1 °C of the desired experimental temperature. The system was calibrated daily with a fresh anoxic solution of 10 mg of sodium sulfite in 1 ml of water (0% O2) and air equilibrated water (100% O2). Once chambers were placed in the recirculating bath, O2 levels of each chamber were read for ~ 25 s in repeating order every 5–15 min for a minimum of 2.5 h. Readings were begun immediately, and an average VO2 was calculated once a steady rate was achieved, approximately 45–60 min after the readings were begun. VO2 was calculated from the decrease in chamber PO2 over time, the oxygen capacitance 





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of the water at the relevant temperature, and the chamber volume minus the volume of the embryo or hatchling, estimated from wet mass. VO2 of empty chambers at each temperature for each chamber size was measured and subtracted from calculated VO2 to control for microorganism respiration in the water (Mueller et al., 2011a). Yolk-free mass-specific VO2 was calculated for individual embryos by dividing VO2 by the dry mass of the embryo (μl O2 h−1 mg embryo−1). Embryos and hatchlings used in the VO2 trials were subsequently fixed in 10% formalin for ≥ 72 h. The 10% formalin was replaced with 70% EtOH, embryos were removed from the chorion, and the yolk and embryo were separated under the AXIO Zoom V16 microscope (Carl Zeiss AG). The embryos were dried in a 70 °C oven for 24 h (model 1500EM, VWR/Sheldon, Cornelius, OR, USA) and weighed to ±0.01 mg (XA105DU; Mettler-Toledo, Columbus, Ohio, USA). Embryos at the end of gastrulation were too small to accurately dissect and weigh, therefore,VO2 is not mass-specific for gastrulation measurements (μl O2 h−1). 





.

VO ( l h-1)

0.04







B

0.05 A

A

0.03 0.02 0.01

Gastrulation 

Fig. 1. Mean VO2 (μl h−1) at the end of gastrulation for embryos incubated at 2 °C, 5 °C or 8 °C (N = 30, each temperature). Uppercase letters denote Tukey post-hoc comparisons (1-way ANOVA, P b 0.05). Error bars are SEM.

3.2. Primordial organogenesis



fH and VO2 critical window treatments that were separately compared were (Price, 1934a,b, 1935): 

1. End of gastrulation (Price stage 128) prior to temperature switch. VO2 only. N = 30, each treatment. 2. End of primordial organogenesis (Price stage 320) prior to temperature switch. N = 18, at 2, 5, or 8 °C control groups, and N = 6, other treatments. 3. Beginning of intermittent fin flutter movement (50% of embryonic development; Price stage 680). N = 6, each treatment. 4. Constant fin flutter movement (Price stage 776). N = 6, each treatment. 5. Pre-hatch embryo (Price stage 800), when ~25% of embryos in each treatment had hatched. N = 6, each treatment. 6. Hatchlings (1 day post-hatch). VO2 only. N = 6, each treatment.

Across the three control temperatures (2, 5, and 8 °C), mean fH at 5 °C and 8 °C were significantly higher than fH at 2 °C (150% increase; Fig. 2A) but did not differ from each other. Mean VO2 in the control groups increased significantly by ~55% for each 3 °C increase in temperature (Fig. 2B; P b 0.05). At 2 °C, mean fH was similar across all three treatments (Fig. 2A). However, mean VO2 was significantly reduced in the 8 → 2 °C treatment, 



A

fH (beats min-1)

2.4. Statistical analyses



In order to determine the potential significance of different constant incubation temperatures, VO2 or fH was compared across the three control temperatures (2, 5, and 8 °C) at each stage of 1–6 above, using a 1-way Analysis of Variance (ANOVA), and a post-hoc Tukey HSD test separated values into distinct subsets (JMP v.11, SAS, Cary, North Carolina). In order to determine the potential significance of thermal shifts between treatments measured in a common temperature for the stages 2–6 of development, above, VO2 and fH were compared between the three or five treatments within each of the constant temperatures (2, 5, or 8 °C) using separate 1-way ANOVAs and a post-hoc Tukey HSD test (JMP v.11). This statistical analysis focuses on whether previous incubation temperature caused persistent changes in VO2 and fH measured subsequently at an identical common temperature, and does not compare shifted temperature treatments measured subsequently at different temperatures. All data sets conformed to assumptions required by ANOVA tests, including normality and homogeneity of variance. Posthoc tests were performed following a significant ANOVA. Throughout the text, data are presented as mean ± SEM, and differences were accepted as statistically significant at α = 0.05. 

20 18 16 14 12 10 8 6 4 2

A

A C

B

A

A

A

B

A

1.2 1.0

B

)



A

0.8

.

VO (

0.6



0.4

AB A

3.1. Gastrulation 

VO2 was measured for constant temperature 2, 5, or 8 °C (N = 30, each), at the end of gastrulation (Table 1). Mean VO2 at 8 °C was 60% higher than the average VO2 measured at 2 and 5 °C (Fig. 1; P b 0.05). 



B

B

AB B

0.2

Gastrulation Organogenesis 

3. Results

B

B

Fig. 2. Mean fH (A; beats·min−1) and mean mass-specific VO2 (B; μl h−1 mg−1) at the end of primordial organogenesis for embryos incubated at 2 °C (N = 18), 5 → 2 °C (N = 6), 8 → 2 °C (N = 6), 5 °C (N = 18), 2 → 5 °C (N = 6), 8 → 5 °C (N = 6), 8 °C (N = 18), 2 → 8 °C (N = 6), and 5 → 8 °C (N = 6). Arrows in incubation temperature treatments indicate groups shifted from one temperature to another at the end of gastrulation. White bars indicate treatments measured at 2 °C, gray bars indicate treatments measured at 5 °C, and black bars indicate treatments measured at 8 °C. Uppercase Latin letters denote Tukey post-hoc comparisons within each set of colored bars, and lowercase Greek letters denote Tukey post-hoc comparisons across the three control temperatures, 2 °C, 5 °C and 8 °C (1-way ANOVA, P b 0.05). Error bars are SEM.

J. Eme et al. / Comparative Biochemistry and Physiology, Part A 179 (2015) 71–80 

compared to the 2 °C control group (Fig. 2B; P b 0.05). At 5 °C, fH and VO2 displayed similar trends, with reduced fH and VO2 in treatments incubated in either higher or lower temperatures through gastrulation (Fig. 2). Both the 2 → 5 °C and 8 → 5 °C treatments had significantly lower fH compared to the 5 °C control group (32% and 12% decrease, respectively). The 2 → 5 °C treatment had a significantly lower VO2 compared to the 5 °C control group (41% decrease), and the 8 → 5 °C treatment had a value statistically similar to the 2 → 5 °C and 5 °C treatments. At 8 °C, fH and VO2 showed similar trends compared to the control 8 °C group, with fH and VO2 reduced by 18% and 45% in the 2 → 8 °C treatment, respectively, and reduced by 23% and 39% in the 5 → 8 °C treatment, respectively (P b 0.05). 







3.3. Intermittent and constant fin flutter Across the three control temperatures (2, 5, and 8 °C) at the intermittent fin flutter stage, mean fH and VO2 were significantly higher at each higher temperature with a N 25% increase between each 3 °C increment (Fig. 3; P b 0.05). Across the three control temperatures at the constant fin flutter stage, fH was significantly higher at each higher temperature with a 38–52% increase between each 3 °C increment (Fig. 4A). 

75

Across the three control temperatures at the constant fin flutter stage, VO2 was statistically similar between 2 °C and 5 °C, which were significantly lower than that at 8 °C (100% decrease; Fig. 4B; P b 0.05). At 2 °C in the intermittent fin flutter stage, mean fH was statistically similar across all five treatments (Fig. 3A). However, mean VO2 was significantly elevated in the 8 → 8 → 2 °C treatment (28% increase), compared to the 2 °C control group (Fig. 3B; P b 0.05). At 5 °C in the intermittent fin flutter stage, fH was significantly depressed in the 2 → 2 → 5 °C treatment (7% decrease), and VO2 was significantly increased in the 8 → 5 → 5 °C treatment compared to the 5 °C control group (81% increase; Fig. 3B; P b 0.05). At 8 °C in the intermittent fin flutter stage, a trend for decreased fH and VO2 existed for treatments compared to the 8 °C control group; however, the only significant changes following ANOVA and post-hoc tests were observed in fH. fH was significantly reduced in the 2 → 2 → 8 °C and the 5 → 5 → 8 °C treatments, compared to the 8 °C control group (Fig. 3; 14% and 11% decrease, respectively; P b 0.05). At 2 °C in the constant fin flutter stage, mean fH was significantly higher in the 8 → 2 → 2° treatment compared to the 2 °C treatment (6% increase; Fig. 4A; P b 0.05). However, mean VO2 was statistically similar across all five 2 °C treatments (Fig. 4B). At 5 °C in the constant 









A

A

50

A

AB C

fH (beats min-1)

40

AB

AB

BC

A

AB C

30 A

ABC AB

BC

C

20

10

B 0.6

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)

0.5 AB

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VO ( l

0.4 0.3 A

A

AB

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0.2 0.1

Gastrulation Organogenesis Hatch 

Fig. 3. Mean fH (A; beats·min−1) and mean mass-specific VO2 (B; μl h−1 mg−1) at the intermittent fin flutter stage for embryos incubated at 2 °C, 5 → 2 → 2 °C, 8 → 2 → 2 °C, 5 → 5 → 2 °C, 8 → 8 → 2 °C, 5 °C, 2 → 5 → 5 °C, 8 → 5 → 5 °C, 2 → 2 → 5 °C, 8 → 8 → 5 °C, 8 °C, 2 → 8 → 8 °C, 5 → 8 → 8 °C, 2 → 2 → 8 °C, and 5 → 5 → 8 °C (N = 6, each treatment). Arrows in the incubation temperature treatments indicate groups shifted from one temperature to another at the end of gastrulation or primordial organogenesis. White bars indicate treatments measured at 2 °C, gray bars indicate treatments measured at 5 °C, and black bars indicate treatments measured at 8 °C. Uppercase Latin letters denote Tukey post-hoc comparisons within each set of colored bars, and lowercase Greek letters denote Tukey post-hoc comparisons across the three control temperatures, 2 °C, 5 °C and 8 °C (1-way ANOVA, P b 0.05). Error bars are SEM.

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A

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fH (beats min-1)

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Fig. 4. Mean fH (A; beats·min−1) and mean mass-specific VO2 (B; μl h−1 mg−1) at the constant fin flutter stage for embryos incubated at 2 °C, 5 → 2 → 2 °C, 8 → 2 → 2 °C, 5 → 5 → 2 °C, 8 → 8 → 2 °C, 5 °C, 2 → 5 → 5 °C, 8 → 5 → 5 °C, 2 → 2 → 5 °C, 8 → 8 → 5 °C, 8 °C, 2 → 8 → 8 °C, 5 → 8 → 8 °C, 2 → 2 → 8 °C, and 5 → 5 → 8 °C (N = 6, each treatment). Arrows in the incubation temperature treatments indicate groups shifted from one temperature to another at the end of gastrulation or primordial organogenesis. White bars indicate treatments measured at 2 °C, gray bars indicate treatments measured at 5 °C, and black bars indicate treatments measured at 8 °C. Uppercase Latin letters denote Tukey post-hoc comparisons within each set of colored bars, and lowercase Greek letters denote Tukey post-hoc comparisons across the three control temperatures, 2 °C, 5 °C and 8 °C (1-way ANOVA, P b 0.05). Error bars are SEM.



fin flutter stage, fH and VO2 were statistically similar across all five 5 °C treatments. At 8 °C in the constant fin flutter stage, fH was statistically similar across all five 8 °C treatments. At 8 °C in the constant fin flutter stage, VO2 was significantly lower in the 5 → 8 → 8 °C treatment compared to the 2 → 8 → 8 °C treatment (28% decrease; P b 0.05). 

and no differences in f H following post-hoc analyses (Fig. 5). At 8 °C in the pre-hatch embryo stage, f H was statistically higher in the 5 → 5 → 8 °C treatment, compared to the 8 °C control group (10% increase; P b 0.05), and a similar, non-significant, trend was observed in VO2 . VO2 was significantly reduced in the 5 → 8 → 8 °C treatment compared to the 8 °C control group (31% decrease; P b 0.05). At 2 °C in the hatchlings, mean VO2 was statistically higher in the 8 → 8 → 2 °C and 5 → 5 → 2 °C treatments, compared to the 2 °C control group (43–65% increase; Fig. 6; P b 0.05). At 5 °C in the hatchlings, VO2 was statistically similar across all five treatments. At 8 °C in the hatchlings, VO2 was significantly reduced in the 5 → 8 → 8 °C treatment compared to the 8 °C control group, similar to the pre-hatch embryo stage (33% decrease; P b 0.05). 





3.4. Pre-hatch embryos and hatchlings Across the three control temperatures (2, 5, and 8 °C) at the prehatch embryo stage, mean fH and VO2 were significantly higher at each temperature increase with a N 20% increase between each 3 °C increment (Fig. 5; P b 0.05). Across the three control temperatures for hatchlings, VO2 was significantly higher at each temperature increase with a 50–120% increase between each 3 °C increment (Fig. 6; P b 0.05). Mean VO2 was 10-fold greater in hatchlings compared to pre-hatch embryos, within each temperature (Figs. 5, 6; 2, 5, and 8 °C). At 2 °C the pre-hatch embryo stage, mean fH and mean VO2 were statistically similar across all five treatments at each temperature. At 5 °C, there were no differences in VO2 of different treatments 













4. Discussion Temperature has pervasive, possibly permanent effects on the embryonic developmental trajectory and resultant physiological phenotypes of ectothermic vertebrates (Scott and Johnston, 2012; Alvine

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A A

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Fig. 5. Mean fH (A; beats·min−1) and mean mass-specific VO2 (B; μl h−1 mg−1) at the pre-hatch stage for embryos incubated at 2 °C, 5 → 2 → 2 °C, 8 → 2 → 2 °C, 5 → 5 → 2 °C, 8 → 8 → 2 °C, 5 °C, 2 → 5 → 5 °C, 8 → 5 → 5 °C, 2 → 2 → 5 °C, 8 → 8 → 5 °C, 8 °C, 2 → 8 → 8 °C, 5 → 8 → 8 °C, 2 → 2 → 8 °C, and 5 → 5 → 8 °C (N = 6, each treatment). Arrows in the incubation temperature treatments indicate groups shifted from one temperature to another at the end of gastrulation or primordial organogenesis. White bars indicate treatments measured at 2 °C, gray bars indicate treatments measured at 5 °C, and black bars indicate treatments measured at 8 °C. Uppercase Latin letters denote Tukey post-hoc comparisons within each set of colored bars, and lowercase Greek letters denote Tukey post-hoc comparisons across the three control temperatures, 2 °C, 5 °C and 8 °C (1-way ANOVA, P b 0.05). Error bars are SEM.

et al., 2013; Schnurr et al., 2014). Our findings suggest that exposure to shifts in temperature at the end of gastrulation or primordial organogenesis caused changes in fH and VO2 of Lake Whitefish embryos and hatchlings, when compared at the same temperature to control embryos incubated under constant thermal regimes. Increased or decreased measurement temperature produced the typical increase or decrease in fH and VO2 , respectively, but interestingly, thermal shifts up or down from the initial incubation temperatures resulted in persistent reduced embryonic fH and VO2 compared to the control embryos when all embryos were measured at common developmental timepoints at the same temperature (2 °C, 5 °C, or 8 °C; Figs. 2–5). However, in hatchlings measured at 2 °C, VO2 was higher in groups incubated in 8 °C or 5 °C through organogenesis (Fig. 6). These findings indicate that early thermal environmental shifts produced changes to cardiovascular physiology and metabolic rate, and that whole animal metabolism changes measured at 2 °C persisted through to hatching for embryos incubated through organogenesis at 8 °C. VO2 and fH for Lake Whitefish embryos were within the range of previously published values for other whitefish and coldwater species. VO2 values for pre-hatch embryos and hatchlings at 2–8 °C were 











slightly lower than previous values for European whitefish (Coregonus lavaretus) measured at warmer temperatures, 10–15 °C (Dabrowski et al., 1984). VO2 for whitefish embryos were also similar to previous values for cod embryos (Gadus morhua) measured at 5 °C (Davenport and Lönning, 1980), and the observed increase in VO2 between prehatch embryos and 1 day hatchlings was similar to that observed for Pacific herring (Clupea pallasi), Lumpfish (Cyclopterus lumpus) and Atlantic salmon (Hayes et al., 1951; Eldridge et al., 1977; Davenport, 1983). VO2 for hatchlings were similar to values for Baltic herring measured at 8 °C (Clupea harengus membras), but higher than those for Pacific herring at 5 °C (Holliday et al., 1964; Eldridge et al., 1977). Heart rate was in the same range as values for brook charr (Salvelinus fontinalis) measured at 6–12 °C (43–73 beats·min− 1) from the eyed stage through hatching (Benfey and Bennett, 2009). In addition, the duration of development at 2 °C, 5 °C or 8 °C was similar to previous data on Lake Whitefish (Brooke, 1975). VO2 or fH for shifted temperature treatments differed from control embryos measured at the organogenesis stage, suggesting that the initial thermal shift following gastrulation revealed a critical window of embryonic phenotypic plasticity that persisted through to 







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3.5

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Fig. 6. Mean mass-specific VO2 (μl h−1 mg−1) for hatchlings incubated as embryos at 2 °C, 5 → 2 → 2 °C, 8 → 2 → 2 °C, 5 → 5 → 2 °C, 8 → 8 → 2 °C, 5 °C, 2 → 5 → 5 °C, 8 → 5 → 5 °C, 2 → 2 → 5 °C, 8 → 8 → 5 °C, 8 °C, 2 → 8 → 8 °C, 5 → 8 → 8 °C, 2 → 2 → 8 °C, and 5 → 5 → 8 °C (N = 6, each treatment). Arrows in the incubation temperature treatments indicate groups shifted from one temperature to another at the end of gastrulation or primordial organogenesis. White bars indicate treatments measured at 2 °C, gray bars indicate treatments measured at 5 °C, and black bars indicate treatments measured at 8 °C. Uppercase Latin letters denote Tukey post-hoc comparisons within each set of colored bars, and lowercase Greek letters denote Tukey post-hoc comparisons across the three control temperatures, 2 °C, 5 °C and 8 °C (1-way ANOVA, P b 0.05). Error bars are SEM.

the organogenesis stage (Fig. 2). Embryos shifted at gastrulation and measured at organogenesis had been in their new shifted thermal treatment for 8–16 days at the time of measurement, and consequently acute responses were not the likely cause of observed differences in fH and VO2 . At 8 °C, fH and VO2 were both lower in treatments where embryos were incubated in 2 °C or 5 °C during gastrulation (Fig. 2). The decrease in fH and VO2 suggests that when shifted from cooler waters to those in the warmer end of normal incubation temperatures, Lake Whitefish embryos had not adjusted metabolic rate in their new environment in 8–10 days or the temperature during gastrulation fixed the metabolic phenotype up to this stage of development. Embryos measured at 5 °C demonstrated the same trend, as embryos incubated at 2 °C during gastrulation displayed a lower fH and VO2 than control 5 °C embryos after 11 days at 5 °C. Interestingly, embryos measured at 2 °C and 5 °C that had been previously incubated at warmer temperatures during gastrulation had lowered VO2 after more than 2 weeks at 2 °C (Fig. 2). Therefore, embryos initially incubated at higher temperatures and shifted to lower temperatures, as well as embryos initially incubated at lower temperatures and shifted to a higher temperatures, displayed lowered metabolism and heart rate compared to embryos constantly incubated at the control temperature. Our finding of altered fH following thermal shifts prior to the formation of the heart is supported by previous research. For example, the myocardium of Atlantic herring (Clupea harengus) hatchlings raised from fertilization to hatching at 5 °C, 8 °C or 12 °C displayed different levels of thermal sensitivity to growth and cellular organization, with 8 °C being optimal for this species (Zummo et al., 1996). Heart rate increases during vertebrate embryonic development, occasionally reaching a plateau late in development, and rapid increases in developmental heart rate can be associated with organogenesis, as seen in the tropical frog (Eleutherodactylus coqui) (Burggren et al., 1990). The initial configuration of the vertebrate heart is a peristaltic tube consisting of a primary myocardium, and the peristaltic tube in fish embryos quickly resembles the formed adult fish heart (Jensen et al., 2013). Prior to gastrulation, heart progenitor cells can be identified in zebrafish embryos, and progenitor cell differentiation into endocardial and myocardial 









cells begins at the formation of the heart tube and continues through differentiation of the tube into the atrium and ventricle (Stainier, 2001). In addition, restriction of progenitor cells to either atrial or ventricular fates occurs by the end of blastula formation (Stainier et al., 1993). Consequently, cells that form the heart tube were present prior to the first whitefish embryo thermal shift at gastrulation, and the initially formed heart tube was undergoing a phase of rapidly increasing physiological and morphological changes that were temperature sensitive. Changes in fH and VO2 following thermal shifts early in development suggest a mechanism whereby the metabolic and cardiovascular phenotype of Lake Whitefish embryos has become altered by initial thermal exposure, possibly through changes in rate-limiting allozymes or isozymes, or fish muscle fiber type, size, and mitochondrial content (Precht, 1958; Vieira and Johnston, 1992; Hazel, 1993; Hochachka and Somero, 2002; Macqueen et al., 2008; Schnurr et al., 2014). At the intermittent and constant fin flutter stages (Figs. 3, 4), fH and VO2 measured at 8 °C were reduced for treatments incubated at initially cooler temperatures, suggesting that embryos had not adjusted their metabolic rate to their new incubation temperature of 8 °C (O'Steen and Janzen, 1999; Nilsson et al., 2010). However, at the pre-hatch stage fH was higher for the 5 → 5 → 8 °C group, compared to the 8 °C treatment, possibly due to prolonged incubation times in these groups (Table 1). For measurements at 5 °C, fH and VO2 were different from the control embryos in shifted embryos only at the intermittent fin flutter stage (Fig. 3), and all values were statistically similar at the constant fin flutter and pre-hatch stages, which indicated that embryos had returned to control values prior to hatching (Figs. 4, 5). A similar trend was observed for 2 °C measurements where initial incubation at warmer temperatures altered fH and VO2 more prominently at the intermittent fin flutter stage (Fig. 3), and values returned to control levels by the pre-hatch stage (Fig. 5). Our findings of altered fH and VO2 is consistent with those for embryos of the annual killifish (Austrofundulus limnaeus) incubated at 25 °C and 30 °C in hypoxic and normoxic water, which displayed peak sensitivity to temperature during somitogenesis and early organogenesis (Anderson and Podrabsky, 2014). In addition, the 









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developmental timing of myogenesis and myogenic fiber type can be irreversibly affected by incubation temperature, and muscle is a dominant cell type in early embryonic development (Temple et al., 2001; Johnston, 2006; Schnurr et al., 2014). Fish embryos raised at different temperatures, even for short periods of time, can have permanent changes in muscle fiber number as well as mitochondrial number (Vieira and Johnston, 1992; Macqueen et al., 2008). Atlantic herring raised throughout embryonic development at increased temperature (15 °C vs 10 °C and 5 °C) displayed increased mitochondrial content in muscle fibers, and three year old Atlantic salmon raised as embryos to the eyed stage at 8 °C and 10 °C showed increased muscle fiber compared to individuals raised to the eyed stage at 2 °C and 5 °C (Vieira and Johnston, 1992; Macqueen et al., 2008). Therefore, throughout the second half of embryonic development, through to 1 day hatchlings, embryos incubated initially in cooler or warmer water may have altered cellular mechanisms that resulted in observed changes to fH and VO2 . Following completion of embryonic development (~60–150 days), 1 day whitefish hatchlings displayed altered VO2 when measured at 8 °C, but incubated initially in cooler water, as well as hatchlings measured at 2 °C but initially incubated in warmer water (Fig. 6). Most notably, for hatchlings measured at 2 °C both treatments incubated in 5 °C or 8 °C through primordial organogenesis displayed markedly increased oxygen consumption, a 43–65% increase. Across the three measurement temperatures (2 °C, 5 °C, 8 °C), VO2 was 10-fold higher in hatchlings, compared to pre-hatch embryos measured at the same temperature, and measurements of hatchling or pre-hatch embryos differed by at most 1 day or a maximal 1.5% difference in total incubation days. Our data show that the chorion represents a significant barrier to oxygen diffusion in late stage fish embryos (Davenport and Lönning, 1980; Ciuhandu et al., 2005), and this barrier may have masked temperature-driven differences between treatment groups prior to hatching. Coupled with previous findings of long-term physiological and metabolic consequences of the thermal incubation environment (Johnston, 2006; Scott and Johnston, 2012; Anderson and Podrabsky, 2014; Schnurr et al., 2014), altered VO2 in hatchling whitefish in the present study suggests that short-term thermal alterations in the initial third of embryonic development can alter hatchling phenotypes (Macqueen et al., 2008). 







4.1. Summary 

The major finding of this study was that VO2 and fH of embryonic or hatchling whitefish measured at identical temperatures were influenced by the temperatures that embryos were incubated in within approximately the first third of embryonic development, through organogenesis. This critical window of embryonic development contains important morphological and physiological milestones, including blastoderm formation, germ layer formation, tissue differentiation, and organogenesis. The observed altered whole-organism VO2 in the present study suggests that cellular or mitochondrial metabolic plasticity exists very early in development, and future investigations will examine enzymatic changes following thermal shifts early in development. However, we did not find a ‘dose-dependent’ response to early embryonic exposure to warmer or cooler temperatures at all of the timepoints measured. The present study utilized a large number of treatments that are compared to control values; however, this experimental design also limited the logistical capacity for large sample sizes within each treatment. Future investigations will focus on the period from fertilization to organogenesis at only 2 °C and 8 °C, and compare control embryos to thermally shifted treatments at multiple measurement temperatures. Overall, our data support the hypothesis that relatively small windows in embryonic development may be periods when permanent shifts in environmental factors could alter the metabolic and cardiovascular phenotype of fish (Macqueen et al., 2008). 

79

Acknowledgments We sincerely thank Chris Thome for the help in acquiring whitefish embryos, and the Ontario Ministry of Natural Resources for the permit to collect whitefish (UGLMU2013-08). J.E. and C.A.M. were supported by a MITACS Accelerate grant to D.R.B and J.Y.W. Funding was provided by Bruce Power®, Inc. and a Collaborative Research and Development Grant from the Natural Sciences and Engineering Research Council of Canada to J.Y.W., R.G.M., and C.M.S.

References Alvine, T., Rhen, T., Crossley II, D.A., 2013. Temperature-dependent sex determination modulates cardiovascular maturation in embryonic snapping turtles Chelydra serpentina. J. Exp. Biol. 216, 751–758. Anderson, S.N., Podrabsky, J.E., 2014. The effects of hypoxia and temperature on metabolic aspects of embryonic development in the annual killifish Austrofundulus limnaeus. J. Comp. Physiol. B 184, 355–370. Benfey, T.J., Bennett, L.E., 2009. Effect of temperature on heart rate in diploid and triploid brook charr, Salvelinus fontinalis, embryos and larvae. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 152, 203–206. Booth, D.T., 1998. Nest temperature and respiratory gases during natural incubation in the broad-shelled river turtle, Chelodina expansa (Testudinata:Chelidae). Aust. J. Zool. 46, 183–191. Brooke, L.T., 1975. Effect of different constant incubation temperatures on egg survival and embryonic development in Lake whitefish (Coregonus clupeaformis). Trans. Am. Fish. Soc. 104, 555–559. Burggren, W.W., Fritsche, R., 1997. Cardiovascular development in amphibians. In: Burggren, W.W., Keller, B. (Eds.), Cardiovascular Development: From Molecules to Organisms. Burggren, W.W., Reyna, K.S., 2011. Developmental trajectories, critical windows and phenotypic alteration during cardio-respiratory development. Respir. Physiol. Neurobiol. 178, 13–21. Burggren, W.W., Wartburton, S., 1994. Patterns of form and function in developing hearts: contributions from non-mammalian vertebrates. Cardioscience 5, 183–191. Burggren, W.W., Infantino, R.L., Townsend, D.S., 1990. Developmental changes in cardiac and metabolic physiology of the direct-developing tropical frog Eleutherodactylus coqui. J. Exp. Biol. 152, 129–147. Burggren, W.W., Christoffels, V.M., Crossley II, D.A., Enok, S., Farrell, A.P., Hedrick, M.S., Hicks, J.W., Jensen, B., Moorman, A.F.M., Mueller, C.A., Skovgaard, N., Taylor, E.W., Wang, T., 2014. Comparative cardiovascular physiology: future trends, opportunities and challenges. Acta Physiol. 210, 257–276. Carlson, B.M., 1988. Patten's Foundations of Embryology, 5 ed. McGraw-Hill, New York. Ciuhandu, C.S., Stevens, E.D., Wright, P.A., 2005. The effect of oxygen on the growth of Oncorhynchus mykiss embryos with and without a chorion. J. Fish Biol. 67, 1544–1551. Dabrowski, K., Kaushik, S., Luquet, P., 1984. Metabolic utilization of body stores during the early life of whitefish, Coregonus lavaretus. J. Fish Biol. 24. Davenport, J., 1983. Oxygen and the developing eggs and larvae of the lumpfish, Cyclopterus lumpus. J. Mar. Biol. Assoc. U. K. 63, 633–640. Davenport, J., Lönning, S., 1980. Oxygen uptake in developing eggs and larvae of the cod, Gadus morhua L. J. Fish Biol. 16, 249–256. Du, W.G., Shine, R., 2008. The influence of hydric environments during egg incubation on embryonic heart rates and offspring phenotypes in a scincid lizard (Lampropholis guichenoti). Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 151, 102–107. Dzialowski, E.M., Von Plettenberg, D., Elmonoufy, N.A., Burggren, W.W., 2002. Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 131, 713–724. Eldridge, M.B., Echeverria, T., Whipple, J.A., 1977. Energetics of pacific herring (Clupea harengus pallasi) embryos and larvae exposed to low concentrations of benzene, a monoaromatic component of crude oil. Trans. Am. Fish. Soc. 106, 452–461. Eme, J., Rhen, T., Tate, K.B., Gruchalla, K., Kohl, Z.F., Slay, C.E., Crossley II, D.A., 2013. Plasticity of cardiovascular function in snapping turtle embryos (Chelydra serpentina): chronic hypoxia alters autonomic regulation and gene expression. Am. J. Physiol. Regul. Integr. Comp. Physiol. 304, R966–R979. Ferguson, M.W.J., 1985. Reproductive biology and embryology of the crocodilians. In: Gans, C., Billett, F., Maderson, P.F.A. (Eds.), Biology of the Reptilia: Development A. Wiley-Interscience Publication, New York, Chichester, Brisbane, Toronto, Singapore, pp. 329–491. Finstad, A.G., Jonsson, B., 2012. Effect of incubation temperature on growth performance in Atlantic salmon. Mar. Ecol. Prog. Ser. 454, 75–82. Garland, T., Kelly, S.A., 2006. Phenotypic plasticity and experimental evolution. J. Exp. Biol. 209, 2344–2361. Hayes, F.R., Wilmont, I.R., Linvingston, D.A., 1951. The oxygen consumption of the salmon egg in relation to development and activity. J. Exp. Zool. 116, 377–395. Hazel, J.R., 1993. Thermal biology. In: Evans, D.H. (Ed.), The Physiology of Fishes. CRC Press, Boca Raton, FL, pp. 427–467. Hochachka, P.W., Somero, G.N., 2002. Biochemical Adaptation: Mechanism and Process in Physiological Evolution. Oxford University Press, New York. Hodin, J., 2000. Plasticity and constraints in development and evolution. J. Exp. Zool. 288, 1–20. Holliday, F.G.T., Blaxter, J.H.S., Lasker, R., 1964. Oxygen uptake of developing eggs and larvae of the herring (Clupea harengus). J. Mar. Biol. Assoc. U. K. 44, 711–723.

80

J. Eme et al. / Comparative Biochemistry and Physiology, Part A 179 (2015) 71–80

Hopkins, G.R., Brodie, E.D., French, S.S., 2014. Developmental and evolutionary history affect survival in stressful environments. PLoS One 9, 1–9. Jensen, B., Wang, T., Christoffels, V.M., Moorman, A.F., 2013. Evolution and development of the building plan of the vertebrate heart. Biochim. Biophys. Acta 1833, 783–794. Johnston, I.A., 2006. Environment and plasticity of myogenesis in teleost fish. J. Exp. Biol. 209, 2249–2264. Johnston, I.A., Lee, H.T., Macqueen, D.J., Paranthaman, K., Kawashima, C., Anwar, A., Kinghorn, J.R., Dalmay, T., 2009. Embryonic temperature affects muscle fibre recruitment in adult zebrafish: genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes. J. Exp. Biol. 212, 1781–1793. Johnston, E.F., Alderman, S.L., Gillis, T.E., 2013. Chronic hypoxia exposure of trout embryos alters swimming performance and cardiac gene expression in larvae. Physiol. Biochem. Zool. 86, 567–575. Macqueen, D.J., Robb, D.H., Olsen, T., Melstveit, L., Paxton, C.G., Johnston, I.A., 2008. Temperature until the ‘eyed stage’ of embryogenesis programmes the growth trajectory and muscle phenotype of adult Atlantic salmon. Biol. Lett. 4, 294–298. Mitz, C., Thome, C., Cybulski, M.E., Laframbo, L., Somers, C.M., Manzon, R.G., Wilson, J.Y., Boreham, D.R., 2014. A self-contained, controlled hatchery system for rearing Lake whitefish embryos for experimental aquaculture. N. Am. J. Aquacult. 76, 179–184. Mueller, C.A., Joss, J.M., Seymour, R.S., 2011a. Effects of environmental oxygen on development and respiration of Australian lungfish (Neoceratodus forsteri) embryos. J. Comp. Physiol. B 1–12. Mueller, C.A., Joss, J.M., Seymour, R.S., 2011b. The energy cost of embryonic development in fishes and amphibians, with emphasis on new data from the Australian lungfish, Neoceratodus forsteri. J. Comp. Physiol. B 1–10. Nilsson, G.E., Ostlund-Nilsson, S., Munday, P.L., 2010. Effects of elevated temperature on coral reef fishes: loss of hypoxia tolerance and inability to acclimate. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 156, 389–393. O'Steen, S., Janzen, F.J., 1999. Embryonic temperature affects metabolic compensation and thyroid hormones in hatchling snapping turtles. Physiol. Biochem. Zool. 72, 520–533.

Precht, H., 1958. Concepts of the Temperature Adaptation of Unchanging Reaction Systems of Cold-blooded Animals. American Physiological Society, Washington, D.C. Price, J.W., 1934a. The embryology of the whitefish Coregonus clupeaformis (Mitchill) I. Ohio J. Sci. 34, 287–305. Price, J.W., 1934b. The embryology of the whitefish Coregonus clupeaformis (Mitchill) II. Ohio J. Sci. 34, 399–414. Price, J.W., 1935. The embryology of the whitefish Coregonus clupeaformis (Mitchill) III. Ohio J. Sci. 34, 40–53. Price, J.W., 1940. Time-temperature relations in the incubation of the whitefish, Coregonus clupeaformis (Mitchill). J. Gen. Physiol. 23, 449–468. Salinas, S., Munch, S.B., 2012. Thermal legacies: transgenerational effects of temperature on growth in a vertebrate. Ecol. Lett. 15, 159–163. Schnurr, M.E., Yin, Y., Scott, G.R., 2014. Temperature during embryonic development has persistent effects on metabolic enzymes in the muscle of zebrafish. J. Exp. Biol. 217, 1370–1380. Scott, G.R., Johnston, I.A., 2012. Temperature during embryonic development has persistent effects on thermal acclimation capacity in zebrafish. Proc. Natl. Acad. Sci. U. S. A. 109, 14247–14252. Stainier, D.Y.R., 2001. Zebrafish genetics and vertebrate heart formation. Nat. Rev. Genet. 2, 39–48. Stainier, D.Y.R., Lee, R.K., Fishman, M.C., 1993. Cardiovascular development in zebrafish I. Myocardial fate map and heart tube formation. Development 119, 31–40. Temple, G.K., Cole, N.J., Johnston, I.A., 2001. Embryonic temperature and the relative timing of muscle-specific genes during development in herring (Clupea harengus L.). J. Exp. Biol. 204, 3629–3637. Vieira, V.I.A., Johnston, I.A., 1992. Influence of temperature on muscle-fibre development in larvae of the herring Clupea harengus. Mar. Biol. 112, 333–341. West-Eberhard, M.J., 2003. Developmental Plasticity and Evolution. Oxford University Press, New York. Zummo, G., Farina, F., Tota, B., Johnston, I.A., 1996. Influence of temperature on the development of the heart ventricle in herring (Clupea harengus) larvae. J. Exp. Zool. 275, 196–203.