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Larval and juvenile air breathing fish may experience nocturnal and/or seasonal aquatic hypoxia. Yet, whether hypoxia induces respiratory developmental plasticity in larval air breathing fish is uncertain. This study predicted that larvae of two closely related anabantid fish-the facultative air breather the Siamese fighting fish (Betta splendens) and the obligate air breathing blue gourami (Trichopodus trichopterus)-show distinct differences in developmental changes in body, gill, and labyrinth morphology because of their differences in levels of dependency upon air breathing and habitat. Larval populations of both species were reared in normoxia or chronic nocturnal hypoxia from hatching through 35-38 days postfertilization. Gill and labyrinth variables were measured at the onset of air breathing. Betta splendens reared in normoxia possessed larger, more developed gills (~3× greater area) than T. trichopterus at comparable stages. Surface area of the emerging labyrinth, the air breathing organ, was ~ 85% larger in normoxic B. splendens compared to T. trichopterus. Rearing in mild hypoxia stimulated body growth in B. splendens, but neither mild nor severe hypoxia affected growth in T. trichopterus. Condition factor, K (~ 1.3 in B. splendens, 0.7 in T. trichopterus) was unaffected by mild hypoxia in either species, but was reduced by severe hypoxia to

[Figure][1] Glenn Tattersall and Warren Burggren discuss the impact of Robert Boutilier and Graham Shelton’s classic 1986 JEB paper ‘Gas exchange, storage and transport in voluntarily diving Xenopus laevis ’. Robert ‘Bob’ Boutilier was a deep and philosophical thinker and an

Transgenerational inheritance from both parental lines can occur by genetic and epigenetic inheritance. Maternal effects substantially influence offspring survival and fitness. However, investigation of the paternal contribution to offspring success has been somewhat neglected. In the present study, adult zebrafish were separated into female and male groups exposed for 21 days to either a control diet or to a diet containing water accommodated fractions of crude oil. Four F1 offspring groups were obtained: (1) control (non-exposed parents), (2) paternally exposed, (3) maternally exposed and (4) dual-parent-exposed. To determine the maternal and paternal influence on their offspring, we evaluated responses from molecular to whole organismal levels in both generations. Growth rate, hypoxia resistance and heart rate did not differ among parental groups. However, global DNA methylation in heart tissue was decreased in oil-exposed fish compared with control parents. This decrease was accompanied by an upregulation of glycine N-methyltransferase. Unexpectedly, maternal, paternal and dual exposure all enhanced survival of F1 offspring raised in oiled conditions. Regardless of parental exposure, however, F1 offspring exposed to oil exhibited bradycardia. Compared with offspring from control parents, global DNA methylation was decreased in the three offspring groups derived from oil-exposed parents. However, no difference between groups was observed in gene regulation involved in methylation transfer, suggesting that the changes observed in the F1 populations may have been inherited from both parental lines. Phenotypic responses during exposure to persistent environmental stressors in F1 offspring appear to be influenced by maternal and paternal exposure, potentially benefitting offspring populations to survive in challenging environments.

Genomic modifications occur slowly across generations, whereas short-term epigenetic inheritance of adaptive phenotypes may be immediately beneficial to large numbers of individuals acting as a bridge for survival when adverse environments occur. Crude oil was used as an example of an environmental stressor. Adult zebrafish (P0) were dietarily-exposed for three weeks to no, low, medium or high concentrations of crude oil. The F1 offspring obtained from the P0 groups were then assessed for transgenerational epigenetic transfer of oil-induced phenotypes. The exposure did not alter body length, body and organ mass or condition factor in the P0. However, when the P0 were bred, the fecundity in both sexes decreased in proportion to the amount of oil fed. Then the F1 larvae from each P0 were exposed from hatch to 5dpf to oil in their ambient water. Remarkably, F1 larvae derived from oil-exposed parents, when reared in oiled water, showed a 30% enhanced survival compared to controls (P

The slow, inexorable rise in annual average global temperatures and acidification of the oceans are often advanced as consequences of global change. However, many environmental changes, especially those involving weather (as opposed to climate), are often stochastic, variable and extreme, particularly in temperate terrestrial or freshwater habitats. Moreover, few studies of animal and plant phenotypic plasticity employ realistic (i.e. short-term, stochastic) environmental change in their protocols. Here, I posit that the frequently abrupt environmental changes (days, weeks, months) accompanying much longer-term general climate change (e.g. global warming over decades or centuries) require consideration of the true nature of environmental change (as opposed to statistical means) coupled with an expansion of focus to consider developmental phenotypic plasticity. Such plasticity can be in multiple forms – obligatory/facultative, beneficial/deleterious – depending upon the degree and rate of environmental variability at specific points in organismal development. Essentially, adult phenotypic plasticity, as important as it is, will be irrelevant if developing offspring lack sufficient plasticity to create modified phenotypes necessary for survival.

A critical window (sensitive period) represents a period during development when an organism's phenotype is responsive to intrinsic or extrinsic (environmental) factors. Such windows represent a form of developmental phenotypic plasticity and result from the interaction between genotype and environment. Critical windows have typically been defined as comprising discrete periods in development with a distinct starting time and end time, as identified by experiments following an on and an off protocol. Yet in reality, periods of responsiveness during development are likely more ambiguous that depicted. Our goal is to extend the concept of the developmental critical window by introducing a three-dimensional construct in which time during development, dose of the stressor applied, and the resultant phenotypic modification can be utilized to more realistically define a critical window. Using the example of survival of the brine shrimp (Artemia franciscana) during exposure to different salinity levels during development, we illustrate that it is not just stressor dose or exposure time but the interaction of these two factors that results in the measured phenotypic change, which itself may vary within a critical window. We additionally discuss a systems approach to critical windows, in which the components of a developing system--whether they be molecular, physiological, or morphological--may show differing responses with respect to time and dose. Thus, the plasticity of each component may contribute to a broader overall system response.

CO2 exposure elicits multiple changes in the acid–base balance and hematology of avian embryos, but the time-specific, dose-dependent effects of graded increases in extrinsic CO2 in a normoxic environment are poorly understood. Consequently, we exposed day 15 chicken embryos to 1, 3, 5, 6 or 10 % CO2 in 20 % O2. We hypothesized that both the magnitude of hypercapnic respiratory acidosis and the resultant metabolic compensation within 24 h of exposure to

Considerable variation is inherent both within and between comparative physiological data sets. Known sources for such variation include diet, gender, time of day and season of experiment, among many other factors, but a meta-analysis of physiological studies shows that surprisingly few studies report controlling for these factors. In fact, less than 3% of comparative physiological papers mention epigenetics. However, our understanding of epigenetic influences on physiological processes is growing rapidly, and it is highly likely that epigenetic phenomena are an additional ‘hidden’ source of variation, particularly in wild-caught specimens. Recent studies have shown epigenetic inheritance of commonly studied traits such as metabolic rate (water fleas Daphnia magna; emu, Dromaius novaellandiae), hypoxic tolerance, cardiac performance (zebrafish, Danio rerio), as well as numerous morphological effects. The ecological and evolutionary significance of such epigenetic inheritance is discussed in a comparative physiological context. Finally, against this context of epigenetic inheritance of phenotype, this essay also provides a number of caveats and warnings regarding the interpretation of transgenerational phenotype modification as a true epigenetic phenomenon. Parental effects, sperm storage, multiple paternity and direct gamete exposure can all be confounding factors. Epigenetic inheritance may best be studied in animal models that can be maintained in the laboratory over multiple generations, to yield parental stock that themselves are free of epigenetic effects from the historical experiences of their parents.

An increase in both vascular circumferential tension and shear stress in the developing vasculature of the chicken embryo has been hypothesized to stimulate angiogenesis in the developing peripheral circulation chorioallantoic membrane (CAM). To test this hypothesis, angiogenesis in the CAM, development, and growth were measured in the early chicken embryo, following acute and chronic topical application of the purely bradycardic drug ZD7288. At hour 56, ZD7288 reduced heart rate (f(H)) by ~30% but had no significant effect on stroke volume (~0.19 ± 0.2 μL), collectively resulting in a significant fall in cardiac output (CO) from ~27 ± 3 to 18 ± 2 μL min(-1). Mean f(H) at 72 h of development was similarly significantly lowered by acute ZD7288 treatment (250 μM) to 128 ± 0.3 beats min(-1), compared with 174.5 ± 0.3 and 174.7 ± 0.8 beats min(-1) in control and Pannett-Compton (P-C) saline-treated embryos, respectively. Chronic dosing with ZD7288-and the attendant decreases in f(H) and CO-did not change eye diameter or cervical flexion (key indicators of development rate) at 120 h but significantly reduced overall growth (wet and dry body mass decreased by 20%). CAM vessel density index (reflecting angiogenesis) measured 200-400 μm from the umbilical stalk was not altered, but ZD7288 reduced vessel numbers-and therefore vessel density-by 13%-16% more distally (500-600 μm from umbilical stalk) in the CAM. In the ZD7288-treated embryos, a decrease in vessel length was found within the second branch order (~300-400 μm from the umbilical stock), while a decrease in vessel diameter was found closer to the umbilical stock, beginning in the first branch order (~200-300 μm). Paradoxically, chronic application of P-C saline also reduced peripheral CAM vessel density index at 500 and 600 μm by 13% and 7%, respectively, likely from washout of local angiogenic factors. In summary, decreased f(H) with reduced CO did not slow development rate but reduced embryonic growth rate and angiogenesis in the CAM periphery. This study demonstrates for the first time that different processes in the ontogeny of the early vertebrate embryo (i.e., hypertrophic growth vs. development) have differential sensitivities to altered convective blood flow.

The avian embryo is a time-honored animal model for understanding vertebrate development. A key area of extensive study using bird embryos centers on developmental phenotypic plasticity of the cardio-respiratory system and how its normal development can be affected by abiotic factors such as temperature and oxygen availability. Through the investigation of the plasticity of development, we gain a better understanding of both the regulation of the developmental process and the embryo's capacity for self-repair. Additionally, experiments with abiotic and biotic stressors during development have helped delineate not just critical windows for avian cardio-respiratory development, but the general characteristics (e.g., timing and dose-dependence) of critical windows in all developing vertebrates. Avian embryos are useful in exploring fetal programming, in which early developmental experiences have implications (usually negative) later in life. The ability to experimentally manipulate the avian embryo without the interference of maternal behavior or physiology makes it particularly useful in future studies of fetal programming. The bird embryo is also a key participant in studies of transgenerational epigenetics, whether by egg provisioning or effects on the germline that are transmitted to the F1 generation (or beyond). Finally, the avian embryo is heavily exploited in toxicology, in which both toxicological testing of potential consumer products as well as the consequences of exposure to anthropogenic pollutants are routinely carried out in the avian embryo. The avian embryo thus proves useful on numerous experimental fronts as an animal model that is concurrently both of adequate complexity and sufficient simplicity for probing vertebrate cardio-respiratory development.

Hypoxic stress can alter conspecific phenotype and additionally alter phenotypes of the filial generation, for example, via maternal or epigenetic processes. Lasting effects can also be seen across development and generations even after stressors have been removed. This study utilized the model of rapidly developing, parthenogenetic Daphnia to examine the intraspecific variability of response of exposure of a parental generation to hypoxia (4 kPa) within a single clone line across development, across broods, and across generations. Body mass across development and reproductive output were monitored in the parental generation and the first three broods of the first filial generation (which were not directly exposed to hypoxia). O(2) consumption across a wide Po(2) range (normoxia to anoxia) was assessed to determine whether exposure of the parental generation to hypoxia conferred hypoxia tolerance on the offspring and whether this transgenerational, epigenetic phenomenon varied intraspecifically. Differences in mass occurred in both the parental generation (hypoxia-exposed smaller during brood 1 and brood 2 neonate production) and the filial generation (e.g., brood 1 and 2 neonates from hypoxic mothers were initially smaller than control neonates). However, differences in mass were not accompanied by changes in reproductive output (assessed by brood number and neonate size). At day 0, first filial generation brood 1 neonates from hypoxia-exposed mothers had a higher metabolic rate than control neonates. However, this effect, like that of body mass, dissipated with development within a brood but also with subsequent broods. An isometric scaling exponent for [Formula: see text] was repeatedly observed across a wide Po(2) range (21-2 kPa) throughout neonatal development.

Intraspecific allometric (scaling) relationships for metabolism, which have received little examination compared to interspecific relationships, reflect a complex interplay of organogenesis, growth, and shifting physiologies. In this study of the silkworm Bombyx mori, we hypothesized that allometric relationships for metabolism both across all developmental stages and within each stage would not reflect conventional scaling coefficients (e.g., b not equal to 0.75). Histology, gross morphology, body surface and cross-sectional area, total lipid content, and cytochrome c oxidase activity levels (as evidence of the total metabolic potential of mitochondria) were determined across development. Also measured were oxygen consumption, carbon dioxide production, and the respiratory exchange ratio. The overall slope, b, in the allometric equation relating to body mass across all developmental stages was 0.82, not greatly different from the value of 0.75 typical of interspecific data. However, within larval instars II-V and in prepupae, b varied between 0.99 and 1.49, far higher than hypothesized. Thus, in B. mori, an analytical approach that lumps all developmental stages hides interinstar variability. Morphological and biochemical data suggest that observed scaling patterns in B. mori are likely correlated with changes in overall mitochondrial density rather than with specific changes in body proportion of tissues with higher intrinsic metabolic intensity.

Evolutionary morphologists and physiologists have long recognized the phylogenetic significance of the ectothermic sauropsids. Sauropids have been classically considered to bridge between early tetrapods, ectotherms, and the evolution of endotherms. This transition has been associated with many modifications in cardiovascular form and function, which have changed dramatically during the course of vertebrate evolution. Most cardiovascular studies have focused upon adults, leaving the development of this critical system largely unexplored. In this essay, we attempt a synthesis of sauropsid cardiovascular development based on the limited literature and indicate fertile regions for future studies. Early morphological cardiovascular development, i.e., the basic formation of the tube heart and the major pulmonary and systemic vessels, is similar across tetrapods. Subsequent cardiac chamber development, however, varies considerably between developing chelonians, squamates, crocodilians, and birds, reflected in the diversity of adult ventricular structure across these taxa. The details of how these differences in morphology develop, including the molecular regulation of cardiac and vascular growth and differentiation, are still poorly understood. In terms of the functional maturation of the cardiovascular system, reflected in physiological mechanisms for regulating heart rate and cardiac output, recent work has illustrated that changes during ontogeny in parameters such as heart rate and arterial blood pressure are somewhat species-dependent. However, there are commonalities, such as a β-adrenergic receptor tone on the embryonic heart appearing prior to 60% of development. Differential gross morphological responses to environmental stressors (oxygen, hydration, temperature) have been investigated interspecifically, revealing that cardiac development is relatively plastic, especially, with respect to change in heart growth. Collectively, the data assembled here reflects the current limited morphological and physiological understanding of cardiovascular development in sauropsids and identifies key areas for future studies of this diverse vertebrate lineage. J. Morphol., 2009. © 2009 Wiley-Liss, Inc.

The extent to which hematocrit (Hct) is regulated and the impact of altered Hct on blood oxygen transport in avian embryos are largely unknown. Consequently, we investigated how acute blood removal or Ringer solution injection modified Hct in day 15 embryos, and how ;blood doping' with erythrocyte-enriched whole blood influenced O(2) consumption in day 15-17 chicken embryos. Mean Hct (+/-s.e.m.) at day 15, 16 and 17 was 26.7+/-0.6%, 28.0+/-0.4% and 30.7+/-0.5%, respectively. Blood withdrawal (19 increments of 125 microl each, separated by 30 min) caused a progressive fall in Hct to approximately 12% at day 15. Hct decline was strictly proportional to the extent of blood withdrawal. Incremental Ringer solution injection over an 8 h period, transiently increasing blood volume up to 85% over initial values, did not decrease Hct, indicating that injected Ringer solution rapidly left the circulating blood compartment. Blood doping with erythrocyte-enriched whole blood artificially elevated Hct from 27% to 38%, but caused no significant change in routine O(2) consumption (0.35-0.39 ml O(2) min(-1) egg(-1)) at any point over the subsequent 6 h period in day 15-17 embryos. We conclude that Hct is not protected acutely in day 15 chicken embryos, with no evidence of erythrocyte sequestration or release. Additionally, at day 15-17, Hct increases of approximately 10% do not enhance embryonic oxygen consumption, suggesting that blood oxygen carrying capacity per se is not limiting to oxygen consumption.

The concept of animal models is well honored, and amphibians have played a prominent part in the success of using key species to discover new information about all animals. As animal models, amphibians offer several advantages that include a well-understood basic physiology, a taxonomic diversity well suited to comparative studies, tolerance to temperature and oxygen variation, and a greater similarity to humans than many other currently popular animal models. Amphibians now account for approximately 1/4 to 1/3 of lower vertebrate and invertebrate research, and this proportion is especially true in physiological research, as evident from the high profile of amphibians as animal models in Nobel Prize research. Currently, amphibians play prominent roles in research in the physiology of musculoskeletal, cardiovascular, renal, respiratory, reproductive, and sensory systems. Amphibians are also used extensively in physiological studies aimed at generating new insights in evolutionary biology, especially in the investigation of the evolution of air breathing and terrestriality. Environmental physiology also utilizes amphibians, ranging from studies of cryoprotectants for tissue preservation to physiological reactions to hypergravity and space exploration. Amphibians are also playing a key role in studies of environmental endocrine disruptors that are having disproportionately large effects on amphibian populations and where specific species can serve as sentinel species for environmental pollution. Finally, amphibian genera such as Xenopus, a genus relatively well understood metabolically and physiologically, will continue to contribute increasingly in this new era of systems biology and "X-omics."

SUMMARYPhysiologists both admire and fear complexity, but we have made relatively few attempts to understand it. Inherently complex systems are more difficult to study and less predictable. However, a deeper understanding of physiological systems can be achieved by modifying experimental design and analysis to account for complexity. We begin this essay with a tour of some mathematical views of complexity. After briefly exploring chaotic systems,information theory and emergent behavior, we reluctantly conclude that, while a mathematical view of complexity provides useful perspectives and some narrowly focused tools, there are too few generally practical take-home messages for physiologists studying complex systems. Consequently, we attempt to provide guidelines as to how complex systems might be best approached by physiologists. After describing complexity based on the sum of a physiological system's structures and processes, we highlight increasingly refined approaches based on the pattern of interactions between structures and processes. We then provide a series of examples illustrating how appreciating physiological complexity can improve physiological research, including choosing experimental models, guiding data collection, improving data interpretations and constructing more rigorous system models. Finally, we conclude with an invitation for physiologists, applied mathematicians and physicists to collaborate on describing, studying and learning from studies of physiological complexity.

In chick embryos, various instantaneous heart rate (IHR) fluctuations (e.g. HR variability, HR irregularities) have been found and developmental patterns of mean heart rate (MHR) have been elucidated. IHR changes have also measured in newly hatched and young chickens in order to investigate the developmental patterns of MHR and any potential diurnal HR rhythmicity such as a circadian rhythm. The present report reviews measurements of embryonic and hatchling HR fluctuations and examine development of HR circadian rhythms during the late stages of incubation and after hatching. Embryonic HR was continuously measured during the last half period of incubation. After hatching, continuous measurements of HR were made for two weeks at the longest in eight chickens ranging in age from 0 to 47 days. During the embryonic development, circadian rhythm of HR was found to be absent until hatching. After hatching, distinct circadian rhythm of HR appeared with increasing daily MHR from day 0 to day 7. During the second week of post-hatch life, the daily scope of MHR (diurnal maximum-nocturnal minimum) reached a maximum level ranging from 200 to 300bpm with constant baseline of nocturnal MHR. In young chicks, the nocturnal HR baseline was the same as that during the fortnight of posthatch life and daily scope of MHR decreased to 100^150bpm.

Embryonic physiology is often viewed as merely those processes understood for the adult but conducted on a smaller physical scale. Yet striking examples of the inaccuracy of this perspective can be identified in the embryonic cardiovascular system. For example, dogma holds that the embryonic heart begins to beat to pump blood for convective transport, just like that of the adult. This is the major assumption inherent in the hypothesis we have called "convective synchronotropy"; that is, the embryonic heart starts to beat synchronously with the need for convective blood flow. However, there is compelling evidence on many fronts that the convective flow of blood generated by the early embryonic vertebrate heart is simply not required for transport of oxygen, nutrients, metabolic wastes, or hormones, all of which can be achieved entirely by diffusion. In fact, fish, amphibian, and bird embryos lacking a functional heart (either through surgical intervention or mutation) or whose oxygen-hemoglobin transport has been chemically eliminated nonetheless continue to function and grow in size for extended periods up to the point at which diffusion alone can no longer serve oxygen transport needs. We advocate the alternative hypothesis of "prosynchronotropy" (i.e., the heart starts to beat well before convective blood flow is needed for bulk transport). So, what is the purpose of the early embryonic heart beat? Evidence is presented herein in support of a morphogenic rationale for prosynchronotropy. Specifically, it appears that the initial rationale for the beat of the vertebrate embryonic heart may be two-fold: to aid in subtle but significant aspects of cardiac growth, shaping, and maturation, and to facilitate cardiac maturation angiogenesis--the formation of new vessels by sprouting from vessel tips. Ultimately, the embryonic cardiovascular system provides a graphic demonstration of how adult physiological functions should not, without verification, be interpolated back to the embryo of that species.

Hypothermic incubation reduces the ability of the late-stage chicken embryo to mount endothermic heat production. This study investigates whether blood O(2) transport is similarly limited by cooler incubation temperatures. Two populations of chicken embryos were incubated at 38 degrees C and 35 degrees C. At Hamilton-Hamburger (HH) developmental stage 41-42, hypothermic incubation had no significant effect on hematocrit ( approximately 26%) or [Hb] (7 g%). However, in the final stages of incubation (stage 43-44), hypothermic incubation reduced hematocrit from approximately 31% at 38 degrees C to approximately 27.5% at 35 degrees C. Hypothermic incubation similarly caused a reduced [Hb] from 8.5 g% (38 degrees C) to 6.25 g% (35 degrees C), indicating a reduction in blood O(2)-carrying capacity in embryos. Incubation temperature had a strong effect on blood-O(2) affinity in late development (stage 43-44), with P(50) at 38 degrees C falling significantly from approximately 6 kPa in 38 degrees C embryos to approximately 4 kPa in 35 degrees C embryos. P(O(2)) values in chorioallantoic arterial blood at HH 41-42 were 4.3 kPa at pH 7.46 (38 degrees C) and 3.4 kPa at pH 7.39 (35 degrees C). In chorioallantoic venous blood these values were 9.1 kPa at pH 7.34 (38 degrees C) and 8.1 kPa at pH 7.42 (35 degrees C). With further development to HH 43-44, chorioallantoic arterial blood oxygenation fell to 2.4 kPa at pH 7.54 (38 degrees C) and 2.8 kPa at pH 7.52 (35 degrees C). Similarly, P(O(2)) in chorioallantoic venous blood fell slightly to 7.7 kPa at pH 7.42 (38 degrees C) and 7.4 kPa at pH 7.48 (35 degrees C). Collectively, these data reveal that beyond HH 41-42, 35 degrees C embryos experience retarded hematological development, and the findings that support the observed delayed metabolic response to acute temperature changes.

Few physiological studies to date have focused on whether variation among sibling groups during development can account for often large, intraspecific physiological variation. In this study, we measured heart rate in the direct-developing frog Eleutherodactylus cooki throughout its embryonic development and examined heart rate variation among egg clutches comprising from 10 to 40 eggs. Clutches were collected in the wild in Yubucoa, Puerto Rico, and individual eggs were maintained under equivalent conditions in the lab. Heart rate showed large increases during development, rising from about 40 beats min(-1) in the earliest stages to about 110 beats min(-1) at hatching. The effect of stage (averaged across clutches) was highly significant (P0.001). However, repeated-measures MANOVA also revealed that there were highly significant effects on heart rate associated with both clutch (variation among clutches averaged across development; P0.001) and clutch-stage interactions (differences among clutches in the developmental change in heart rate; P0.0001). These effects and interactions reveal that throughout development, heart rate in siblings is much more similar than in nonsiblings and that sib groups follow different heart rate trajectories during their development. Collectively, these data indicate that "clutch effects" caused by genetic and/or maternal influences can strongly affect patterns of heart function during development within cave coqui populations. This phenomenon also occurs in bird eggs and armadillo neonates, suggesting that physiological variation attributable to clutch effects might be a widespread phenomenon in vertebrates.

Summary There is currently tremendous interest in how the physiology of individual animals changes and develops during ontogeny. One of the key areas is the extent to which the timing and/or rate of physiological development is fixed within an individual and to what extent can it be altered. We propose that plasticity in the timing of the onset of a particular physiological regulatory system during an individuals development be referred to as physiological heterokairy (to clearly distinguish this phenomenon from physiological heterochrony, which is an evolutionary pattern), and we marshal evidence for three different patterns of heterokairy: 1. altering relative position in the physiological itinerary; 2. altering overall rate of development per se and; 3. a combination of 1 and 2. Using these patterns as a starting point, we develop a framework for investigating physiological heterokairy which takes cognizance of the facts that multiple components of each regulatory system could appear at different times and multiple regulatory systems could come ‘on-line’ at different times. We finish by placing physiological heterokairy in the wider context of its ecological and evolutionary implications and its relationship to physiological genomics and heterochrony.

Epigenetic studies of both intragenerational and transgenerational epigenetic phenotypic modifications have proliferated in the last few decades. However, the strong reductionist focus on mechanism that prevails in many epigenetic studies to date has diverted attention away what might be called the ‘dynamics’ of epigenetics and its role in comparative biology. Epigenetic dynamics describes how both transgenerational and intragenerational epigenetic phenotypic modifications change in non-linear patterns over time. Importantly, a dynamic perspective suggests that epigenetic phenomena should not be regarded as ‘digital’ (on–off), in which a modified trait necessarily suddenly disappears between one generation and the next. Rather, dynamic epigenetic phenomena may be better depicted by graded, time-related changes that can potentially involve the ‘washout’ of modified phenotype both within and across generations. Conceivably, an epigenetic effect might also ‘wash-in’ over multiple generations, and there may be unexplored additive effects resulting from the pressures of environmental stressors that wax, wane and then wax again across multiple generations. Recognition of epigenetic dynamics is also highly dependent on the threshold for detection of the phenotypic modification of interest, especially when phenotypes wash out or wash in. Thus, studies of transgenerational epigenetic effects (and intragenerational effects, for that matter) that search for persistence of the phenomenon are best conducted with highly sensitive, precise quantitative methods. All of the scenarios in this review representing epigenetic dynamics are possible and some even likely. Focused investigations that concentrate on the time course will reveal much about both the impact and mechanisms of epigenetic phenomena.

A 3D conceptual framework of 'critical windows' was used to examine whether the morphometry of Artemia franciscana is altered by salinity exposure during certain key periods of development. Artemia franciscana were hatched at 20 ppt (designated control salinity) and were then exposed to 10, 30, 40 or 50 ppt either chronically (days 1-15) or only on days 1-6, 7-9, 10-12 or 13-15. On day 15, maturity was assessed and morphometric characteristics, including mass, total body length, tail length and width, length of the third swimming appendage and eye diameter, were measured. Maturation and morphometry on day 15 were influenced by the exposure window and salinity dose. Artemia franciscana were generally larger following exposure to 10 and 40 ppt during days 1-6 and 7-9 when compared with days 10-12 and 13-15, in part due to a higher percentage of mature individuals. Exposure to different salinities on days 1-6 produced the greatest differences in morphometry, and thus this appears to be a period in development when A. franciscana is particularly sensitive to salinity. Viewing the developmental window as three-dimensional allowed more effective visualization of the complex interactions between exposure window, stressor dose and the magnitude of morphometric changes in A. franciscana.

The past decade has seen an explosion of articles in scientific journals involving non-genetic influences on phenotype through modulation of gene function without changes in gene sequence. The excitement in modern molecular biology surrounding the impact exerted by the environment on development of the phenotype is focused largely on mechanism and has not incorporated questions asked (and answers provided) by early philosophers, biologists, and psychologists. As such, this emergence of epigenetic studies is somewhat “old wine in new bottles” and represents a reformulation of the old debate of preformationism versus epigenesis—one resolved in the 1800s. Indeed, this tendency to always look forward, with minimal concern or regard of what has gone before, has led to the present situation in which “true” epigenetic studies are believed to consist of one of two schools. The first is primarily medically based and views epigenetic mechanisms as pathways for disease (e.g., “the epigenetics of cancer”). The second is primarily from the basic sciences, particularly molecular genetics, and regards epigenetics as a potentially important mechanism for organisms exposed to variable environments across multiple generations. There is, however, a third, and separate, school based on the historical literature and debates and regards epigenetics as more of a perspective than a phenomenon. Against this backdrop, comparative integrative biologists are particularly well-suited to understand epigenetic phenomena as a way for organisms to respond rapidly with modified phenotypes (relative to natural selection) to changes in the environment. Using evolutionary principles, it is also possible to interpret “sunsetting” of modified phenotypes when environmental conditions result in a disappearance of the epigenetic modification of gene regulation. Comparative integrative biologists also recognize epigenetics as a potentially confounding source of variation in their data. Epigenetic modification of phenotype (molecular, cellular, morphological, physiological, and behavioral) can be highly variable depending upon ancestral environmental exposure and can contribute to apparent “random” noise in collected datasets. Thus, future research should go beyond the study of epigenetic mechanisms at the level of the gene and devote additional investigation of epigenetic outcomes at the level of both the individual organism and how it affects the evolution of populations. This review is the first of seven in this special issue of Integrative and Comparative Biology that addresses in detail these and other key topics in the study of epigenetics.

The heart beat of vertebrate embryos has been assumed to begin when convective bulk transport by blood takes over from transport by simple diffusion. To test this hypothesis, we measured eye growth, cervical flexure and rates of oxygen consumption ( V(O2)) in day 3-4 chick embryos denied cardiac output by ligation of the outflow tract and compared them with those of embryos with an intact cardiovascular system. Eye diameter, used as the index for embryonic growth, increased at a rate of approximately 4.5-5 % h(-)(1) during the observation period. There was no significant difference (P>0.1) in the rate of increase in eye diameter between control (egg opened), sham-ligated (ligature present but not tied) and ligated embryos. Similarly, the normal progression of cervical flexure was not significantly altered by ligation (P>0.1). V(O2) (ml O(2 )g(-)(1 )h(-)(1)) at 38 degrees C, measured by closed respirometry, was not significantly different (P>0.1) on day 3 in sham-ligated (14.5+/-1.9 ml O(2 )g(-)(1 )h(-)(1)) and ligated 17.6+/-1.8 ml O(2 )g(-)(1 )h(-)(1)) embryos. Similarly, on day 4, V(O2) in sham-ligated and ligated embryos was statistically the same (sham-ligated 10. 5+/-2.9 ml O(2 )g(-)(1 )h(-)(1); ligated 9.7+/-2.9 ml O(2 )g(-)(1 )h(-)(1)). Expressed as a linear function of body mass (M), V(O2) in sham-ligated embryos was described by the equation V(O2)=-0.48M+24.06 (r(2)=0.36, N=18, P

The role of genetics on neonatal physiological variability was examined in the nine-banded armadillo (Dasypus novemcinctus). Since armadillos give birth to only monozygous quadruplets, the genetic variation within litters is essentially zero. Quadruplets born in captivity were isolated and weighed within 8 h of birth. Oxygen consumption (V.(O2)) was measured in resting neonates by flow-through respirometry, heart rate obtained from an electrocardiogram and ventilation was measured by impedance techniques. Following the measurements, neonates were returned to the mother. Measurements were repeated at 4 and 8 days after birth. Mean heart rate significantly increased from 132 beats min(−1) on the day of birth to 169 beats min(−1) on day 8. Mean ventilation rate significantly decreased from 81 breaths min(−1) on the day of birth to 54 breaths min(−1) on day 8. During this same developmental period, mean mass significantly increased from 100 g to 129 g, and mean mass-specific oxygen consumption significantly decreased from 32.2 ml O(2)kg(−1)min(−1) to 28.6 ml O(2)kg(−1) min(−1). For all variables measured, within-litter variability was always significantly less than between-litter variability, confirming a ‘sibling effect’ that we attribute to the genetic components determining physiological characters.

Normal heart rate (HR), and the HR responses to hypoxia and hyperoxia during early heart development in chick embyros have not been studied in detail, particularly in undisturbed embryos within the intact egg. HR was measured in day 3-9 chick embryos at 38 degrees C using relatively noninvasive impedance cardiography. Embryos were exposed to air (control) and to hypoxic (10% O2) or hyperoxic (100% O2) gas for a 2-h or 4-h period, during which HR was continually monitored. Control (normoxic) HR increased from about 150 beats per min (bpm) on day 3 to about 240 bpm on days 7-9. HR in very early embryos showed a variety of moderate responses to hypoxia (all survived), but as development progressed beyond day 6, hypoxic exposure induced a profound bradycardia that frequently terminated in death before the end of the measurement period. In contrast to the marked developmental changes in hypoxic sensitivity, HR showed little response to hyperoxia throughout development, suggesting no "hypoxic drive" to HR. We speculate that hypoxia has little effect early in development because of the embryo's small absolute O2 demand, but as the embryo grows, hypoxia represents a progressively more severe perturbation. Although general trends were identified, there was considerable variation in both HR and HR responses to ambient O2 changes between individuals of the same developmental stage.

The present study investigates the ontogeny of cardio-respiratory physiology in Xenopus laevis where O2 transport is obstructed. Animals were raised from eggs (NF stage 1) to metamorphic climax (NF stage 63), while maintained either in air or in chronic 2 kPa CO, which functionally ablates O2 transport by hemog.lobin (Hb). Whole-animal rate of oxygen consumption , whole-body lactate concentration, individual mass, heart rate (fH) and stroke volum. e (VS) were measured. Additionally, cardiac output (Q̇) and the ratio of the rate of oxygen consumption to the total rate at which oxygen is transported in the blood were calculated to determine limitations imparted when O2 transport is impaired. Our data on early development suggest that the onset of convective blood flow occurs prior to the absolute need for convection to supplement diffusive transport. Values for , whole-body lactate concentration, mass and fHdid not differ significantly between controls and CO-exposed animals. However, CO-exposed animals showed a. significant (P

Arterial hemolymph flow was measured in restrained crayfish (Procambarus clarkii) and lobsters (Homarus americanus). Implanted pulsed Doppler flow transducers were used to measure arterial flows in the anterior aorta, posterior aorta, sternal artery, lateral artery, ventral thoracic artery and ventral abdominal artery, allowing determination of flow simultaneously in several arteries over a period of 4 days. Calculated Doppler hemolymph flow showed a strong correlation (P0.05) with 'pumped' hemolymph flow as determined by in situ calibration. Arterial flow patterns remained constant during quiet conditions. In crayfish, cardiac output was 7.5plusmn;1.1 ml min-1 (252 ml kg-1 min-1), of which the anterior aorta received 1.3plusmn;0.15 ml min-1 (20.1plusmn;4.0 %), the posterior aorta received 0.8plusmn;0.1 ml min-1 (12.3plusmn;2.7 %) and the sternal artery received 5.2plusmn;1.4 ml min-1 (67.5plusmn;37.0 %). Mean heart frequency at rest was 125.6plusmn;5.2 beats min-1 and stroke volume was 0.06plusmn;0.01 ml beat-1 (1.98 ml kg-1 beat-1). In lobsters, cardiac output was 60.8plusmn;4.4 ml min-1 (93.6plusmn;6.8 ml kg-1 min-1), with the anterior aorta receiving 7.8plusmn;0.8 ml min-1 (12.8plusmn;2.7 %), the lateral arteries receiving 0.6plusmn;0.2 ml min-1 (1.0plusmn;0.5 %), the posterior aorta receiving 12.6plusmn;1.0 ml min-1 (20.7plusmn;3.3 %) and the sternal artery receiving 38.9plusmn;4.1 ml min-1 (64.0plusmn;13.4 %). Flows in the branches of the sternal artery were 0.3plusmn;0.05 ml min-1 (0.5plusmn;2 %) in the ventral abdominal artery and 4.0plusmn;0.1 ml min-1 (6.5plusmn;0.3 %) in the ventral thoracic artery. Lobster heart rate was 82.5plusmn;2.9 beats min-1 and stroke volume was 0.7plusmn;0.05 ml beat-1. Periods of constant hemolymph flow were interrupted by tail flexions (abdominal flexion) and, in lobsters, periods of cardiac/respiratory pause. Tail movement increased flow (peak height and minimum flow values) in both crayfish and lobsters, although the general wave form of hemolymph flow and pressure did not change. In lobsters, periodic respiratory pauses were observed during which all arteries received hemolymph, despite the low heart rate.

All-trans-retinoic acid (tRA), an active metabolite of vitamin A, directly influences the developing kidney, and is a major regulatory signal during vertebrate organogenesis. The aim of the current study was to specifically target potential critical windows in renal development, and assess altered renal function through disruptions in embryonic fluid compartments. In addition, the effect of exogenous tRA administration on embryonic growth and metabolism was determined. Embryos were exposed to 0.1 or 0.3 mg tRA on embryonic day 8. Morphological and physiological measurements were made on days 12, 14, 16 and 18. Embryo wet mass on day 18 was reduced by 23 % (0.1 mg tRA) and 44 % (0.3 mg tRA). tRA exposure elevated mass-specific oxygen consumption in embryos exposed to 0.1 mg (21.2 ± 0.3 μL(-1) g(-1) min(-1)) and 0.3 mg (23.4 ± 0.4 μL(-1) g(-1) min(-1)) when compared to sham (18.9 ± 0.6 μL(-1) g(-1) min(-1)) on day 14, but not subsequent incubation days. Osmolality of blood plasma was transiently lowered in embryos exposed to 0.3 mg tRA between days 14 and 16. Allantoic fluid osmolality was significantly elevated by tRA to ~220 mmol L(-1) from days 16 to 18 compared to controls. Blood plasma [Na(+)] was reduced by ~17 % over the same period, while allantoic fluid [Na(+)] was elevated in tRA-treated embryos compared to control embryos. Collectively, our data indicates that exogenous administration of tRA produces significant alterations to the developmental trajectory of the developing embryonic chicken.

Well-developed larval Xenopus laevis (NF stages 58shy;66) are oxygen regulators, at least during mild hypoxia. When and how they change from oxygen conformers (the presumed condition of the fertilized egg) to oxygen regulators is unknown. Also unknown is how anaerobic metabolic capabilities change during development, especially in response to acute hypoxia, and to what extent, if any, anaerobiosis is used to supplement aerobic metabolism. Consequently, we have investigated resting rates of oxygen consumption (M.O2) and concentrations of whole-body lactate (lactic acid) during development in normoxia and in response to acute hypoxia in Xenopus laevis. M.O2 increased in an episodic, non-linear fashion during development. Resting, normoxic M.O2 increased about tenfold (to approximately 0.20micro;mol g-1 h-1) between NF stages 1shy;39 and 40shy;44, and then another tenfold between NF stages 45shy;48 and 49shy;51 (to approximately 2.0micro;mol g-1 h-1), remaining at about 2micro;mol g-1 h-1 for the remainder of larval development. M.O2 reached its highest level in newly metamorphosed frogs (nearly 4micro;mol g-1 h-1), before decreasing to about 1.0micro;mol g-1 h-1 in large adults. X. laevis embryos and larvae up to NF stage 54shy;57 were oxygen conformers when exposed to variable levels of acute hypoxia. The only exception was NF stage 45shy;48 (external gills present yet body mass still very small), which showed some capability of oxygen regulation. All larvae older than stage 54shy;57 and adults were oxygen regulators and had the lowest values of Pcrit (the oxygen partial pressure at which M.O2 begins to decline). Whole-body lactate concentration in normoxia was about 1micro;mol g-1 for all larval groups, rising to about 12micro;mol g-1 in adults. Concentrations of lactic acid in NF stages 1shy;51 were unaffected by even severe ambient hypoxia. However, whole-body lactate levels in NF stages 52shy;66 increased in response to severe hypoxia, indicating that some anaerobic metabolism was being used to supplement diminishing aerobic metabolism. The largest increases in concentration of lactate occurred in late larvae and adults.

Hypoxia inhibits avian embryonic development, as well as increases embryonic mortality. However, the key organ systems affected by hypoxia, and their critical windows for development, are poorly understood. Consequently, chicken embryos were continuously exposed to 3 levels of oxygen (21, 15, or 13% O2) throughout d 0 to 10, d 11 to 18, or d 0 to 18 of incubation, followed by morphometric and blood physiological measurements. Hypoxia occurring early during incubation (d 0 to 10) had larger effects on embryonic mortality and organ growth than hypoxia occurring at later stages (d 10 to 18). Growth of the heart and chorioallantoic membrane was stimulated by chronic hypoxia, whereas the lung, brain, eye, liver, stomach, beak, and toes showed no disruption. Sustained hypoxia from the beginning of incubation decreased blood hemoglobin, hematocrit, and red blood cell concentration of embryos at d 10, but the values among hypoxic and normoxic groups were not significantly different at d 18. Blood partial pressure of O2 and partial pressure of CO2 were dependent upon incubation O2 level at a given day of development. These results indicated that either modest hypoxia (15% O2) throughout development, or hypoxia at any level during the late stages (d 11 to 18), increased the heart and chorioallantoic membrane weight, which partly compensated for the detrimental effects of hypoxia on embryonic development. We conclude that the first half of embryonic development contained the critical windows for the detrimental effects of hypoxia, and the second half contained the critical windows for the compensatory response of hypoxia in key organs.

The ontogeny of cardiac hypoxic responses, and how such responses may be modified by rearing environment, are poorly understood in amphibians. In this study, cardiac performance was investigated in Xenopus laevis from 2 to 25 days post-fertilization (dpf). Larvae were reared under either normoxia or moderate hypoxia (PO2 = 110 mmHg), and each population was assessed in both normoxia and acute hypoxia. Heart rate (f h ) of normoxic-reared larvae exhibited an early increase from 77 ± 1 beats min−1 at 2 dpf to 153 ± 1 beats min−1 at 4 dpf, followed by gradual decreases to 123 ± 3 beats min−1 at 25 dpf. Stroke volume (SV), 6 ± 1 nl, and cardiac output (CO), 0.8 ± 0.1 μl min−1, at 5 dpf both increased by more than 40-fold to 25 dpf with rapid larval growth (~30-fold increase in body mass). When exposed to acute hypoxia, normoxic-reared larvae increased f h and CO between 5 and 25 dpf. Increased SV in acute hypoxia, produced by increased end-diastolic volume (EDV), only occurred before 10 dpf. Hypoxic-reared larvae showed decreased acute hypoxic responses of EDV, SV and CO at 7 and 10 dpf. Over the period of 2–25 dpf, cardiac scaling with mass showed scaling coefficients of −0.04 (f h ), 1.23 (SV) and 1.19 (CO), contrary to the cardiac scaling relationships described in birds and mammals. In addition, f h scaling in hypoxic-reared larvae was altered to a shallower slope of −0.01. Collectively, these results indicate that acute cardiac hypoxic responses develop before 5 dpf. Chronic hypoxia at a moderate level can not only modulate this cardiac reflex, but also changes cardiac scaling relationship with mass.

SummaryMaternal influences are a potentially important component of transgenerational transfer of phenotype in vertebrates. This study on zebrafish (Danio rerio) examined how chronic hypoxic exposure on adults affected the phenotype of their offspring. Separate adult populations were exposed to hypoxia (13.1 kPa O2) or normoxia (21.1 kPa O2) for periods ranging from 1 to 12 weeks. Adults were then returned to normoxia and bred within experimental groups. Adult fecundity and egg characteristics (volume of egg, yolk and perivitelline fluid) were assessed. Subsequently, larval body length, time to loss of equilibrium in severe hypoxia (~4 kPa O2), and critical thermal minima (CTMin) and maxima (CTMax) were measured at 6, 9, 12, 15, 18, 21 and 60 days post fertilization (dpf). Adult fecundity was depressed by hypoxic exposure. Egg component volumes were also depressed in adults exposed to 1-2 weeks of hypoxia, but returned to control levels following longer hypoxic exposure. Adult hypoxic exposures of >1 week resulted in longer body lengths in their larval offspring. Time to loss of equilibrium in severe hypoxia (i.e. hypoxic resistance) in control larvae decreased from 6 to 12 dpf, remaining constant thereafter. Notably, hypoxic resistance from 6-18 dpf was ~15% lower in larvae whose parents were exposed to just 1 week of chronic hypoxia, but resistance was significantly increased by ~24-30% in 6-18 dpf in larvae from adults exposed to 2, 3 or 4 weeks of hypoxia. CTMin (~39.5°C) and CTMax (~10-12 °C) were unchanged by parental hypoxic exposure. This study demonstrates that parental hypoxic exposure in adult zebrafish has profound epigenetic effects on the morphological and physiological phenotype of their offspring.

Traditionally the process of respiration is divided into three phases: (1) cellular respiration, (2) transport of respiratory gases and (3) ventilation of the gas exchange organs (breathing). Thereby organisms assimilate chemical energy from the environment, and within their cells transfer it from molecule to molecule in a stepwise fashion. Although studied separately, these phases represent a continuum and cellular respiration in all life forms has much in common. Ironically, these respiratory foci have been artificially delineated by their own practitioners, who tend to publish in their own journals, and attend their own conferences. The goal of modern respiratory biology should be to understand biological connectivity and complexity by viewing an organism as a series of interconnecting systems from molecule to ecosystem. The future of science in general, and biology in particular, lies in disciplinary networking: combining the results of traditional disciplines to better understand the whole. Because of its universality, Respiratory Biology can best provide this bridge and improve interdisciplinary studies in biology generally. To this end, the First International Congress of Respiratory Biology was held from August 14 to 16, 2006, at Bonn, Germany. As evident from the success of this inaugural meeting, these are exciting times for Respiratory Biology. The explosion of "X-omics" and systems biology, the powerful genetic approaches to disease treatment, and the long-standing and newly emerging questions in evolutionary biology and ecology; all portend a continuing role of respiratory biology as a key integrative discipline.

SUMMARY Maternal effects are important in epigenetic determination of offspring phenotypes during all life stages. In the chicken (Gallus gallus domesticus), transgenerational transfer of egg yolk factors may set the stage for morphological and physiological phenotypic differences observed among breeds. To investigate the effect of breed-specific yolk composition on embryonic broiler and layer chicken phenotypes, we employed an ex ovo, xenobiotic technique that allowed the transfer of broiler and layer chicken embryos from their natural yolks to novel yolk environments. Embryonic day two broiler embryos developing on broiler yolk culture medium (YCM) had significantly higher heart rates than layer embryos developing on layer YCM (176±7 beats min–1 and 147±7 beats min–1, respectively). Broiler embryos developing on layer YCM exhibited heart rates typical of layer embryos developing normally on layer YCM. However, layer embryo heart rates were not affected by development on broiler YCM. Unlike O2 consumption, development rate and body mass of embryos were significantly affected by exposure to different yolk types, with both broiler and layer embryos displaying traits that reflected yolk source rather than embryo genotype. Analysis of hormone concentrations of broiler and layer egg yolks revealed that testosterone concentrations were higher in broiler yolk (4.63±2.02 pg mg–1vs 3.32±1.92 pg mg–1), whereas triiodothyronine concentrations were higher in layer yolk (1.05±0.18 pg mg–1vs 0.46±0.22 pg mg–1). Thus, a complex synergistic effect of breed-specific genotype and yolk environment exists early in chicken development, with yolk thyroid hormone and yolk testosterone as potential mediators of the physiological and morphological effects.

The influence of acetylcholine (ACh) on cardiac performance of larval (Taylor Kollros [TK] stages II-XVIII) and postmetamorphic (3–609 g) Rana catesbeiana was analyzed in situ (circulatory system intact) and in vitro (isolated heart or ventricular strip preparations). Topical application of ACh to the heart in situ resulted in a dose-dependent decrease in heart rate and in a slight decrease in systolic ventricular pressure in all developmental stages. Injection of acetylcholine in to the ventricle lumen in situ caused a dose-dependent transient decrease in systolic ventricular pressure, with little heart rate effect. Intraventricular ACh injection also changed the hemodynamic coupling between ventricle and conus arteriosus, generating a biphasic pressure profile in the conus due to sequential contractions of the ventricle and of the conus. In situ the sensitivity of the ventricle to ACh decreased during larval development, with the lowest sensitivity in small postmetamorphic adults. ACh applied in vitro to cardiac muscle strips or small hearts produced a negative inotropic effect. The ACh dose necessary to induce a 50% reduction in muscle strip contraction force in vitro decreased substantially during larval development, indicating an increase in ACh sensitivity with development. The effects of ACh both in vitro and in situ were diminished or eliminated by topical application or injection of atropine, suggesting the presence of muscarinic cholinergic receptors. After preincubation with the acetylcholinesterase blocker eserine, injection of ACh in to the conus arteriosus decreased systolic ventricular pressure with a delay of 4–10 seconds, probably representing the minimum blood circulation time. The observed inotropic and chronotropic responses result from the action of ACh on cardiac muscle, primarily affecting systolic ventricular pressure, and on the cardiac pacemaker, mainly influencing heart rate. These responses occur as early as TK, stage II, indicating a well-developed set of mechanisms to regulate cardiovascular performance early in development. © 1993 Wiley-Liss, Inc.

Metabolic rate, branchial morphology, and modes of gill ventilation were studied in young (2-10 g) North American paddlefish, Polyodon spathula, with anatomical, behavioral, and physiological methods. Polyodon lacks the oral and opercular valves that are typical for fishes that rely on a buccal pump system to ventilate the gills, and the jaw opening system of Polyodon is poorly suited for regular pumping movements. Unrestrained, undisturbed juvenile paddlefishes swim constantly at a mean speed of 1.1-1.5 body lengths · $s^{-1}$ (bls). The maximum speed sustainable for > 10 min is 1.6-1.8 bls. When forced to swim at slow speeds in flow tanks or water tunnels, ventilation of the gills by buccal pumping occurs at a frequency of 50-80 · $min^{-1}$. As swimming speed increases, buccal ventilation becomes intermittent and continuous ram ventilation occurs above 0.6-0.8 bls, which means that Polyodon is essentially an obligate ram ventilator under normal conditions. Oxygen consumption ($\dot{M}O_{2}$), carbon di...

Summary Epigenetics, the transgenerational transfer of phenotypic characters without modification of gene sequence, is a burgeoning area of study in many disciplines of biology. However, the potential impact of this phenomenon on the physiology of animals is not yet broadly appreciated, in part because the phenomenon of epigenetics is not typically part of the design of physiological investigations. Still enigmatic and somewhat ill defined is the relationship between the overarching concept of epigenetics and interesting transgenerational phenomena (e.g. ‘maternal/parental effects’) that alter the physiological phenotype of subsequent generations. The lingering effect on subsequent generations of an initial environmental disturbance in parent animals can be profound, with genes continuing to be variously silenced or expressed without an associated change in gene sequence for many generations. Known epigenetic mechanisms involved in this phenomenon include chromatin remodeling (DNA methylation and histone modification), RNA-mediated modifications (non-coding RNA and microRNA), as well as other less well studied mechanisms such as self-sustaining loops and structural inheritance. In this review we: (1) discuss how the concepts of epigenetics and maternal effects both overlap with, and are distinct from, each other; (2) analyze examples of existing animal physiological studies based on these concepts; and (3) offer a construct by which to integrate these concepts into the design of future investigations in animal physiology.

The duration of natural intertidal air exposure was determined for a population of Chiton stokesii from the rocky intertidal zone of Naos Island, Panama. On-site measurement indicated that chitons seek protected locations during air exposure and are little affected by increased air temperatures. Construction of an artificial tidal system allowed hemolymph sampling and assessment of physiological compensation of chitons during air exposure in the laboratory. During air exposure chitons showed significant hemoconcentration associated with reduction in heart rate and intracardiac hemolymph pressure. Air exposure was also associated with the buildup of hemolymph Pco₂ and the development of significant but largely compensated hemolymph acidosis. No consequent decrease in postbranchial hemolymph oxygenation occurred; indeed, the partial pressure of O₂ in postbranchial hemolymph (Pao₂) had increased slightly but significantly at 17 h air exposure. This increased oxygenation was not associated with significant ch...

The neotropical frog Eleutherodactylus coqui (Thomas) undergoes direct development within the egg, emerging at hatching as a juvenile frog. The aim of this study was to investigate cardiorespiratory changes that accompany development, as well as to determine how the developmental physiology of direct-developing anurans differs from anurans with free-living larval stages. Heart rate (fH), oxygen uptake , vitelline fluid pH, and the protein and lactate concentrations of vitelline fluid were measured as a function of development. At 24–25 °C, fH doubled from 50 to 100 beats min−1 during the early stages of heart development, then increased more slowly with later embryonic development to 120 beats min−1 at hatching. The act of hatching itself was accompanied by a transient increase in fH to 155 beats min−1, this mild tachycardia lasting for at least 2 h. In juveniles and adults, heart rate decreased with increasing body mass, falling to about 80beats min−1 in 6 g frogs at 24–25°C. at 24–25 °C increased from about 2μmol g egg−1h−1 for the earliest embryos examined to about 12μmolg egg−1h−1 at hatching. in adults decreased with increasing body size, ranging from 4 to 5μmolg−1h−1 in the largest adults examined (6g). Both fH and were also measured at 18°C. The Q10 for/it was between 1.5 and 2 throughout development. The Q10 for in embryos ranged between 3.5 and 6.5 until hatching, falling to 1.6–2.0 in juveniles and small adults. Vitelline fluid pH (24–25°C) decreased from 5.2’ in early embryonic stages to 4.7 at the time of hatching. Egg dehydration caused a significant decrease in the pH of vitelline fluid and was associated with a significant rise in fH. Vitelline fluid protein concentration was about 5–6mgdl−1, and showed no consistent change with development. Whole-egg lactate concentration ranged from 4 to 11 μ g g egg−1, and also showed no consistent change during development. These findings indicate considerable changes in cardiac and metabolic physiology associated with opment in Eleutherodactylus coqui. These changes appear to be associated with both organogenesis (primary influence before hatching) and body mass changes (primary influence after hatching).

Hemolymph volume in restrained land crabs (Cardisoma guanhumi) was altered in 5% increments by rapid infusion of filtered seawater into, or rapid withdrawal of hemolymph from, an infrabranchial sinus. Associated changes in intracardiac hemolymph pressure and in heart rate were measured. Elevation of hemolymph volume resulted in a brief (

There has been considerable recent interest in the development of the circulation in the zebrafish. Optical techniques typically used to visualize changes in heart size allow measurement of stroke volume during early vertebrate development, but this approach is complicated in zebrafish larvae because of the heart's irregular shape and its significant change in morphology during the first 6 d of development. By use of a three-dimensional integration of the early zebrafish heart and vessels, we have greatly reduced measurement error of stroke volume and cardiac output and have determined the cross-sectional growth of major vessels in the developing zebrafish larvae. A dramatic 500%-600% increase in cardiac output (from 10 to 50-60 nL min(-1)) occurs on days 5 and 6 postfertilization in Danio rerio. Cross-sectional area of key vessels (dorsal artery, caudal artery, dorsal vein) as well as between-individual variation significantly decreased over the first 6 d of development. Associated with the decrease in cross-sectional area is a significant increase in red blood cell velocity on days 5 and 6 postfertilization. Together, the three-dimensional data of the cardiac and vascular systems have shown that the most profound physiological and developmental changes occur in days 5 and 6, which corresponds with the appearance of the adult form of the heart and the transition from diffusive to convective O2 supply to internal tissues.

Toads experiencing dehydrating conditions exhibit complex physiological and behavioral responses, some of which can potentially impact cutaneous gas exchange, an important component of total gas exchange. We measured the effect of dehydration on cutaneous gas exchange in the xeric-adapted toad Bufo woodhousei. First, two pharmacological agents were used to stimulate cutaneous blood flow--phentolamine (an alpha-blocker) and isoproterenol, a beta-stimulant and powerful cardio-accelerator--to determine a relationship between cutaneous blood flow and water loss. Both drugs increased heart rate and blood pressure, and caused visually evident extensive vasodilation of the skin. Untreated toads in a dry air stream took an average of 10.1+/-0.7 h to dehydrate to 80% body mass, while animals treated with isoproterenol and phentolamine requires only 7.2+/-0.8 h and 7.4+/-0.9 h, respectively. Rehydration, which was more rapid than dehydration, was similarly accelerated in pharmacologically treated toads. Cutaneous gas exchange (M(O2), M(CO2)) and C18O diffusing capacity (D(Skin)C18O) were then examined in unanesthetized toads under different states of body hydration. Blood gases and hematocrit were measured separately but under identical conditions. In fully hydrated toads at 23-25 degrees C, cutaneous gas exchange values were: M(O2) = 1.43+/-0.47 micromol g(-1) h(-1), M(CO2) = 1.75+/-0.85 micromol g(-1) h(-1), and the respiratory exchange ratio R = 1.36+/-0.56 (N=6, mean + 1 S.D.). D(Skin)C18O was 0.48+/-0.03 micromol g body mass(-1) h(-1) kPa. Following an enforced 20-25% loss of body water, D(Skin)C18O fell by nearly 50% to 0.28+/-0.09 micromol g(-1) h(-1) kPa. However, cutaneous M(O2), M(CO2) and R were unchanged at 1.48+/-0.15 micromol g(-1) h(-1), 1.72+/-0.29 micromol g(-1) h(-1) and 1.13+/-0.08 micromol g(-1) h(-1), respectively. Partial pressure of arterial (sciatic) oxygen, Pa(O2), normally about 12-13 kPa, remained unchanged by dehydration, but Pa(CO2) increased about 250% from 0.93+/-0.27 up to 2.27+/-0.93 kPa. The fall in D(Skin)C18O during dehydration presumably results at least in part from decreased cutaneous blood flow, possibly in an attempt to reduce the transcutaneous water loss that would otherwise result during dehydrating conditions. Concurrently, cutaneous M(CO2) is maintained under dehyrdating conditions by a greatly increased Pa(CO2) diffusion gradient across the skin. Thus, Bufo woodhousei appears able to restrict cutaneous blood flow without compromising vital cutaneous CO2 loss.

SUMMARY Our understanding of avian embryonic cardiovascular regulation has been based on studies in chickens. The present study was undertaken to determine if the patterns established in chickens are generally applicable to the emu, a ratite bird species. We studied cardiovascular physiology over the interval from 60% to 90% of the emu's 50-day incubation period. During this period,embryonic emus exhibit a slight fall in resting heart rate (from 171 beats min-1 to 154 beats min-1) and a doubling of mean arterial pressure (from 1.2 kPa to 2.6 kPa). Exposures to 15% or 10%O2 initially decreased heart rate during the first period of emu incubation studied [60% of incubation (60%I)] but increased heart rate in the 90%I group. Arterial pressure responded to hypoxia with an initial depression(-1.6 kPa) at 60%I and 70%I but showed no response during the later periods of incubation (80%I and 90%I). In addition, tonic stimulation of both cholinergic and adrenergic (α and β) receptors was present on heart rate at 70%I, with the cholinergic and β-adrenergic tone increasing in strength by 90%I. Arterial pressure was dependent on a constant β-adrenergic and constant α-adrenergic tone from 60%I to 90%I. A comparison with embryonic white leghorn chickens over a similar window of incubation revealed that emus and white leghorn chickens both possess an adrenergic tone on heart rate and pressure but that only emus possess a cholinergic tone on heart rate. Collectively, these data indicate that the maturation of cardiovascular control systems differs between white leghorn chickens and emus, inviting investigation of additional avian species to determine other patterns.

SUMMARYLarval zebrafish (Danio rerio) of three different age classes (‘yolk-sac’ larvae, 96 h; ‘swim-up’ larvae, 9 days old; and ‘free-swimming’ larvae, 21 days old) were trained for 2, 6 and 11 days, respectively, to swim at 0 body lengths per second (BL s–1), 2 BL s–1 and 5 BL s–1. Survival was significantly diminished in larvae trained at 5 BL s–1 compared to controls (0 BL s–1). Although training produced no significant differences in mass and length, the youngest larvae absorbed their yolk at a faster rate during training. Routine oxygen consumption (ṀO2r) and mass-specific routine oxygen consumption (ṀO2r,m) were not significantly affected by chronic training in the yolk-sac larvae and swim-up larvae. However, trained free-swimming larvae had a significantly higher ṀO2r (after 11 days of training) and ṀO2r,m (after 8 and 11 days of training) compared to control larvae. Trained free-swimming larvae consumed significantly less oxygen during swimming compared to control larvae, as measured by closed-system respirometry. Trained yolk-sac larvae exposed to increasing hypoxia levels were more effective O2 regulators. Additionally, training enhanced survival during exposure to extreme hypoxia in all age groups. Thus, physiological acclimation to chronic swimming occurs in the earliest stages of life in the zebrafish.

While an extensive literature on cardiovascular development exists for insects, almost all studies focus on in vitro preparations, and very few report on more than a single developmental stage. The present study examines in vivo cardiac performance in the intact, unanesthetized larvae, pupae and adults of the tobacco hornworm Manduca sexta. For all three stages, electrode pairs of fine steel wire were inserted subcuticularly at two dorsal abdominal locations. Impedance signals produced by contraction of the dorsal abdominal vessel (tube heart) were amplified and recorded. In addition to providing heart rate, a comparison of the relative timing of the signal from each electrode pair allowed the calculation of the propagation velocity and direction of heart contraction. Experimental treatments of intact animals included exposure to hypoxia and hyperoxia (21 %, 15 %, 10 %, 5 %, 0 % and 100 % O(2)), to hypercapnia (0 %, 4 %, 8 %, 16 %, 20 % and 24 % CO(2)), to temperature variation (10, 20 and 30 degrees C) and to 2 min periods of forced activity. The pattern of contraction of the dorsal abdominal vessel of M. sexta changed substantially with developmental stage. Larvae showed a relatively simple, invariably posterior-to-anterior pattern (mean rate 34.8+/−1.16 beats min(−)(1)). The heart rate pattern in pupal M. sexta displayed great variability in rate, amplitude and direction. Periods of regular heart beats (21.5+/−1.09 beats min(−)(1)) were frequently and irregularly interrupted by periods of cardiac arrests ranging from a few seconds to over 20 min. Adults showed a highly stereotypic but complex pattern, with periods of ‘fast forward’ (FF; rate 47.6+/−2.6 beats min(−)(1)), ‘slow forward’ (SL; 32.8+/−3.0 beats min(−)(1)) and ‘reversed’ (R; 32.2+/−2.4 beats min(−)(1)) beating. The contraction propagation velocity in larvae and pupae averaged 5. 52+/−0.36 and 2.03+/−0.11 cm s(−)(1), respectively. The SF, R and FF phases of the adults had average propagation velocities of 5.52+/−0. 51, 5.05+/−0.52 and 5.43+/−0.37 cm s(−)(1), respectively. Heart rate and contraction propagation velocity were remarkably resistant to ambient hypoxia and hypercapnia at all developmental stages, decreasing significantly only at 0 % O(2) or 24 % CO(2). As expected, the heart rates of all three developmental stages increased significantly with increasing temperature, with heart rate Q(10) values for larvae, pupae and adults of 2.33, 3.14 and 1.61, respectively, between 10 and 20 degrees C. Corresponding Q(10) values for these stages between 20 and 30 degrees C were 2.22, 2.03 and 2.29. Larval heart rates showed no significant response to forced activity induced by prodding. In contrast, adult heart rate increased nearly fivefold from 50.1 beats min(−)(1) during rest to 223.5 beats min(−)(1) after 1 min of prodding. The activity-induced tachycardia in adults ceased within 10–12 min. Patterns of cardiac contraction in larval, pupal and adult M. sexta were as dissimilar as their morphological appearances and revealed a gradation from simple to complex. These developmentally based distinctive cardiac patterns are undoubtedly related to developmental differences in both morphology and life-style. Larvae are anatomically ‘homogeneous’ compared with other stages, with no distinct head, thorax and abdominal region (or wings) that might require selective perfusion or drainage. The far more complex pattern of heart activity seen in pupae probably relates to the dramatic changes in internal morphology during this stage. Simultaneous degradation and synthesis of tissues throughout the body may expose the heart to numerous peptides or neurohormones that affect cardiac activity. In adult moths, the complex and repetitive pattern of cardiac activity is reflected in the previously described complexity of hemolymph movement, together with thermoregulatory capabilities in this species that depend on well-regulated hemolymph movements between the thorax, wings and abdomen.

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