Forelimb muscle activation patterns in American alligators: Insights into the evolution of limb posture and powered flight in archosaurs.

The evolution of archosaurs provides an important context for understanding the mechanisms behind major functional transformations in vertebrates, such as shifts from sprawling to erect limb posture and the acquisition of powered flight. While comparative anatomy and ichnology of extinct archosaurs have offered insights into musculoskeletal and gait changes associated with locomotor transitions, reconstructing the evolution of motor control requires data from extant species. However, the scarcity of electromyography (EMG) data from the forelimb, especially of crocodylians, has hindered understanding of neuromuscular evolution in archosaurs. Here, we present EMG data for nine forelimb muscles from American alligators during terrestrial locomotion. Our aim was to investigate the modulation of motor control across different limb postures and examine variations in motor control across phylogeny and locomotor modes. Among the nine muscles examined, m. pectoralis, the largest forelimb muscle and primary shoulder adductor, exhibited significantly smaller mean EMG amplitudes for steps in which the shoulder was more adducted (i.e., upright). This suggests that using a more adducted limb posture helps to reduce forelimb muscle force and work during stance. As larger alligators use a more adducted shoulder and hip posture, the sprawling to erect postural transition that occurred in the Triassic could be either the cause or consequence of the evolution of larger body size in archosaurs. Comparisons of EMG burst phases among tetrapods revealed that a bird and turtle, which have experienced major musculoskeletal transformations, displayed distinctive burst phases in comparison to those from an alligator and lizard. These results support the notion that major shifts in body plan and locomotor modes among sauropsid lineages were associated with significant changes in muscle activation patterns.

Crocodile perception of distress in hominid baby cries.

It is generally argued that distress vocalizations, a common modality for alerting conspecifics across a wide range of terrestrial vertebrates, share acoustic features that allow heterospecific communication. Yet studies suggest that the acoustic traits used to decode distress may vary between species, leading to decoding errors. Here we found through playback experiments that Nile crocodiles are attracted to infant hominid cries (bonobo, chimpanzee and human), and that the intensity of crocodile response depends critically on a set of specific acoustic features (mainly deterministic chaos, harmonicity and spectral prominences). Our results suggest that crocodiles are sensitive to the degree of distress encoded in the vocalizations of phylogenetically very distant vertebrates. A comparison of these results with those obtained with human subjects confronted with the same stimuli further indicates that crocodiles and humans use different acoustic criteria to assess the distress encoded in infant cries. Interestingly, the acoustic features driving crocodile reaction are likely to be more reliable markers of distress than those used by humans. These results highlight that the acoustic features encoding information in vertebrate sound signals are not necessarily identical across species.

Histology, ultrastructure, and differential gene expression in relation to seasonal sperm storage in the oviduct of the Chinese alligator, Alligator sinensis.

Although oviductal sperm storage are essential steps in reproduction for female animals with internal fertilisation, no systematic study on the identification of genes involving sperm storage has been performed in crocodilian species. In the present research, the relationship between morphological variation related to sperm storage in the oviduct and gene expression patterns derived from RNA sequencing analyses between active period (AP), breeding period (BP), and hibernation period (HP) were investigated. The corresponding results indicated that sperm were observed not only in the ciliated cells within infundibulum and mucosal layer of uterus during BP, but also been detected in the spermatosperm storage tube (SST) in the anterior uterus at HP stage. The further transmission electron microscopy analysis indicated that the differences in the number and activity of the secretory cells likely to attributed to the seasonal variation of microenvironment related to the sperm storage. Based on the RNA-sequecing, 13147 DEGs related to the Peroxisome proliferator-activated receptors (PPARs) and FOXO signalling were identified, including these, the down-regulated ATG12 and BCL2L11 in the HP group may thus constitute an important point of convergence between autophagy and apoptosis involving the FOXO1 pathway. The genes involved in the PPARs pathway might modulate the immune response and thereby contribute to prolong the life span of stored spermatozoa in Alligator sinensis . The outcomes of this study provide fundamental insights into the mechanism of sperm storage in A. sinensis .

Three-dimensional polygonal muscle modelling and line of action estimation in living and extinct taxa.

Biomechanical models and simulations of musculoskeletal function rely on accurate muscle parameters, such as muscle masses and lines of action, to estimate force production potential and moment arms. These parameters are often obtained through destructive techniques (i.e., dissection) in living taxa, frequently hindering the measurement of other relevant parameters from a single individual, thus making it necessary to combine multiple specimens and/or sources. Estimating these parameters in extinct taxa is even more challenging as soft tissues are rarely preserved in fossil taxa and the skeletal remains contain relatively little information about the size or exact path of a muscle. Here we describe a new protocol that facilitates the estimation of missing muscle parameters (i.e., muscle volume and path) for extant and extinct taxa. We created three-dimensional volumetric reconstructions for the hindlimb muscles of the extant Nile crocodile and extinct stem-archosaur Euparkeria, and the shoulder muscles of an extant gorilla to demonstrate the broad applicability of this methodology across living and extinct animal clades. Additionally, our method can be combined with surface geometry data digitally captured during dissection, thus facilitating downstream analyses. We evaluated the estimated muscle masses against physical measurements to test their accuracy in estimating missing parameters. Our estimated muscle masses generally compare favourably with segmented iodine-stained muscles and almost all fall within or close to the range of observed muscle masses, thus indicating that our estimates are reliable and the resulting lines of action calculated sufficiently accurately. This method has potential for diverse applications in evolutionary morphology and biomechanics.

Cardiac structures in marine animals provide insight on potential directions for interventions for pediatric congenital heart defects.

Despite recent advances in pediatric diagnosis and surgical intervention, mortality and morbidity continue to be a prevalent issue in both tetralogy of Fallot (ToF) and hypoplastic left heart syndrome (HLHS). Therefore, novel approaches to studying both of these conditions are warranted. Investigating cardiac anatomical features of different species in the animal kingdom that are similar to the defects and complications present in ToF and HLHS (as well as others) could serve as a new avenue for improving the management of congenital heart diseases (CHD). This review reveals that although some structures found in HLHS and ToF are pathological, similar structures can be found in diving mammals and reptiles that are adaptive. Pathological aortic dilation in CHD resembles the aortic bulb present in diving mammals, but the latter is more elastic and distensible compared with the former. The unrepaired HLHS heart resembles the univentricular heart of non-crocodilian reptiles. Right ventricle hypertrophy is pathological in HLHS and ToF, but in contrast, adaptive in crocodilians and diving mammals. Lastly, the increased pulmonary resistance due to pulmonary stenosis in ToF is comparable with increased pulmonary resistance in crocodilians due to the presence of an active valve proximal to the pulmonary valve. Some of these anatomical structures could potentially be adapted for palliative surgery in children with HLHS or ToF. Moreover, further investigating the underlying molecular signals responsible for the adaptive tissue responses seen in other species may also be useful for developing novel strategies for preventing some of the complications that occur after surgical repair in both of these congenital heart diseases.

Nest temperature assessment in an American crocodile (Crocodylus acutus) population on the central coast of Oaxaca, Mexico.

The temperatures at which eggs of crocodilians are incubated plays an important role in embryo survival, rate of embryonic development and sex definition. The aim of this study was to assess the nest temperatures of an American crocodile (Crocodylus acutus) population on the central coast of Oaxaca state in Mexico. The fieldwork was carried out from February to June 2018 at Palmasola Lagoon, Oaxaca. Ten natural nests of C. acutus were carefully excavated to determine clutch size. When putting the eggs back in the nests, we placed a data logger in the center of the egg mass to determine the temperature parameters in the nest chamber environment, as well as the variation in temperature during the incubation period. All nests were revisited to count the number of hatched eggs (NHE) and to determine the hatching success (HS), along with the duration of the incubation period (IP). Hatching success was 89.04%. The mean clutch size in the American crocodile nests was 30.7 ± 7.83 eggs (ranging from 17 to 46 eggs), and the mean incubation period was 77.6 ± 5.89 days. The mean nest incubation temperature throughout the reproductive season was significantly different among nests. Based on the average temperature during the middle third of the incubation period, the nests should have produced both sexes, but with a higher proportion of males. This study tried to elucidate the impact of nest temperatures during the incubation period on embryo survival, as well as hatchling sex ratio in a local climate on the central coast of Oaxaca.

American crocodiles (Crocodylus acutus) as restoration bioindicators in the Florida Everglades.

The federally threatened American crocodile (Crocodylus acutus) is a flagship species and ecological indicator of hydrologic restoration in the Florida Everglades. We conducted a long-term capture-recapture study on the South Florida population of American crocodiles from 1978 to 2015 to evaluate the effects of restoration efforts to more historic hydrologic conditions. The study produced 10,040 crocodile capture events of 9,865 individuals and more than 90% of captures were of hatchlings. Body condition and growth rates of crocodiles were highly age-structured with younger crocodiles presenting with the poorest body condition and highest growth rates. Mean crocodile body condition in this study was 2.14±0.35 SD across the South Florida population. Crocodiles exposed to hypersaline conditions (> 40 psu) during the dry season maintained lower body condition scores and reduced growth rate by 13% after one year, by 24% after five years, and by 29% after ten years. Estimated hatchling survival for the South Florida population was 25% increasing with ontogeny and reaching near 90% survival at year six. Hatchling survival was 34% in NE Florida Bay relative to a 69% hatchling survival at Crocodile Lake National Wildlife Refuge and 53% in Flamingo area of Everglades National Park. Hypersaline conditions negatively affected survival, growth and body condition and was most pronounced in NE Florida Bay, where the hydrologic conditions have been most disturbed. The American crocodile, a long-lived animal, with relatively slow growth rate provides an excellent model system to measure the effects of altered hydropatterns in the Everglades landscape. These results illustrate the need for continued long-term monitoring to assess system-wide restoration outcomes and inform resource managers.

Exposure to xenoestrogens alters the expression of key morphoregulatory proteins of oviduct adenogenesis in the broad-snouted caiman (Caiman latirostris).

Endocrine disrupting compounds (EDCs) are contaminants ubiquitously found in the environment, which pose a potential threat to aquatic and wetland ecosystems. Caiman latirostris, a crocodilian species that inhabits South American wetlands, is highly sensitive to EDC exposure. Previously, we reported that early postnatal exposure to EDCs such as Bisphenol A (BPA) and 17ß-Estradiol (E2) alters C. latirostris oviduct differentiation. The aim of this work was to elucidate the molecular mechanisms behind this alteration. To accomplish this, we established the ontogenic changes in histological features and the expression of Wnt-7a, Wnt-5a, ß-catenin, FoxA2, desmin, and alpha smooth muscle actin (α-SMA) in the oviduct of C. latirostris. Then, we evaluated the effects of BPA and E2 exposure on these histological features and protein expressions. Our results showed that during the postnatal differentiation of the oviduct the presence of histological features related to adenogenesis is associated with the levels of expression of FoxA2, ß-catenin, Wnt-5a and Wnt-7a. Early postnatal exposure to BPA and E2 decreased the presence of histological features related to adenogenesis and altered the levels of expression of FoxA2, ß-catenin, Wnt-5a and Wnt-7a, as well as the desmin/α-SMA ratio. These findings suggest that altered levels of Wnt-7a, Wnt-5a, ß-catenin and FoxA2 could play a role in the BPA and E2-induced alteration in oviduct differentiation in C. latirostris. Thus, impaired adenogenesis and, probably, impaired reproduction in wildlife naturally exposed to BPA and other estrogenic agonists cannot be completely ruled out.

Musculoskeletal modelling of the Nile crocodile (Crocodylus niloticus) hindlimb: Effects of limb posture on leverage during terrestrial locomotion.

We developed a three-dimensional, computational biomechanical model of a juvenile Nile crocodile (Crocodylus niloticus) pelvis and hindlimb, composed of 47 pelvic limb muscles, to investigate muscle function. We tested whether crocodiles, which are known to use a variety of limb postures during movement, use limb orientations (joint angles) that optimise the moment arms (leverages) or moment-generating capacities of their muscles during different limb postures ranging from a high walk to a sprawling motion. We also describe the three-dimensional (3D) kinematics of the crocodylian hindlimb during terrestrial locomotion across an instrumented walkway and a treadmill captured via X-ray Reconstruction of Moving Morphology (biplanar fluoroscopy; 'XROMM'). We reconstructed the 3D positions and orientations of each of the hindlimb bones and used dissection data for muscle lines of action to reconstruct a focal, subject-specific 3D musculoskeletal model. Motion data for different styles of walking (a high, crouched, bended and two types of sprawling motion) were fed into the 3D model to identify whether any joints adopted near-optimal poses for leverage across each of the behaviours. We found that (1) the hip adductors and knee extensors had their largest leverages during sprawling postures and (2) more erect postures typically involved greater peak moment arms about the hip (flexion-extension), knee (flexion) and metatarsophalangeal (flexion) joints. The results did not fully support the hypothesis that optimal poses are present during different locomotory behaviours because the peak capacities were not always reached around mid-stance phase. Furthermore, we obtained few clear trends for isometric moment-generating capacities. Therefore, perhaps peak muscular leverage in Nile crocodiles is instead reached either in early/late stance or possibly during swing phase or other locomotory behaviours that were not studied here, such as non-terrestrial movement. Alternatively, our findings could reflect a trade-off between having to execute different postures, meaning that hindlimb muscle leverage is not optimised for any singular posture or behaviour. Our model, however, provides a comprehensive set of 3D estimates of muscle actions in extant crocodiles which can form a basis for investigating muscle function in extinct archosaurs.

Variations in the cerebrospinal fluid dynamics of the American alligator (Alligator mississippiensis).

BACKGROUND: Studies of mammalian CSF dynamics have been focused on three things: paravascular flow, pressure and pulsatility, and "bulk" flow; and three (respective) potential motive forces have been identified: vasomotor, cardiac, and ventilatory. There are unresolved questions in each area, and few links between the different areas. The American alligator (Alligator mississippiensis) has pronounced plasticity in its ventilatory and cardiovascular systems. This study was designed to test the hypothesis that the greater cardiovascular and ventilatory plasticity of A. mississippiensis would result in more variation within the CSF dynamics of this species. METHODS: Pressure transducers were surgically implanted into the cranial subarachnoid space of 12 sub-adult alligators; CSF pressure and pulsatility were monitored along with EKG and the exhalatory gases. In four of the alligators a second pressure transducer was implanted into the spinal subarachnoid space. In five of the alligators the CSF was labeled with artificial microspheres and Doppler ultrasonography used to quantify aspects of the spinal CSF flow. RESULTS: Both temporal and frequency analyses of the CSF pulsations showed highly variable contributions of both the cardiac and ventilatory cycles. Unlike the mammalian condition, the CSF pressure pulsations in the alligator are often of long (~ 3 s) duration, and similar duration CSF unidirectional flow pulses were recorded along the spinal cord. Reduction of the duration of the CSF pulsations, as during tachycardia, can lead to a "summation" of the pulsations. There appears to be a minimum duration (~ 1 s) of isolated CSF pulsations. Simultaneous recordings of cranial and spinal CSF pressures reveal a 200 ms delay in the propagation of the pressure pulse from the cranium to the vertebral canal. CONCLUSIONS: Most of the CSF flow dynamics recorded from the alligators, are similar to what has been reported from studies of the human CSF. It is hypothesized that the link between ventilatory mechanics and CSF pulsations in the alligator is mediated by displacement of the spinal dura. The results of the study suggest that understanding the CSF dynamics of Alligator may provide unique insights into the evolutionary origins and functional regulation of the human CSF dynamics.

Effects of change in temperature on the cardiac contractility of broad-snouted caiman (Caiman latirostris) during digestion.

In many reptiles, digestion has been associated with the selection of higher body temperatures, the so-called post-prandial thermophilic response. This study aimed to investigate the excitation-contraction (E-C) coupling in postprandial broad-snouted caimans (Caiman latirostris) in response to acute warming within a preferred body temperature range of crocodiles. Isometric preparations subjected to a temperature transition from 25°C to 30°C were used to investigate myocardial contractility of postprandial caimans, that is, 48 h after the animals ingested a rodent meal corresponding to 15% of body mass. The caiman heart exhibits a negative force-frequency relationship that is independent of the temperature. At 25°C, cardiac muscle was able to maintain a constant force up to 36 bpm, above which it decreased significantly, reaching minimum values at the highest frequency of 84 bpm. Moreover, E-C coupling is predominantly dependent on transsarcolemmal Ca2+ transport denoted by the lack of significant ryanodine effects on force generation. On the contrary, ventricular strips at 30°C were able to sustain the cardiac contractility at higher pacing frequencies (from 12 to 144 bpm) due to an important role of Na+ /Ca2+ exchanger in Ca2+ cycling, as indicated by the decay of the post-rest contraction, and a significant contribution of the sarcoplasmic reticulum above 72 bpm. Our results demonstrated that the myocardium of postprandial caimans exhibits a significant degree of thermal plasticity of E-C coupling during acute warming. Therefore, myocardial contractility can be maximized when postprandial broad-snouted caimans select higher body temperatures (preferred temperature zone) following feeding.

A new role for joint mobility in reconstructing vertebrate locomotor evolution.

Reconstructions of movement in extinct animals are critical to our understanding of major transformations in vertebrate locomotor evolution. Estimates of joint range of motion (ROM) have long been used to exclude anatomically impossible joint poses from hypothesized gait cycles. Here we demonstrate how comparative ROM data can be harnessed in a different way to better constrain locomotor reconstructions. As a case study, we measured nearly 600,000 poses from the hindlimb joints of the Helmeted Guineafowl and American alligator, which represent an extant phylogenetic bracket for the archosaurian ancestor and its pseudosuchian (crocodilian line) and ornithodiran (bird line) descendants. We then used joint mobility mapping to search for a consistent relationship between full potential joint mobility and the subset of joint poses used during locomotion. We found that walking and running poses are predictably located within full mobility, revealing additional constraints for reconstructions of extinct archosaurs. The inferential framework that we develop here can be expanded to identify ROM-based constraints for other animals and, in turn, will help to unravel the history of vertebrate locomotor evolution.

Alligator presence influences colony site selection of long-legged wading birds through large scale facilitative nest protector relationship.

Positive ecological relationships, such as facilitation, are an important force in community organization. The effects of facilitative relationships can be strong enough to cause changes in the distributions of species and in many cases have evolved as a response to predation pressure, however, very little is known about this potential trend in vertebrate facilitative relationships. Predation is an important selective pressure that may strongly influence breeding site selection by nesting birds. The American Alligator (Alligator mississippiensis) facilitates a safer nesting location for wading birds (Ciconiiformes and Pelecaniformes) by deterring mammalian nest predators from breeding sites. However, alligators do not occur throughout the breeding range of most wading birds, and it is unclear whether alligator presence affects colony site selection. We predicted that nesting wading birds change colony site preferences when alligators are not present to serve as nest protectors. Within the northern fringe of alligator distribution we compared colony characteristics in locations where alligator presence was either likely or unlikely while controlling for availability of habitat. Wading birds preferred islands that were farther from the mainland and farther from landmasses > 5 ha when alligator presence was unlikely compared to when alligators were likely. These findings indicate that wading birds are seeking nesting locations that are less accessible to mammalian predators when alligators are not present, and that this requirement is relaxed when alligators are present. This study illustrates how a landscape-scale difference between realized and fundamental niche can result from a facilitative relationship in vertebrates.

Aerobic scope and climate warming: Testing the "plastic floors and concrete ceilings" hypothesis in the estuarine crocodile (Crocodylus porosus).

Ectotherms are predicted to show a reduction in absolute aerobic scope (AAS = maximum - standard metabolic rates) if habitat temperatures surpass optima. However, thermal phenotypic plasticity may play a protective role in the maintenance of AAS. In fishes, resting physiological rates ("physiological floors," e.g., standard metabolic rates [SMR]) are typically thermally phenotypically plastic whilst maximum physiological rates ("physiological ceilings," e.g., maximum metabolic rate [MMR]) are typically fixed. This observation led to the "plastic floors and concrete ceilings" hypothesis. The applicability of this hypothesis to nonavian reptiles remains untested, despite this group being at risk of climate warming-induced extinction. We tested this hypothesis in juvenile estuarine crocodiles (Crocodylus porosus) by maintaining animals at a water temperature indicative of current summer conditions (28°C) or at a water temperature reflecting a high magnitude of warming (34°C; i.e., thermal acclimation treatments) for 6 months. Metabolic traits (SMR, MMR, and AAS) were subsequently quantified between 28-36°C. A twofold increase in SMR was observed between 28°C and 36°C in both thermal acclimation treatments (pooled Q10 = 3.2). MMR was thermally insensitive between 28°C and 36°C in 28°C-acclimated crocodiles but doubled between 28°C and 36°C in 34°C-acclimated crocodiles. These findings demonstrate thermal phenotypic plasticity in a "physiological ceiling" (MMR) and rigidity in a "physiological floor" (SMR), showing the opposite pattern to many fishes. Overall, crocodiles displayed impressive aerobic capacity at temperatures reflecting climate warming scenarios. AAS remained unchanged across an 8°C temperature range in 28°C-acclimated animals and doubled in 34°C-acclimated animals.

Glans inflation morphology and female cloaca copulatory interactions of the male American alligator phallus†.

The phallic glans of the American alligator (Alligator mississippiensis) is the distal termination of the semen-conducting sulcus spermaticus and during copulation has the closest, most intimate mechanical interactions with the female urodeum, the middle cloacal chamber that contains the opening to the vaginal passages and oviducts. However, the details of this interface leading to insemination and gamete uptake are unclear. Here, we: (1) histologically characterize the underlying tissue types and morphologically quantify the shape changes associated with glans inflation into the copulatory conformation, (2) digitally reconstruct from MRI the 3D shape of functional tissue compartments, and (3) diffusible iodine-based contrast-enhanced computed tomography image the copulatory fit between male phallus and female cloaca. We discuss these results in relation to tissue type material properties, the transfer on intromittent forces, establishing potential copulatory lock, inflated glans volume scaling with body mass/length, the mechanics of semen targeting and insemination, and potential female cryptic choice impacting multiple clutch paternity. In part, this study further clarifies the phallic morphological variation observed among crocodylians and begins to investigate the role(s) these divergent male forms play during copulation interacting with female cloacal forms to increase reproductive success.

Molecular cloning, characterization, and gene expression behavior of glucocorticoid and mineralocorticoid receptors from the Chinese alligator (Alligator sinensis).

The Chinese alligator is an endemic crocodilian species in China. We isolated and obtained the glucocorticoid and mineralocorticoid receptor genes coding from the kidney of Alligator sinensis by nested polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The glucocorticoid receptor (GR) gene has 2343 base pairs encoding 780 amino acids, while the mineralocorticoid receptor (MR) gene is 2958 bp in length encoding 985 amino acids. Quantitative real-time PCR was used to detect the distribution of messenger RNA (mRNA) levels. The maximum mRNA expressions were observed in the ovary and kidney, suggesting that these receptors may be involved in basic cellular functions or stress response of alligators. Besides this, RT-qPCR was performed to analyze the abundance of GR and MR mRNA transcripts in early embryonic development of the Chinese alligator in the kidney, liver, and heart. The mRNA levels of GR and MR at earlier stages in kidney, liver, and heart indicates that they might involve in the transcriptional regulation of early embryos and activate many precise developmental effects in fetal tissues. We also measured the protein expression in the liver embryonic developmental stages and found that the GR and MR proteins were restricted to both the nuclei and cytoplasm. The protein expression levels in the liver at different embryonic developmental stages have extremely prominent differences. Taken together, our results showed the full coding regions of GR and MR, their characteristics, and embryonic developmental mRNA and protein expressions of both genes in A. sinensis. This study could provide the necessary information for further investigating the diverse functions of GR and MR in A. sinensis.

Looking for genetic effects of polluted anthropized environments on Caiman crocodilus crocodilus (Reptilia, Crocodylia): A comparative genotoxic and chromosomal analysis.

The Amazon aquatic ecosystems have been modified by the human population growth, going through changes in their water bodies and aquatic biota. The spectacled alligator (Caiman crocodilus crocodilus) has a wide distribution and adaptability to several environments, even those polluted ones. This study aimed to investigate if a Caiman species living in urban streams of Manaus city (Amazonas State, Brazil) is affected by environmental pollution. For that, it was used classical and molecular cytogenetic procedures, in addition to micronucleus and comet assays. Although the karyotype macrostructure remains unaltered (2 n = 42 chromosomes; 24 t + 18 m/sm; NF = 60), the genotoxic analysis and the cytogenetic mapping of repetitive DNA sequences demonstrated that polluted environments alter the genome of the specimens, affecting both the chromosomal organization and the genetic material.

Molecular mechanism of Chinese alligator (Alligator sinensis) adapting to hibernation.

Hibernation is a physiological state for Chinese alligators to cope with cold weather. In mammals, gene expression changes during hibernation and their regulatory mechanisms have been extensively studied, however, these studies in reptiles are still rare. Here, integrated analysis of messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA) reveals the molecular mechanisms of the hypothalamus, liver, and skeletal muscle in hibernating and active individuals. During hibernation, the number of genes increased in the hypothalamus, liver, and skeletal muscle was 585, 282, and 297, while the number of genes decreased was 215, 561, and 627, respectively, as compared with active individuals. Through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, the differential expressed genes were mainly enriched in DNA damage repair, biological rhythm, energy metabolism, myoprotein degradation, and other related items and pathways. Besides, 4740 miRNAs were identified in three tissues. Through the comprehensive analysis of miRNA and mRNA abundance profiles, 12,291, 6997, and 8232 miRNA-mRNA pairs all showed a negative correlation in the hypothalamus, liver, and skeletal muscle, respectively. Some miRNA target genes were related tobiological rhythm and energy metabolism, suggesting that miRNA may play an important role in the physiological metabolism of the hibernating adaptability of Chinese alligators. Moreover, 402, 230, and 130 differentially expressed lncRNAs were identified in the hypothalamus, liver, and skeletal muscle, respectively. The targeting relationship of four lncRNA-mRNA pairs were predicted, with the main function of target genes involved in the amino acid transportation. These results are helpful to further understand the molecular regulatory basis of the hibernation adaptation in Chinese alligators.

Anatomical and histological analyses reveal that tail repair is coupled with regrowth in wild-caught, juvenile American alligators (Alligator mississippiensis).

Reptiles are the only amniotes that maintain the capacity to regenerate appendages. This study presents the first anatomical and histological evidence of tail repair with regrowth in an archosaur, the American alligator. The regrown alligator tails constituted approximately 6-18% of the total body length and were morphologically distinct from original tail segments. Gross dissection, radiographs, and magnetic resonance imaging revealed that caudal vertebrae were replaced by a ventrally-positioned, unsegmented endoskeleton. This contrasts with lepidosaurs, where the regenerated tail is radially organized around a central endoskeleton. Furthermore, the regrown alligator tail lacked skeletal muscle and instead consisted of fibrous connective tissue composed of type I and type III collagen fibers. The overproduction of connective tissue shares features with mammalian wound healing or fibrosis. The lack of skeletal muscle contrasts with lizards, but shares similarities with regenerated tails in the tuatara and regenerated limbs in Xenopus adult frogs, which have a cartilaginous endoskeleton surrounded by connective tissue, but lack skeletal muscle. Overall, this study of wild-caught, juvenile American alligator tails identifies a distinct pattern of wound repair in mammals while exhibiting features in common with regeneration in lepidosaurs and amphibia.

Assessing the Ability of Developmentally Precocious Estrogen Signaling to Recapitulate Ovarian Transcriptomes and Follicle Dynamics in Alligators from a Contaminated Lake.

BACKGROUND: Concern has grown in recent decades over anthropogenic contaminants that interfere with the functioning of endocrine hormones. However, mechanisms connecting developmental processes to pathologies associated with endocrine-disrupting chemical (EDC) exposure are poorly understood in naturally exposed populations. OBJECTIVES: We sought to a) characterize divergence in ovarian transcriptomic and follicular profiles between alligators originating from a historically EDC-contaminated site, Lake Apopka, and a reference site; b) test the ability of developmentally precocious estrogen exposure to recapitulate site-associated patterns of divergence; and c) test whether treatment with exogenous follicle-stimulating hormone (FSH) is capable of rescuing phenotypes associated with contaminant exposure and/or embryonic estrogen treatment. METHODS: Alligators eggs were collected from a contaminated site and a reference site, and a subset of eggs from the reference site were treated with estradiol (E2) during embryonic development prior to gonadal differentiation. After hatching, alligators were raised under controlled laboratory settings for 5 months. Juveniles from both sites were divided and treated with exogenous FSH. Histological analyses and RNA-sequencing were conducted to characterize divergence in ovarian follicle dynamics and transcriptomes between sites, between reference and E2-treated animals, and between FSH-treated and nontreated animals. RESULTS: We observed broad site-of-origin divergence in ovarian transcriptomes and reductions in ovarian follicle density between juvenile alligators from Lake Apopka and the reference site. Treating embryos from the reference site with E2 overwhelmingly recapitulated transcriptional and histological alterations observed in Lake Apopka juveniles. Ovarian phenotypes observed in Lake Apopka alligators or resulting from estrogen treatment were only partially rescued by treatment with exogenous FSH. DISCUSSION: Recapitulation of ovarian abnormalities by precocious E2 revealed a relatively simple mechanism underlying contaminant-induced pathologies in a historical example of environmental endocrine disruption. Findings reported here support a model where the developmental timing of estrogen signaling has the potential to permanently alter ovarian organization and function. https://doi.org/10.1289/EHP6627.