There is no limitation for CO2 excretion across the lung in exercising American alligators (Alligator mississippiensis).

Vertebrates utilize various respiratory organs such as gills, lungs and skin in combination with diverse cardiovascular structures, including single-, three- and four-chambered hearts, to enable oxygen delivery and carbon dioxide removal. They also exhibit differences in aerobic and anaerobic metabolism during exertion, but the cardiorespiratory gas transport of all vertebrates is a four-step process governed by Fick's Principle and Fick's Law of Diffusion over the entire range of metabolic rates. Hillman et al. (2013) suggested that previous exercise studies have focused too narrowly on mammals and proposed that the cardiorespiratory system's excess capacity serves an evolutionary role in enhancing CO2 excretion in non-mammalian vertebrates. In contrast, an analysis by Hicks and Wang (2021) concluded that vertebrates maintain effective gas exchange even at peak activity, finding no evidence of arterial hypercapnia at maximal oxygen consumption and thus challenging the proposal of significant limitations to pulmonary or branchial CO2 efflux. In the present study, we investigated the limits for CO2 exchange in exercising American alligators (Alligator mississippiensis) and provide evidence that the cardiorespiratory system is adequately built to sustain CO2 excretion during strenuous exercise and maintain arterial PCO2, with no evidence of diffusion limitation for pulmonary CO2 excretion.

Temperature effects on blood gases in embryonic American alligators (Alligator mississippiensis).

Numerous studies report on the influence of temperature on blood gases in ectothermic vertebrates, but there is merely a cursory understanding of these effects in developing animals. Animals that develop in eggs are at the mercy of environmental temperature and are expected to lack the capacity to regulate gas exchange and may regulate blood gases by means of altered conductance for gas exchange. We, therefore, devised a series of studies to characterize the developmental changes in blood gases when embryonic alligators were exposed to 25, 30 and 35 °C. To determine how blood parameters were impacted by changes in embryonic temperature, blood was sampled from the chorioallantoic membrane artery. The blood in the chorioallantoic membrane artery is a mixture of oxygen-poor and oxygen-rich blood, which based on the embryonic vascular anatomy may reflect blood that perfuses the chemoreceptors of the developing animal. Our findings indicate that following a 48 h exposure to 25 °C or 35 °C, there was a positive relationship between CAM artery blood PO2, PCO2 and glucose. However, blood pH suggests embryonic alligators lack an acute regulatory mechanism for adjusting blood pH.

Short communication: Characterizing arterial and venous blood gases over the gas exchange surface, the chorioallantoic membrane, of embryonic American alligators (Alligator mississippiensis) at two points of development.

Assessments of arterial and venous blood gases are required to understand the function of respiratory organs in animals at different stages of development. We measured blood gases in the arteries entering and veins leaving the chorioallantoic membrane (CAM) in embryonic alligators (Alligator mississippiensis). The CAM accounts for virtually all gas exchange in these animals, and we hypothesized that the CAM vasculature would be larger in eggs incubated in hypoxia (10% O2 for 50% or 70% of incubation), which would be reflected in a lower partial pressure of CO2 (PCO2). Contrary to this hypothesis, our measurements revealed no effects of hypoxic incubation on PCO2, and seemingly no increase in vascularization of the CAM in response to incubation in 10% O2. PCO2 was lower on the venous side, but only significantly different from arterial blood at 70% of incubation. The calculated blood flow to the CAM increased with development and was lower in both groups of alligators that had been incubated in hypoxia. Future studies should include measurements of blood parameters taken from embryos held in conditions that mirror incubation O2 levels, in combination with direct measurements of CAM artery blood flow.

Validating osteological correlates for the hepatic piston in the American alligator (Alligator mississippiensis).

Unlike the majority of sauropsids, which breathe primarily through costal and abdominal muscle contractions, extant crocodilians have evolved the hepatic piston pump, a unique additional ventilatory mechanism powered by the diaphragmaticus muscle. This muscle originates from the bony pelvis, wrapping around the abdominal viscera, extending cranially to the liver. The liver then attaches to the caudal margin of the lungs, resulting in a sub-fusiform morphology for the entire "pulmo-hepatic-diaphragmatic" structure. When the diaphragmaticus muscle contracts during inspiration, the liver is pulled caudally, lowering pressure in the thoracolumbar cavity, and inflating the lungs. It has been established that the hepatic piston pump requires the liver to be displaced to ventilate the lungs, but it has not been determined if the lungs are freely mobile or if the pleural tissues stretch ventrally. It has been hypothesized that the lungs are able to slide craniocaudally with the liver due to the smooth internal ceiling of the thoracolumbar cavity. We assess this through ultrasound video and demonstrate quantitatively and qualitatively that the pulmonary tissues are sliding craniocaudally across the interior thoracolumbar ceiling in actively ventilating live juvenile, sub-adult, and adult individuals (n = 7) of the American alligator (Alligator mississippiensis) during both natural and induced ventilation. The hepatic piston is a novel ventilatory mechanism with a relatively unknown evolutionary history. Questions related to when and under what conditions the hepatic piston first evolved have previously been left unanswered due to a lack fossilized evidence for its presence or absence. By functionally correlating specific characters in the axial skeleton to the hepatic piston, these osteological correlates can be applied to fossil taxa to reconstruct the evolution of the hepatic piston in extinct crocodylomorph archosaurs.

Hypoxic incubation at 50% of atmospheric levels shifts the cardiovascular response to acute hypoxia in American alligators, Alligator mississippiensis.

We designed a series of studies to investigate whether hypoxia (10% O2) from 20% of incubation to hatching, or from 20 to 50% of incubation, affects cardiovascular function when juvenile American alligators reached an age of 4-5 years compared to juveniles that were incubated in 21% O2. At this age, we measured blood flows in all the major arteries as well as heart rate, blood pressure, and blood gases in animals in normoxia and acute hypoxia (10% O2 and 5% O2). In all three groups, exposure to acute hypoxia of 10% O2 caused a decrease in blood O2 concentration and an increase in heart rate in 4-5-year-old animals, with limited effects on blood flow in the major outflow vessels of the heart. In response to more acute hypoxia (5% O2), where blood O2 concentration decreased even further, we measured increased heart rate and blood flow in the right aorta, subclavian artery, carotid artery, and pulmonary artery; however, blood flow in the left aorta either decreased or did not change. Embryonic exposure to hypoxia increased the threshold for eliciting an increase in heart rate indicative of a decrease in sensitivity. Alligators that had been incubated in hypoxia also had higher arterial PCO2 values in normoxia, suggesting a reduction in ventilation relative to metabolism.

Exposure to hypoxia during embryonic development affects blood flow patterns and heart rate in juvenile American alligators during digestion.

The developmental environment can alter an organism's phenotype through epigenetic mechanisms. We incubated eggs from American alligators in 10% O2 (hypoxia) to investigate the functional plasticity of blood flow patterns in response to feeding later in life. Digestion is associated with marked elevations of metabolism, and we therefore used the feeding-induced stimulation of tissue O2 demand to determine whether there are lasting effects of developmental hypoxia on the cardiovascular response to digestion later in life. In all animals studied, digestion elicited tachycardia and an elevation of blood flow in the right aorta, left aorta, and the pulmonary artery, whereas flows in the carotid and subclavian artery did not change. We found that heart rate and systemic blood flow remained elevated for a longer time period in juvenile alligators that had been incubated in hypoxia; we also found that the pulmonary blood flow was elevated at 24, 36, and 48 h. Collectively, our findings demonstrate that exposure to hypoxia during incubation has lasting effects on the hemodynamics of juvenile alligators 4 years after hatching.

A rare case of partial skull and brain duplication in a hatchling Alligator mississippiensis.

Errors in development occur in all vertebrates. When severe, these anomalies are lethal and frequently escape attention. In rare cases, animals with profound malformations are born and can provide a glimpse into structures and their respective function that would otherwise go unnoticed. A rare abnormality in a hatchling Alligator mississippiensis is described in which duplication of the skull, face, and brain was incomplete. The rostral skull, face, and associated forebrain, including the olfactory apparatus, were duplicated. However, the caudal skull and brainstem were not. These observations were made with advanced imaging using both computed tomography and magnetic resonance coupled with gross brain dissections. These abnormal features emphasize the complex and intertwined relationship between the development of the brain, face, and skull which are influenced by certain signaling molecules, possible gene mutation(s), and potential environmental factors.

Xenobiotic estradiol-17ß alters gut microbiota of hatchling American alligators (Alligator mississippiensis).

Environmental oestrogens pose serious concerns for ecosystems through their effects on organismal survival and physiology. The gut microbiome is highly vulnerable to environmental influence, yet the effects of oestrogens on gut homeostasis are unknown because they are poorly studied in wildlife populations. To determine the influence of environmental oestrogens (i.e., xenoestrogens) on the diversity and abundance of gut microbiota, we randomly assigned 23 hatchling American alligators (Alligator mississippiensis) to three ecologically relevant treatments (control, low, and high oestrogen concentrations) for 10 weeks. We predicted that xenoestrogen exposure would decrease microbial diversity and abundance within the digestive tract and that this effect would be dose-dependent. Microbial samples were collected following diet treatments and microbial diversity was determined using 16S rRNA gene-sequencing. Individuals in oestrogen-treatment groups had decreased microbial diversity, but a greater relative abundance of operational taxonomic units than those in the control group. In addition, this effect was dose-dependent; as individuals were exposed to more oestrogen, their microbiome became less diverse, less rich and less even. Findings from this study suggest that oestrogen contamination can influence wildlife populations at the internal microbial-level, which may lead to future deleterious health effects.

Developmental oxygen preadapts ventricular function of juvenile American alligators, Alligator mississippiensis.

Developmental oxygen is a powerful stressor that can induce morphological and functional changes in the cardiovascular systems of embryonic and juvenile vertebrates. This plasticity has been ascribed, at least in part, to the unique status of the developing cardiovascular system, which undergoes organogenesis while meeting the tissue oxygen demands of the embryo. We have previously reported an array of functional and morphological changes in embryonic American alligators that persist into juvenile life. Most notably, cardiac enlargement as well as functional parameters of anesthetized juvenile alligators remains after embryonic hypoxic exposure. Because the effects of developmental oxygen in crocodilians have only been investigated in anesthetized animals, we explored the pressure dynamics of both ventricles as well as systemic pressure in response to stressors of acute hypoxia and swimming. Our current findings demonstrate that developmental programming of cardiac function (intraventricular pressure and heart rate) does persist into juvenile life, but it is chamber-specific and depends on the experimental manipulation. Acute hypoxic exposure revealed that juvenile alligators that had experienced 10% O2 as embryos maintain right ventricle function and increase left ventricle function during exposure. Finally, the data indicate blood flow in the left aorta must originate from the left ventricle during acute hypoxia and swimming.

Ontogeny of the trigeminal system and associated structures in Alligator mississippiensis.

From the appearance of the vertebrate head, the trigeminal system has played a role in behavioral and ecological adaptation. The trigeminal nerve is the primary cranial somatosensory nerve, also innervating the jaw muscles. In crocodylians, the trigeminal nerve plays a role in modulating the high bite force and unique integumentary sensation. In association with these behaviors, crocodylians are known for large trigeminal nerves, a high volume of trigeminal-innervated musculature, and densely packed, specialized sensory receptors. These innovations also occurred in concert with a restructuring of the lateral braincase wall. These morphologies have previously been investigated in phylogenetic and evolutionary contexts, but an ontogenetic, whole-system investigation of trigeminal tissue and associated musculature, cartilage, and bone is lacking, as is an understanding of developmental timing of morphologies significant to hypotheses of homology. Here, we use contrast-enhanced computed tomography imaging to provide description and analysis of the trigeminal system in an ontogenetic series of Alligator mississippiensis from embryonic to adult form. We explore growth rates and allometric relationships of structures and discuss the significance to hypotheses of homology. We find a high growth rate and allometric trajectory of the trigeminal nerve in comparison to other cranial nerves, likely associated with the large volume of trigeminal musculature and high densities of sensory receptors. We identify a similar trend in the pterygoideus dorsalis muscle, the highest contributor to bite force. We narrow ontogenetic timing of features related to the trigeminal topological paradigm and the undeveloped epipterygoid. Overall, we provide a basis for understanding trigeminal development in crocodylians, which upon comparison across reptiles will reveal ontogenetic origins of morphological variation.

The developing bird pelvis passes through ancestral dinosaurian conditions.

Living birds (Aves) have bodies substantially modified from the ancestral reptilian condition. The avian pelvis in particular experienced major changes during the transition from early archosaurs to living birds1,2. This stepwise transformation is well documented by an excellent fossil record2-4; however, the ontogenetic alterations that underly it are less well understood. We used embryological imaging techniques to examine the morphogenesis of avian pelvic tissues in three dimensions, allowing direct comparison with the fossil record. Many ancestral dinosaurian features2 (for example, a forward-facing pubis, short ilium and pubic 'boot') are transiently present in the early morphogenesis of birds and arrive at their typical 'avian' form after transitioning through a prenatal developmental sequence that mirrors the phylogenetic sequence of character acquisition. We demonstrate quantitatively that avian pelvic ontogeny parallels the non-avian dinosaur-to-bird transition and provide evidence for phenotypic covariance within the pelvis that is conserved across Archosauria. The presence of ancestral states in avian embryos may stem from this conserved covariant relationship. In sum, our data provide evidence that the avian pelvis, whose early development has been little studied5-7, evolved through terminal addition-a mechanism8-10 whereby new apomorphic states are added to the end of a developmental sequence, resulting in expression8,11 of ancestral character states earlier in that sequence. The phenotypic integration we detected suggests a previously unrecognized mechanism for terminal addition and hints that retention of ancestral states in development is common during evolutionary transitions.

Tissue Distribution of Mercury in the Bodies of Wild American Alligators (Alligator mississippiensis) from a Coastal Marsh in Louisiana (USA).

Total mercury (THg) concentrations were measured in wild alligators inhabiting a coastal marsh in southern Louisiana, to determine the tissue distribution of THg among various body organs and tissue compartments. Concentrations of THg in claws and dermal tail scutes were compared to those in blood, brain, gonad, heart, kidney, liver, and skeletal muscle to determine if the former tissues, commonly available by non-lethal sampling, could be used as measures of body burdens in various internal organs. Mercury was found in all body organs and tissue compartments. However, overall, THg concentrations measured in alligators were below the FDA action level for fish consumption and were comparable to previous data reported from southwestern Louisiana. Our results suggest consumption of meat from alligators found in this region may be of little public health concern. However, the extended period of time between sampling (in this study) and the present-day highlight the need for continuous, additional, and more recent sampling to ensure consumer safety. Total mercury concentrations were highest in the kidney (3.18 ± 0.69 mg/kg dw) and liver (3.12 ± 0.76 mg/kg dw). THg levels in non-lethal samples (blood, claws, and dermal tail scutes) were positively correlated with all tissue THg concentrations (blood: R2 = 0.513-0.988; claw: R2 = 0.347-0.637, scutes: R2 = 0.333-0.649). Because THg concentrations from blood, claws, and scutes were correlated with those of the internal organs, non-lethal sampling methods may be a viable method of estimating levels of THg in other body tissues.

Ontogeny of the nasolacrimal apparatus and nasal sensory systems of the American alligator (Alligator mississippiensis).

The nasolacrimal apparatus (NLA) is a feature common to many sauropsid amniotes. It consists of an orbital Harderian gland (HG)whose secretions drain into the nasal cavity, in the vicinity of the vomeronasal organ (VNO), an accessory olfactory organ derived from the olfactory epithelium, and a connecting nasolacrimal duct (NLD). Though not all features are present in all posthatchling sauropsids (i.e., no VNO in crocodilomorphs), it is not clear if this system either never existed or failed to develop during the embryonic stages. The purpose of this study is to histologically describe the ontogeny of the NLA and the main olfactory organ in Alligator mississippiensis. Alligator specimens, from embryonic stage 9 to hatchling, were serially histologically sectioned, stained, photographed, and segmented into different tissues using Abobe Photoshop and then reconstructed using Amira for 3D analysis and quantitative nasal epithelial distribution. Though there was no evidence of a VNO, the rest of the NLA was present. The development of the NLA could be subdivided into four phases: (1) inception of NLD, (2) establishment of orbitonasal connections of NLD, (3) bone development, and (4) nasal cavity growth. Glands mature during this last phase and the nasal region rapidly grows, rotates, and is displaced anteriorly. The gradual proportional increase in nonolfactory epithelial distribution during ontogeny is consistent with the literature. Alligator embryonic nasal and NLD growth differs from that of mammals and squamates. The NLD is connected to the anterior third of the nasal region during its initial attachment, but as anterior nasal growth exceeds posterior growth, it is gradually displaced into the posterior third of the nasal region by hatching. It is unknown whether this is a derived archosaur condition or just another example of the morphological variation seen within sauropsid amniotes.

Ontogeny of the American alligator (Alligator mississippiensis) prenatal head: A morphometric approach.

Variation in vertebrate cranial morphology is both extensive and functionally significant. Morphometric analysis attempts to explain such variation in form in evolutionary and functional contexts. Developmental morphometric analyses of vertebrate crania are less common, and many taxa are underrepresented. For example, the published morphometric studies of crocodilian cranial development focus mainly on posthatchling head development, with few, incomplete morphometric analyses of prehatchling heads. To further explore ontogenetic changes in the alligator head, we recorded and analyzed six linear cranial measurements in 77 preserved embryonic and hatchling American alligators (Alligator mississippiensis) representing 20 different stages. Examination of individual measurements revealed nonlinear changes in growth rate during development, suggesting a level of dynamic complexity worthy of further analysis. Using principal components analysis, we identified three distinct phases in prenatal alligator head growth. The second (intermediate) phase disrupts an otherwise monotonic developmental trajectory and represents a phase of reduced snout growth. Although this is a detailed description of prehatchling ontogenetic trajectory of the alligator head, further studies in other crocodilians are needed to assess evolutionary patterns among crocodilians.

Ontogenetic changes in limb posture, kinematics, forces and joint moments in American alligators (Alligator mississippiensis).

As animals increase in size, common patterns of morphological and physiological scaling may require them to perform behaviors such as locomotion while experiencing a reduced capacity to generate muscle force and an increased risk of tissue failure. Large mammals are known to manage increased mechanical demands by using more upright limb posture. However, the presence of such size-dependent changes in limb posture has rarely been tested in animals that use non-parasagittal limb kinematics. Here, we used juvenile to subadult American alligators (total length 0.46-1.27 m, body mass 0.3-5.6 kg) and examined their limb kinematics, forces, joint moments and center of mass (CoM) to test for ontogenetic shifts in posture and limb mechanics. Larger alligators typically walked with a more adducted humerus and femur and a more extended knee. Normalized peak joint moments reflected these postural patterns, with shoulder and hip moments imposed by the ground reaction force showing relatively greater magnitudes in the smallest individuals. Thus, as larger alligators use more upright posture, they incur relatively smaller joint moments than smaller alligators, which could reduce the forces that the shoulder and hip adductors of larger alligators must generate. The CoM shifted nonlinearly from juveniles through subadults. The more anteriorly positioned CoM in small alligators, together with their compliant hindlimbs, contributes to their higher forelimb and lower hindlimb normalized peak vertical forces in comparison to larger alligators. Future studies of alligators that approach maximal adult sizes could give further insight into how animals with non-parasagittal limb posture modulate locomotor patterns as they increase in mass and experience changes in the CoM.

Scaling of axial muscle architecture in juvenile Alligator mississippiensis reveals an enhanced performance capacity of accessory breathing mechanisms.

Quantitative functional anatomy of amniote thoracic and abdominal regions is crucial to understanding constraints on and adaptations for facilitating simultaneous breathing and locomotion. Crocodilians have diverse locomotor modes and variable breathing mechanics facilitated by basal and derived (accessory) muscles. However, the inherent flexibility of these systems is not well studied, and the functional specialisation of the crocodilian trunk is yet to be investigated. Increases in body size and trunk stiffness would be expected to cause a disproportionate increase in muscle force demands and therefore constrain the basal costal aspiration mechanism, necessitating changes in respiratory mechanics. Here, we describe the anatomy of the trunk muscles, their properties that determine muscle performance (mass, length and physiological cross-sectional area [PCSA]) and investigate their scaling in juvenile Alligator mississippiensis spanning an order of magnitude in body mass (359 g-5.5 kg). Comparatively, the expiratory muscles (transversus abdominis, rectus abdominis, iliocostalis), which compress the trunk, have greater relative PCSA being specialised for greater force-generating capacity, while the inspiratory muscles (diaphragmaticus, truncocaudalis ischiotruncus, ischiopubis), which create negative internal pressure, have greater relative fascicle lengths, being adapted for greater working range and contraction velocity. Fascicle lengths of the accessory diaphragmaticus scaled with positive allometry in the alligators examined, enhancing contractile capacity, in line with this muscle's ability to modulate both tidal volume and breathing frequency in response to energetic demand during terrestrial locomotion. The iliocostalis, an accessory expiratory muscle, also demonstrated positive allometry in fascicle lengths and mass. All accessory muscles of the infrapubic abdominal wall demonstrated positive allometry in PCSA, which would enhance their force-generating capacity. Conversely, the basal tetrapod expiratory pump (transversus abdominis) scaled isometrically, which may indicate a decreased reliance on this muscle with ontogeny. Collectively, these findings would support existing anecdotal evidence that crocodilians shift their breathing mechanics as they increase in size. Furthermore, the functional specialisation of the diaphragmaticus and compliance of the body wall in the lumbar region against which it works may contribute to low-cost breathing in crocodilians.

Correlations between environmental salinity levels, blood biochemistry parameters, and steroid hormones in wild juvenile American alligators (Alligator mississippiensis).

American alligators (Alligator mississippiensis) inhabit freshwater wetlands that are vulnerable to salinization caused by anthropogenic alterations to freshwater flow, in addition to storm surges, sea level rise, and droughts. Salinization of coastal freshwater habitats is a growing concern in a changing climate due to increased frequency and intensity of storm surges and drought conditions. This study opportunistically sampled juvenile male and female wild alligators in various salinities each month excluding November, December, and January for one year at Rockefeller Wildlife Refuge in coastal Louisiana. Blood plasma biochemistry parameters including electrolyte levels were subsequently measured. In addition, levels of various renin-angiotensin-aldosterone system hormones, glucocorticoids, androgens, estrogens, and progestogens were analyzed using liquid chromatography and tandem mass spectrometry. Only males were sampled in hyperosmotic environments (> 10‰) during dry conditions in late summer 2018. In juvenile males, plasma Na+, Cl-, and the progestogen 17α,20ß-dihydroxypregnenone were significantly and positively correlated with environmental salinity. However, variation in glucocorticoids, androgens, and estrogens were not associated with hypersaline water while sex steroids showed significant seasonal variation. This study demonstrated significant correlation of environmental salinity with electrolyte levels and a sex steroid in wild juvenile alligators, and to our knowledge represents the first measurement of 17α,20ß-dihydroxypregnenone in alligators.

Arterial wall thickening normalizes arterial wall tension with growth in American alligators, Alligator mississippiensis.

Arterial wall tension increases with luminal radius and arterial pressure. Hence, as body mass (Mb) increases, associated increases in radius induces larger tension. Thus, it could be predicted that high tension would increase the potential for rupture of the arterial wall. Studies on mammals have focused on systemic arteries and have shown that arterial wall thickness increases with Mb and normalizes tension. Reptiles are good models to study scaling because some species exhibit large body size range associated with growth, thus, allowing for ontogenetic comparisons. We used post hatch American alligators, Alligator mississippiensis, ranging from 0.12 to 6.80 kg (~ 60-fold) to investigate how both the right aortic arch (RAo) and the left pulmonary artery (LPA) change with Mb. We tested two possibilities: (i) wall thickness increases with Mb and normalizes wall tension, such that stress (stress = tension/thickness) remains unchanged; (ii) collagen content scales with Mb and increases arterial strength. We measured heart rate and systolic and mean pressures from both systemic and pulmonary circulations in anesthetized animals. Once stabilized alligators were injected with adrenaline to induce a physiologically relevant increase in pressure. Heart rate decreased and systemic pressures increased with Mb; pulmonary pressures remained unchanged. Both the RAo and LPA were fixed under physiological hydrostatic pressures and displayed larger radius, wall tension and thickness as Mb increased, thus, stress was independent from Mb; relative collagen content was unchanged. We conclude that increased wall thickness normalizes tension and reduces the chances of arterial walls rupturing in large alligators.