Avenues of extrapulmonary oxygen uptake in western painted turtles (Chrysemys picta belli) at 10 degrees C

The major avenues of extrapulmonary oxygen uptake were determined on submerged western painted turtles (Chrysemys picta bellii) at 10 degrees C by selectively blocking one or more potential pathways for exchange. Previous work indicated that the skin, the cloaca, and the buccopharyngeal cavity can all contribute significantly in various species of turtles. O(2) uptake was calculated from the rate of fall in water P(O(2)) in a closed chamber. Two series of experiments were conducted: in Series 1, each of the potential avenues was mechanically blocked either singly or in combination; in Series 2, active cloacal and buccal pumping were prevented pharmacologically using the paralytic agent rocuronium. In addition in Series 2, N(2)-breathing preceded submergence in some animals and in one set of Series 2 experiments arterial blood was sampled and analyzed for pH, lactate, P(O(2)), and P(CO(2)). Results in both Series 1 and Series 2 revealed that prevention of cloacal and/or buccopharyngeal exchange did not significantly affect total O(2) uptake. Interfering with skin diffusion in Series 1, however, significantly reduced O(2) uptake by 50%. N(2)-breathing prior to submergence in Series 2 did not affect O(2) uptake in paralyzed turtles but significantly increased uptake in unparalyzed turtles without catheters. Blood analysis revealed that all submerged turtles developed lactic acidosis, but the rate of rise in lactate was significantly lower in paralyzed animals. We conclude that passive diffusion through the integument is the principal avenue of aquatic O(2) uptake in this species.     

Hemoglobins of reptiles. Expression of alpha-D-genes in the turtles, Chrysemys picta bellii and Phrynops hilarii (Testudines)

The hemoglobins of two turtles (Testudines)--Chrysemys picta bellii (suborder Cryptodira) and Phrynops hilarii (suborder Pleurodira)--were investigated. In both specimens we found two hemoglobin components with two distinct alpha-chains. The alpha-chains of the component HbD of Chrysemys picta bellii and of the component CII of Phyrynops hilarii belong to the alpha D-type, which has so far been reported to occur only in birds. The complete amino-acid sequences of both alpha D-chains are presented. Our further investigations on hemoglobins of other reptiles (Crocodilia, Lacertilia, Serpentes) did not give any evidence for the expression of alpha D-globin genes in the species examined. These findings are discussed with especial reference to the physiology of respiration. It is supposed that alpha D-genes were of certain significance in earlier times. There are findings suggesting that alpha D-genes are embryonic genes with persistent expression in many adult birds and turtles.     

Reproductive variation and the egg size-clutch size trade-off within and among populations of painted turtles (Chrysemys picta bellii)

Interpopulation variation in egg size, clutch size and clutch mass was studied 3 years in four populations of painted turtles (Chrysemys picta bellii) from western Nebraska. Body size varied among all populations and was larger in two large (56–110 ha), sandhills lake populations than in two populations in smaller habitats (1.5–3.6 ha) of the Platte River floodplain. Reproductive parameters (egg mass, clutch mass, and clutch size) generally increased with maternal body size within populations. Clutch wet and dry mass varied among populations but largely as a function of maternal body size. Clutch size was largest in the sandhills lake populations, both absolutely and relative to maternal body size. Egg mass was smallest in the sandhills lakes and varied annually in one population. Over all populations, an egg sizeclutch size trade-off was detected (a negative correlation between egg mass and clutch size) after statistically removing maternal body size effects. Egg wet mass and clutch size were negatively correlated over all years within the sandhills populations and in some years in three populations. Although egg size varied within populations, egg size and clutch size covaried as expected by optimal offspring size models. Thus, patterns of egg size variation should be interpreted in the context of proximate or adaptive maternal body size and temporal effects. Comparisons among populations suggest that large egg size relative to maternal body size may occur when juvenile growth potential is poor and mean maternal body size is small.     

Behavioral responses of hatchling painted turtles (Chrysemys picta) and snapping turtles (Chelydra serpentina) at subzero temperatures

We monitored behavioral responses of cold-acclimated hatchling painted turtles (Chrysemys picta) indigenous to Nebraska and hatchling snapping turtles (Chelydra serpentina) indigenous to Nebraska and Arkansas during cooling (0.1°C/min) to temperatures as low as −19°C. All turtles made exploratory movements during cooling and locomotion occurred at temperatures as low as −2 to −4°C, but C. picta maintained relatively higher levels of locomotor activity than C. serpentina, and no differences in motility occurred between northern and southern groups of C. serpentina. Slow movements of the head and limbs were observed in supercooled hatchling C. picta at temperatures as low as −10°C, whereas at about −5°C, C. serpentina exhibited an increase in spontaneous motor activity followed by muscle contracture, immobility, and spontaneous freezing. C. picta spontaneously froze at about −16°C without exhibiting cold contracture, suggesting that they are better adapted to survive exposure to extreme cold.     

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.     

Metabolic rate of feeding and fasting juvenile midland painted turtles, Chrysemys picta marginata

1. Resting metabolic rates at 25 degrees C were determined for juvenile midland painted turtles that had recently been fed or fasted for 1, 2, 4, 6, 10, 14 or 19 days. 2. Recently fed turtles had an oxygen consumption rate of 211 microliter O2/g/hr. This decreased by 32% on the first day of the fast and by 69% by the 19th day. 3. Mass of the turtles (4.91-14.30 g) did not affect the rate of oxygen consumption (VO2).     

Lactic acid buffering by bone and shell in anoxic softshell and painted turtles

We tested two hypotheses: first, that the inferior anoxia tolerance of the softshell turtle, Apalone spinifera, compared to the western painted turtle, Chrysemys picta bellii, is related to its less mineralized shell, and second, that turtle bone, like its shell, stores lactate during prolonged anoxia. Lactate concentrations of blood, hindlimb bone, and shell were measured on normoxic Apalone and Chrysemys and after anoxic submergence at 10 degrees C for 2 and 9 d, respectively. Blood and shell concentrations of Ca(2+), Mg(2+), Na(+), K(+), and inorganic phosphate (P(i); for shell only) were also measured. Because a preliminary study indicated lactate distribution in Chrysemys throughout its skeleton during anoxia at 20 degrees C, we used hindlimb bones as representative skeletal samples. Apalone shell, though a similar percentage of body mass as Chrysemys shell, had higher water content (76.9% vs. 27.9%) and only 20%-25% as much Ca(2+), Mg(2+), CO(2), and P(i). When incubated at constant pH of 6.0 or 6.5, Apalone shell powder released only 25% as much buffer per gram wet weight as Chrysemys shell. In addition, plasma [Ca(2+)] and [Mg(2+)] increased less in Apalone during anoxia at an equivalent plasma lactate concentration. Lactate concentrations increased in the shell and skeletal bone in both species. Despite less mineralization, Apalone shell took up lactate comparably to Chrysemys. In conclusion, a weaker compensatory response to lactic acidosis in Apalone correlates with lower shell mineralization and buffer release and may partially account for the poorer anoxia tolerance of this species.     

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.     

High energy organophosphate levels in the myocardia of cold acclimated and cold-hypoxic freshwater turtles,Chrysemys picta

• 1.1. Hearts of active, cold-acclimated, and cold-acclimated hypoxic turtles were assayed for their content of ATP, ADP, AMP and phosphocreatine.
• 2.2. Energy levels showed a significant discharge during the first 2 weeks of cold and cold-hypoxia, but remained relatively stable during an additional 2 weeks of cold exposure.
• 3.3. The functional importance of these changes is discussed.

     

Hemoglobins of reptiles. The primary structure of the major and minor hemoglobin component of adult Western Painted Turtle (Chrysemys picta bellii)

Red blood cells of adult Western Painted Turtles (Chrysemys picta bellii) contain two hemoglobin components: HbA (alpha A2 beta 2) and HbD (alpha D2 beta 2). We present the complete amino-acid sequences of the alpha A-chains from the major component and of the beta-chains common to both components. Structural features are discussed with respect to the animals extreme tolerance of severe hypoxic conditions during hibernation which is accompanied by a high oxygen affinity of the hemoglobin. The strong ATP dependence of Western Painted Turtle hemoglobin oxygen affinity is contrasted by the loss of one ATP-binding site, beta 143(H21)-Arg----Leu. The primary structure of the beta-chains excludes an allosteric control mechanism by hydrogencarbonate as it was found in crocodiles. Except in turtles a hemoglobin pattern with HbA and HbD sharing the same beta-subunits has been found only in birds. In comparison to other vertebrate hemoglobins there is a surprising similarity of the sequences to those of bird hemoglobins. alpha A- as well as alpha D-chains show larger homologies to chains of the same type in different species than alpha A- and alpha D-chains to each other in the same species. This indicates a duplication of the alpha-gene preceding the divergence of turtles and birds.     

Effects of developmental exposure to bisphenol A and ethinyl estradiol on spatial navigational learning and memory in painted turtles (Chrysemys picta)

Developmental exposure of turtles and other reptiles to endocrine disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE2, estrogen present in birth control pills), can induce partial to full gonadal sex-reversal in males. No prior studies have considered whether in ovo exposure to EDCs disrupts normal brain sexual differentiation. Yet, rodent model studies indicate early exposure to these chemicals disturbs sexually selected behavioral traits, including spatial navigational learning and memory. Thus, we sought to determine whether developmental exposure of painted turtles (Chrysemys picta) to BPA and EE2 results in sex-dependent behavioral changes. At developmental stage 17, turtles incubated at 26⁰C (male-inducing temperature) were treated with 1) BPA High (100 μg /mL), 2) BPA Low (0.01 μg/mL), 3) EE2 (0.2 μg/mL), or 4) vehicle or no vehicle control groups. Five months after hatching, turtles were tested with a spatial navigational test that included four food containers, only one of which was baited with food. Each turtle was randomly assigned one container that did not change over the trial period. Each individual was tested for 14 consecutive days. Results show developmental exposure to BPA High and EE2 improved spatial navigational learning and memory, as evidenced by increased number of times spent in the correct target zone and greater likelihood of solving the maze compared to control turtles. This study is the first to show that in addition to overriding temperature sex determination (TSD) of the male gonad, these EDCs may induce sex-dependent behavioral changes in turtles.     

Daily and annual patterns of thermoregulation in painted turtles (Chrysemys picta marginata) living in a thermally variable marsh in Northern Michigan

The capacity for an ectothermic reptile to thermoregulate has implications for many components of its life history. Over two years, we studied thermoregulation in a population of Midland painted turtles (Chrysemys picta marginata) in a shallow, thermally variable wetland during summer in Northern Michigan. Mean body temperature (T_b) of free-ranging turtles was greater in 2008 (25.8 °C) than in 2010 (19.7 °C). Laboratory determined thermoregulatory set point (T_set) ranged from 25 °C (T_set-min) to 31 °C (T_set-max) and was lower during the fall (17–26 °C). Deviations of T_b distributions from field measured operative temperatures (T_e) and indices of thermoregulation indicated that C. picta marginata were capable of a limited degree of thermoregulation. Operative temperatures and thermal quality (d_e=|T_set-min−T_e| and |T_e−T_set-max|) cycled daily with maximal thermal quality occurring during late morning and late afternoon. The accuracy of thermoregulation (d_b=|T_set-min−T_b| and |T_b−T_set-max|) was maximal (d_b values were minimal) as T_b declined and traversed T_set during the late afternoon–early evening hours and was higher on cloudy days than on sunny days because relatively low T_e values decreased the number of T_b values that were above T_set. Our index of thermal exploitation (E_x=frequency of T_b observations within T_set) was 36%, slightly lower than that reported for an Ontario population of C. picta marginata. Regression of d_b (thermal accuracy) on d_e (thermal quality) indicated that turtles invested more in thermoregulation when thermal quality was low and when water levels were high than when they were low. There were no intersexual differences in mean T_b throughout the year but females had relatively high laboratory determined T_b values in the fall, perhaps reflecting the importance of maintaining ovarian development prior to winter.