Herpesvirus-like infection in a painted turtle (Chrysemys picta)

A painted turtle (Chrysemys picta) which died in captivity had marked necrosis in the liver and lungs with numerous intranuclear inclusion bodies in hepatocytes and respiratory epithelial cells. Electron microscopy revealed herpesvirus-like particles in cells in affected tissues.     

The physiology of hibernation among painted turtles: the Eastern painted turtle Chrysemys picta picta.

Eastern painted turtles (Chrysemys picta picta) from Connecticut were submerged at 3 degrees C in normoxic and anoxic water to simulate potential respiratory environments within their hibernacula. Those in normoxic water could survive submergence for at least 150 d, while those in anoxic water could survive for a maximum of about 125 d. Turtles in normoxic water developed a slight metabolic acidosis as plasma lactate accumulated to about 50 mM in 150 d, while anoxic turtles developed a severe lactic acidosis as plasma lactate reached about 200 mM in 125 d; there was no respiratory acidosis in either group. Plasma [Na+] changed little in either group, [Cl-] fell by about one-third in both, and [K+] increased by about fourfold in anoxic turtles but only slightly in those in normoxic water. Total plasma magnesium and calcium increased profoundly in anoxic turtles but moderately in those in normoxic water. Consideration of charge balance indicates that all major ions were measured in both groups. Plasma glucose remained unchanged in anoxic turtles until after about 75 d of submergence, when it increased and continued to increase with the duration of anoxia, with much variation among individuals; glucose remained unchanged throughout in turtles in normoxic water. Hematocrit doubled in 150 d in turtles in normoxic water; in anoxic turtles, an initial increase was no longer significant by day 100. Plasma osmolality increased markedly in anoxic turtles, largely because of accumulation of lactate, but anoxic turtles only gained about half the mass of turtles in normoxic water, who showed no increase in osmolality. The higher weight gain in the latter group is attributed to selective perfusion and ventilation of extrapulmonary gas exchange surfaces, resulting in a greater osmotic influx of water. The physiologic responses to simulated hibernation of C. picta picta are intermediate between those of Chrysemys picta bellii and Chrysemys picta dorsalis, which correlates with the severity of the winter each subspecies would be expected to encounter.     

Geographic variation of the physiological response to overwintering in the painted turtle (Chrysemys picta)

We compared the physiological responses of latitudinal pairings of painted turtles submerged in normoxic and anoxic water at 3 degrees C: western painted turtles (Chrysemys picta bellii) from Wisconsin (WI) versus southern painted turtles (Chrysemys picta dorsalis) from Louisiana (LA), Arkansas (AR), and Alabama (AL), and eastern painted turtles (Chrysemys picta picta) from Connecticut (CT) versus C. p. picta from Georgia (GA). Turtles in normoxic water accumulated lactate, with C. p. bellii accumulating less than (20 mmol/L) the other groups (44-47 mmol/L), but with relatively minor acid-base and ionic disturbances. Chrysemys picta bellii had the lowest rate of lactate accumulation over the first 50 d in anoxic water (1.8 mmol/d vs. 2.1 for AR C. p. dorsalis, 2.4 mmol/d for GA C. p. picta, and 2.5 mmol/d for CT C. p. picta after 50 d and 2.6 mmol/d for AL C. p. dorsalis after 46 d). Northern turtles in both groups survive longer in anoxia than their southern counterparts. The diminished viability in C. p. dorsalis versus C. p. bellii can be partially explained by an increased rate of lactate accumulation and a decreased buffering capacity, but for the CT and GA C. p. picta comparison, only buffering capacity differences are seen to influence survivability.     

Normal embryonic stages of the western painted turtle, Chrysemys picta bellii

Twenty-three stages in the embryonic development of the western painted turtle, Chrysemys picta bellii are described. The staging is based primarily on morphological changes, as well as age and size of the embryo.     

Seasonal variations in oviductal morphology of the painted turtle,Chrysemys picta

Histology of each of the five segments of the oviduct of the female turtle Chrysemys picta was described for successive intervals throughout their annual cycle. Uterine and glandular segments showed marked seasonal variations in the extent and content of the submucosal and epithelial glands. Submucosal glands were most prominent in preovulatory and postovulatory animals (May to June), regressing in late summer (oviposited animals) and recrudescing the following spring. These changes correlated with variations in the muscularis layer, the number of uterine epithelial blebs, oviductal vascularity, and the presence of eosinophils in cervical segment cross-sections. These cyclic seasonal changes are discussed in relationship to reported seasonal changes in gonadal steroids in this species. Hormonal control was corroborated by oviductal response to estradiol-17β injected (1 mg/kg daily for 2 weeks) into mature, reproductively inactive (winter) animals. This treatment induced increases in glandular activity, vascularity, and distribution of eosinophils comparable to those of reproductively active (summer) animals.     

Accumulation of lactate by supercooled hatchlings of the painted turtle (Chrysemys picta): implications for overwinter survival

Hatchlings of the North American painted turtle (Chrysemys picta) typically spend their first winter of life inside the shallow, subterranean nest where they completed embryogenesis the preceding summer. Neonates at northern localities consequently may be exposed during winter to subzero temperatures and frozen soil. Hatchlings apparently survive exposure to such conditions by supercooling, but the physiological consequences of this adaptive strategy have not been examined. We measured lactate in hatchling painted turtles after exposure to each of three temperatures (0 °C, −4 °C, and −8 °C) for three time periods (5 days, 15 days, and 25 days) to determine the extent to which overwintering hatchlings might rely on anaerobic metabolism to regenerate ATP. Whole-body lactate increased with increasing duration of exposure and decreasing temperature, and the highest levels were associated with the group that experienced the highest mortality. These results indicate that animals may develop a considerable lactic acidosis during a winter in which temperatures fall below 0 °C for weeks or months and that accumulation of lactate may contribute to mortality of overwintering animals. Accepted: 20 October 1999     

Effects of hibernation on mitochondrial regulation and metabolic capacities in myocardium of painted turtle (Chrysemys picta)

Painted turtles hibernating during winter may endure long-term exposure to low temperature and anoxia. These two conditions may affect the aerobic capacity of a tissue and might be of particular importance to the cardiac muscle normally highly reliant on aerobic energy production. The present study addressed how hibernation affects respiratory characteristics of mitochondria in situ and the metabolic pattern of turtle myocardium. Painted turtles were acclimated to control (25 degrees C), cold (5 degrees C) normoxic and cold anoxic conditions. In saponin-skinned myocardial fibres, cold acclimation increased mitochondrial respiratory capacity and decreased apparent ADP-affinity. Concomitant anoxia did not affect this. Creatine increased the apparent ADP-affinity to similar values in the three acclimation groups, suggesting a functional coupling of creatine kinase to mitochondrial respiration. As to the metabolic pattern, cold acclimation decreased glycolytic capacity in terms of pyruvate kinase activity and increased lactate dehydrogenase (LHD) activity. Concomitant anoxia counteracted the cold-induced decrease in pyruvate kinase activity and increased creatine kinase activity. In conclusion, cold acclimation seems to increase aerobic and decrease anaerobic energy production capacity in painted turtle myocardium. Importantly, anoxia does not affect the mitochondrial functional integrity but seems to increase the capacity for anaerobic energy production and energy buffering.     

Effects of road proximity on genetic diversity and reproductive success of the painted turtle (Chrysemys picta)

Roads have a severe impact on wildlife. Reptiles are particularly susceptible due to their attraction to roads and their low car-avoidance capacity. For example, a high number of road killed freshwater turtles resulted from females selecting the unpaved side of roads as nesting sites. However, roads are harmful not only for adults, but are also expected to affect egg survival and recruitment. In this work, we indirectly determined whether the proximity to roads affects the reproductive success of freshwater turtles. The painted turtle (Chrysemys picta) was chosen for its population density, which is higher than most turtle species considered endangered. Locations near roads (<100 m) and in natural areas (>500 m) were sampled in three geographically distant ecoregions. We estimated the diversity of microsatellite loci from nuclear and mitochondrial genomes to assess the size of the kin groups as a proxy of the reproductive success of females. Similar diversity at nuclear markers suggested a comparable historical and demographic background among populations. However, lower mitochondrial diversity, higher mean and variance in the size of kin groups as well as a lower number of kin groups were strongly associated with the proximity to roads. Results indicated that a lower proportion of females participated in the recruitment of populations close to the roads than in natural areas, resulting in fewer but larger families near roads. We expect similar results for species nesting on the roadside. Barriers or fences that prevent individuals from reaching the road may help reduce their impacts on these populations.     

Maternally derived yolk hormones vary in follicles of the painted turtle,Chrysemys picta

The transfer of hormones from a female to her offspring is known to occur in egg laying vertebrates, and the potential for these early, maternally derived hormones to influence sex determination in reptiles with temperature-dependent sex determination is intriguing. In the present study, we examine variation in the concentrations of progesterone, testosterone, and estradiol among three follicle size classes within a female painted turtle (Chrysemys picta) and among females across four periods that span the pre- to post-nesting season. Females were collected, and both follicles and shelled eggs (when present) were harvested for hormone analysis. Progesterone levels did not vary seasonally. However, the concentration of progesterone did vary among and within follicle classes, and was primarily dependent upon ovulatory state: Recently ovulated follicles (as yolks within shelled eggs) contained significantly more progesterone than unovulated follicles. Concentrations of testosterone were low and did not vary either among size classes or across the season. Estradiol levels decreased with increasing follicle size and were higher later in the nesting season. Thus, hormone concentrations varied among follicle sizes and states but in patterns that differed among hormones. This variation has the potential to influence sex determination.     

Physiological responses to supercooling and hypoxia in the hatchling painted turtle, Chrysemys picta

We investigated physiological responses to supercooling in hatchling painted turtles (Chrysemys picta) which remain in their natal nests over winter and therefore may become exposed to subzero temperatures. These turtles are freeze tolerant but also must rely on supercooling to survive exposure to the lower temperatures occurring in nests during winter. We compared whole-body concentrations of lactate, glucose, glycerol, and ATP in turtles chilled at 0 degrees C, -4 degrees C, or -6 degrees C for 5 days, or at 6 degrees C for 19 days. In a companion experiment, we measured metabolite concentrations in turtles exposed to a hypoxic environment for 1 day, 4 days, or 8 days. Supercooling and hypoxia exposure were both associated with an increase in concentrations of lactate and glucose and a decrease in glycerol concentrations (albeit no change in the ATP pool), suggesting that supercooling induces functional hypoxia. We conclude that hypoxia tolerance may be an important pre-adaptation for surviving exposure to subzero temperatures in hatchling C. picta.     

Proteomic changes in the brain of the western painted turtle (Chrysemys picta bellii) during exposure to anoxia

During anoxia, overall protein synthesis is almost undetectable in the brain of the western painted turtle. The aim of this investigation was to address the question of whether there are alterations to specific proteins by comparing the normoxic and anoxic brain proteomes. Reductions in creatine kinase, hexokinase, glyceraldehyde‐3‐phosphate dehydrogenase, and pyruvate kinase reflected the reduced production of adenosine triphosphate (ATP) during anoxia while the reduction in transitional endoplasmic reticulum ATPase reflected the conservation of ATP or possibly a decrease in intracellular Ca²⁺. In terms of neural protection programed cell death 6 interacting protein (PDCD6IP; a protein associated with apoptosis), dihydropyrimidinase‐like protein, t‐complex protein, and guanine nucleotide protein G_(o) subunit alpha (G_o alpha; proteins associated with neural degradation and impaired cognitive function) also declined. A decline in actin, gelsolin, and PDCD6IP, together with an increase in tubulin, also provided evidence for the induction of a neurological repair response. Although these proteomic alterations show some similarities with the crucian carp (another anoxia‐tolerant species), there are species‐specific responses, which supports the theory of no single strategy for anoxia tolerance. These findings also suggest the anoxic turtle brain could be an etiological model for investigating mammalian hypoxic damage and clinical neurological disorders.     

Molecular systematics,phylogeography, and the effects of Pleistocene glaciation in the painted turtle (Chrysemys picta) complex

Abstract.— The painted turtle, Chrysemys picta , is currently recognized as a continentally distributed polytypic species, ranging across North America from southern Canada to extreme northern Mexico. We analyzed variation in the rapidly evolving mitochondrial control region (CR) in 241 turtles from 117 localities across this range to examine whether the painted turtle represents a continentally distributed species based on molecular analysis. We found strong support for the novel hypothesis that C. p. dorsalis is the sister group to all remaining Chrysemys , with the remaining Chrysemys falling into a single, extremely wide-ranging and genetically undifferentiated species. Given our goal of an evolu-tionarily accurate taxonomy, we propose that two evolutionary lineages be recognized as species within Chrysemys : C. dorsalis (Agassiz 1857) in the southern Mississippi drainage region, and C. picta (Schneider 1783) from the rest of the range of the genus. Neither molecular nor recent morphological analyses argue for the hybrid origin of C. p. marginata as previously proposed. Within C. picta , we find evidence of at least two independent range expansions into previously glaciated regions of North America, one into New England and the other into the upper Midwest. We further find evidence of a massive extinction/recolonization event across the Great Plains/Rocky Mountain region encompassing over half the continental United States. The timing and extent of this colonization is consistent with a recently proposed regional aridification as the Laurentide ice sheets receded approximately 14,000 years ago, and we tentatively propose this paleoclimatological event as a major factor shaping genetic variation in Chrysemys .     

Steroid biosynthesis by reptilian adrenals

Incubations of whole homogenates of. the tiju lizard ($\text{Tupinambis sp}$.) adrenals tissue were carried out using ¹⁴C-labelled progesterone^1*, pregnenolone and cholesterol. ¹⁴C-progesterone was metabolized to labelled 18-hydroxycorticosterone, aldosterone, corticosterone and 11-deoxycorticosterone. Identical metabolites plus ¹⁴C-progesterone were obtained from pregnenolone. Cholesterol-4-¹⁴C was transformed into products similar to those obtained from progesterone. In all these studies the elaboration of cortisol or any other 17-hydroxylated steroids could not be demonstrated. In another set of experiments, whole homogenate preparations from adrenals of the green lizard ($\text{lacerta viridis}$) were incubated with ¹⁴C-labelled androstenedione and testosterone. Ahdrostenedione was converted to testosterone and 11β-hydroxyandrostenedione. Testosterone was metabolized to 11β-hydroxyandrostenedione and androstenedione. The results indicate that the $\text{in vitro}$ transformation of C-27 or C-21 radioactive substrate by lizard adrenals is similar to the other reptiles studied. However, it appears to possess 17β-hydroxysteroid oxido-reductase, though the adrenal tissue itself lacks 17α-hydroxylase activity.     

Accumulation of lactate by frozen painted turtles (Chrysemys picta) and its relationship to freeze tolerance

Hatchling painted turtles (Chrysemys picta) survived freezing at -2 degrees C for 4 d, few recovered from freezing lasting 6 d, and none survived being frozen for 8 d. Whole-body glucose and lactate were low in animals that had not been subjected to cold and ice but increased precipitously in animals that were frozen for 2 d. Both metabolites continued to increase, but at a somewhat lower rate, in animals frozen for 4, 6, or 8 d. The increase in whole-body lactate reflects a reliance by frozen hatchlings on anaerobiosis, whereas the increase in glucose presumably results from mobilization of glycogen reserves to support anaerobic metabolism. Mortality of frozen hatchlings is correlated with the increase in whole-body lactate. Factors that may contribute to the observed correlation include a compromised capacity for individual organs to cope with the lactic acidosis that accompanies anaerobic metabolism and organ-specific depletion of energy reserves. Individual organs must rely on buffering and glucose reserves available in situ because blood of frozen hatchlings does not circulate. Thus, buffer from the shell cannot be transported to other organs, lactate cannot be sequestered in the shell, and glucose mobilized from liver glycogen is not available to supplement glucose reserves of other tissues. This integrated suite of physiological disruptions may limit tolerance of freezing to conditions with little or no ecological relevance.     

Hatchling painted turtles (Chrysemys picta) survive exposure to subzero temperatures during hibernation by avoiding freezing

Hatchling painted turtles (Chrysemys picta) were placed individually into artificial nests constructed in jars of damp soil and then were cooled slowly to temperatures between-7.7 and-12.7 °C. Distinct exotherms were recorded in all jars when water in the soil began to freeze at temperatures between-0.9 and-2.4 °C. A second (animal) exotherm was subsequently detected in some of the jars when water in hatchlings also began to freeze. An animal exotherm occurred in the temperature records for all 23 hatchlings that died in tests terminating at temperatures between-7.7 and-10.8 °C, but no such exotherm was apparent in the temperature records for the 23 turtles that survived these treatments. Moreover, the 4 hatchlings that produced exotherms in tests terminating between-11.5 and-12.7 °C failed to survive, but 5 of 7 hatchlings that produced no exotherm in these tests also died. Thus, turtles that die at subzero temperatures above-11 °C apparently succumb to freezing when ice propagates across their integument from the frozen soil, but animals that die at temperatures below-11 °C generally perish from some other cause. These findings indicate that hatchling painted turtles overwintering inside their shallow, subterranean nests survive exposure to subzero temperatures by avoiding freezing instead of by tolerating freezing.     

Insulin cells are found in the main and accessory urinary bladders of the painted turtle, Chrysemys picta

Insulin has been localized immunocytochemically to cells in the main and accessory urinary bladders of the painted turtle, Chrysemys picta, and represents an unusual addition to the specturm of regulatory peptides associated with the urinary bladder. These stellate to fibroblastoid cells often possess neural-like processes and are similar in morphology to neurotensin cells found in Chrysemys and Pseudemys urinary bladders. Radioimmunoassay of 2M acetic acid extracts of bladder tissue indicate that the insulin concentration of accessory bladder is several-fold greater than main bladder but considerably lower than the insulin content of pancreas. Pieces of accessory bladder incubated in vitro exhibit a stable insulin release into the medium over 1 hour, but release is unaltered by known insulin secretagogues. It is tempting to postulate an endocrine or paracrine regulatory function for these cells, but at present their role in Chrysemys bladder function remains unknown.     

The role of the integument as a barrier to penetration of ice into overwintering hatchlings of the painted turtle (Chrysemys picta)

Hatchlings of the North American painted turtle (Chrysemys picta) spend their first winter of life inside a shallow, subterranean hibernaculum (the natal nest) where they may be exposed for extended periods to ice and cold. Hatchlings seemingly survive exposure to such conditions by becoming supercooled (i.e., by remaining unfrozen at temperatures below the equilibrium freezing point for body fluids), so we investigated the role of their integument in preventing ice from penetrating into body compartments from surrounding soil. We first showed that hatchlings whose epidermis has been damaged are more likely to be penetrated by growing crystals of ice than are turtles whose cutaneous barrier is intact. We next studied integument from a forelimb by light microscopy and discovered that the basal part of the alpha-keratin layer of the epidermis contains a dense layer of lipid. Skin from the forelimb of other neonatal turtles lacks such a layer of lipid in the epidermis, and these other turtles also are highly susceptible to inoculative freezing. Moreover, epidermis from the neck of hatchling painted turtles lacks the lipid layer, and this region of the skin is readily penetrated by growing crystals of ice. We therefore conclude that the resistance to inoculation imposed by skin on the limbs of hatchling painted turtles results from the presence of lipids in the alpha-keratin layer of the epidermis. Neonates apparently are able to avoid freezing during winter by drawing much of the body inside the shell, leaving only the ice-resistant integument of the limbs exposed to ice in the environment. The combination of behavior and skin morphology enables overwintering hatchlings to exploit an adaptive strategy based on supercooling.     

Excurrent duct system of the male turtle Chrysemys picta

The epididymis and efferent duct system of the turtle Chrysemys picta were examined. Seminiferous tubules are drained by a series of ducts that form a rete exterior to the tunica albuginea. The rete is located lateral to the testis and consists of anastamosing tubules of varying diameters, lined by a simple epithelium consisting of squamous to cuboidal cells. The rete is highly vascularized. A series of tubules (efferent ductules) connect the rete to the epididymis proper. The efferent ductules are highly convoluted, running between the epididymal tubules and are of varying diameters. The simple columnar epithelium lining these tubules possesses tight junctions, with every third or fourth cell possessing long cilia that protrude into the lumen. The cytoplasm of these epithelial cells contains abundant mitochondria. In the central portion of the efferent ductule, epithelial cells possess granules that appear to be secreted into the lumen by an apocrine process. The epididymis proper is a single, long, highly convoluted tubule that receives efferent ductules along its entire length. It is lined by a pseudostratified epithelium containing several cell types. The most abundant cell (vesicular cell) lacks cilia, but has a darkly staining apical border due to numerous small vesicles immediately beneath the luminal membrane. The small vesicles appear to fuse with each other basally to form larger vesicles. These cells appear to have an absorptive function, and occasionally sperm are embedded in their cytoplasm. The second-most abundant cell is a basal cell found along the basement membrane. The number of these cells fluctuates throughout the year, being most abundant in late summer and early fall. A small narrow cell with an oval nucleus and darkly staining cytoplasm, extending from the basement membrane to the apical surface, is present in small numbers, particularly in the caudal regions of the epididymis. This cell is frequently found in association with another narrow cell having a rounded nucleus and abundant mitochondria in its cytoplasm.