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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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 .     

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.     

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     

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.     

The relationship between gut contents and supercooling capacity in hatchling painted turtles (Chrysemys picta)

Painted turtles (Chrysemys picta) typically spend their first winter of life in a shallow, subterranean hibernaculum (the natal nest) where they seemingly withstand exposure to ice and cold by resisting freezing and becoming supercooled. However, turtles ingest soil and fragments of eggshell as they are hatching from their eggs, and the ingestate usually contains efficient nucleating agents that cause water to freeze at high subzero temperatures. Consequently, neonatal painted turtles have only a modest ability to undergo supercooling in the period immediately after hatching. We studied the limit for supercooling (SCP) in hatchlings that were acclimating to different thermal regimes and then related SCPs of the turtles to the amount of particulate matter in their gastrointestinal (GI) tract. Turtles that were transferred directly from 26 degrees C (the incubation temperature) to 2 degrees C did not purge soil from their gut, and SCPs for these animals remained near -4 degrees C for the 60 days of the study. Animals that were held at 26 degrees C for the duration of the experiment usually cleared soil from their GI tract within 24 days, but SCPs for these turtles were only slightly lower after 60 days than they were at the outset of the experiment. Hatchlings that were acclimating slowly to 2 degrees C cleared soil from their gut within 24 days and realized a modest reduction in their SCP. However, the limit of supercooling in the slowly acclimating animals continued to decline even after all particulate material had been removed from their GI tract, thereby indicating that factors intrinsic to the nucleating agents themselves also may have been involved in the acclimation of hatchlings to low temperature. The lowest SCPs for turtles that were acclimating slowly to 2 degrees C were similar to SCPs recorded in an earlier study of animals taken from natural nests in late autumn, so the current findings affirm the importance of seasonally declining temperatures in preparing animals in the field to withstand conditions that they will encounter during winter.     

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.     

Succinate and alanine as anaerobic end-products in the diving turtle (Chrysemys picta bellii)

The western painted turtle is an extremely anoxia-tolerant vertebrate capable of tolerating blood lactate levels of 150-200 mM. Since lactate increases to such high levels, other fermentation end-products such as succinate and alanine, which have not been previously measured in this species, might also be expected to increase. Therefore, I measured turtle heart, liver, and blood concentrations of lactate, succinate, and alanine following a 28-day anoxic dive at 5 degrees C. Succinate and lactate concentrations increased significantly in all three compartments while alanine increased significantly in the liver only. Lactate was found to accumulate by a similar amount in all three compartments (66.4-80.5 micromol g or ml(-1) in the blood compartment) and was used as a reference to which alanine and succinate concentrations could be compared. Succinate and alanine levels increased by 2 and 0.9% of lactate in liver, approximately 0.3 and 0.04% of lactate in blood, and 0.6 and 0.07% of lactate in heart, respectively. The contribution of each to the total anoxic heat production was calculated and accounted for an additional 1.5% of the previously measured exothermic gap. I conclude that succinate and alanine concentrations do increase in the anoxic turtle but are minor anaerobic end-products.     

Ultrastructural changes in granulosa cells and plasma steroid levels after administration of luteinizing hormone-releasing hormone in the Western painted turtle, Chrysemys picta

In this study we investigated the effects of treatment by luteinizing hormone-releasing hormone (LHRH) on the morphology and steroid release of ovarian tissues in the Western painted turtle, (Chrysemys picta). In Experiment I, four adult female turtles were injected with synthetic mammalian LHRH (i.p., 500 pg/g bodyweight) and four with saline 2-3 weeks prior to ovulation. Granulosa cells from LHRH-treated turtles vs controls contained both preovulatory follicles (16-20 mm in diameter) and small follicles (0.5-1.00mm in diameter) with increased RER, free ribosomes and mitochondria with swollen cristae. An increase in the amount of cytoskeletal material (microfilaments) was observed in granulosa cells of the experimental turtles compared to the controls. Cytoplasmic extensions of the oocyte and granulosa cells were longer in the small follicles of treated animals, accounting for the observed increase in the thickness of the zona pellucida (ZP) over the controls. In Experiment II, administration of LHRH (i.p.) to 10 turtles during the same period triggered a substantial increase in plasma progesterone and estradiol-17beta levels over the 10 saline-injected controls. This supports the idea that in this species, as in mammals, steroidogenic activity in the ovarian follicles are under the control of the hypothalamic-pituitary axis. The ultrastructure and hormonal levels of the experimental animals were typical of untreated turtles just prior to ovulation. In this species the development of follicles and steroidogenesis can be stimulated prematurely by a releasing hormone from a nonreptilian origin.