The Pulmonary Anatomy of Alligator mississippiensis and Its Similarity to the Avian Respiratory System

Using gross dissections and computed tomography we studied the lungs of juvenile American alligators (Alligator mississippiensis). Our findings indicate that both the external and internal morphology of the lungs is strikingly similar to the embryonic avian respiratory system (lungs + air sacs). We identified bronchi that we propose are homologous to the avian ventrobronchi (entobronchi), laterobronchi, dorsobronchi (ectobronchi), as well as regions of the lung hypothesized to be homologous to the cervical, interclavicular, anterior thoracic, posterior thoracic, and abdominal air sacs. Furthermore, we suggest that many of the features that alligators and birds share are homologous and that some of these features are important to the aerodynamic valve mechanism and are likely plesiomorphic for Archosauria. Anat Rec, 2012. © 2012 Wiley Periodicals Inc.     

Keratinization and ultrastructure of the epidermis of late embryonic stages in the alligator (Alligator mississippiensis)

Using specific anti-beta keratin and general anti-alpha keratin antibodies, keratins were located in the epidermis of the alligator during the final developmental stages by ultrastructural and immunocytochemical methods. The maturation of the bilayered periderm (= embryonic epidermis) coincides with the disappearance of cell organelles, including the 25-35-nm-thick coarse filaments, and the coalescing of alpha-keratin filaments into a compact mass. The plasmalemma of peridermal cells forms a 15-25-nm-thick electron-dense corneous envelope. These changes start at stage 25, about 3 weeks before hatching, and continue until hatching when the embryonic epidermis is shed. Immature beta-keratogenic cells beneath the embryonic epidermis accumulate immunolabelled beta-filaments which are packed into thin, electron-pale beta-keratogenic cells in the corneous layer. Together, electron-pale and electron-dense materials form a compact 3-4-nm filament pattern of beta-keratin. Melanosomes from epidermal melanocytes, incorporated into beta-cells, give rise to the banded skin pattern of hatchlings. Beta-keratin production is much reduced in the hinge regions, where many alpha-filaments remain packed together with lipid droplets or mucous granules into thinner, more electron-dense, alpha-cells. The keratinaceous material of the alpha-cells is mostly concentrated along the cell membrane, while the lipid/mucous material remains centrally located, as in sebokeratinocytes of the apteric areas of avian skin. Some lipid and mucus is also incorporated into typical beta-cells of the outer scale surface, so that lipids are part of the fully keratinized hard keratin layer of the alligator. Lipids within beta-cells of outer scale surfaces and alpha-cells of the hinge region are probably responsible for limiting water loss and ion movements across the skin. Neither typical mammalian keratohyalin granules nor lepidosaurian keratohyalin-like granules were detected anywhere in alligator epidermis. The combination of anti-beta and anti-alpha keratin antibodies revealed different distributions of beta- and alpha-keratins. In late embryonic stages (25-26 to hatching), beta-keratin occurs only in the upper suprabasal cells, in prekeratinized and keratinized layers, whereas alpha-keratin bundles (tonofilaments) remain only in the lowest layers. The cross-reactivity of the beta-antibody, produced against a chick scale keratin, further shows that avian and crocodilian hard (beta) keratins share common antigenic sites, reflecting a phylogenetic affinity between these taxa.     

Development of the pulmonary vein in the American alligator (Alligator mississippiensis)

The origin of the embryonic common pulmonary vein in terrestrial vertebrates is still uncertain. Most earlier studies in nonhuman embryos describe the vein as entering the sinus venosus. The currently prevailing view, however, based largely on the study of human material, is that the embryonic common pulmonary vein is associated with the left atrium from its inception. We recently observed the pulmonary vein entering the sinus venous part of the right atrium in several normal dog embryos of a stage comparable to horizon XIV in man (Streeter: Contrib. Embryol. Carnegie Inst. Wash., 31:53, 1945). In slightly older specimens the vein entered the left atrium just to the left of septum primum. This observation, and the fact that some atrial septal and pulmonary venous anomalies in man still await a plausible pathogenetic explanation, stimulated a restudy of the origin of the vein. The alligator was used because we already had prepared a large number of closely graded serially sectioned embryos for other purposes. Wax plate reconstructions clearly showed that the common pulmonary vein entered the left side of the sinus venosus. With the formation of the atrial septum, this part of the sinus venosus is "pinched off" and becomes incorporated into the left atrium, thus transferring the pulmonary venous ostium to that atrium.     

Differential Limb Scaling in the American Alligator (Alligator mississippiensis) and Its Implications for Archosaur Locomotor Evolution

Bipedalism evolved multiple times within archosaurs, and relatively shorter forelimbs characterize both crocodyliforms and nonavian dinosaurs. Analysis of a comprehensive ontogenetic sequence of specimens (embryo to adult) of the sauropodomorph Massospondylus has shown that bipedal limb proportions result from negative forelimb allometry. We ask, is negative forelimb allometry a pattern basal to archosaurs, amplified in certain taxa to produce bipedalism? Given the phylogenetic position of extant crocodylians and their relatively shorter forelimb, we tested the hypothesis that prevalent negative forelimb allometry is present in Alligator mississippiensis from a sample of wild specimens from embryonic to adult sizes. Long bone lengths (humerus, radius, ulna, femur, tibia, fibula, third metapodials) were measured with their epiphyseal cartilage intact at all sizes. Our results show an overall isometric pattern for most elements regressed on femur length, humerus length, or total limb length. However, negative allometry was prevalent for the ulna, and the third metapodials scale with positive allometry embryonically. These data suggest that the general forelimb proportions in relation to the hindlimb do not change significantly with increasing size in A. mississippiensis. The negative allometry of the ulna and embryonicaly positive allometry of the third metapodials appears to be related to maintaining the functional integrity of the limbs. We show that this pattern is different from that of the sauropodomorph Massospondylus, and we suggest that if bipedalism in archosaurs is tied, in part, to negative forearm allometry, it was either secondarily lost through isometric scaling, or never developed in the ancestor of A. mississippiensis. Anat Rec, 292:787–797, 2009. © 2009 Wiley‐Liss, Inc.     

Histological organization and its relationship to function in the femur of Alligator mississippiensis

Histological analysis of a growth series of alligator femora tests the correlation between strain milieu and microstructure. From mid‐diaphyseal cross‐sections of these femora (n = 7), vascular canal orientation and density as well as collagen fibre organization were recorded. Throughout ontogeny, the proportion of transverse–spiral (TS) collagen in the dorsal cortex is significantly greater than it is in the ventral cortex (P = 0.008). This regional difference in the proportion of TS collagen is correlated with a regional difference in the state of peak principal strain (compressive or tensile). Nevertheless, the predominant orientation of collagen fibres is longitudinal, which is inconsistent with biomechanical hypotheses that involve peak principal or shear strains. Although the density and orientation of vascular canals do not show significant regional differences (P = 0.26 and P = 0.26, respectively), as with collagen orientation, the vascular canal orientation is predominantly longitudinal. The longitudinal organization of both the vascular canals and the collagen fibres is probably a consequence of longitudinal shifting of subperiosteal osteoid during femoral lengthening. When taken together, these data suggest that growth dynamics is the dominant influence on the histological organization of primary bony tissues in alligator femora.     

Natural development of dermal ectopic bone in the american alligator (Alligator mississippiensis) resembles heterotopic ossification disorders in humans

Heterotopic ossification (HO) occurs when soft tissues are inappropriately converted to bony tissue. Several human diseases result in HO with few reliable treatment options. Animal models that naturally produce dermal ectopic bone (i.e., osteoderms), such as crocodilians, have never been utilized as models for studying these disorders in humans. Here, a histological evaluation and staging criteria for osteoderm development is described for the first time in the American alligator (Alligator mississipiensis). Differential staining and immunohistochemistry of alligator scales depict a progressive change during development, where woven bone forms from the differentiated dermis. Bone formation proceeds via intramembranous ossification, which is initiated in part by endothelial cell precursors that undergo endothelial‐to‐mesenchymal transition and eventually acquire an osteoblast phenotype. As such, the development of osteoderms in the American alligator bears morphological and mechanistic similarities to HO in humans, presenting a potential model for future study of soft tissue mineralization pathologies and providing insight into the morphological and molecular development of osteoderms in other vertebrate lineages. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:56–76, 2018. © 2017 Wiley Periodicals, Inc.     

Low cost of ventilation in the vagotomised alligator (Alligator mississippiensis)

Pulmonary ventilation requires energy, but the estimated costs of breathing in reptiles vary from 1 to 30% of resting metabolic rate. The low values have been estimated from changes in oxygen uptake during hypoxia or hypercapnia, but it remains possible that these treatments affected metabolism. We equipped alligators with masks for simultaneous measurements of ventilation and oxygen uptake during hypercapnia, hypoxia and bilateral vagotomy. Hypercapnia and hypoxia caused a marked increase in total ventilation, but oxygen uptake remained unchanged indicating a very low energetic cost of breathing. Upon vagotomy, breathing pattern changed to occasional and exceedingly deep breaths (76.1+/-11.1 ml kg(-1)) followed by buccal oscillations and shallower breaths (22.2+/-2.3 ml kg(-1)) interspersed between long non-ventilatory periods. This change in breathing pattern did not affect oxygen uptake. The duration of inspiration increased substantially upon vagotomy, so that inspiratory flow rate did not increase proportionally to tidal volume. These prolonged inspiratory times leads to less flow resistance and may explain that tidal volume could be increased substantially without a measurable energetic cost.     

Structural design and mechanical behavior of alligator (Alligator mississippiensis) osteoderms

Alligator is a well-adapted living fossil covered with dorsal armor. This dermal shield consists of bony plates, called osteoderms, interconnected by sutures and non-mineralized collagen fibers, providing a dual function of protection and flexibility. Osteoderm features a sandwich structure, combining an inner porous core and an outer dense cortex, to offer enhancements for stiffness and energy absorbance. In this study, we investigated the multi-scale structure and mechanical behaviors of the American alligator (Alligator mississippiensis) osteoderm. Microcomputed tomography was applied to reveal the complex neurovascular network. Through the observation under optical and scanning electron microscopes, the osteoderm was found to consist of woven bone in the dorsal region and lamellar-zonal bone in the ventral region. Nanoindentation and compressive tests were performed to evaluate the mechanical properties of osteoderms. The varying mineral contents and porosity result in a graded mechanical property: a hard and stiff dorsal cortex gradually transform to a more compliant ventral base. Three protective mechanisms optimized for alligator osteoderms were proposed and elucidated.     

Fine structure of the developing epidermis in the embryo of the American alligator (Alligator mississippiensis,Crocodilia, Reptilia)

The morphological transition from the simple epidermis that contacts the amniotic fluid of embryonic crocodilians to the adult epidermis required in a terrestrial environment has never been described. We used light and electron microscopy to study the development, differentiation and keratinisation of the epidermis of the American alligator, Alligator mississippiensis, between early and late stages of embryonic skin formation. In early embryonic development, the epidermis consists of a flat bilayer. As it develops, the bilayered epidermis comes to lie beneath the peridermis. Glycogen is almost absent from the bilayered epidermis but increases in basal and suprabasal cells when scales form. Glycogen disappears from suprabasal cells that accumulate keratin. The peridermis and 1 or 2 subperidermal layers form an embryonic epidermis that is partially or totally lost before hatching. These cells accumulate coarse filaments and form reticulate bodies. Mucous and lamellate granules are produced in the Golgi apparatus and are partly secreted extracellularly. The embryonic cells darken with the formation of larger reticulate bodies that aggregate with intermediate filaments and other cell organelles, as their nuclear chromatin condenses. Thin β‐cells resembling those of scutate scales of birds develop beneath the embryonic epidermis and form a stratified β‐layer that varies in thickness in different body regions. The epidermis differentiates first in the back, tail and belly. At the beginning of β‐cell differentiation, the cytoplasm contains sparse bundles of α‐keratin filaments, glycogen and lipid droplets or vacuoles apparently derived from the endoplasmic reticulum and Golgi apparatus. These organelles disappear rapidly as irregular bundles of electron‐dense β‐keratin filaments accumulate and form larger bundles. The larger bundles consist of 3 nm thick electron‐pale keratin microfibrils and are derived from the assemblage of β‐keratin molecules produced by ribosomes. While in mammals the epidermal barrier is formed by α‐keratinocytes, in the alligator the barrier is formed by β‐keratin cells. The β‐layer is reduced or absent from the small hinge region between scales. In the latter areas the barrier is made of a or a mixture of α/β keratinocytes. Thus alligators resemble birds where the β‐keratin molecules are deposited directly over an α‐keratin scaffold, rather than an initial production of β‐keratin packets which then merge with α‐keratin, as occurs in the Chelonia and Lepidosauria. The pigmentation of the epidermis of embryos is mostly derived from epidermal melanocytes.     

Histology-based morphology of the neurocentral synchondrosis in Alligator mississippiensis (Archosauria, Crocodylia)

Morphology of the neurocentral synchondroses--thin cartilaginous layers between centra and neural arches--are documented in the extant crocodilian, Alligator mississippiensis (Archosauria, Crocodylia). Examination of dry skeletons demonstrates that neurocentral suture closure occurs in very late postnatal ontogeny (after reaching sexual maturity and/or body size ca. 40% from the upper range). Before sexual maturity (body length (BL) ≥ ca. 1.80 m), completely fused centra and neural arches are restricted to the caudal vertebral series. In contrast, the presacral vertebrae often remain unfused throughout postnatal ontogeny, retaining open sutures in very mature individuals (BL ≥ 2.80 m). These unfused centra and neural arches are structurally supported by the relatively large surface area of the neurocentral junctions, which results from primarily horizontal (mediolateral) increases with strong positive allometry. Cleared and stained specimens show that the cartilaginous neurocentral synchondrosis starts to form after approximately 40 embryonic days. Histological examination of the neurocentral junction in dorsal and anterior caudal vertebrae of six individuals (BL = 0.28-3.12 m) shows : (1) neurocentral fusion is the result of endochondral ossification of the neurocentral synchondrosis, (2) the neurocentral synchondrosis exhibits bipolar organization of three types of cartilaginous cells, and (3) complex neurocentral sutures (i.e., curved, zigzagged, and/or interdigitated boundaries) come from clumping of bone cells of the neural arches and centra into the neurocentral synchondrosis. The last two morphological features can be advantageous for delaying neurocentral fusion, which seems to be unique in crocodilians and possibly their close relatives, including nonavian dinosaurs and other Mesozoic archosaurs.     

Temporal Profile of Nerve Growth Factor Expression in the Partial Central Nervous System of the Yangtze Alligator Alligator sinensis (Reptilia,Crocodylia) During Early Postnatal Growth

Expression of nerve growth factor (NGF) in structures of the partial central nervous system of the Yangtze alligator, Alligator sinensis (Reptilia, Crocodylia) was examined during early postnatal growth using immunohistochemistry and Western blot assays. In animals 0–2 years of age NGF‐positive cells in the cerebral cortex increased gradually in number and size, and were predominantly distributed in the molecular layer. NGF‐positive cells in the midbrain showed similar increases but with predominant distribution in the ependymal layer. NGF‐positive cells increased in the cerebellum between 0 and 1 years of age, with increased NGF expression being seen during the first 2 years of life mostly in the ependymal layer. NGF‐positive cells were mainly found in the gray matter of the spinal cord with decreasing cell numbers, NGF expression levels being seen from 0 to 2 years and small processes without synaptic connection from 1 to 2 years. These results suggest that NGF is involved in the early postnatal growth of several structures of Yangtze alligator partial central nervous system, suggesting a possible role of NGF in the Yangtze alligator partial central nervous system. Anat Rec, 296:840–845, 2013. © 2013 Wiley Periodicals, Inc.     

Detection of antibodies to a pathogenic mycoplasma in American alligators (Alligator mississippiensis), broad-nosed Caimans (Caiman latirostris), and Siamese crocodiles (Crocodylus siamensis)

An epidemic of pneumonia with fibrinous polyserositis and multifocal arthritis emerged in captive American alligators (Alligator mississippiensis) in Florida, United States, in 1995. Mycoplasma alligatoris sp. nov. was cultured from multiple organs, peripheral blood, synovial fluid, and cerebrospinal fluid of affected alligators. In a subsequent experimental inoculation study, the Henle-Koch-Evans postulates were fulfilled for M. alligatoris as the etiological agent of fatal mycoplasmosis of alligators. That finding was remarkable because mycoplasmal disease is rarely fatal in animals. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies produced by alligators in response to M. alligatoris exposure was developed by using plasma obtained from naturally infected alligators during the original epidemic. The assay was validated by using plasma obtained during an experimental dose-response study and applied to analyze plasma obtained from captive and wild crocodilian species. The ELISA reliably detected alligator seroconversion (P < 0.05) beginning 6 weeks after inoculation. The ELISA also detected seroconversion (P < 0.05) in the relatively closely related broad-nosed caiman Caiman latirostris and the relatively distantly related Siamese crocodile Crocodylus siamensis following experimental inoculation with M. alligatoris. The ELISA may be used to monitor exposure to the lethal pathogen M. alligatoris among captive, repatriated, and wild crocodilian species.     

Three‐dimensional skeletal kinematics of the shoulder girdle and forelimb in walking Alligator

Crocodylians occupy a key phylogenetic position for investigations of archosaur locomotor evolution. Compared to the well‐studied hindlimb, relatively little is known about the skeletal movements and mechanics of the forelimb. In this study, we employed manual markerless XROMM (X‐ray Reconstruction Of Moving Morphology) to measure detailed 3‐D kinematics of the shoulder girdle and forelimb bones of American alligators (Alligator mississippiensis) walking on a treadmill. Digital models of the interclavicle, scapulocoracoid, humerus, radius and ulna were created using a 3‐D laser scanner. Models were articulated and aligned to simultaneously recorded frames of fluoroscopic and standard light video to reconstruct and measure joint motion. Joint coordinate systems were established for the coracosternal, glenohumeral and elbow joints. Our analysis revealed that the limb joints only account for about half of fore/aft limb excursion; the remaining excursion results from shoulder girdle movements and lateral bending of the vertebral column. Considerable motion of each scapulocoracoid relative to the vertebral column is consistent with coracosternal mobility. The hemisellar design of the glenohumeral joint permits some additional translation, or sliding in the fore‐aft plane, but this movement does not have much of an effect on the distal excursion of the bone.     

Localization of insulin-like growth factor-I-like immunoreactivity in the reproductive tract of the vitellogenic female American alligator,Alligator mississippiensis

Insulin-like growth factor I (IGF-I) is a 70 amino acid, mitogenic polypeptide, which, in mammals, acts through an endocrine, paracrine, and/or autocrine pathway to regulate growth and development. The primary goal of this study was to determine whether or not IGF-I-like immunoreactivity is present in the oviduct of the vitellogenic American alligator, Alligator mississippiensis, and if immunoreactivity patterns vary among the three functional oviducal regions: the albumen-secreting tube region, the anterior, fiber-secreting uterus, and the posterior, calcium-secreting uterus. Immunolocalization of IGF-I-like immunoreactivity was accomplished using a polyclonal antihuman rabbit antiserum with an immunoperoxidase staining system. IGF-I-like immunoreactivity was detected in all three oviducal regions of the vitellogenic alligator. The presence of IGF-I-like immunoreactivity in the oviduct suggests this hormone could function in the growth and proliferation of the alligator oviduct. Furthermore, the presence of IGF-I-like immunoreactivity in the tubal glands, which secrete components of the egg white, suggests that growth factors such as IGF-I may be synthesized by these glands and incorporated into the albumen during egg formation. © 1993 Wiley-Liss, Inc.     

Alligator wrestling: morphological,molecular, and phylogenetic data on Odhneriotrema incommodum (Leidy, 1856) (Digenea: Clinostomidae) from Alligator mississippiensis Daudin, 1801 in Mississippi,USA

Parasitology Research - Based on specimens collected from harvested American alligator Alligator mississippiensis Daudin, 1801 in Mississippi, USA, novel molecular data for both nuclear ribosomal...     

The fine structure and elemental analysis of keratinized epithelium of the filiform papillae analysis on the dorsal tongue in the American alligator (Alligator mississippiensis).

The filiform papillae of subadult alligator (120 cm-260 cm total length) tongues are examined by scanning electron microscopy and electron microprobe analysis. The filiform papillae form cone-like structures and are observed over the entire dorsal surface of a relatively long tongue with a round tip. The filiform papillae are composed of four layers; outer, upper intermediate, lower intermediate, and basal layer. The keratinized epithelial cells are analysed by the sulphur (S), and nitrogen (N) content levels. The S content is higher than that of N. In the anterior side of the filiform papillae, this content is lower than that in the posteriors. The S content is highest of all at about 2 microns in depth from the surface. These results suggest that the use of S concentration measurements may serve to be an effective tool for a simple, offhand evaluation of keratinization.