Effects of temperature and moisture during incubation on carcass composition of hatchling snapping turtles (Chelydra serpentina)

Summary Flexible-shelled eggs of common snapping turtles (Chelydra serpentina) were incubated on each of two substrates (vermiculite, sand) at each of three temperatures (26.0°C, 28.5°C, 31.0°C) and three moisture regimes (wet, intermediate, dry). Embryos developing in cool, wet environments mobilized the largest amounts of protein from their yolk and attained the largest size before hatching, whereas turtles developing in warm, dry environments mobilized the smallest quantities of protein and were the smallest in body size at hatching. Embryos on wet substrates mobilized more lipid from their yolk than did embryos on dry media, but ambient temperature had no demonstrable influence on patterns of lipid mobilization. The total reserve of neutral lipid available in residual yolk plus carcass to sustain neonates in the interval prior to the beginning of feeding was largest in hatchlings from dry environments and smallest in animals from wet environments, but was unaffected by temperature during incubation. Hydration of tissues in hatchlings was higher when incubation was in cool, moist conditions than when incubation was in warm, dry settings, thereby indicating that some of the effects of moisture and temperature on mobilization of nutrients by embryos may be mediated by differences in intracellular water. Patterns of response to temperature and moisture recorded for turtles emerging from eggs on sand were similar to those recorded for hatchlings on vermiculite, so no important conclusion would have been affected by incubating eggs on one medium instead of the other.     

Wetness of the nest environment influences cardiac development in pre- and post-natal snapping turtles (Chelydra serpentina)

We dissected hearts from near-term embryos and hatchlings of common snapping turtles (Chelydridae: Chelydra serpentina) whose eggs had incubated on wet or dry substrates, and then dried and individually weighed the heart and yolk-free carcass from each animal. Hearts and carcasses of prenatal and neonatal animals grew at different rates, and the patterns of growth by both heart and carcass differed between wet and dry environments. Hearts grew faster, both in actual mass and in mass adjusted for variation in body size, in embryos and hatchlings whose eggs were incubated on dry substrates than in animals whose eggs were held on wet media. This finding is consistent with a hypothesis that embryos incubating in dry settings experience hypovolemia secondary to dehydration and that enlargement of the heart compensates, in part, for the associated increase in viscosity of the blood. Embryonic turtles seemingly exhibit the same plasticity and response that would be expected from other vertebrate ectotherms subjected to the physiological challenges associated with desiccation and an associated reduction in blood volume.     

Availability of water affects organ growth in prenatal and neonatal snapping turtles (Chelydra serpentina)

We manipulated the amount of water that was available to prenatal and neonatal snapping turtles (Chelydra serpentina) in order to assess the impact of water on growth by different organs in these animals. Three treatments were used: (1) turtles that completed their incubation on a wet substrate, (2) turtles that completed their incubation on a dry substrate, and (3) turtles that spent a few days in water after completing incubation on a dry substrate. Turtles hatching on a dry substrate (treatment 2) were smaller than animals in the other two treatments (which did not differ in size), so data for mass of different organs were adjusted by ANCOVA to remove effects of body size. Scaled masses of liver, stomach, lungs, kidneys, and small intestine did not differ between turtles emerging in wet environments and those hatching in dry environments, but hearts of turtles hatching in dry settings were substantially larger than those of animals hatching in wet ones. Thus, the mass of most organs in turtles developing in wet and dry environments scaled to body size, whereas the heart was hypertrophied in embryos developing in dry environments. Turtles that spent a few days in water after hatching from eggs in dry environments grew rapidly in size, and the increase in body size was accompanied by disproportionately rapid growth in the liver, stomach, lungs, kidneys, and small intestine. The heart did not increase in size during this period, despite the substantial increase in body mass over that at hatching. The enlarged heart of turtles hatching on dry substrates may have been caused by a circulatory hypovolemia late in incubation; the rapid growth of organs other than the heart when these animals were placed in water may reflect a release from constraints on growth once circulatory volume was restored. Accepted: 2 November 1999     

Does the hydric environment affect the incubation of small rigid-shelled turtle eggs?

Hydric environments are hypothesized to have minor effects on the embryonic development of rigid-shelled turtle eggs due to the low water permeability of the eggshell. However, the water reserve in the eggs may still influence their resistance to environmentally induced dehydration. We incubated rigid-shelled turtle eggs (Pelodiscus sinensis) on different moist substrates (from ? 12 to ? 750 kPa) to test the hypothesis that small rigid-shelled eggs would be sensitive to hydric environments. The hydric treatment significantly affected the incubation period, with eggs incubated in the moistest and driest substrates taking longer to hatch than those on the medium-moisture substrates. Hatching success was slightly lower for eggs incubated in dry conditions than those in wet conditions, but the difference was not statistically significant. The heart rates of early embryos were lower on moist substrates than those on dry substrates, but this difference disappeared in late embryos. Hatchlings from the moistest substrate were larger (in carapace length and width) and heavier than those from drier substrates. However, the dry body mass of the hatchlings did not differ among the hydric treatments. The functional performance (righting response) of the hatchlings was affected by the hydric environment. The time to right was shorter for the hatchlings from the substrate of ? 12 kPa than those from ? 220 kPa. These results are consistent with the hypothesis that the hydric environment may significantly affect developing embryos and the resulting hatchlings in turtle species, such as P. sinensis, with small rigid-shelled eggs.     

Sources and timing of calcium mobilization during embryonic development of the corn snake, Pantherophis guttatus

Embryos of oviparous Reptilia (=turtles, lepidosaurs, crocodilians and birds) extract calcium for growth and development from reserves in the yolk and eggshell. Yolk provides most of the calcium to embryos of lizards and snakes. In contrast, the eggshell supplies most of the calcium for embryonic development of turtles, crocodilians and birds. The yolk sac and chorioallantoic membrane of birds recover and transport calcium from the yolk and eggshell and homologous membranes of squamates (lizards and snakes) probably transport calcium from these two sources as well. We studied calcium mobilization by embryos of the snake Pantherophis guttatus during the interval of greatest embryonic growth and found that the pattern of calcium transfer was similar to other snakes. Calcium recovery from the yolk is relatively low until the penultimate embryonic stage. Calcium removal from the eggshell begins during the same embryonic stage and total eggshell calcium drops in each of the final 2 weeks prior to hatching. The eggshell supplies 28% of the calcium of hatchlings. The timing of calcium transport from the yolk and eggshell is coincident with the timing of growth of the yolk sac and chorioallantoic membrane and expression of the calcium binding protein, calbindin-D28K, in these tissues as reported in previous studies. In the context of earlier work, our findings suggest that the timing and mechanism of calcium transport from the yolk sac of P. guttatus is similar to birds, but that both the timing and mechanism of calcium transport by the chorioallantoic membrane differs. Based on the coincident timing of eggshell calcium loss and embryonic calcium accumulation, we also conclude that recovery of eggshell calcium in P. guttatus is regulated by the embryo.     

Some analyses of artificially incubated eggs and hatchlings of Green and Loggerhead sea turtles

Eggs of the Green sea turtle ( Chelonia mydas ) and the Loggerhead sea turtle ( Caretta caretta ) were incubated in coral sand or silica sand moistened with distilled water. The eggs and hatchlings were analysed for calcium, magnesium and phosphorus. There was less magnesium but more calcium in the hatchlings than in the original egg contents but this was not influenced by the type of sand used during incubation. The results are interpreted as indicating that the eggshell is the major source of calcium for the developing embryos. The Logger-head sea turtle shows a greater efficiency in the incorporation of yolk magnesium and phosphorus into the embryo than does the Green sea turtle.     

Calcium mobilization, water balance, and growth in embryos of the agamid lizard Amphibolurus barbatus

Embryos of the agamid lizard Amphibolurus barbatus are at developmental stage 29 of Dufaure and Hubert at the time of oviposition. Mobilization of calcium and other nutrients from the yolk proceeds slowly for the first half of incubation, during which time embryonic growth also proceeds slowly. During the second half of incubation, however, embryos withdraw calcium and other nutrients from the yolk very rapidly, and growth rates are correspondingly high. Approximately 60% of the calcium used by developing embryos is obtained from the yolk, but fully 40% of their requirement is met by calcium mobilized from the eggshell. Very little calcium remains in residual yolk of hatchlings, so this yolk must be used in maintenance metabolism rather than in growth of neonates. No dichotomy exists among oviparous, amniotic vertebrates with respect to sources of calcium used by developing embryos, but one does exist with respect to patterns of mobilization of this element. Whereas calcium is extracted from yolk of embryonic reptiles throughout incubation, it actually is deposited in yolk of embryonic birds after the midpoint in development.     

Vitamin D and chick embryonic yolk calcium mobilization: identification and regulation of expression of vitamin D-dependent Ca2(+)-binding protein, calbindin-D28K, in the yolk sac

The developing chick embryo acquires calcium from two sources. Until about Day 10 of incubation, the yolk is the only source; thereafter, calcium is also mobilized from the eggshell. We have previously shown that during normal chick embryonic development, vitamin D is involved in regulating yolk calcium mobilization, whereas vitamin K is required for eggshell calcium translocation by the chorioallantoic membrane. We have studied here the biochemical action of 1,25-dihydroxy vitamin D3 in the yolk sac by examining the expression and regulation of the cytosolic vitamin D-dependent calcium-binding protein, calbindin-D28K. Two types of embryos are used for this study, normal embryos developing in ovo and embryos maintained in long-term shell-less culture ex ovo, the latter being dependent solely on the yolk as their calcium source. Our findings are (1) calbindin-D28K is expressed in the embryonic yolk sac, detectable at incubation Days 9 and 14; (2) the embryonic yolk sac calbindin-D28K resembles that of the adult duodenum in both molecular weight (Mr 28,000) and isoelectric point, as well as the presence of E-F hand Ca2(+)-binding structural domains; (3) systemic calcium deficiency caused by shell-less culture of chick embryos results in enhanced expression of calbindin-D28K in the yolk sac during late development; (4) yolk sac calbindin-D28K expression is inducible by 1,25-dihydroxy vitamin D3 treatment in vivo and in vitro; and (5) immunohistochemistry revealed that yolk sac calbindin-D28K is localized exclusively to the cytoplasm of the yolk sac endoderm. These findings indicate that the chick embryonic yolk sac is a genuine target tissue of 1,25-dihydroxy vitamin D3.     

Accumulation of calcium and phosphorus in pigeon (Columba livia) embryos

Summary Calcium and phosphorus were measured in the yolk and albumen of fertile pigeon (Columba livia) eggs incubated for 0–17 days, and in embryos and hatchlings. Shell provided most of the calcium for skeletal mineralization of the embryos, whereas phosphorus was derived from the yolk and albumen. Mobilization of calcium from the shell to the embryo commenced at approximately day 11 of incubation, accumulating both in the embryo and the yolk sac. There was 1.4 times more calcium in squab yolk sacs than that contained in newly laid egg yolks. The results suggest that whereas general patterns of calcium and phosphorus accumulation during embryogenesis in altricial birds closely resemble those of precocial birds, calcium mobilization from the shell begins later, proceeds at a slower rate and results in a less mineralized hatchling.CIDA/NSERC Visiting Research Associate Permanent address: Department of Animal Science, University of Peradeniya, Peradeniya, Sri Lanka     

Influence of water availability during incubation on hatchling size, body composition, desiccation tolerance, and terrestrial locomotor performance in the snapping turtle Chelydra serpentina

The effects of water availability during incubation on the water contents of neonatal snapping turtles at hatching were examined, along with the influence of hatchling water content on desiccation tolerance and terrestrial locomotor performance. The water contents of hatchlings from eggs incubated on wet substrates were both absolutely and proportionally greater than were those of hatchlings from eggs incubated on dry substrates. Hatchlings with greater water contents at hatching were able to survive longer and to lose more water before physiological performance was adversely affected by desiccation. Increased water contents in hatchlings with greater water availability during incubation may enhance survival by increasing the amount of water the animal can afford to lose before dehydration begins to adversely affect whole animal performance.     

Incubation of rigid-shelled turtle eggs: do hydric conditions matter?

Rigid-shelled eggs of the broad-shelled river turtle Chelodina expansa were incubated at 28 degrees C in wet (-100 kPa), intermediate (-350 kPa) and dry (-750 kPa) conditions. Incubation period was influenced by clutch of origin, but was independent of incubation water potential. Rates of water gained from the environment and pre-pipping egg mass were influenced by incubation water potential -- eggs incubating at higher (less negative) water potentials absorbing more water from their environment. Hatchlings from wet conditions had greater mass but a smaller amount of residual yolk than hatchlings from dry conditions and it is suggested that the amount of yolk converted to tissue is influenced by the amount of water absorbed by the egg during incubation. Water content of yolk-free hatchlings from the -100-kPa treatment was greater than those from the -350-kPa and -750-kPa treatments, but the water content of residual yolks was similar across all hydric conditions.     

Fetal nutrition in lecithotrophic squamate reptiles: Toward a comprehensive model for evolution of viviparity and placentation

The primary pattern of embryonic nutrition for squamate reptiles is lecithotrophy; with few exceptions, all squamate embryos mobilize nutrients from yolk. The evolution of viviparity presents an opportunity for an additional source of embryonic nutrition through delivery of uterine secretions, or placentotrophy. This pattern of embryonic nutrition is thought to evolve through placental supplementation of lecithotrophy, followed by increasing dependence on placentotrophy. This review analyzes the relationship between reproductive mode and pattern of embryonic nutrition in three lecithotrophic viviparous species, and oviparous counterparts, for concordance with a current model for the evolution of viviparity and placentation. The assumptions of the model, that nutrients for oviparous embryos are mobilized from yolk, and that this source is not disrupted in the transition to viviparity, are supported for most nutrients. In contrast, calcium, an essential nutrient for embryonic development, is mobilized from both yolk and eggshell by oviparous embryos and reduction of eggshell calcium is correlated with viviparity. If embryonic fitness is compromised by disruption of a primary source of calcium, selection may not favor evolution of viviparity, yet viviparity has arisen independently in numerous squamate lineages. Studies of fetal nutrition in reproductively bimodal species suggest a resolution to this paradox. If uterine calcium secretion occurs during prolonged intrauterine egg retention, calcium placentotrophy evolves prior to viviparity as a replacement for eggshell calcium and embryonic nutrition will not be compromised. This hypothesis is integrated into the current model for evolution of viviparity and placentation to address the unique attributes of calcium nutrition. The sequence of events requires a shift in timing of uterine calcium secretion and the embryonic mechanism of calcium retrieval to be responsive to calcium availability. Regulation of uterine calcium secretion and the mechanism of embryonic uptake of calcium are important elements to understanding evolution of viviparity and placentation. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Influence of the nest environment on bone mineral content in hatchling painted turtles (Chrysemys picta)

We performed an experiment at a field site in north-central Nebraska to assess the role of the nest environment in inducing variation in bone mineral content in hatchling painted turtles Chrysemys picta (Schneider 1783). The contents of several newly constructed nests were manipulated by reciprocal transplant, after which the eggs were allowed to incubate for 8 wk under natural conditions. The nests were then excavated, and the eggs were brought into the laboratory to complete incubation and hatch under standard conditions of temperature and moisture. The hatchlings were killed, and their carcasses and residual yolks were analyzed separately for calcium and phosphorus. More of the random variation in carcass calcium and phosphorus was related to the nest in which eggs incubated (37% and 42%, respectively) than was associated with the clutch of origin (21% and 37%). Moreover, hatchlings from some nests contained substantially more calcium and phosphorus than did hatchlings from other nests, both in terms of the absolute amounts of the elements in their carcasses (pointing to variation in body size) and in terms of the concentrations of those elements (pointing to variation in bone density). The amounts of calcium and phosphorus in carcasses of hatchlings were positively correlated with changes in mass of their eggs during the 8 wk that the eggs incubated in nests in the field, thereby indicating that the influence of the nest environment on developing embryos probably was mediated by water exchanges experienced by the eggs. These findings indicate that developmental plasticity underlies a major fraction of the variation in mineral content of hatchling painted turtles emerging from nests in the field. Phenotypic variation attributable to plasticity consequently needs to be addressed in models for life-history evolution of painted turtles and other chelonians producing eggs with soft, flexible shells.     

Patterns of mobilization of calcium,magnesium, and phosphorus by embryonic yellow-headed blackbirds(Xanthocephalus xanthocephalus)

Summary Embryonic blackbirds(Xanthocephalus xanthocephalus) obtain most of their calcium from the eggshell (85 90%), but all of their phosphorus comes from reserves in the yolk (80–85%) and albumen (15–20%). Approximately equal amounts of magnesium are supplied by the eggshell, the yolk, and the albumen. Yolk is depleted of magnesium and phosphorus during embryogenesis, but excess calcium absorbed from the eggeshell is stored in the yolk. Consequently reserves of calcium in the yolk actually increase 8-fold during embryonic development. Our results reveal that altricial birds manifest patterns of mobilization and deposition of calcium and other elements similar to those described for precocial species. Evolution of altriciality from precocity evidently did not entail major changes in how embryonic birds meet the challenge of obtaining the calcium, magnesium, and phosphorus required for development.     

Action of 1,25-dihydroxyvitamin D3 and parathyroid hormone on 45calcium uptake by the yolk sac membrane of chick embryos

During development, the chick embryo mobilizes the calcium it needs from two extraembryonic sources, initially from the yolk and later from the eggshell. Calcium may be hormonally regulated during avian embryogenesis, but details of this regulation are lacking. We investigated the effects of 1,25-dihydroxycholecalciferol [1,25(OH)2D3], bovine parathyroid hormone [bPTH], and vehicle [ethanol or saline] on blood calcium values and incorporation of 45Ca into the yolk sac membrane of 9, 12, and 15 day chick embryos. Control data were also collected from uninjected 6 day embryos. Solutions were injected directly into the yolk sac compartment 48 and 24 hours prior to the experiment. Exogenous 1,25(OH)2D3 induced hypercalcemia in all age groups examined, while exogenous PTH induced hypercalcemia in day 12 and 15 embryos. Small disks of yolk sac membrane were incubated in medium to which 45Ca was added and assayed for 45Ca content at various intervals after start of incubation. In control yolk sac tissue, the uptake of 45Ca was greatest in younger embryos with decreasing uptake at developmentally more advanced ages; 1,25(OH)2D3 treatment significantly enhanced the uptake of 45Ca into yolk sac tissue in all groups (9, 12, and 15 day embryos). PTH treatment caused a significant elevation in 45Ca uptake in the day 12 and 15 embryos.     

Placental calcium provision in a lizard with prolonged oviductal egg retention

A prominent scenario for the evolution of viviparity and placentation in reptiles predicts a step-wise pattern with an initial phase of prolonged oviductal egg retention accompanied by progressive reduction in eggshell thickness culminating in viviparity; calcium placentotrophy evolves secondarily to viviparity. Saiphos equalis is an Australian scincid lizard with a reproductive mode that is uncommon for squamates because eggs are retained in the oviduct until late developmental stages, and the embryonic stage at oviposition varies geographically. We studied calcium mobilization by embryos in two populations with different oviductal egg retention patterns to test the hypothesis that the pattern of nutritional provision of calcium is independent of the embryonic stage at oviposition. Females from one population are viviparous and oviposit eggs containing fully formed embryos, whereas embryos in oviposited eggs of the second population are morphologically less mature, and these eggs hatch several days later. The reproductive mode of this population is denoted as prolonged oviductal egg retention. Yolk provided the highest proportion of calcium to hatchlings in both populations. Eggs of both populations were enclosed in calcified eggshells, but shells of the population with prolonged egg retention had twice the calcium content of the viviparous population and embryos recovered calcium from these eggshells. Placental transfer accounted for a substantial amount of calcium in hatchlings in both populations. Hatchling calcium concentration was higher in the population with prolonged egg retention because these embryos mobilized calcium from yolk, the eggshell and the placenta. This pattern of embryonic calcium provision in which both a calcified eggshell and placentotrophy contribute to embryonic nutrition is novel. The reproductive pattern of S. equalis illustrates that calcified eggshells are compatible with prolonged oviductal egg retention and that viviparity is not requisite to calcium placentotrophy.     

Changes in mass of eggs of softshell turtless (Trionyx spiniferus) incubated under hydric conditions simulating those of natural nests

Rigid-shelled eggs of softshell turtles ( Trionyx spiniferus ) incubated at 29C under hydric conditions simulating those at the interior of natural nests (i.e. eggs not contacting the substrate) declined in mass during incubation owing to the continuous escape of water vapour to air trapped inside the chambers, with eggs incubated in relatively dry chambers declining more in mass than eggs held in relatively wet chambers. Eggs held under hydric conditions approximating those at the periphery of natural nests (i.e. eggs partly buried in the substrate) also declined in mass during incubation, but at lower rates than characterized eggs not contacting the substrate, indicating that absorption of liquid water from the substrate compensated for part of the water vapour escaping to the air inside the chambers. There was no relationship between hatching success and either the position of eggs in the chambers or the wetness of substrates, nor was there any variation among experimental groups in the size of hatchlings. Thus, development of embryos of Trionyx spiniferus seems not to be closely coupled with the hydric environment of the nest, a situation contrasting with that of embryos of those species of turtles producing pliable-shelled eggs.     

Egg environments have large effects on embryonic development,but have minimal consequences for hatchling phenotypes in an invasive lizard

Plastic responses of embryos to developmental environments can shape phenotypes in ways that impact fitness. The mechanisms by which developmental conditions affect offspring phenotypes vary substantially among taxa and are poorly understood in most systems. In this study, we evaluate the effects of thermal and hydric conditions on patterns of egg water uptake, embryonic development and yolk metabolism in embryos of the lizard Anolis sagrei to gain insights into how these factors shape morphological variation in hatchlings. Our 3 × 2 experimental design (3 thermal and 2 hydric conditions) revealed that developmental temperature has strong effects on rates of development and yolk metabolism, but the impacts of moisture were minimal. Increased water uptake by eggs under relatively wet conditions resulted in larger hatchlings with less internalized residual yolk than hatchlings from dry‐incubated eggs. However, the relatively small phenotypic differences among treatments may have small fitness consequences. These results demonstrate that embryos of A. sagrei can tolerate a broad range of environmental conditions without substantial impacts on critical morphological traits. Such embryonic tolerances may facilitate colonization and establishment in novel environments. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 25–41.     

Structure of shells from eggs of kinosternid turtles

Shells from eggs of five species of kinosternid turtle (Sternotherus minor, Kinosternon flavescens, K. baurii, K. Hirtipes, and K. alamosae) were examined with light and scanning electron microscopy. Except for possible differences among species in thickness of eggshells, structure of shells from all eggs was similiar. In general, kinosternid turtles lay eggs having a rigid calcareous layer composed of calcium carbonate in the form of aragonite. The calcareous layer is organized into individual shell units with needlelike crystallites radiating from a common center. Most of the thickness of the eggshell is attributable to the calcareous layer, with the fibrous shell membrane comprising only a small fraction of shell thickness. Pores are found in the calcareous layer, but they are not numereous. The outer surface of the eggshells is sculptured and may have a thick, organic layer in places. The outer surface of the shell membrane of decalcified eggshells is studded with spherical cores which presumably nucleate growth of shell units during shell formation. The shell membrane detaches from eggs incubated to hatching, carrying with it remnants of the calcareous layer. Such changes in shell structure presumably reflect withdrawal of calcium from the eggshell by developing embryos.     

Can natural phenotypic variances be estimated reliably under homogeneous laboratory conditions?

The phenotypic variance is assumed to be greater in a more heterogeneous environment. The validity of this assumption is important for microevolutionists to extrapolate results from the laboratory to field environments. We subjected clutches of eggs from common snapping turtles (Chelydra serpentina) to a split-family design to evaluate the variability in incubation time and four size traits of neonates from eggs incubated in the laboratory and those left in situ. Mean size measurements were similar between the laboratory and the field, but incubation time was systematically longer in the field. We found no tendency among clutches for hatchlings resulting from eggs incubated in laboratory or field environments to demonstrate greater variability. Also contrary to expectation, clutches that experienced greater thermal variation in the field did not exhibit greater variation in phenotypic traits. Consequently, extrapolating results from the laboratory to the field may not always be problematic for microevolutionary analyses.