Effective practices for thermal tolerance polygon experiments using mottled catfish Corydoras paleatus.

Temperature is an important environmental factor that affects how organisms allocate metabolic resources to physiological processes. Laboratory experiments that determine absolute thermal limits for representative species are important for understanding how fishes are affected by climate change. Critical Thermal Methodology (CTM) and Chronic Lethal Methodology (CLM) experiments were utilized to construct a complete thermal tolerance polygon for the South American fish species, Mottled catfish (Corydoras paleatus). Mottled catfish showed Chronic Lethal Maxima (CLMax) of 34.9 ± 0.52 °C and Chronic Lethal Minima (CLMin) of 3.8 ± 0.08 °C. Fish were chronically acclimated (∼2 weeks) to 6 temperatures ranging from 7.2 ± 0.05 °C →32.2 ± 0.16 °C (7 °C, 12 °C, 17 °C, 22 °C, 27 °C, and 32 °C), and CTM used to estimate upper and lower acute temperature tolerance. Linear regressions of Critical Thermal Maxima (CTMax) and Minima (CTMin) data with each acclimation temperature were used along with CLMax and CLMin to create a complete thermal tolerance polygon. The highest CTMax was 38.4 ± 0.60 °C for fish acclimated to 32.2 ± 0.16 °C, and the lowest CTMin was 3.36 ± 1.84 °C for fish acclimated to 7.2 ± 0.05 °C. Mottled catfish have a polygon measuring 785.7°C2, and the slope of the linear regressions showed the species gained 0.55 °C and 0.32 °C of upper and lower tolerance per degree of acclimation temperature, respectively. We compared slopes of CTMax or CTMin regression lines to each other using a set of comparisons between 3, 4, 5, or 6 acclimation temperatures. Our data demonstrated that 3 acclimation temperatures were as sufficient as 4 â†’ 6 to pair with estimates of chronic upper and lower thermal limits for accurately determining a complete thermal tolerance polygon. Construction of this species' complete thermal tolerance polygon provides a template for other researchers. The following is sufficient to generate a complete thermal tolerance polygon: Three chronic acclimation temperatures that are spread somewhat evenly across a species' thermal range, include an estimation of CLMax and CLMin, and are followed by CTMax and CTMin measurements.

Cardiovascular responses to putative chemoreceptor stimulation of embryonic common snapping turtles (Chelydra serpentina) chronically incubated in hypoxia (10% O2).

Developmental hypoxia has been shown to result in significant changes in cardiovascular development of American alligators and common snapping turtles. These include similar effects on cardiac mass and aspects of cardiovascular function. However, given the distant phylogenetic relationship between crocodilians and chelonians, we hypothesized that snapping turtles would also exhibit differences in the effects of developmental hypoxia on cardiovascular regulation. This hypothesis was based in part on prior studies that documented differences in plasticity of vagal tone on the heart between alligators and snapping turtles incubated in hypoxic conditions. To test this hypothesis, we investigated how 10% O2 exposure over final 80% of incubation altered the heart rate and blood pressure response to two chemical manipulations of the "chemoreflex" in common snapping turtles at 70% and 90% of incubation. NaCN injections produced a dose dependent bradycardia that was mediated by cholinergic receptor stimulation. This reflex was relatively unaffected by hypoxic incubation conditions in snapping turtle embryos. Injections of the 5-HT3 agonist phenylbiguanide (PBG) caused a pronounced bradycardia that decreased in intensity at 90% of incubation in embryos from the normoxic group while the heart rate response was unchanged in the hypoxic group. This differs from the previously reported diminished heart rate response of embryonic alligators incubated in 10% O2, suggesting plasticity in this chemoreflex response differs between the species. Our data also indicate the cardiovascular response is mediated by a secondary cholinergic receptor stimulation however the inability of ganglionic blockade to inhibit the PBG response leaves the location of the receptors antagonized by PBG in question in embryonic snapping turtles. Primarily, our findings refute the hypothesis that hypoxic incubation decreases the "chemoreflex' response of snapping turtle embryos.

Thermal tolerance and routine oxygen consumption of convict cichlid, Archocentrus nigrofasciatus, acclimated to constant temperatures (20 °C and 30 °C) and a daily temperature cycle (20 °C → 30 °C).

Organismal temperature tolerance and metabolic responses are correlated to recent thermal history, but responses to thermal variability are less frequently assessed. There is great interest in whether organisms that experience greater thermal variability can gain metabolic or tolerance advantages through phenotypic plasticity. We compared thermal tolerance and routine aerobic metabolism of Convict cichlid acclimated for 2 weeks to constant 20 °C, constant 30 °C, or a daily cycle of 20 → 30 °C (1.7 °C/h). Acute routine mass-specific oxygen consumption ([Formula: see text]O2) and critical thermal maxima/minima (CTMax/CTMin) were compared between groups, with cycle-acclimated fish sampled from the daily minimum (20 °C, 0900 h) and maximum (30 °C, 1600 h). Cycle-acclimated fish demonstrated statistically similar CTMax at the daily minimum and maximum (39.0 °C, 38.6 °C) but distinct CTMin values, with CTMin 2.4 °C higher for fish sampled from the daily 30 °C maximum (14.8 °C) compared to the daily 20 °C minimum (12.4 °C). Measured acutely at 30 °C, [Formula: see text]O2 decreased with increasing acclimation temperature; 20 °C acclimated fish had an 85% higher average [Formula: see text]O2 than 30 °C acclimated fish. Similarly, acute [Formula: see text]O2 at 20 °C was 139% higher in 20 °C acclimated fish compared to 30 °C acclimated fish. Chronic [Formula: see text]O2 was measured in separate fish continually across the 20 → 30 °C daily cycle for all 3 acclimation groups. Chronic [Formula: see text]O2 responses were very similar between groups between average individual hourly values, as temperatures increased or decreased (1.7 °C/h). Acute [Formula: see text]O2 and thermal tolerance responses highlight "classic" trends, but dynamic, chronic trials suggest acclimation history has little effect on the relative change in oxygen consumption during a thermal cycle. Our results strongly suggest that the minimum and maximum temperatures experienced more strongly influence fish physiology, rather than the thermal cycle itself. This research highlights the importance of collecting data in both cycling and static (constant) thermal conditions, and further research should seek to understand whether ectotherm metabolism does respond uniquely to fluctuating temperatures.

Temperature tolerance and oxygen consumption of two South American tetras, Paracheirodon innessi and Hyphessobrycon herbertaxelrodi.

Temperature is a primary factor affecting species' ability to thrive in a particular ecological niche, but thermal conditions have changed dramatically in recent decades. Fishes shift their thermal tolerance range with a maximum and minimum temperature correlated to their recent thermal acclimation history, and species can show a reduced temperature quotient (Q10) following chronic thermal acclimation. Neon tetra (Paracheirodon innesi) and Black Neon tetra (Hyphessobrycon herbertaxelrodi) are popular hobbyist aquarium fishes, and both species are examples of freshwater teleosts native to South American river systems that are potentially affected by changing thermal conditions. We acclimated these species to three different constant temperatures (26 °C, 29 °C, and 31 °C) for 15.4 ±â€¯2.1 days, then measured acute critical thermal maxima (CTMax) and acute oxygen consumption rate (Mo2) at each acclimation temperature. We also estimated chronic lethal thermal maximum (CLT) for both species following a 2-week acclimation to 30.4 °C. Mean CTMax of both species were found to increase with acclimation temperature from 38.5 to 39.6 °C for Neon tetra and from 39.5 to 41.0 °C for Black Neon tetra, gaining 0.24 (Neon tetra) or 0.29 °C (Black Neon tetra) of tolerance per 1 °C of acclimation. However, Black Neon tetra demonstrated consistently higher CTMax (1.0-1.4 °C). CLT was lower for Neon tetra (33.5 °C), compared to Black Neon tetra (35.9 °C). Mean Mo2 were statistically similar across acclimation temperatures within species; Q10 between 26-31 °C were 1.92 and 1.22 for Neon and Black Neon tetra, respectively. Neon and Black Neon tetras physiologically acclimated to changing thermal demands, and although they demonstrate robust CTMax responses, CLT responses indicated both species are unable to survive temperatures 4-5 °C above current average natural values. The demonstrated metabolic plasticity and CTMax values provide a moderate cushion for both species to combat changing temperatures due to climate change, but CLT values suggest vulnerability to projected climate trends.

Scaling of major organs in hatchling female American alligators (Alligator mississippiensis).

Allometric equations represent relationships between morphological/physiological traits and body mass Y = aMb , where Y is the trait, a is elevation, b is the exponent describing the shape of the line, and M is body mass. We measured visceral organ masses in hatchling alligators (Alligator mississippiensis) from five clutches from approximately 45 to 500 g wet body mass. The interaction between initial egg mass and clutch identity was significant for initial hatchling mass, but only egg mass, not clutch, had a significant effect on initial snout-vent and head length. Kidney and liver mass showed biphasic scaling with body mass, as determined by "breakpoint" analyses, with the breakpoint at 120 g wet body mass. Kidney and liver wet mass showed slopes b > 1.0 as animals increased approximately 45-120 g, with significantly lower b approximately 0.8-0.9 for alligators 120-500 g. Within kidney and liver mass, below and above the breakpoint, organ mass slopes tended to be similar across clutches. Lung and heart wet mass did not show biphasic scaling, with b approximately 0.8-0.9. Within lung and heart mass, clutches had statistically identical slopes. Combined clutch data for wet mass showed distinct regressions with b > 1.4 for approximately 45-120 g alligators' kidney and liver mass, compared with approximately 120-500 g alligators' kidney, liver, lung, and heart mass b < 1.0. Alligators show rapid kidney and liver growth following hatching, with higher rates than lung or heart tissue. Clutch, egg mass, and hatchling size influence organ size, and each factor should be accounted for in future studies exploring reptile morphology and physiology to assess environmental versus clutch contributions.

Daily, repeating fluctuations in embryonic incubation temperature alter metabolism and growth of Lake whitefish (Coregonus clupeaformis).

Lake whitefish (Coregonus clupeaformis) utilize overwintering embryonic development (up to 180 days), and such stenothermic, cold-water embryos may be particularly susceptible to thermal shifts. We incubated whitefish embryos in temperature treatments that were constant temperature (2.0 ±â€¯0.1 °C, 5.0 ±â€¯0.1 °C, and 8.0 ±â€¯0.1 °C; mean ±â€¯SD) or variable temperature (VT, mean = 5.0 ±â€¯0.3 °C). In the VT, a daily 2 °C temperature change followed a continuous pattern throughout development: 2-4-6-8-6-4-2 °C. Hatchling survival proportion from fertilization to hatch was significantly impacted by incubation temperature (P < 0.001): 2 °C (0.88 ±â€¯0.01) and 5 °C (0.91 ±â€¯0.01) showed higher survival than both the VT (0.83 ±â€¯0.02) and 8 °C groups (0.15 ±â€¯0.06), which were statistically distinct from each other. Time to hatch (dpf) was significantly different across all treatments (P < 0.001): 8 °C (68 ±â€¯2 dpf), VT (111 ±â€¯4 dpf), 5 °C (116 ±â€¯4 dpf), 2 °C (170 ±â€¯3 dpf). Likewise, hatchling yolk-free dry mass (mg) and total body length (mm) were significantly different across all treatments (P < 0.001): 8 °C (0.66 ±â€¯0.08 mg; 11.1 ±â€¯0.08 mm), VT (0.97 ±â€¯0.06 mg; 11.7 ±â€¯0.05 mm), 5 °C (1.07 ±â€¯0.03 mg; 12.0 ±â€¯0.02 mm), 2 °C (1.36 ±â€¯0.04 mg; 12.8 ±â€¯0.05 mm). Oxygen consumption rate (V̇o2) was significantly affected by the interaction between treatment and measurement temperature (P < 0.001). Hatchling VT whitefish showed mean V̇o2 that was higher compared to the 2 °C group measured at 2 °C, and lower compared to the 2 °C and 5 °C group measured at 8 °C. This study demonstrates that the VT incubation treatment produced fewer (increased mortality), smaller embryos that hatched earlier than 2 °C and 5 °C embryos. The plasticity of V̇o2 for this stenothermic-incubating fish species under variable incubation conditions reveals a metabolic cost to cycling thermal incubation conditions.

Chronic captopril treatment reveals the role of ANG II in cardiovascular function of embryonic American alligators (Alligator mississippiensis).

Angiotensin II (ANG II) is a powerful vasoconstrictor of the renin-angiotensin system (RAS) that plays an important role in cardiovascular regulation in adult and developing vertebrates. Knowledge of ANG II's contribution to developmental cardiovascular function comes from studies in fetal mammals and embryonic chickens. This is the first study to examine the role of ANG II in cardiovascular control in an embryonic reptile, the American alligator (Alligator mississippiensis). Using chronic low (~ 5-mg kg embryo-1), or high doses (~ 450-mg kg embryo-1) of captopril, an angiotensin-converting enzyme (ACE) inhibitor, we disrupted the RAS and examined the influence of ANG II in cardiovascular function at 90% of embryonic development. Compared to embryos injected with saline, mean arterial pressure (MAP) was significantly reduced by 41 and 72% under low- and high-dose captopril treatments, respectively, a greater decrease in MAP than observed in other developing vertebrates following ACE inhibition. Acute exogenous ANG II injection produced a stronger hypertensive response in low-dose captopril-treated embryos compared to saline injection embryos. However, ACE inhibition with the low dose of captopril did not change adrenergic tone, and the ANG II response did not include an α-adrenergic component. Despite decreased MAP that caused a left shifted baroreflex curve for low-dose captopril embryos, ANG II did not influence baroreflex sensitivity. This study demonstrates that ANG II contributes to cardiovascular function in a developing reptile, and that the RAS contributes to arterial blood pressure maintenance during development across multiple vertebrate groups.

Metabolic responses to chronic hypoxic incubation in embryonic American alligators (Alligator mississippiensis).

Chronic hypoxic incubation is a common tool used to study developmental changes in reduced O2 conditions, and it has been useful for identifying phenotypically plastic periods during ontogeny in laboratory settings. Reptilian embryos can be subjected to natural hypoxia due to nesting strategy, and recent studies have been important in establishing the phenotypic responses of several species to low developmental oxygen. In particular, the cardiovascular responses of American alligators (Alligator mississippiensis) to low developmental oxygen have been detailed, including a substantial cardiac enlargement that may support a higher mass specific metabolic rate. However, embryo mass-specific metabolic demands of hypoxic incubated alligator embryos have not been measured. In this study, alligator eggs were incubated in 10% O2 (H) or 21% O2 (N) environments for the entire course of embryonic development. Acute metabolic measures in 21% and 10% O2 were taken for both H and N groups. We hypothesized that acute 10% O2 exposure has no impact on metabolic rate of embryonic alligators, and that metabolic rate is unaffected by chronic hypoxic incubation when studied in embryos measured at 21% O2. Our findings suggest phenotypic changes resulting from hypoxic incubation early in incubation, in particular relative cardiac enlargement, enable embryonic alligators to sustain metabolic rate during acute hypoxic exposure.

Lipid content and fatty acid profile during lake whitefish embryonic development at different incubation temperatures.

Lipids serve as energy sources, structural components, and signaling molecules during fish embryonic development, and utilization of lipids may vary with temperature. Embryonic energy utilization under different temperatures is an important area of research in light of the changing global climate. Therefore, we examined percent lipid content and fatty acid profiles of lake whitefish (Coregonus clupeaformis) throughout embryonic development at three incubation temperatures. We sampled fertilized eggs and embryos at gastrulation, eyed and fin flutter stages following chronic incubation at temperatures of 1.8, 4.9 and 8.0°C. Hatchlings were also sampled following incubation at temperatures of 3.3, 4.9 and 8.0°C. Fertilized eggs had an initial high percentage of dry mass composed of lipid (percent lipid content; ~29%) consisting of ~20% saturated fatty acids (SFA), ~32% monounsaturated fatty acids (MUFA), ~44% polyunsaturated fatty acids (PUFA), and 4% unidentified. The most abundant fatty acids were 16:0, 16:1, 18:1(n-9c), 20:4(n-6), 20:5(n-3) and 22:6(n-3). This lipid profile matches that of other cold-water fish species. Percent lipid content increased during embryonic development, suggesting protein or other yolk components were preferentially used for energy. Total percentage of MUFA decreased during development, which indicated MUFA were the primary lipid catabolized for energy during embryonic development. Total percentage of PUFA increased during development, driven largely by an increase in 22:6(n-3). Temperature did not influence percent lipid content or percent MUFA at any development stage, and had inconsistent effects on percent SFA and percent PUFA during development. Thus, lake whitefish embryos appear to be highly adapted to low temperatures, and do not alter lipids in response to temperature within their natural incubation conditions.

Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis).

During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50-70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70-90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered ß-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator.

The effects of increased constant incubation temperature and cumulative acute heat shock exposures on morphology and survival of Lake Whitefish (Coregonus clupeaformis) embryos.

Increasing incubation temperatures, caused by global climate change or thermal effluent from industrial processes, may influence embryonic development of fish. This study investigates the cumulative effects of increased incubation temperature and repeated heat shocks on developing Lake Whitefish (Coregonus clupeaformis) embryos. We studied the effects of three constant incubation temperatures (2°C, 5°C or 8°C water) and weekly, 1-h heat shocks (+3°C) on hatching time, survival and morphology of embryos, as these endpoints may be particularly susceptible to temperature changes. The constant temperatures represent the predicted magnitude of elevated water temperatures from climate change and industrial thermal plumes. Time to the pre-hatch stage decreased as constant incubation temperature increased (148d at 2°C, 92d at 5°C, 50d at 8°C), but weekly heat shocks did not affect time to hatch. Mean survival rates and embryo morphometrics were compared at specific developmental time-points (blastopore, eyed, fin flutter and pre-hatch) across all treatments. Constant incubation temperatures or +3°C heat-shock exposures did not significantly alter cumulative survival percentage (~50% cumulative survival to pre-hatch stage). Constant warm incubation temperatures did result in differences in morphology in pre-hatch stage embryos. 8°C and 5°C embryos were significantly smaller and had larger yolks than 2°C embryos, but heat-shocked embryos did not differ from their respective constant temperature treatment groups. Elevated incubation temperatures may adversely alter Lake Whitefish embryo size at hatch, but weekly 1-h heat shocks did not affect size or survival at hatch. These results suggest that intermittent bouts of warm water effluent (e.g., variable industrial emissions) are less likely to negatively affect Lake Whitefish embryonic development than warmer constant incubation temperatures that may occur due to climate change.

Phenotypic plasticity in the common snapping turtle (Chelydra serpentina): long-term physiological effects of chronic hypoxia during embryonic development.

Studies of embryonic and hatchling reptiles have revealed marked plasticity in morphology, metabolism, and cardiovascular function following chronic hypoxic incubation. However, the long-term effects of chronic hypoxia have not yet been investigated in these animals. The aim of this study was to determine growth and postprandial O2 consumption (V̇o2), heart rate (fH), and mean arterial pressure (Pm, in kPa) of common snapping turtles (Chelydra serpentina) that were incubated as embryos in chronic hypoxia (10% O2, H10) or normoxia (21% O2, N21). We hypothesized that hypoxic development would modify posthatching body mass, metabolic rate, and cardiovascular physiology in juvenile snapping turtles. Yearling H10 turtles were significantly smaller than yearling N21 turtles, both of which were raised posthatching in normoxic, common garden conditions. Measurement of postprandial cardiovascular parameters and O2 consumption were conducted in size-matched three-year-old H10 and N21 turtles. Both before and 12 h after feeding, H10 turtles had a significantly lower fH compared with N21 turtles. In addition, V̇o2 was significantly elevated in H10 animals compared with N21 animals 12 h after feeding, and peak postprandial V̇o2 occurred earlier in H10 animals. Pm of three-year-old turtles was not affected by feeding or hypoxic embryonic incubation. Our findings demonstrate that physiological impacts of developmental hypoxia on embryonic reptiles continue into juvenile life.

Critical Windows of Cardiovascular Susceptibility to Developmental Hypoxia in Common Snapping Turtle (Chelydra serpentina) Embryos.

Environmental conditions fluctuate dramatically in some reptilian nests. However, critical windows of environmental sensitivity for cardiovascular development have not been identified. Continuous developmental hypoxia has been shown to alter cardiovascular form and function in embryonic snapping turtles (Chelydra serpentina), and we used this species to identify critical periods during which hypoxia modifies the cardiovascular phenotype. We hypothesized that incubation in 10% O2 during specific developmental periods would have differential effects on the cardiovascular system versus overall somatic growth. Two critical windows were identified with 10% O2 from 50% to 70% of incubation, resulting in relative heart enlargement, either via preservation of or preferential growth of this tissue, while exposure to 10% O2 from 20% to 70% of incubation resulted in a reduction in arterial pressure. The deleterious or advantageous aspects of these embryonic phenotypes in posthatching snapping turtles have yet to be explored. However, identification of these critical windows has provided insight into how the developmental environment alters the phenotype of reptiles and will also be pivotal in understanding its impact on the fitness of egg-laying reptiles.

Embryonic critical windows: changes in incubation temperature alter survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis).

The timing, success and energetics of fish embryonic development are strongly influenced by temperature. However, it is unclear if there are developmental periods, or critical windows, when oxygen use, survival and hatchling phenotypic characteristics are particularly influenced by changes in the thermal environment. Therefore, we examined the effects of constant incubation temperature and thermal shifts on survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis) embryos. We incubated whitefish embryos at control temperatures of 2, 5, or 8 °C, and shifted embryos across these three temperatures at the end of gastrulation or organogenesis. We assessed hatch timing, mass at hatch, and yolk conversion efficiency (YCE). We determined cost of development, the amount of oxygen required to build a unit of mass, for the periods from fertilization-organogenesis, organogenesis-fin flutter, fin flutter-hatch, and for total development. An increase in incubation temperature decreased time to 50 % hatch (164 days at 2 °C, 104 days at 5 °C, and 63 days at 8 °C), survival decreased from 55 % at 2 °C, to 38 % at 5 °C, and 17 % at 8 °C, and hatchling yolk-free dry mass decreased from 1.27 mg at 2 °C to 0.61 mg at 8 °C. Thermal shifts altered time to 50 % hatch and hatchling yolk-free dry mass and revealed a critical window during gastrulation in which a temperature change reduced survival. YCE decreased and cost of development increased with increased incubation temperature, but embryos that hatched at 8 °C and were incubated at colder temperatures during fertilization-organogenesis had reduced cost. The relationship between cost of development and temperature was altered during fin flutter-hatch, indicating it may be a critical window during which temperature has the greatest impact on energetic processes. The increase in cost of development with an increase in temperature has not been documented in other fishes and suggests whitefish embryos are more energy efficient at colder temperatures.

Chronic hypercapnic incubation increases relative organ growth and reduces blood pressure of embryonic American alligator (Alligator mississippiensis).

Reptilian nests can experience natural hypoxic and hypercapnic conditions. We incubated alligator eggs at a female-only producing temperature (30°C) in three conditions: 21% O2/0.04% CO2, 21% O2/3.5% CO2 and 21% O2/7% CO2. Alligator embryos chronically incubated in high CO2 were markedly hypotensive (blood pressure reduced by 46%) and had relatively (mass-specific) enlarged hearts (dry mass increased by 20%), lungs (dry mass increased by 17%), and kidneys (dry mass increased by 14%). This study is the first to chronically incubate reptilian eggs in hypercapnia and suggests that high CO2 alters the cardiovascular phenotype of alligator embryos (low blood pressure, relatively enlarged hearts), as well as the relative size of the organs primarily responsible for acid base balance, lungs and kidneys. The lungs and kidneys are largely non-functional during embryonic development, and the embryonic phenotype of increased relative mass may be a predictive-adaptation to metabolic or respiratory acidosis, such as during exercise or high respiratory CO2. This study demonstrates that phenotypic plasticity of alligator embryos incubated in high CO2 may result in either preferential organ growth, or maintenance of organ growth with reduced somatic growth.

Adjustments in cholinergic, adrenergic and purinergic control of cardiovascular function in snapping turtle embryos (Chelydra serpentina) incubated in chronic hypoxia.

Adenosine is an endogenous nucleoside that acts via G-protein coupled receptors. In vertebrates, arterial or venous adenosine injection causes a rapid and large bradycardia through atrioventricular node block, a response mediated by adenosine receptors that inhibit adenylate cyclase and decrease cyclic AMP concentration. Chronic developmental hypoxia has been shown to alter cardioregulatory mechanisms in reptile embryos, but adenosine's role in mediating these responses is not known. We incubated snapping turtle embryos under chronic normoxic (N21; 21 % O2) or chronic hypoxic conditions (H10; 10 % O2) beginning at 20 % of embryonic incubation. H10 embryos at 90 % of incubation were hypotensive relative to N21 embryos in both normoxic and hypoxic conditions. Hypoxia caused a hypotensive bradycardia in both N21 and H10 embryos during the initial 30 min of exposure; however, f H and P m both trended towards increasing during the subsequent 30 min, and H10 embryos were tachycardic relative to N21 embryos in hypoxia. Following serial ≥1 h exposure to normoxic and hypoxic conditions, a single injection of adenosine (1 mg kg(-1)) was given. N21 and H10 embryos responded to adenosine injection with a rapid and large hypotensive bradycardia in both normoxia and hypoxia. Gene expression for adenosine receptors were quantified in cardiac tissue, and Adora1 mRNA was the predominant receptor subtype with transcript levels 30-82-fold higher than Adora2A or Adora2B. At 70 % of incubation, H10 embryos had lower Adora1 and Adora2B expression compared to N21 embryos. Expression of Adora1 and Adora2B decreased in N21 embryos during development and did not differ from H10 embryos at 90 % of incubation. Similar to previous results in normoxia, H10 embryos in hypoxia were chronically tachycardic compared to N21 embryos before and after complete cholinergic and adrenergic blockade. Chronic hypoxia altered the development of normal cholinergic and adrenergic tone, as well as adenosine receptor mRNA levels. This study demonstrates that adenosine may be a major regulator of heart rate in developing snapping turtle embryos, and that chronic hypoxic incubation alters the response to hypoxic exposure.

Chronic hypoxic incubation blunts thermally dependent cholinergic tone on the cardiovascular system in embryonic American alligator (Alligator mississippiensis).

Environmental conditions play a major role in shaping reptilian embryonic development, but studies addressing the impact of interactions between chronic and acute environmental stressors on embryonic systems are lacking. In the present study, we investigated thermal dependence of cholinergic and adrenergic cardiovascular tone in embryonic American alligators (Alligator mississippiensis) and assessed possible phenotypic plasticity in a chronic hypoxic incubation treatment. We compared changes in heart rate (f H) and mean arterial blood pressure (P M) for chronically hypoxic and normoxic-incubated embryos after cholinergic and adrenergic blockade following three different acute temperature treatments: (1) 30 °C (control incubation temperature), (2) acute, progressive decrease 30-24 °C then held at 24 °C, and (3) acute, progressive increase 30-36 °C then held at 36 °C. f H progressively fell in response to decreasing temperature and rose in response to increasing temperature. P M did not significantly change with decreasing temperature, but was lowered significantly with increasing acute temperature in the normoxic group at 90 % of development only. Propranolol administration (ß adrenergic antagonist) produced a significant f H decrease at 24, 30, and 36 °C that was similar at all temperatures for all groups. For normoxic-incubated embryos at 90 % of development, atropine administration (cholinergic antagonist) significantly increased f H in both 24 and 36 °C treatments, but not in the 30 °C control treatment. This atropine response at 24 and 36 °C demonstrated acute thermally dependent cholinergic tone on f H late in development for normoxic-incubated, but not chronically hypoxic-incubated embryos. Collectively, data indicated that cardiovascular control mechanisms in embryonic alligators may be activated by thermal extremes, and the maturation of control mechanisms was delayed by chronic hypoxia.

Plasticity of cardiovascular function in snapping turtle embryos (Chelydra serpentina): chronic hypoxia alters autonomic regulation and gene expression.

Reptile embryos tolerate large decreases in the concentration of ambient oxygen. However, we do not fully understand the mechanisms that underlie embryonic cardiovascular short- or long-term responses to hypoxia in most species. We therefore measured cardiac growth and function in snapping turtle embryos incubated under normoxic (N21; 21% O2) or chronic hypoxic conditions (H10; 10% O2). We determined heart rate (fH) and mean arterial pressure (Pm) in acute normoxic (21% O2) and acute hypoxic (10% O2) conditions, as well as embryonic responses to cholinergic, adrenergic, and ganglionic pharmacological blockade. Compared with N21 embryos, chronic H10 embryos had smaller bodies and relatively larger hearts and were hypotensive, tachycardic, and following autonomic neural blockade showed reduced intrinsic fH at 90% of incubation. Unlike other reptile embryos, cholinergic and ganglionic receptor blockade both increased fH. ß-Adrenergic receptor blockade with propranolol decreased fH, and α-adrenergic blockade with phentolamine decreased Pm. We also measured cardiac mRNA expression. Cholinergic tone was reduced in H10 embryos, but cholinergic receptor (Chrm2) mRNA levels were unchanged. However, expression of adrenergic receptor mRNA (Adrb1, Adra1a, Adra2c) and growth factor mRNA (Igf1, Igf2, Igf2r, Pdgfb) was lowered in H10 embryos. Hypoxia altered the balance between cholinergic receptors, α-adrenoreceptor and ß-adrenoreceptor function, which was reflected in altered intrinsic fH and adrenergic receptor mRNA levels. This is the first study to link gene expression with morphological and cardioregulatory plasticity in a developing reptile embryo.

Development of sympathetic cardiovascular control in embryonic, hatchling, and yearling female American alligator (Alligator mississippiensis).

We used arterial tyramine injections to study development of sympathetic actions on in vivo heart rate and blood pressure in embryonic, hatching and yearling female American alligators. Tyramine is a pharmacological tool for understanding comparative and developmental sympathetic regulation of cardiovascular function, and this indirect sympathomimetic agent causes endogenous neuronal catecholamine release, increasing blood pressure and heart rate. Arterial tyramine injection in hatchling and yearling alligators caused the typical vertebrate response - rise in heart rate and blood pressure. However, in embryonic alligators, tyramine caused a substantial and immediate bradycardia at both 70% and 90% of embryonic development. This embryonic bradycardia was accompanied by hypotension, followed by a sustained hypertension similar to the hatchling and juvenile responses. Pretreatment with atropine injection (cholinergic receptor blocker) eliminated the embryonic hypotensive bradycardia, and phentolamine pretreatment (α-adrenergic receptor blocker) eliminated the embryonic hypotensive and hypertensive responses but not the bradycardia. In addition, hexamethonium pretreatment (nicotinic receptor blocker) significantly blunted embryos' bradycardic tyramine response. However, pretreatment with 6-hydroxydopamine, a neurotoxin that destroys catecholaminergic terminals, did not eliminate the embryonic bradycardia. Tyramine likely stimulated a unique embryonic response - neurotransmitter release from preganglionic nerve terminals (blocked with hexamethonium) and an acetylcholine mediated bradycardia with a secondary norepinephrine-dependent sustained hypertension. In addition, tyramine appears to stimulate sympathetic nerve terminals directly, which contributed to the overall hypertension in the embryonic, hatchling and yearling animals. Data demonstrated that humoral catecholamine control of cardiovascular function was dominant over the immature parasympathetic nervous system in developing alligator embryos, and suggested that sympathetic and parasympathetic nerve terminals were present and developing in ovo but were not tonically active.

Effects of dehydration on cardiovascular development in the embryonic American alligator (Alligator mississipiensis).

Effects of dehydration on reptilian embryonic cardiovascular function are unknown. Here, we present the first morphological and physiological data quantifying the cumulative effects of four acute dehydration events on the embryonic American alligator, Alligator mississipiensis. We hypothesized that dehydration would alter embryonic morphology, reduce blood volume and augment the response to angiotensin II (Ang II), a key osmotic and blood volume regulatory response element in adult vertebrates. Drying events at 30%, 40%, 50%, and 60% of embryonic incubation reduced total egg water content by 14.43 ± 0.37 g, a 3.4 fold increase relative to controls. However, embyronic blood volume was greater in the dehydration group at 70% of embryonic incubation compared to controls (0.39 ± 0.044 mLg(-1) and 0.22 ± 0.03 mLg(-1), respectively), however, both groups were similar at 90% of incubation (0.18 ± 0.02 mLg(-1) in the controls and 0.23 ± 0.03 mLg(-1) in the dehydrated group). Dehydration altered the morphological phenotype and resulted in an overall reduction in embryonic mass at both incubation time points measured. Dehydration also altered the physiological phenotype, resulting in embryonic alligators that were relatively bradycardic at 90% of incubation. Arterial Ang II injections resulted in a dose dependent hypertension, which increased in intensity over the span of incubation studied. While progressive incubation altered the Ang II response, dehydration had no impact on the cardiovascular responses to the peptide. Quantification of Ang II type-1 receptor protein using western blot analysis illustrated that dehydration condition and incubation time point did not alter protein quantity. Collectively, our results show that dehydration during embryonic development of the American alligator alters embryonic morphology and baseline heart rate without altering arterial pressure and response to Ang II.