Temperature and hypoxia trigger developmental phenotypic plasticity of cardiorespiratory physiology and growth in the parthenogenetic marbled crayfish, Procambarus virginalis Lyko, 2017.

Attempting to differentiate phenotypic variation caused by environmentally-induced alterations in gene expression from that caused by actual allelic differences can be experimentally difficult. Environmental variables must be carefully controlled and then interindividual genetic differences ruled out as sources of phenotypic variation. We investigated phenotypic variability of cardiorespiratory physiology as well as biometric traits in the parthenogenetically-reproducing marbled crayfish Procambarus virginalis Lyko, 2017, all offspring being genetically identical clones. Populations of P. virginalis were reared from eggs tank-bred at four different temperatures (16, 19, 22 and 25 °C) or two different oxygen levels (9.5 and 20 kPa). Then, at Stage 3 and 4 juvenile stages, physiological (heart rate, oxygen consumption) and morphological (carapace length, body mass) variables were measured. Heart rate and oxygen consumption measured at 23 °C showed only small effects of rearing temperature in Stage 3 juveniles, with larger effects evident in older, Stage 4 juveniles. Additionally, coefficients of variation were calculated to compare our data to previously published data on P. virginalis as well as sexually-reproducing crayfish. Comparison revealed that carapace length, body mass and heart rate (but not oxygen consumption) indeed showed lower, yet notable coefficients of variation in clonal crayfish. Yet, despite being genetically identical, significant variation in their morphology and physiology in response to different rearing conditions nonetheless occurred in marbled crayfish. This suggests that epigenetically induced phenotypic variation might play a significant role in asexual but also sexually reproducing species.

Short communication: Baroreflex function in embryonic emus (Dromiceius novaehollandiae).

The baroreflex involves cardiovascular homeostatic mechanisms that buffer the system against acute deviations in arterial blood pressure. It is comprised of the cardiac limb which involves adjustments in heart rate and the peripheral limb which involves adjustments in vascular resistance. This negative feedback loop mechanism has been investigated in numerous species of adult vertebrates, however our understanding of the maturation and functional importance of the reflex in developing animals remains poorly understood. In egglaying species, our knowledge of this mechanism is limited to the domestic chicken embryo and the embryonic alligator. While each of these species possess a cardiac baroreflex prior to hatching, they differ in the timing when it becomes functional, with the embryonic chicken possessing the reflex at 90% of incubation, while the alligator possesses the reflex at 70% of incubation. In an effort to determine if bird species might share similar patterns of active baroreflex function, we studied embryonic emus (Dromiceius novaehollandiae). However, we hypothesized that emus would possess a pattern of baroreflex function similar to that of the American alligator given the emu embryo possesses functional vagal tone at 70% of incubation, possibly indicating a more mature collection of cardiovascular control mechanism than those found in embryonic chickens. Our findings illustrate that emu embryos possess a hypotensive baroreflex at 90% of incubation. Therefore, our data fail to support our original hypothesis. While only two species of birds have been studied in this context, it could indicate that baroreflex function is not essential for cardiovascular homeostasis in birds for the majority of in ovo development.

Insufficient reporting of experimental variables as a cause for nonreproducibility in animal physiology? A case study.

Nonreproducibility in scientific investigations has been explained by inadequately reporting methodology, honest error, and even misconduct. We hypothesized that, within the field of animal physiology, the most parsimonious explanation for nonreproducibility is inadequate reporting of key methodological details. We further hypothesized that implementation of relatively recently released reporting guidelines has positively impacted journal article quality, as measured by completeness of the methodology descriptions. We analyzed 84 research articles published in five primarily organismal animal physiology journals in 2008-2010 (generally before current guidelines) and 2018-2020. Compliance for reporting 34 variables referring to biology, experiments, and data collection was assessed. Reporting compliance was just ∼61% in 2008-2010, rising only slightly to 67.5% for 2018-2020. Only 21% of the reported variables showed significant differences across the period from 2008-2020. We conclude that, despite attempts by societies and journals to promote greater reporting compliance, such efforts have so far been relatively unsuccessful in the field of animal physiology.

Hypoxia- and hyperoxia-related gene expression dynamics during developmental critical windows of the tropical gar Atractosteus tropicus.

Aquatic hypoxia is both a naturally-occurring and anthropogenically-generated event. Fish species have evolved different adaptations to cope with hypoxic environments, including gill modifications and air breathing. However, little is known about the molecular mechanisms involved in the respiration of embryonic and larval fishes during critical windows of development. We assessed expression of the genes hif-1α, fih-1, nhe1, epo, gr and il8 using the developing tropical gar as a piscine model during three developmental periods (fertilization to hatch, 1 to 6 days post hatch (dph) and 7 to 12 dph) when exposed to normoxia (~7.43 mg/L DO), hypoxia (~2.5 mg/L DO) or hyperoxia (~9.15 mg/L DO). All genes had higher expression when fish were exposed to either hypoxia or hyperoxia during the first two developmental periods. However, fish continuously exposed to hypoxia had increased expression of the six genes by hatching and 6 dph, and by 12 dph only hif-1α still had increased expression. The middle developmental period was the most hypoxia-sensitive, coinciding with several changes in physiology and morphology. The oldest larvae were the most resilient to gene expression change, with little variation in expression of the six genes compared. This study is the first to relate the molecular response of an air-breathing fish to oxygen availability to developmental critical windows and contributes to our understanding of some molecular responses of developing fish to changes in oxygen availability.

Putting the August Krogh principle to work in developmental physiology.

The August Krogh principle has guided many comparative physiological studies, being especially useful for developmental physiology. Several attributes of unusual, if not unique, animals enable researchers to understand developmental phenomena more generally - the essence of the Krogh principle. This article provides examples of unusual traits of animals currently being used to understand development and reproduction. 1) Accelerated development greatly minimizes time spent examining how animals develop across time from egg to adult. For example, the tropical gar begins to breath air within as little as 2.5 days after hatching - much faster than other air-breathing fishes - facilitating study of the development of respiratory reflexes in fishes. 2) Transparency of the body wall has been exploited to image cardiac output in near-microscopic larvae of the zebrafish and mahi mahi, and to capitalize on bacterial biosensors to investigate development of in vivo digestive function in Caenorhabditis elegans. 3) Gigantism, as in the chicken-sized embryos of the emu, or the larvae of the paradoxical frog, allows surgeries not otherwise feasible. 4) Reproductive traits such as polyembryony in armadillos and parthenogenesis in planaria have informed us about classic gene vs. environment questions. Finally, 5) large body mass range enables clearer allometric analyses. Insects like the silk moth, show a more than a 1000-fold difference between eggs and adults. The August Krogh principle, then, is not simply to justify the study of exotic animals (as interesting as that is!), but has been used to generate a broader synthesis and understanding of all taxa.

Exposure to Crude Oil Induces Retinal Apoptosis and Impairs Visual Function in Fish.

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil are known to impair visual development in fish. However, the underlying mechanism of PAH-induced toxicity to the visual system of fish is not understood. Embryonic zebrafish (Danio rerio) at 4 h post fertilization were exposed to weathered crude oil and assessed for visual function using an optokinetic response, with subsequent samples taken for immunohistochemistry and gene expression analysis. Cardiotoxicity was also assessed by measuring the heart rate, stroke volume, and cardiac output, as cardiac performance has been proposed to be a contributing factor to eye-associated malformations following oil exposure. Larvae exposed to the highest concentrations of crude oil (89.8 µg/L) exhibited an increased occurrence of bradycardia, though no changes in stroke volume or cardiac output were observed. However, genes important in eye development and phototransduction were downregulated in oil-exposed larvae, with an increased occurrence of cellular apoptosis, reduced neuronal connection, and reduced optokinetic behavioral response in zebrafish larvae.

Mahi-mahi (Coryphaena hippurus) life development: morphological, physiological, behavioral and molecular phenotypes.

BACKGROUND: Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important fish species that is widely distributed in tropical and subtropical waters. Biological attributes and reproductive capacities of mahi-mahi make it a tractable model for experimental studies. In this study, life development of cultured mahi-mahi from the zygote stage to adult has been described. RESULTS: A comprehensive developmental table has been created reporting development as primarily detailed observations of morphology. Additionally, physiological, behavioral, and molecular landmarks have been described to significantly contribute in the understanding of mahi life development. CONCLUSION: Remarkably, despite the vast difference in adult size, many developmental landmarks of mahi map quite closely onto the development and growth of Zebrafish and other warm-water, active Teleost fishes.

Inadequacy of typical physiological experimental protocols for investigating consequences of stochastic weather events emerging from global warming.

Increasingly variable, extreme, and nonpredictable weather events are predicted to accompany climate change, and such weather events will especially affect temperate, terrestrial environments. Yet, typical protocols in comparative physiology that examine environmental change typically employ simple step-wise changes in the experimental stressor of interest (e.g., temperature, water availability, oxygen, nutrition). Such protocols fall short of mimicking actual natural environments and may be inadequate for fully exploring the physiological effects of stochastic, extreme weather events. Indeed, numerous studies from the field of thermal biology, especially, indicate nonlinear and sometimes counterintuitive findings associated with variable and fluctuating (but rarely truly stochastic) protocols for temperature change. This Perspective article suggests that alternative experimental protocols should be employed that go beyond step-wise protocols and even beyond variable protocols employing circadian rhythms, for example, to those that actually embrace nonpredictable elements. Such protocols, though admittedly more difficult to implement, are more likely to reveal the capabilities (and, importantly, the limitations) of animals experiencing weather, as distinct from climate. While some possible protocols involving stochasticity are described as examples to stimulate additional thought on experimental design, the overall goal of this Perspective article is to encourage comparative physiologists to entertain incorporation of nonpredictable experimental conditions as they design future experimental protocols.

Parental stressor exposure simultaneously conveys both adaptive and maladaptive larval phenotypes through epigenetic inheritance in the zebrafish (Danio rerio).

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. In the present study, crude oil was used as an example of an environmental stressor. Adult zebrafish (P0) were dietarily exposed for 3 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 groups. However, the P0 fecundity of both sexes decreased in proportion to the amount of oil fed. The F1 larvae from each P0 were then exposed from 3 hpf to 5 dpf 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 with controls (P<0.001). Unexpectedly, from day 3 to 5 of exposure, F1 larvae from oil-exposed parents showed poorer survival in clean water (up to 55% decreased survival). Additionally, parental oil exposure induced bradycardia (presumably maladaptive) in F1 larvae in both clean and oiled water. We conclude that epigenetic transgenerational inheritance can lead to an immediate and simultaneous inheritance of both beneficial and maladaptive traits in a large proportion of the F1 larvae. The adaptive responses may help fish populations survive when facing transient environmental stressors.

Metabolic physiology of the Mayan cichlid fish (Mayaheros uropthalmus): Re-examination of classification as an oxyconformer.

The Mayan cichlid (Mayaheros uropthalmus) is a freshwater fish inhabiting warm, potentially hypoxic and/or brackish waters, in Mexico and Central America. Despite its description as highly hypoxia tolerant, M. uropthalmus has been classified physiologically as an 'oxyconformer', which would place it in a very small (and shrinking) category of fishes that purportedly cannot maintain oxygen consumption (MO2) as ambient PO2 falls. However, hypoxia tolerance is often associated with strong oxyregulation, not oxyconformation as described for M. uropthalmus. To resolve these inconsistencies, we measured MO2, the ambient PO2 at which MO2 begins to decline as PO2 falls (PCrit), and gill ventilation rate (fG) in the Mayan cichlid. Variables were measured at 23o, 28 o and 33 °C and temperature sensitivity (Q10) calculated for each function. MO2 at air saturation was 2.9 ±â€¯0.2, 4.3 ±â€¯0.4, and 5.9 ±â€¯0.3 µmol O2/g/h at 23o, 28o and 33 °C, respectively. PCrits were low at 2.6 ±â€¯0.8 kPa, 3.2 ±â€¯0.8 kPa and 4.7 ±â€¯0.9 kPa at 23o, 28o and 33 °C, respectively. Q10 values for MO2 were 2.56 ±â€¯0.21 (23-28 °C), 1.89 ±â€¯0.15 (28-33 °C) and 2.2 ±â€¯0.1 (full temperature range of 23-33 °C), suggesting overall Q10s typical for tropical freshwater fish. fG was 39 ±â€¯3, 45 ±â€¯4, and 53 ±â€¯6 breaths/min at 23o, 28o and 33 °C, respectively, and increase 2-3 fold in severe hypoxia at each temperature. Experiments employing hyperoxia up to 35 kPa indicate a strong 'hypoxic drive' for gill ventilation. Collectively, these data show that, in contrast to a previous characterization, the Mayan cichlid is a strong oxyregulator exhibiting attributes (e.g. very low PCrit) typical of very hypoxia-tolerant fishes.

Developmental changes in oxygen consumption and hypoxia tolerance in the heat and hypoxia-adapted tabasco line of the Nile tilapia Oreochromis niloticus, with a survey of the metabolic literature for the genus Oreochromis.

The genus Oreochromis is among the most popular of the tilapiine cichlid tribe for aquaculture. However, their temperature and hypoxia tolerance, if tested at all, is usually tested at temperatures of 20-25°C, rather than at the considerably higher temperatures of 30-35°C typical of tropical aquaculture. We hypothesized that both larvae and adults of the heat and hypoxia-adapted Tabasco-line of the Nile tilapia Oreochromis niloticus would be relatively hypoxia-tolerant. Oxygen consumption rate ( M ˙ O 2 ), Q10 and aquatic surface respiration (ASR) was measured using closed respirometry at 2 (c. 0.2 g), 30 (c. 2-5 g), 105 c. (10-15 g) and 240 (c. 250 g) days of development, at 25°C, 30°C and 35°C. M ˙ O 2 at 30°C was inversely related to body mass: c. 90 µM O2 g-1 /h in larvae down to c. 1 µM O2 g-1 /h in young adults. Q10 for M ˙ O 2 was typical for fish over the range 25-35°C of 1.5-2.0. ASR was exhibited by 50% of the fish at pO2 of 15-50 mmHg in a temperature-dependent fashion. However, the largest adults showed notable ASR only when pO2 fell to below 10 mmHg. Remarkably, pcrit for M ˙ O 2 was 12-17 mmHg at 25-30°C and still only 20-25 mmHg across development at 35°C. These values are among the lowest measured for teleost fishes. Noteworthy is that all fish maintain equilibrium, ventilated their gills and showed routine locomotor action for 10-20 min after M ˙ O 2 ceased at near anoxia and when then returned to oxygenated waters, all fish survived, further indicating a remarkable hypoxic tolerance. Remarkably, data assembled for M ˙ O 2 from >30 studies showed a > x2000 difference, which we attribute to calculation or conversion errors. Nonetheless, pcrit was very low for all Oreochromis sp. and lowest in the heat and hypoxia-adapted Tabasco line.

Hematology from embryo to adult in the bobwhite quail (Colinus virginianus): Differential effects in the adult of clutch, sex and hypoxic incubation.

Hematology and its regulation in developing birds have been primarily investigated in response to relatively short-term environmental challenges in the embryo. Yet, whether any changes induced in the embryo persist into adulthood as a hematological form of "fetal programming" is unknown. We hypothesized that: 1) chronic as opposed to acute hypoxic incubation will alter hematological respiratory variables in embryos of bobwhite quail (Colinus virginianus), and 2) alterations first appearing in the embryo will persist into hatchlings through into adulthood. To test these hypotheses, we first developed an embryo-to-adult profile of normal hematological development by measuring hematocrit (Hct), red blood cell concentration ([RBC]), hemoglobin concentration ([Hb]), mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration, as well plasma osmolality. Hct, [RBC] and [Hb] in normoxic-incubated birds (controls) steadily increased from ~22%, ~1.6 × 106 µL-1 and ~7 g% in day 12 embryos to almost double the values at maturity in adult birds. Both cohort and sex affected hematology of normoxic-incubated birds. A second population, incubated from day 0 (d0) in 15% O2, surprisingly revealed little or no significant difference from controls in hematology in embryos. In hatchlings and adults, hypoxic incubation caused no significant modification to any variables. Compared to major hematological effects caused by hypoxic incubation in chickens, the hematology of the bobwhite quail embryo appears to be minimally affected by hypoxic incubation, with very few effects induced during hypoxic incubation actually persisting into adulthood.

Incubation relative humidity induces renal morphological and physiological remodeling in the embryo of the chicken (Gallus gallus domesticus).

The metanephric kidneys of the chicken embryo, along with the chorioallantoic membrane, process water and ions to maintain osmoregulatory homeostasis. We hypothesized that changes in relative humidity (RH) and thus osmotic conditions during embryogenesis would alter the developmental trajectory of embryonic kidney function. White leghorn chicken eggs were incubated at one of 25-30% relative humidity, 55-60% relative humidity, and 85-90% relative humidity. Embryos were sampled at days 10, 12, 14, 16, and 18 to examine embryo and kidney mass, glomerular characteristics, body fluid osmolalities, hematological properties, and whole embryo oxygen consumption. Low and especially high RH elevated mortality, which was reflected in a 10-20% lower embryo mass on D18. Low RH altered several glomerular characteristics by day 18, including increased numbers of glomeruli per kidney, increased glomerular perfusion, and increased total glomerular volume, all indicating potentially increased functional kidney capacity. Hematological variables and plasma and amniotic fluid osmolalities remained within normal physiological values. However, the allantoic, amniotic and cloacal fluids had a significant increase in osmolality at most developmental points sampled. Embryonic oxygen consumption increased relative to control at both low and high relative humidities on Day 18, reflecting the increased metabolic costs of osmotic stress. Major differences in both renal structure and performance associated with changes in incubation humidity occurred after establishment of the metanephric kidney and persisted into late development, and likely into the postnatal period. These data indicate that the avian embryo deserves to be further investigated as a promising model for fetal programming of osmoregulatory function, and renal remodeling during osmotic stress.

Developmental cardiorespiratory physiology of the air-breathing tropical gar, Atractosteus tropicus.

The physiological transition to aerial breathing in larval air-breathing fishes is poorly understood. We investigated gill ventilation frequency (fG), heart rate (fH), and air breathing frequency (fAB) as a function of development, activity, hypoxia, and temperature in embryos/larvae from day (D) 2.5 to D30 posthatch of the tropical gar, Atractosteus tropicus, an obligate air breather. Gill ventilation at 28°C began at approximately D2, peaking at ∼75 beats/min on D5, before declining to ∼55 beats/min at D30. Heart beat began ∼36-48 h postfertilization and ∼1 day before hatching. fH peaked between D3 and D10 at ∼140 beats/min, remaining at this level through D30. Air breathing started very early at D2.5 to D3.5 at 1-2 breaths/h, increasing to ∼30 breaths/h at D15 and D30. Forced activity at all stages resulted in a rapid but brief increase in both fG and fH, (but not fAB), indicating that even in these early larval stages, reflex control existed over both ventilation and circulation prior to its increasing importance in older fishes. Acute progressive hypoxia increased fG in D2.5-D10 larvae, but decreased fG in older larvae (≥D15), possibly to prevent branchial O2 loss into surrounding water. Temperature sensitivity of fG and fH measured at 20°C, 25°C, 28°C and 38°C was largely independent of development, with a Q10 between 20°C and 38°C of ∼2.4 and ∼1.5 for fG and fH, respectively. The rapid onset of air breathing, coupled with both respiratory and cardiovascular reflexes as early as D2.5, indicates that larval A. tropicus develops "in the fast lane."

Salt sensitivity of the morphometry of Artemia franciscana during development: a demonstration of 3D critical windows.

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.

Dynamics of epigenetic phenomena: intergenerational and intragenerational phenotype 'washout'.

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.

Dynamics of blood viscosity regulation during hypoxic challenges in the chicken embryo (Gallus gallus domesticus).

Hypoxia in chicken embryos increases hematocrit (Hct), blood O2 content, and blood viscosity. The latter may limit O2 transport capacity (OTC) via increased peripheral resistance. Hct increase may result from increased nucleated red blood cell concentration ([RBC]) and mean corpuscular volume (MCV) or reduced plasma volume. We hypothesized changes in Hct, hemoglobin concentration ([Hb]), [RBC] and MCV and their effects on viscosity would reduce OTC. Five experimental treatments that increase Hct were conducted on day 15 embryos: 60min water submergence with 60min recovery in air; exposure to 15% O2 with or without 5% CO2 for 24 h with 6 h recovery; or exposure to 10% O2 with or without 5% CO2 for 120 min with 120 min recovery. Control Hct, [Hb], [RBC], MCV, and viscosity were approximately 26%, 9g%, 2.0 10(6)µL(-1), 130µm(3), and 1.6mPas, respectively. All manipulations increased Hct and blood viscosity without changing blood osmolality (276mmolkg(-1)). Increased viscosity was attributed to increased [RBC] and MCV in submerged embryos, but solely MCV in embryos experiencing 10% O2 regardless of CO2. Blood viscosity in embryos exposed to 15% O2 increased via increased MCV alone, and viscosity was constant during recovery despite increased [RBC]. Consequently, blood viscosity was governed by MCV and [RBC] during submergence, while MCV was the strongest determinant of blood viscosity in extrinsic hypoxia with or without hypercapnia. Increased Hct and blood O2 content did not compensate for the effect of increased viscosity on OTC during these challenges.

Epigenetics as a source of variation in comparative animal physiology - or - Lamarck is lookin' pretty good these days.

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.

The actions of the renin-angiotensin system on cardiovascular and osmoregulatory function in embryonic chickens (Gallus gallus domesticus).

Using embryonic chickens (Gallus gallus domesticus), we examined the role of the renin-angiotensin system (RAS) in cardiovascular and osmotic homeostasis through chronic captopril, an angiotensin-converting enzyme (ACE) inhibitor. Captopril (5 mg kg⁻¹ embryo wet mass) or saline (control) was delivered via the egg air cell daily from embryonic day 5-18. Mean arterial pressure (MAP), heart rate (ƒ(H)), fluid osmolality and ion concentration, and embryonic and organ masses were measured on day 19. Exogenous angiotensin I (ANG I) injection did not change MAP or ƒ(H) in captopril-treated embryos, confirming ACE inhibition. Captopril-treated embryos were significantly hypotensive, with MAP 15% lower than controls, which we attributed to the loss of vasoconstrictive ANG II action. Exogenous ANG II induced a relatively greater hypertensive response in captopril-treated embryos compared to controls. Changes in response to ANG II following pre-treatment with phentolamine (α-adrenergic antagonist) indicated a portion of the ANG II response was due to circulating catecholamines in captopril-treated embryos. An increase in MAP and ƒ(H) in response to hexamethonium indicated vagal tone was also increased in the absence of ACE activity. Captopril-treated embryos had lower osmolality, lower Na⁺ and higher K⁺ concentration in the blood, indicating osmoregulatory changes. Larger kidney mass in captopril-treated embryos suggests disrupting the RAS may stimulate kidney growth by decreasing resistance at the efferent arteriole and increasing the fraction of cardiac output to the kidneys. This study suggests that the RAS, most likely through ANG II action, influences the development of the cardiovascular and osmoregulatory systems.