Lipids in the American Alligator stratum corneum provide insights into the evolution of vertebrate skin.

In terrestrial vertebrates, the outermost layer of the skin, the stratum corneum (SC), provides a durable and flexible interface with the environment and is comprised of corneocytes embedded in lipids. However, the morphology and lipid composition of the SC varies throughout evolutionary history. Because crocodilians and birds phylogenetically bracket the Archosaurian clade, lipid composition in crocodilian SC may be compared with that of birds and other vertebrates to make inferences about broader phylogenetic patterns within Archosaurs while highlighting adaptations in vertebrate skin. We identified and quantified lipid classes in the SC of the American Alligator (Alligator mississippiensis) from three skin regions varying in mobility. Our results find similarities in lipid composition between alligator and avian SC, including a high percentage of cerebrosides, a polar lipid previously found only in the SC of birds and bats. Furthermore, polar lipids were more abundant in the most mobile region of the SC. Because polar lipids bind with water to increase skin hydration and therefore its pliability under physical stress, we hypothesize that selection for lipids in Archosaurian SC was driven by the unique distribution of proteins in the SC of this clade, and cerebrosides may have served as pre-adaptations for flight.

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.

Maternal provisioning interacts with incubation temperature to affect hatchling mercury exposure in an oviparous reptile.

The thermal environment experienced by developing embryos can influence the utilization of maternally provisioned resources. Despite being particularly consequential for oviparous ectotherms, these dynamics are largely unexplored within ecotoxicological frameworks. Here, we test if incubation temperature interacts with maternally transferred mercury to affect subsequent body burdens and tissue distributions of mercury in hatchling American alligators (Alligator mississippiensis). Nine clutches of alligator eggs were collected from a mercury-contaminated reservoir and incubated at either female- or male-promoting temperatures. Total mercury (THg) concentration was measured in egg yolk collected during incubation and in a suite of tissues collected from hatchlings. THg concentrations in residual yolk and blood were higher in hatchlings incubated at cooler, female-promoting temperatures compared to the warmer, male-promoting temperatures. THg concentrations in most tissues were positively correlated with THg concentrations in blood and dermis, and egg yolk THg concentration was the best predictor of THg concentration in many resultant tissues. Our results highlight a hereto unknown role of the developmental environment in mediating tissue specific uptake of contaminants in an oviparous reptile.

Social structure and habitat design affect the impact of a novel feeding enrichment for alligators.

Providing enrichment that expands the range of behavioral opportunities associated with food acquisition and environmental exploration is an important contributing factor to the well-being of zoo animals. These behaviors can be difficult to promote in carnivores, given their foraging strategies and the logistical, ethical, and financial challenges of providing live prey. In this study, we introduced a novel feeding enrichment to Jacksonville Zoo and Gardens' five adult American alligators (Alligator mississippiensis) in an attempt to simulate a live prey organism within the exhibit and promote natural hunting behaviors like chasing and lunging, as well as increase daily activity levels. The enrichment promoted some behavioral goals for two of the alligators, but it did not promote behavioral goals for the other three alligators. This could have been due to a variety of factors including an existing dominance hierarchy amongst the group's females and the resulting spatial distribution of individuals across a habitat with only one water feature. Our results suggest that female alligators may carve out territories and avoid overlapping space usage with other females during the warmest months of the year. Given the outcomes and limitations of this enrichment strategy, we provide recommendations for this group specifically as well as future enrichment efforts in the general captive crocodilian population.

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.

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.

Early life differences in behavioral predispositions in two Alligatoridae species.

Behavioral predispositions are innate tendencies of animals to behave in a given way without the input of learning. They increase survival chances and, due to environmental and ecological challenges, may vary substantially even between closely related taxa. These differences are likely to be especially pronounced in long-lived species like crocodilians. This order is particularly relevant for comparative cognition due to its phylogenetic proximity to birds. Here we compared early life behavioral predispositions in two Alligatoridae species. We exposed American alligator and spectacled caiman hatchlings to three different novel situations: a novel object, a novel environment that was open and a novel environment with a shelter. This was then repeated a week later. During exposure to the novel environments, alligators moved around more and explored a larger range of the arena than the caimans. When exposed to the novel object, the alligators reduced the mean distance to the novel object in the second phase, while the caimans further increased it, indicating diametrically opposite ontogenetic development in behavioral predispositions. Although all crocodilian hatchlings face comparable challenges, e.g., high predation pressure, the effectiveness of parental protection might explain the observed pattern. American alligators are apex predators capable of protecting their offspring against most dangers, whereas adult spectacled caimans are frequently predated themselves. Their distancing behavior might be related to increased predator avoidance and also explain the success of invasive spectacled caimans in the natural habitats of other crocodilians.

Preferential Liver Accumulation of Mercury Explains Low Concentrations in Muscle of Caiman yacare (Alligatoridae) in Upper Amazon.

Caiman yacare is considered one of the top predators in the Amazon basin, and understanding pollutant distribution within its tissues may help its sustainable management. As a top predator, C. yacare should have the highest mercury concentrations, but has lower Hg concentrations than carnivorous fish (Rivera et al. 2016), which are part of their diet. We compared total Hg among liver, kidney, fat, and muscle of C. yacare, and whether trends in the distribution of Hg among tissues were like other crocodilians, aquatic birds, omnivorous, and carnivorous fish. Fat had the lowest concentrations (0.025 ± 0.03 mg kg-1) followed by muscle (0.15 ± 0.06 mg kg-1), kidney (0.57 ± 0.30 mg kg-1) and liver (1.81 ± 0.80 mg kg-1). Such preferential accumulation makes C. yacare meat a safer alternative for human consumption than carnivorous fish. The relation between Hg accumulation in liver and muscle is highest in crocodilians, which has evolutive and environmental implications.

Spatial and temporal variation in nest temperatures forecasts sex ratio skews in a crocodilian with environmental sex determination.

Species displaying temperature-dependent sex determination (TSD) are especially vulnerable to the effects of a rapidly changing global climate due to their profound sensitivity to thermal cues during development. Predicting the consequences of climate change for these species, including skewed offspring sex ratios, depends on understanding how climatic factors interface with features of maternal nesting behaviour to shape the developmental environment. Here, we measure thermal profiles in 86 nests at two geographically distinct sites in the northern and southern regions of the American alligator's (Alligator mississippiensis) geographical range, and examine the influence of both climatic factors and maternally driven nest characteristics on nest temperature variation. Changes in daily maximum air temperatures drive annual trends in nest temperatures, while variation in individual nest temperatures is also related to local habitat factors and microclimate characteristics. Without any compensatory nesting behaviours, nest temperatures are projected to increase by 1.6-3.7°C by the year 2100, and these changes are predicted to have dramatic consequences for offspring sex ratios. Exact sex ratio outcomes vary widely depending on site and emission scenario as a function of the unique temperature-by-sex reaction norm exhibited by all crocodilians. By revealing the ecological drivers of nest temperature variation in the American alligator, this study provides important insights into the potential consequences of climate change for crocodilian species, many of which are already threatened by extinction.

Detection and analysis of autophagy in the American alligator (Alligator mississippiensis).

In response to environmental temperature depression in the fall and winter, American alligators (Alligator mississippiensis) brumate. Brumation is characterized by lethargy, fasting, decreased metabolism, and decreased body temperature. During brumation, alligators will periodically emerge for basking or other encounters when environmental conditions permit. This sporadic activity and lack of nutrient intake may place strain on nutrient reserves. Nutrient scarcity, at the cellular and/or organismal level, promotes autophagy, a well-conserved subcellular catabolic process used to maintain energy homeostasis during periods of metabolic or hypoxic stress. An analysis of the putative alligator autophagy-related proteins has been conducted, and the results will be used to investigate the physiological role of autophagy during the brumation period. Using published genomic data, we have determined that autophagy is highly conserved, and alligator amino acid sequences exhibit a high percentage of identity with human homologs. Transcriptome analysis conducted using liver tissue derived from alligators confirmed the expression of one or more isoforms of each of the 34 autophagy initiation and elongation genes assayed. Five autophagy-related proteins (ATG5, ATG9A, BECN1, ATG16L1, and MAP1-LC3B), with functions spanning the major stages of autophagy, have been detected in alligator liver tissue by western blot analysis. In addition, ATG5 was detected in alligator liver tissue by immunohistochemistry. This is the first characterization of autophagy in crocodylians, and the first description of autophagy-related protein expression in whole blood.

Identification and characterization of microRNAs (miRNAs) and their transposable element origins in the saltwater crocodile, Crocodylus porosus.

MicroRNAs (miRNAs) are 18-24 nucleotide regulatory RNAs. They are involved in the regulation of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Data suggests a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to birds, the closest extant relatives of crocodilians. Understanding gene regulation in the saltwater crocodile in the context of relatively slow genomic change thus holds potential for the investigation of genomics, evolution, and adaptation. Utilizing eleven tissue types and sixteen small RNA libraries, we report 644 miRNAs in the saltwater crocodile with >78% of miRNAs being novel to crocodilians. We also identified potential targets for the miRNAs and analyzed the relationship of the miRNA repertoire to transposable elements (TEs). Results suggest an increased association of DNA transposons with miRNAs when compared to retrotransposons. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile. In addition, the data suggests a supporting role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation.

Microbiota of Four Tissue Types in American Alligators (Alligator mississippiensis) Following Extended Dietary Selenomethionine Exposure.

Selenium represents an essential trace nutrient that is necessary for biological functions. Deficiencies can induce disease, but excess can induce toxicity. Selenium deficiency is a major concern in underdeveloped countries, while also posing as a toxic pollutant in waterways surrounding landfills, agricultural areas, and fossil fuel production sites. We examined the microbiome of selenomethionine (SeMet) fed American alligators (Alligator mississippiensis) at the beginning and end of a 7-week exposure experiment. Alligators were randomly divided into three groups: control and 1000 or 2000 ppm SeMet. DNA from before exposure (oral and cloaca swabs) and post-exposure (oral, cloaca, small & large intestines) sampling were extracted and amplified for bacterial 16 s rRNA. While treatment did not seem to have much effect, we observed a predominance of Fusobacteriaceae and Porpyromonodaceae across all tissue types. Cetobacterium and Clostridium are the most abundant genera as potential indicators of the aquatic and carrion feeding lifestyle of alligators.

Embryonic developmental oxygen preconditions cardiovascular functional response to acute hypoxic exposure and maximal ß-adrenergic stimulation of anesthetized juvenile American alligators (Alligator mississippiensis).

The effects of the embryonic environment on juvenile phenotypes are widely recognized. We investigated the effect of embryonic hypoxia on the cardiovascular phenotype of 4-year-old American alligators (Alligator mississippiensis). We hypothesized that embryonic 10% O2 preconditions cardiac function, decreasing the reduction in cardiac contractility associated with acute 5% O2 exposure in juvenile alligators. Our findings indicate that dobutamine injections caused a 90% increase in systolic pressure in juveniles that were incubated in 21% and 10% O2, with the 10% O2 group responding with a greater rate of ventricular relaxation and greater left ventricle output compared with the 21% O2 group. Further, our findings indicate that juvenile alligators that experienced embryonic hypoxia have a faster rate of ventricular relaxation, greater left ventricle stroke volume and greater cardiac power following ß-adrenergic stimulation, compared with juvenile alligators that did not experience embryonic hypoxia. When juveniles were exposed to 5% O2 for 20 min, normoxic-incubated juveniles had a 50% decline in left ventricle maximal rate of pressure development and maximal pressure; however, these parameters were unaffected and decreased less in the hypoxic-incubated juveniles. These data indicate that embryonic hypoxia in crocodilians alters the cardiovascular phenotype, changing the juvenile response to acute hypoxia and ß-adrenergic stimulation.

A novel accessory respiratory muscle in the American alligator ( Alligator mississippiensis).

The muscles that effect lung ventilation are key to understanding the evolutionary constraints on animal form and function. Here, through electromyography, we demonstrate a newly discovered respiratory function for the iliocostalis muscle in the American alligator ( Alligator mississippiensis). The iliocostalis is active during expiration when breathing on land at 28°C and this activity is mediated through the uncinate processes on the vertebral ribs. There was also an increase in muscle activity during the forced expirations of alarm distress vocalizations. Interestingly, we did not find any respiratory activity in the iliocostalis when the alligators were breathing with their body submerged in water at 18°C, which resulted in a reduced breathing frequency. The iliocostalis is an accessory breathing muscle that alligators are able to recruit in to assist expiration under certain conditions.

Hemodynamics of tonic immobility in the American alligator (Alligator mississippiensis) identified through Doppler ultrasonography.

American alligators (Alligator mississippiensis) held inverted exhibit tonic immobility, combining unresponsiveness with flaccid paralysis. We hypothesize that inverting the alligator causes a gravitationally promoted increase in right aortic blood flowing through the foramen of Panizza, with a concurrent decrease in blood flow through the primary carotid, and thereby of cerebral perfusion. Inverting the alligator results in displacement of the liver, post-pulmonary septum, and the heart. EKG analysis revealed a significant decrease in heart rate following inversion; this decrease was maintained for approximately 45 s after inversion which is in general agreement with the total duration of tonic immobility in alligators (49 s). Doppler ultrasonography revealed that following inversion of the alligator, there was a reversal in direction of blood flow through the foramen of Panizza, and this blood flow had a significant increase in velocity (compared to the foraminal flow in the prone alligator). There was an associated significant decrease in the velocity of blood flow through the primary carotid artery once the alligator was held in the supine position. Tonic immobility in the alligator appears to be a form of vasovagal syncope which arises, in part, from the unique features of the crocodilian heart.

Contribution of active atrial contraction to cardiac output in anesthetized American alligators (Alligator mississippiensis).

Ventricular filling may occur directly from the venous circulation during early diastole or via atrial contraction in late diastole. The contribution of atrial contraction to ventricular filling is typically small in mammals (10-40%), but has been suggested to predominate in reptiles. We investigated the importance of atrial contraction in filling of the ventricle in American alligators (Alligator mississippiensis) by bypassing both atria (with the use of ligatures to prevent atrial filling) and measuring the resultant effects on cardiac output in anesthetized animals. Atrial ligation had no significant effects on total systemic blood flow before or after adrenaline injection. Unexpectedly, pulmonary flow was increased following atrial ligation prior to adrenaline treatment, but was unaffected after it. These findings suggest that the atria are non-essential (i.e. redundant) for ventricular filling in alligators, at least under anesthesia, but may serve as important volume reservoirs.

Vascular patterns in the heads of crocodilians: blood vessels and sites of thermal exchange.

Extant crocodilians are a highly apomorphic archosaur clade that is ectothermic, yet often achieve large body sizes that can be subject to higher heat loads. Therefore, the anatomical and physiological roles that blood vessels play in crocodilian thermoregulation need further investigation to better understand how crocodilians establish and maintain cephalic temperatures and regulate neurosensory tissue temperatures during basking and normal activities. The cephalic vascular anatomy of extant crocodilians, particularly American alligator (Alligator mississippiensis) was investigated using a differential-contrast, dual-vascular injection technique and high resolution X-ray micro-computed tomography (µCT). Blood vessels were digitally isolated to create representations of vascular pathways. The specimens were then dissected to confirm CT results. Sites of thermal exchange, consisting of the oral, nasal, and orbital regions, were given special attention due to their role in evaporative cooling and cephalic thermoregulation in other diapsids. Blood vessels to and from sites of thermal exchange were studied to detect conserved vascular patterns and to assess their ability to deliver cooled blood to neurosensory tissues. Within the orbital region, both the arteries and veins demonstrated consistent branching patterns, with the supraorbital, infraorbital, and ophthalmotemporal vessels supplying and draining the orbit. The venous drainage of the orbital region showed connections to the dural sinuses via the orbital veins and cavernous sinus. The palatal region demonstrated a vast plexus that comprised both arteries and veins. The most direct route of venous drainage of the palatal plexus was through the palatomaxillary veins, essentially bypassing neurosensory tissues. Anastomotic connections with the nasal region, however, may provide an alternative route for palatal venous blood to reach neurosensory tissues. The nasal region in crocodilians is probably the most prominent site of thermal exchange, as it offers a substantial surface area and is completely surrounded by blood vessels. The venous drainage routes from the nasal region offer routes directly to the dural venous sinuses and the orbit, offering evidence of the potential to directly affect neurosensory tissue temperatures. The evolutionary history of crocodilians is complex, with large-bodied, terrestrial, and possibly endothermic taxa that may have had to deal with thermal loads that likely provided the anatomical building-blocks for such an extensive vascularization of sites of thermal exchange. A clear understanding of the physiological abilities and the role of blood vessels in the thermoregulation of crocodilians neurosensory tissues is not available but vascular anatomical patterns of crocodilian sites of thermal exchange indicate possible physiological abilities that may be more sophisticated than in other extant diapsids.

Methyltestosterone alters sex determination in the American alligator (Alligator mississippiensis).

Effects of xenobiotics can be organizational, permanently affecting anatomy during embryonic development, and/or activational, influencing transitory actions during adulthood. The organizational influence of endocrine-disrupting contaminants (EDC's) produces a wide variety of reproductive abnormalities among vertebrates that exhibit temperature-dependent sex determination (TSD). Typically, such influences result in subsequent activational malfunction, some of which are beneficial in aquaculture. For example, 17-αmethyltestosterone (MT), a synthetic androgen, is utilized in tilapia farming to bias sex ratio towards males because they are more profitable. A heavily male-biased hatchling sex ratio is reported from a crocodile population near one such tilapia operation in Guanacaste, Costa Rica. In this study we test the effects of MT on sexual differentiation in American alligators, which we used as a surrogate for all crocodilians. Experimentally, alligators were exposed to MT in ovo at standard ecotoxicological concentrations. Sexual differentiation was determined by examination of primary and secondary sex organs post hatching. We find that MT is capable of producing male embryos at temperatures known to produce females and demonstrate a dose-dependent gradient of masculinization. Embryonic exposure to MT results in hermaphroditic primary sex organs, delayed renal development and masculinization of the clitero-penis (CTP).

Size, sex and individual-level behaviour drive intrapopulation variation in cross-ecosystem foraging of a top-predator.

Large-bodied, top-predators are often highly mobile, with the potential to provide important linkages between spatially distinct food webs. What biological factors contribute to variation in cross-ecosystem movements, however, have rarely been examined. Here, we investigated how ontogeny (body size), sex and individual-level behaviour impacts intrapopulation variation in cross-ecosystem foraging (i.e. between freshwater and marine systems), by the top-predator Alligator mississippiensis. Field surveys revealed A. mississippiensis uses marine ecosystems regularly and are abundant in estuarine tidal creeks (from 0·3 to 6·3 individuals per km of creek, n = 45 surveys). Alligator mississippiensis captured in marine/estuarine habitats were significantly larger than individuals captured in freshwater and intermediate habitats. Stomach content analysis (SCA) showed that small juveniles consumed marine/estuarine prey less frequently (6·7% of individuals) than did large juveniles (57·8%), subadult (73%), and adult (78%) size classes. Isotopic mixing model analysis (SIAR) also suggests substantial variation in use of marine/estuarine prey resources with differences among and within size classes between sexes and individuals (range of median estimates for marine/estuarine diet contribution = 0·05-0·76). These results demonstrate the importance of intrapopulation characteristics (body size, sex and individual specialization) as key determinants of the strength of predator-driven ecosystem connectivity resulting from cross-ecosystem foraging behaviours. Understanding the factors, which contribute to variation in cross-ecosystem foraging behaviours, will improve our predictive understanding of the effects of top-predators on community structure and ecosystem function.

Reference levels for corticosterone and immune function in farmed saltwater crocodiles (Crocodylus porosus) hatchlings using current Code of Practice guidelines.

To determine reference levels for on-farm stressors on immune responsiveness and growth rate, 253 hatchling crocodiles from 11 known breeding pairs were repeatedly measured and blood sampled during their first year. Plasma corticosterone (CORT) was used to quantify baseline stress levels in captive animals and were found to be lower (mean 1.83±SE 0.16 ng/mL) than previously reported in saltwater crocodile hatchlings. Two tests of immune function were also conducted. Innate constitutive immunity was assessed using bacterial killing assays (BKA) against two bacterial species: Escherichia coli and Providencia rettgeri, whereby the latter causes considerable economic loss to industry from septicaemic mortalities. Although the bactericidal capabilities were different at approximately 4 months old (32±3% for E. coli and 16±4% for P. rettgeri), the differences had disappeared by approximately 9 months old (58±2% and 68±6%, respectively). To assess immune responsiveness to a novel antigen, the inflammatory swelling response caused by phytohaemagglutinin (PHA) injection was assessed but was only significantly different between Samplings 1 and 3 (5% LSD). There were no significant clutch effects for CORT or PHA but there were for both BKA traits. CORT was not significantly associated with growth (head length) or the immune parameters except for P. rettgeri BKA where higher CORT levels were associated with better bactericidal capability. As such, these results suggest that the crocodiles in this study are not stressed, therefore endorsing the management strategies adopted within the Australian industry Code of Practice.