Clutch may predict growth of hatchling Burmese pythons better than food availability or sex.

Identifying which environmental and genetic factors affect growth pattern phenotypes can help biologists predict how organisms distribute finite energy resources in response to varying environmental conditions and physiological states. This information may be useful for monitoring and managing populations of cryptic, endangered, and invasive species. Consequently, we assessed the effects of food availability, clutch, and sex on the growth of invasive Burmese pythons (Python bivittatus Kuhl) from the Greater Everglades Ecosystem in Florida, USA. Though little is known from the wild, Burmese pythons have been physiological model organisms for decades, with most experimental research sourcing individuals from the pet trade. Here, we used 60 hatchlings collected as eggs from the nests of two wild pythons, assigned them to High or Low feeding treatments, and monitored growth and meal consumption for 12 weeks, a period when pythons are thought to grow very rapidly. None of the 30 hatchlings that were offered food prior to their fourth week post-hatching consumed it, presumably because they were relying on internal yolk stores. Although only two clutches were used in the experiment, we found that nearly all phenotypic variation was explained by clutch rather than feeding treatment or sex. Hatchlings from clutch 1 (C1) grew faster and were longer, heavier, in better body condition, ate more frequently, and were bolder than hatchlings from clutch 2 (C2), regardless of food availability. On average, C1 and C2 hatchling snout-vent length (SVL) and weight grew 0.15 cm d-1 and 0.10 cm d-1, and 0.20 g d-1 and 0.03 g d-1, respectively. Additional research may be warranted to determine whether these effects remain with larger clutch sample sizes and to identify the underlying mechanisms and fitness implications of this variation to help inform risk assessments and management. This article has an associated First Person interview with the first author of the paper.

Development of hemipenes in the ball python snake Python regius.

Within amniotes, external copulatory organs have undergone extensive morphological diversification. One of the most extreme examples is squamate (lizards and snakes) hemipenes, which are paired copulatory organs that extend from the lateral margins of the cloaca. Here, we describe the development of hemipenes in a basal snake, the ball python (Python regius). Snake hemipenes arise as a pair of lateral swellings on either side of the caudal part of the cloaca, and these paired outgrowths persist to form the left and right hemipenes. In non-squamate amniotes, external genitalia form from paired swellings that arise on the anterior side of the cloaca, which then fuse medially to form a single genital tubercle, the anlagen of the penis or clitoris. Whereas in non-squamate amniotes, Sonic hedgehog (Shh)-expressing cells of the cloacal endoderm form the urethral or sulcus epithelium and are required for phallus outgrowth, the hemipenes of squamates lack an endodermal contribution, and the sulcus does not express Shh. Thus, snake hemipenes differ from the genital tubercles of non-squamate amniotes both in their embryonic origins and in at least part of patterning mechanisms, which raises the possibility that hemipenes may not be direct homologs of the unpaired amniote penis. Nonetheless, we find that some developmental genes show similar expression patterns in snake hemipenes buds and non-squamate genital tubercles, suggesting that homologous developmental mechanisms are involved in aspects of external genital development across amniotes, even when these structures may have different developmental origins and may have arisen independently during evolution.

Can reptile embryos influence their own rates of heating and cooling?

Previous investigations have assumed that embryos lack the capacity of physiological thermoregulation until they are large enough for their own metabolic heat production to influence nest temperatures. Contrary to intuition, reptile embryos may be capable of physiological thermoregulation. In our experiments, egg-sized objects (dead or infertile eggs, water-filled balloons, glass jars) cooled down more rapidly than they heated up, whereas live snake eggs heated more rapidly than they cooled. In a nest with diel thermal fluctuations, that hysteresis could increase the embryo's effective incubation temperature. The mechanisms for controlling rates of thermal exchange are unclear, but may involve facultative adjustment of blood flow. Heart rates of snake embryos were higher during cooling than during heating, the opposite pattern to that seen in adult reptiles. Our data challenge the view of reptile eggs as thermally passive, and suggest that embryos of reptile species with large eggs can influence their own rates of heating and cooling.

Maternal influences on early development: preferred temperature prior to oviposition hastens embryogenesis and enhances offspring traits in the Children's python, Antaresia childreni.

Embryonic life is particularly sensitive to its surroundings, and the developmental environment can have long-lasting effects on offspring. In oviparous species, the impacts of the developmental environment on offspring traits are mostly examined during development within the egg. However, as more than 25% of the development of squamate reptiles can occur prior to oviposition, we explored the effect of thermal conditions on development prior to oviposition in an oviparous snake species, the Children's python (Antaresia childreni). We housed gravid female pythons under three thermal cycles: an optimal regime that reflected maternal preference in a non-constrained environment (constant preferred body temperature of gravid females, T(set)=31.5°C) and two mildly suboptimal regimes that shared the same mean temperature of 27.7°C, but differed in the duration at T(set). In one of the constraining regimes, females had access to T(set) for 4 h daily whereas in the other regime, females never reached T(set) (maximal temperature of 29.0°C). Thermal treatments were maintained throughout gravidity in all three groups, but, after oviposition, all eggs were incubated at T(set) until hatching. Compared with the optimal regime, the two suboptimal regimes had a longer duration of gravidity, which resulted in delayed hatching. Between the two suboptimal regimes, gravidity was significantly shorter in the treatment that included time at T(set). Furthermore, suboptimal regimes influenced offspring traits at hatching, including body morphology, antipredator behavior, strength and metabolism. However, partial access to maternal T(set) significantly enhanced several offspring traits, including performance. Our results demonstrate the importance of time at T(set) on early development and suggest an adaptive significance of maternal thermoregulation prior to oviposition.

Unicuspid and bicuspid tooth crown formation in squamates.

The molecular and developmental factors that regulate tooth morphogenesis in nonmammalian species, such as snakes and lizards, have received relatively little attention compared to mammals. Here we describe the development of unicuspid and bicuspid teeth in squamate species. The simple, cone-shaped tooth crown of the bearded dragon and ball python is established at cap stage and fixed in shape by the differentiation of cells and the secretion of dental matrices. Enamel production, as demonstrated by amelogenin expression, occurs relatively earlier in squamate teeth than in mouse molars. We suggest that the early differentiation in squamate unicuspid teeth at cap stage correlates with a more rudimentary tooth crown shape. The leopard gecko can form a bicuspid tooth crown despite the early onset of differentiation. Cusp formation in the gecko does not occur by the folding of the inner enamel epithelium, as in the mouse molar, but by the differential secretion of enamel. Ameloblasts forming the enamel epithelial bulge, a central swelling of cells in the inner enamel epithelium, secrete amelogenin at cap stage, but cease to do so by bell stage. Meanwhile, other ameloblasts in the inner enamel epithelium continue to secrete enamel, forming cusp tips on either side of the bulge. Bulge cells specifically express the gene Bmp2, which we suggest serves as a pro-differentiation signal for cells of the gecko enamel organ. In this regard, the enamel epithelial bulge of the gecko may be more functionally analogous to the secondary enamel knot of mammals than the primary enamel knot.

A network of Wnt, hedgehog and BMP signaling pathways regulates tooth replacement in snakes.

Most dentate vertebrates, from fish to humans, replace their teeth and yet the molecular basis of tooth replacement is poorly understood. Canonical Wnt signaling regulates tooth number in mice and humans, but it is unclear what role it plays in tooth replacement as it naturally occurs. To clarify this, we characterized Wnt signaling activity in the dental tissues of the ball python Python regius. This species replaces teeth throughout life (polyphyodonty) and in the same manner as in humans, i.e., sequential budding of teeth from the tip of the dental lamina. From initiation stage onwards, canonical Wnt read-out genes (Lef1 and Axin2) are persistently expressed by cells in the dental lamina tip and surrounding mesenchyme. This implies that molecular signaling at work during dental initiation carries over to tooth replacement. We show that canonical Wnt signaling promotes cell proliferation in python dental tissues and that by confining Wnt activity in the dental lamina the structure extends instead of thickens. Presumably, lamina extension creates space between successive tooth buds, ensuring that tooth replacement occurs in an ordered manner. We suggest that hedgehog signaling confines Wnt activity in the dental epithelium by direct planar repression and, during tooth replacement stages, by negatively regulating BMP levels in the dental mesenchyme. Finally, we propose that Wnt-active cells at the extending tip of the python dental lamina represent the immediate descendents of putative stem cells housed in the lingual face of the lamina, similar to what we have recently described for another polyphyodont squamate species.

Initiation and patterning of the snake dentition are dependent on Sonic hedgehog signaling.

Here we take the first look at cellular dynamics and molecular signaling in the developing snake dentition. We found that tooth formation differs from rodents in several respects. The majority of snake teeth bud off of a deep, ribbon-like dental lamina rather than as separate tooth germs. Prior to and after dental lamina ingrowth, we observe asymmetries in cell proliferation and extracellular matrix distribution suggesting that localized signaling by a secreted protein is involved. We cloned Sonic hedgehog from the African rock python Python sebae and traced its expression in the species as well as in two other snakes, the closely-related Python regius and the more derived corn snake Elaphe guttata (Colubridae). We found that expression of Shh is first confined to the odontogenic band and defines the position of the future dental lamina. Shh transcripts in pythons are progressively restricted to the oral epithelium on one side of the dental lamina and remain in this position throughout the prehatching period. Shh is expressed in the inner enamel epithelium and the stellate reticulum of the tooth anlagen, but is absent from the outer enamel epithelium and its derivative, the successional lamina. This suggests that signals other than Shh are responsible for replacement tooth formation. Functional studies using cyclopamine to block Hh signaling during odontogenesis prevented initiation and extension of the dental lamina into the mesenchyme, and also affected the directionality of this process. Further, blocking Hh signaling led to disruptions of the inner enamel epithelium. To explore the role of Shh in lamina extension, we looked at its expression in the premaxillary teeth, which form closer to the oral surface than elsewhere in the mouth. Oral ectodermal Shh expression in premaxillary teeth is lost soon after the teeth form reinforcing the idea that Shh is controlling the depth of the dental lamina. In summary, we have found diverse roles for Shh in patterning the snake dentition but, have excluded the participation of this signal in replacement tooth formation.

Alternating egg-brooding behaviors create and modulate a hypoxic developmental micro-environment in Children's pythons (Antaresia childreni).

Parental care is a widespread and ecologically relevant adaptation known to enhance the developmental environment of offspring. Parental behaviors, however, may entail both costs and benefits for developing offspring. In Children's pythons (Antaresia childreni), we monitored both maternal egg-brooding behavior and intra-clutch oxygen partial pressure (PO2) in real-time to assess the effects of various brooding behaviors on PO2 in the clutch micro-environment at three stages of development. Furthermore, at the same developmental stages, we measured O2 consumption rates (VO2) of eggs at varying PO2 to determine their critical oxygen tension (i.e. the minimal PO2 that supports normal respiratory gas exchange) and to predict the impact that naturally brooded intra-clutch PO2 has on embryonic metabolism. At all three stages of development, a tightly coiled brooding posture created an intra-clutch PO2 that was significantly lower than the surrounding nest environment. Maternal postural adjustments alleviated this hypoxia, and the magnitude of such corrections increased with developmental stage. Mean intra-clutch PO2 decreased with stage of development, probably because of increasing egg VO2. Additionally, embryo critical oxygen tension increased with developmental stage. Together, these results suggest that python embryos are unable to maintain normal metabolism under brooded conditions during the final 10% of incubation. These results demonstrate that specific parental behaviors can impose obligatory costs to developing offspring and that balancing these behaviors can mediate deleterious consequences.

Embryonic development of Python sebae - I: Staging criteria and macroscopic skeletal morphogenesis of the head and limbs.

This study explores the post-ovipositional craniofacial development of the African Rock Python (Python sebae). We first describe a staging system based on external characteristics and next use whole-mount skeletal staining supplemented with Computed tomography (CT) scanning to examine skeletal development. Our results show that python embryos are in early stages of organogenesis at the time of laying, with separate facial prominences and pharyngeal clefts still visible. Limb buds are also visible. By 11 days (stage 3), the chondrocranium is nearly fully formed; however, few intramembranous bones can be detected. One week later (stage 4), many of the intramembranous upper and lower jaw bones are visible but the calvaria are not present. Skeletal elements in the limbs also begin to form. Between stages 4 (day 18) and 7 (day 44), the complete set of intramembranous bones in the jaws and calvaria develops. Hindlimb development does not progress beyond stage 6 (33 days) and remains rudimentary throughout adult life. In contrast to other reptiles, there are two rows of teeth in the upper jaw. The outer tooth row is attached to the maxillary and premaxillary bones, whereas the inner row is attached to the pterygoid and palatine bones. Erupted teeth can be seen in whole-mount stage 10 specimens and are present in an unerupted, mineralized state at stage 7. Micro-CT analysis reveals that all the young membranous bones can be recognized even out of the context of the skull. These data demonstrate intrinsic patterning of the intramembranous bones, even though they form without a cartilaginous template. In addition, intramembranous bone morphology is established prior to muscle function, which can influence bone shape through differential force application. After careful staging, we conclude that python skeletal development occurs slowly enough to observe in good detail the early stages of craniofacial skeletogenesis. Thus, reptilian animal models will offer unique opportunities for understanding the early influences that contribute to perinatal bone shape.

Embryonic development of Python sebae - II: Craniofacial microscopic anatomy, cell proliferation and apoptosis.

This study explores the microscopic craniofacial morphogenesis of the oviparous African rock python (Python sebae) spanning the first two-thirds of the post-oviposition period. At the time of laying, the python embryo consists of largely undifferentiated mesenchyme and epithelium with the exception of the cranial base and trabeculae cranii, which are undergoing chondrogenesis. The facial prominences are well defined and are at a late stage, close to the time when lip fusion begins. Later (11-12d), specializations in the epithelia begin to differentiate (vomeronasal and olfactory epithelia, teeth). Dental development in snakes is different from that of mammals in several aspects including an extended dental lamina with the capacity to form 4 sets of generational teeth. In addition, the ophidian olfactory system is very different from the mammalian. There is a large vomeronasal organ, a nasal cavity proper and an extraconchal space. All of these areas are lined with a greatly expanded olfactory epithelium. Intramembranous bone differentiation is taking place at stage 3 with some bones already ossifying whereas most are only represented as mesenchymal condensations. In addition to routine histological staining, PCNA immunohistochemistry reveals relatively higher levels of proliferation in the extending dental laminae, in osseous mesenchymal condensations and in the olfactory epithelia. Areas undergoing apoptosis were noted in the enamel organs of the teeth and osseous mesenchymal condensations. We propose that localized apoptosis helps to divide a single condensation into multiple ossification centres and this is a mechanism whereby novel morphology can be selected in response to evolutionary pressures. Several additional differences in head morphology between snakes and other amniotes were noted including a palatal groove separating the inner and outer row of teeth in the upper jaw, a tracheal opening within the tongue and a pharyngeal adhesion that closes off the pharynx from the oral cavity between stages 1 and 4. Our studies on these and other differences in the python will provide valuable insights into in developmental, molecular and evolutionary mechanisms of patterning.

Distribution of lipids from the yolk to the tissues during development of the water python (Liasis fuscus).

Energy metabolism during embryonic development of snakes differs in several respects from the patterns displayed by other reptiles. There are, however, no previous reports describing the main energy source for development, the yolk lipids, in snake eggs. There is also no information on the distribution of yolk fatty acids to the tissues during snake development. In eggs of the water python ( Liasis fuscus), we report that triacylglycerol, phospholipid, cholesteryl ester and free cholesterol, respectively, form 70.3%, 14.1%, 5.7% and 2.1% of the total lipid. The main polyunsaturate of the yolk lipid classes is 18:2n-6. The yolk phospholipid contains 20:4n-6 and 22:6n-3 at 13.0% and 3.6% (w/w), respectively. Approximately 10% and 30% of the initial egg lipids are respectively recovered in the residual yolk and the fat body of the hatchling. A major function of yolk lipid is, therefore, to provision the neonate with large energy reserves. The proportion of 22:6n-3 in brain phospholipid of the hatchling is 11.1% (w/w): this represents only 0.24% of the amount of 22:6n-3 originally present in the egg. This also contrasts with values for free-living avian species where the proportion of DHA in neonatal brain phospholipid is 16-19%. In the liver of the newly hatched python, triacylglycerol, phospholipid and cholesteryl ester, respectively, form 68.2%, 7.7% and 14.3% of total lipid. This contrasts with embryos of birds where cholesteryl ester forms up to 80% of total liver lipid and suggests that the mechanism of lipid transfer in the water python embryo differs in some respects from the avian situation.

Epidermal differentiation during ontogeny and after hatching in the snake Liasis fuscus (Pythonidae, Serpentes, Reptilia), with emphasis on the formation of the shedding complex.

Differentiation and localization of keratin in the epidermis during embryonic development and up to 3 months posthatching in the Australian water python, Liasis fuscus, was studied by ultrastructural and immunocytochemical methods. Scales arise from dome-like folds in the skin that produce tightly imbricating scales. The dermis of these scales is completely differentiated before any epidermal differentiation begins, with a loose dermis made of mesenchymal cells beneath the differentiating outer scale surface. At this stage (33) the embryo is still unpigmented and two layers of suprabasal cells contain abundant glycogen. At Stage 34 (beginning of pigmentation) the first layers of cells beneath the bilayered periderm (presumptive clear and oberhautchen layers) have not yet formed a shedding complex, within which prehatching shedding takes place. At Stage 35 the shedding complex, consisting of the clear and oberhautchen layers, is discernible. The clear layer contains a fine fibrous network that faces the underlying oberhautchen, where the spinulae initially contain a core of fibrous material and small beta-keratin packets. Differentiation continues at Stage 36 when the beta-layer forms and beta-keratin packets are deposited both on the fibrous core of the oberhautchen and within beta-cells. Mesos cells are produced from the germinal layer but remain undifferentiated. At Stage 37, before hatching, the beta-layer is compact, the mesos layer contains mesos granules, and cells of the alpha-layer are present but are not yet keratinized. They are still only partially differentiated a few hours after hatching, when a new shedding complex is forming underneath. Using antibodies against chick scale beta-keratin resolved at high magnification with immunofluorescent or immunogold conjugates, we offer the first molecular confirmation that in snakes only the oberhautchen component of the shedding complex and the underlying beta cells contain beta-keratin. Initially, there is little immunoreactivity in the small beta-packets of the oberhautchen, but it increases after fusion with the underlying cells to produce the syncytial beta layer. The beta-keratin packets coalesce with the tonofilaments, including those attached to desmosomes, which rapidly disappear in both oberhautchen and beta-cells as differentiation progresses. The labeling is low to absent in forming mesos-cells beneath the beta-layer. This study further supports the hypothesis that the shedding complex in lepidosaurian reptiles evolved after there was a segregation between alpha-keratogenic cells from beta-keratogenic cells during epidermal renewal.

Developmental basis of limblessness and axial patterning in snakes.

The evolution of snakes involved major changes in vertebrate body plan organization, but the developmental basis of those changes is unknown. The python axial skeleton consists of hundreds of similar vertebrae, forelimbs are absent and hindlimbs are severely reduced. Combined limb loss and trunk elongation is found in many vertebrate taxa, suggesting that these changes may be linked by a common developmental mechanism. Here we show that Hox gene expression domains are expanded along the body axis in python embryos, and that this can account for both the absence of forelimbs and the expansion of thoracic identity in the axial skeleton. Hindlimb buds are initiated, but apical-ridge and polarizing-region signalling pathways that are normally required for limb development are not activated. Leg bud outgrowth and signalling by Sonic hedgehog in pythons can be rescued by application of fibroblast growth factor or by recombination with chick apical ridge. The failure to activate these signalling pathways during normal python development may also stem from changes in Hox gene expression that occurred early in snake evolution.