SUMMARYEmbryos of the annual killifish Austrofundulus limnaeus enter a state of developmental arrest termed diapause as part of their normal developmental program. Diapause can occur at two distinct developmental stages in this species, termed diapause II and III. When incubated at 25°C, most embryos enter diapause II, whereas a small percentage of 'escape' embryos develop continuously past diapause II and enter diapause III. Control of entry into diapause II can be altered by maternal influences and the incubation environment experienced by the embryos. Young females produce a higher proportion of escape embryos than do older females. In addition, increasing the incubation temperature from 25 to 30°C induces all embryos to escape from diapause. Surprisingly, escape embryos follow a different morphological and physiological developmental trajectory than do embryos that enter diapause II. Development of anterior structures is advanced compared with that of posterior structures in escape embryos when compared with embryos that will enter diapause II. The difference in timing of development for these two trajectories is consistent with changes observed between two species but is very atypical of variation observed within a species. Importantly, the two developmental pathways diverge early in development, during the segmentation period, when, according to evolutionary theory, constraint on developmental pathways should be relatively high. The possession of alternative developmental pathways in a vertebrate embryo is a novel finding, the ecological and evolutionary importance of which is still unknown, but potentially significant in terms of life-history evolution.

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. β-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 β-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.

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.

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 β-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.

Amphibian pulmonary and systemic vascular circuits are arranged in parallel, with potentially important consequences for resistance (R) to blood flow. The contribution of the parallel anatomic arrangement to total vascular R (R T), independent of blood viscosity, is unknown. We measured pulmonary (R P) and systemic (R S) vascular R with an in situ Ringer's solution perfusion technique using anesthetized anuran and urodele species to determine: (1) relative contributions of vascular anatomy and blood viscosity to R T; (2) distensibility index (%Δ flow kPa(-1)) of the pulmonary and systemic vascular circuits; and (3) interspecific correlates of variation in these parameters with red blood cell size, cardiac power output, and aerobic capacities. R P was lower than R S in anurans, while R P of the urodeles was greater than R S and significantly greater than anuran R P. Anuran R T was lowest and did not vary interspecifically, whereas urodele R T was significantly greater than anuran, and varied interspecifically. Pulmonary and systemic circuit distensibility differences may explain cardiac shunt patterns in toads with changes in cardiac output from rest to activity. When blood viscosity was taken into account, vascular resistance accounted for about 25 % of R T while blood viscosity accounted for the remaining 75 %. Owing to lower R T, terrestrial anuran species required lower cardiac power outputs when moving fluid through their vasculature compared to aquatic species. These results indicate that physical characteristics of the vasculature can account for interspecific differences in cardiovascular physiology and suggest a co-evolution of cardiac and vascular anatomy among amphibians.

The nests of embryonic turtles naturally experience elevated CO 2 (hypercarbia), which leads to increased blood P CO2 and a respiratory acidosis, resulting in reduced blood pH [extracellular pH ( pH e )]. Some fishes preferentially regulate tissue pH [intracellular pH ( pH i )] against changes in pH e ; this has been proposed to be associated with exceptional CO 2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO 2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pH i , conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa P CO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pH e but not pH i , at all developmental ages. At 70% of incubation, pH e was depressed by 0.324 pH units while pH i of brain, white muscle and lung increased; heart, liver and kidney pH i remained unchanged. At 90% of incubation, pH e was depressed by 0.352 pH units but heart pH i increased with no change in pH i of other tissues. Yearlings exhibited a pH e reduction of 0.235 pH units but had no changes in pH i of any tissues. The results indicate common snapping turtles preferentially regulate pH i during development, but the degree of response is reduced throughout development. This is the first time preferential pH i regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general.

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Amphibians have a single ventricle and common conus arteriosus that produces an equal pressure to the parallel pulmocutaneous and systemic vascular circuits. The distribution of blood flows between the pulmocutaneous (Q pul ) and systemic (Q sys ) circuits (net cardiac shunt) varies with a number of environmental conditions and behaviours; although autonomic regulation of pulmonary vascular resistance conductance has been emphasized, little attention has been paid to the possible contribution of the passive physical characteristics of the two circuits to pressure changes associated with variation in cardiac output. In this study, we re-analysed three recent studies that recorded net cardiac shunts in the cane toad (Rhinella marina) under a variety of conditions and treatments. In all three studies, Q pul and Q sys were linearly related to cardiac output (Q tot ), but the slope was threefold higher for Q pul compared with Q sys as predicted by relative conductance increases associated with increases in pressure from perfused preparations where autonomic regulation and humoral control were eliminated. Our analysis indicates that the net cardiac shunt in the cane toad is predicted primarily by the physical, rather than physiological, characteristics of the parallel pulmonary and systemic vascular circuits.