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Oxidative cost of breathing in the turtle Chrysemys picta bellii.

We estimated the cost of breathing of turtles by measuring ventilation and oxygen consumption during air breathing and CO2 breathing. We assumed that any increment in oxygen consumption due to hypercapnic hyperpnea was due to the metabolic cost of the increased breathing. Six turtles were studied while breathing air and then 5% CO2 in air after at least 12 h breathing each gas. For the measurements, the turtles were submerged unrestrained in water at 20 degrees C and were free to raise their heads into a ventilated chamber. Tidal volumes were measured by the pressure changes in the chamber, and oxygen consumption was measured by conventional open-circuit respirometry. Ventilation increased markedly during CO2 breathing up to 50 times the control level, but oxygen consumption increased only slightly. Assuming no depression in nonventilatory metabolism, our data indicate an oxidative cost of breathing on the order of 1% of the total metabolic rate at rest. This is far less than the 15-20% cost predicted from published work (Kinney et al., Respir. Physiol. 31: 327-332, 1976) on a closely related species of turtle and is consistent with earlier work in our laboratory. We conclude that the cost of breathing in turtles is low, similar to other air-breathing vertebrates, and therefore the existing notion that turtle breathing is expensive and inefficient should be discarded.

Animals↗

Cerebral anoxia tolerance in turtles: regulation of intracellular calcium and pH.

To investigate mechanisms of cerebral anoxia tolerance, cerebrocortical intracellular calcium ([Ca2+]i) and pH (pHi) regulation were compared in turtles (Trachemys scripta) and laboratory rats. [Ca2+]i and pHi in living 200 to 300-microns-thick cortical brain slices were measured with the fluorescent indicators fura-2/acetoxymethyl ester (AM) and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein during exposure to anoxia. Within 5 min, [Ca2+]i increased to > 1,000 nM in rat brain slices exposed to anoxia but [Ca2+]i was normal even after 5 h of anoxia in turtles. ATP levels remained normal in anoxic turtle brain but fell rapidly in rats. During anoxia, pHi fell by 0.25 +/- 0.08 pH units in rats but only 0.10 +/- 0.04 in turtles (P < 0.05). Inhibition of glycolysis in anoxic turtle brain with iodoacetate resulted in large increases in [Ca2+]i but prior exposure of slices to anoxia resulted in greatly attenuated calcium entry. The reduction in calcium flux was greater with increasing exposure to anoxia, suggesting progressive arrest of calcium channel activity. Tolerance of cerebral anoxia in turtles may be related to anaerobic ATP production, arrest of calcium channels, and attenuation of changes in pHi.

Adenosine Triphosphate↗

Inhibition of brain calcium channels by plasma proteins from anoxic turtles.

Recent studies suggest that turtles avoid disturbances in brain ionic regulation during anoxia by reducing the activity of brain calcium and sodium channels. Because glutamate released during anoxia may cause cytotoxic elevations in intracellular calcium, blockade of glutamate-mediated calcium channels may be essential for cellular survival. Elevations in intracellular calcium, measured with the fluorescent dye fura 2, were used to assay glutamate-induced activation of calcium channels in cerebrocortical brain slices from rats and turtles. Fourteen hours of anoxia produced long-lasting reduction in glutamate-mediated calcium flux in the turtle brain. Furthermore, a plasma protein from turtles maintained under anoxic conditions produced blockade of glutamate-mediated calcium flux in cortical brain slices from both turtles and rats. These results suggest that long-lasting modulation of brain calcium channels as well as blockade of calcium channel activity by regulatory proteins may play important roles in reducing transcellular ion fluxes in turtles during anoxia.

Animals↗

Reduced ionic conductance in turtle brain.

Whole cell recording techniques were employed to measure whole cell (Gw) and specific membrane (Gm) conductance in turtle and rat pyramidal neurons in slices. Results indicate that rat neurons are 4.2 times more conductive compared with turtle neurons at 25 degrees C, which is accentuated by temperature, so that rat neurons at 37 degrees C are 22 times more conductive than turtle neurons at 15 degrees C. A conductance Q10 of 1.9 was measured for both turtle (15-25 degrees C) and rat (25-35 degrees C) pyramidal neurons. Conductance measurements of turtle pyramidal neurons over 6-9 h of anoxia indicate no statistical difference between Gm or Gw from normoxic control measures. These results indirectly support the concept of low ATP-dependent ion pump activity in the turtle brain as one mechanism for reduced energy expenditure in the normoxic state.

Animals↗

Comparative brain oxygenation and mitochondrial redox activity in turtles and rats.

Comparisons were made between brain oxygenation and cytochrome c oxidase (cytochrome aa3) redox status in turtles (Pseudymys scripta) and rats. Although average brain O2 tension (PtO2) during normoxia was similar in turtle telencephalon and rat parietal cortex, a greater frequency of high and low values of PtO2 occurred in rats, which may indicate a steeper O2 gradient between capillaries and cells. When the fractional inspired O2 concentration (FIO2) was increased, PtO2 was elevated and cytochrome aa3 became more oxidized, whereas decreased FIo2 produced opposite changes in both species. In turtle brain, however, the relationship between PtO2 and cytochrome aa3 redox state was nearly linear over a wide range of PtO2 values. In rats, this relationship was steeper at PtO2 below normoxia and approached a plateau at Pto2 values above normoxia. Turtle brain cytochrome aa3 appeared more reduced in normoxia, which may be due either to lower affinity for O2 or to enhanced substrate supply. These results indicate that differences in resistance to hypoxia/anoxia observed between rats and turtles are probably not due to differences in O2 availability. Rather, it is likely that differences in redox activities of cytochrome aa3 and/or in substrate use play a role in the relative insensitivity of turtle brain to O2 deprivation.

Animals↗

Frequency potentiation in the medial cortex of young turtle brains in vitro.

Turtle brains have a relatively primitive cortex. Glutamate receptors in the cortex of turtles include N-methyl-D-aspartate (NMDA) and DL-alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA). Our aim was to determine whether the medial cortex in turtles, like the cortex and hippocampus in mammals exhibits frequency potentiation, a non-lasting form of synaptic plasticity, and, if so, to identify the involved receptors. Our results indicate that (1) the medial cortex exhibits this phenomenon with septal stimulation at 2 Hz, the frequency with maximum power spectral density in the electrocorticogram of turtles, showing an increase in both the excitatory postsynaptic potential and the evoked potential amplitudes; (2) the frequency potentiation of the medial cortex in turtles is mediated by AMPA type glutamate receptors; (3) the dynamics of frequency potentiation development in turtles show a number of differences from that in mammals. In summary, the cortex in this group of reptiles exhibits a functional trait of the cortex in mammals that is related to learning and memory; this trait, frequency potentiation, may have appeared as an independent specialization in both groups.

Animals↗

Chemosensory orientation behavior in juvenile sea turtles.

It has been widely believed for several decades that hatchling sea turtles imprint to chemical cues characteristic of their natal beach and use this information as part of a repertoire of mechanisms enabling their return to the same beach for mating and nesting. This has proven very difficult to test. Although the imprinting theory is conceptually simple, functionally it is quite complex. This involves not only chemical imprinting of nestlings but growth and migration to habitats where the adults are found, long-term memory of their earlier chemical exposure, reproductive maturation, and homing. A few studies have been conducted to examine these elements of the imprinting theory. Experiments involving the exposure of embryos and hatchlings to chemicals suggest that juvenile turtles 'imprint' to the chemical environment of their nest. This can be termed chemical imprinting. Loggerhead turtles, Caretta caretta, and ridley turtles, Lepidochelys kempi, appear to be attracted to chemicals (morpholine and natural seawater, respectively) to which they were exposed as embryos. The strongest support for chemical imprinting is that six-month-old green turtles, Chelonia mydas, exposed to either morpholine or 2-phenylethanol in the nest and for a period of time after hatching, respond similarly to the chemical to which they were exposed as nestlings. Although chemical imprinting does not 'prove' the imprinting theory of turtle homing, it is a necessary component of the theory not previously examined.

Animals↗

Comparison of olfactory bulb projections in pigeons and turtles.

The projection targets of the olfactory bulb in pigeons and turtles were investigated using autoradiographic techniques. Despite the relatively smaller size of the olfactory bulbs in pigeons, the projection targets of the olfactory bulb are very similar to those in turtles. In both pigeons and turtles, the olfactory bulb projects to the entire rostrocaudal extent of a portion of the dorsolateral telencephalon (which is here recognized as the pyriform cortex in both birds and reptiles) and to portions of the medial telencephalic wall including the medial septal region. In addition, a projection to the olfactory tubercle of the ventral telencephalon is clearly present in turtles and also appears to be present in pigeons. Pigeons and turtles do differ significantly, however, in the extent of the projection to the amygdaloid region. In turtles, olfactory bulb input encompasses the entire mediolateral and rostrocaudal extent of the amygdaloid region, while in pigeons the input is restricted to a small dorsomedial portion of the amygdala termed nucleus taeniae of the archistriatum. The present results suggest that the olfactory bulb projections in birds are generally similar to those in reptiles, with the exception that secondary olfactory bulb projections to the amygdala may be much reduced in birds compared to those in reptiles. The functional significance of the reduction in olfactory input to the amygdala is presently uncertain.

Animals↗

Seaward orientation of hatchling turtles: turning systems in the optic tectum.

This paper reports studies on the mechanisms underlying seaward orientation in hatchling turtles. The particular aim was to investigate whether activity in different regions of the retina and associated tectal areas, as assessed by some comparator mechanism, results in turning in different directions. Hatchling green turtles (Chelonia mydas) were tested for sea-finding ability in a natural situation on the beach following lesions of the optic tectum. Asymmetrical bilateral lesions resulted in a number of turtles making circles in the direction of the tectum with the posterior lesion and in other turtles deviated in this direction. Bilaterally lesioned turtles were also slower and less consistent in their sea-finding behaviour. No major disruptions of sea finding were detected in animals with unilateral tectal lesions. By suspending lights in the nasal visual field of unilaterally blindfolded green and leatherback turtles (Dermochelys coriacea) it was possible to produce circling in the direction of the covered eye; in contrast, with the light suspended in the temporal field, turning was always in the direction of the uncovered eye. The results are consistent with the view that sea finding depends on a complex phototropotactic system with stimulation in different parts of a single retina being associated with turning in opposite directions.

Animals↗

Effects of swimming on metabolic recovery from anoxia in the painted turtle.

Anoxic submergence in the Western painted turtle results in a severe metabolic acidosis characterized by high plasma lactate and depressed arterial pH, a response similar to that seen in other vertebrates following exhaustive exercise. We tested the hypothesis that 1 or 2 h of aerobic swimming following anoxic submergence would enhance the rate of lactate disappearance from the blood just as sustained aerobic exercise does in mammals and fishes following strenuous exercise. Following 2 h of anoxic submergence at 25 degrees C and 1 h of recovery, the pattern of plasma lactate disappearance in turtles previously trained to swim in a flume and swum aerobically (2-3x resting V(O(2))) for 1 or 2 h did not differ significantly from that in trained and untrained non-swimming turtles. Turtles were fully recovered by 7-10 h post-anoxia. The response patterns also did not differ between treatments for arterial P(O(2)), P(CO(2)), pH, and plasma glucose and HCO(3)(-). Blood pH and plasma HCO(3)(-) recovered by 1 and 4 h, respectively. Despite the large lactate load, painted turtles are able to sustain periods of continuous swimming for at least 2 h without compromising metabolic recovery. Although this activity did not consistently enhance recovery, the rate of lactate disappearance was positively correlated with oxygen consumption rate in actively and passively recovering turtles. We suggest that active recovery was not a more important enhancer of recovery either because swimming may have had an inhibitory effect on hepatic gluconeogenesis or that there is variation in fuel utilization during the swimming period.

Analysis of Variance↗

Aquatic turning performance of painted turtles (Chrysemys picta) and functional consequences of a rigid body design.

The ability to capture prey and avoid predation in aquatic habitats depends strongly on the ability to perform unsteady maneuvers (e.g. turns), which itself depends strongly on body flexibility. Two previous studies of turning performance in rigid-bodied taxa have found either high maneuverability or high agility, but not both. However, examinations of aquatic turning performance in rigid-bodied animals have had limited taxonomic scope and, as such, the effects of many body shapes and designs on aquatic maneuverability and agility have yet to be examined. Turtles represent the oldest extant lineage of rigid-bodied vertebrates and the only aquatic rigid-bodied tetrapods. We evaluated the aquatic turning performance of painted turtles, Chrysemys picta (Schneider, 1783) using the minimum length-specific radius of the turning path (R/L) and the average turning rate (omega(avg)) as measures of maneuverability and agility, respectively. We filmed turtles conducting forward and backward turns in an aquatic arena. Each type of turn was executed using a different pattern of limb movements. During forward turns, turtles consistently protracted the inboard forelimb and held it stationary into the flow, while continuing to move the outboard forelimb and both hindlimbs as in rectilinear swimming. The limb movements of backward turns were more complex than those of forward turns, but involved near simultaneous retraction and protraction of contralateral fore- and hindlimbs, respectively. Forward turns had a minimum R/L of 0.0018 (the second single lowest value reported from any animal) and a maximum omega(avg) of 247.1 degrees. Values of R/L for backward turns (0.0091-0.0950 L) were much less variable than that of forward turns (0.0018-1.0442 L). The maneuverability of turtles is similar to that recorded previously for rigid-bodied boxfish. However, several morphological features of turtles (e.g. shell morphology and limb position) appear to increase agility relative to the body design of boxfish.

Animals↗

Maintenance of neutral buoyancy by depth selection in the loggerhead turtle Caretta caretta.

Time-series data of swimming speed and dive depth were recorded in six female loggerhead turtles Caretta caretta during the internesting period. The dive profiles of all animals indicated that they stayed at particular depths without swimming and that these depths were correlated with dive duration. These results support the hypothesis that lung air is used to achieve neutral buoyancy in the loggerhead turtle. To test this hypothesis, female turtles were equipped with lead weights and time/depth recorders. The residence depth of the turtles increased when their specific gravity was artificially decreased. This indicates that they control depth rather than lung volume, suggesting that the residence depth of loggerhead turtles during the internesting period is not determined actively. They presumably remain at a particular depth exclusively to save energy for egg maturation during the internesting period. Lung volume was estimated from the change in depth of weighted animals to be 50-150 ml kg(-1). The resulting residence depth of all turtles was within the range at which they maintained the neutral buoyancy.

Air↗

Seasonal change in the capacity for supercooling by neonatal painted turtles.

Hatchlings of the North American painted turtle (Chrysemys picta) typically spend their first winter of life inside the shallow, subterranean nest where they completed incubation the preceding summer. This facet of their natural history commonly causes neonates in northerly populations to be exposed in mid-winter to ice and cold, which many animals survive by remaining unfrozen and supercooled. We measured the limit of supercooling in samples of turtles taken shortly after hatching and in other samples after 2 months of acclimation (or acclimatization) to a reduced temperature in the laboratory or field. Animals initially had only a limited capacity for supercooling, but they acquired an ability to undergo deeper supercooling during the course of acclimation. The gut of most turtles was packed with particles of soil and eggshell shortly after hatching, but not after acclimation. Thus, the relatively high limit of supercooling for turtles in the days immediately after hatching may have resulted from the ingestion of soil (and associated nucleating agents) by the animals as they were freeing themselves from their eggshell, whereas the relatively low limit of supercooling attained by acclimated turtles may have resulted from their purging their gut of its contents. Parallels may, therefore, exist between the natural-history strategy expressed by hatchling painted turtles and that expressed by numerous terrestrial arthropods that withstand the cold of winter by sustaining a state of supercooling.

Acclimatization↗

Effects of organochlorine contaminants on loggerhead sea turtle immunity: comparison of a correlative field study and in vitro exposure experiments.

Several laboratory and field studies indicate that organochlorine contaminants (OCs), such as polychlorinated biphenyls (PCBs) and pesticides, modulate immune responses in rodents, wildlife, and humans. In the present study we examined the effects of OCs on immunity in free-ranging loggerhead sea turtles (Caretta caretta). Mitogen-induced lymphocyte proliferation responses, lysozyme activity, and OC concentrations were measured from blood samples. Mitogens chosen in the lymphocyte proliferation assay were phytohemagglutinin (PHA) and concanavalin A (ConA) for T-lymphocyte stimulation, and lipopolysaccharide (LPS) and phorbol 12,13-dibutyrate (PDB) for B-lymphocyte stimulation. Lysozyme activity was significantly and negatively correlated with whole-blood concentrations of 4,4 -dichlorodiphenyldichloroethylene (4,4 -DDE) and the sum of chlordanes. Lymphocyte proliferation responses stimulated by PHA, LPS, and PDB were significantly and positively correlated with concentrations of the sum of PCBs measured in whole blood. LPS- and PDB-induced proliferation were also significantly and positively correlated with 4,4 -DDE blood concentrations. These correlative observations in free-ranging turtles suggest that current, chronic exposure to OCs may suppress innate immunity and enhance certain lymphocyte functions of loggerhead sea turtles. To further test this hypothesis, lymphocyte proliferation was measured after in vitro exposure of peripheral blood leukocytes from 16 turtles to Aroclor 1254 (0-13.5 microg/mL) or 4,4 -DDE (0-13.4 microg/mL). Both contaminants increased PHA- and PDB-induced proliferation at concentrations below those that affected cell viability. Moreover, the concentrations that enhanced PDB-induced proliferation in vitro were similar to concentrations measured in turtles with the highest proliferative responses. The similarities between the in vitro experiments and the correlative field study suggest that OC exposure modulates immunity in loggerhead turtles.

Animals↗

Presence of a neurophysin-like precursor in the green turtle (Chelonia mydas).

A glycoprotein of neurohypophysial origin was found to have cofractionated with FSH prepared from pituitary glands of the green turtle, Chelonia mydas. Antiserum raised against this preparation contained high antibody titres and affinity for the neurohypophysial component and allowed development of a specific radioimmunoassay to monitor its purification and distribution in the brain. Immunocytochemistry revealed that the glycoprotein was concentrated in the pars nervosa and associated nerve tracts passing through the median eminence to the supraoptic and paraventricular nuclei; similar distributions were observed in turtles and rats. The antiserum to the turtle material bound radiolabelled rat vasopressin (VP)-neurophysin and precipitated precursors of this neurophysin, but it did not cross-react with rat oxytocin-neurophysin. An amino-terminal alanine was also consistent with the structure of rat VP-neurophysin, but the turtle molecule was larger than the corresponding rat molecule. Limited tryptic digests of the turtle glycoprotein contained two components, one of which bound to lysine VP. Both components contained carbohydrate, but only the one which bound to VP cross-reacted in a radioimmunoassay for rat VP-neurophysin. The apparent surge in plasma immuno-FSH at the time of oviposition previously described in the turtle probably represented release of a neurophysin-like 'carrier' molecule associated with secretion of the neurohypophysial hormone (e.g. arginine vasotocin; AVT) responsible for oviduct contractility. These data suggest that the neurohypophysial glycoprotein represents a partially processed AVT precursor and provide the first biochemical evidence of a mammalian-like biosynthetic pathway for neurohypophysial hormones in a non-mammalian species.

Amino Acids↗

Organochlorine contaminants in sea turtles: correlations between whole blood and fat.

Monitoring toxic organochlorine (OC) compounds is an important aspect in wildlife studies, especially in protected species such as sea turtles. The goal of this study was to determine whether blood OC concentrations can predict those in adipose tissue of sea turtles. Blood offers many benefits for monitoring OCs. It can be collected nondestructively from live turtles and can be sampled repeatedly for continuous monitoring. Organochlorine concentrations in blood may better represent the exposure levels of target tissues, but blood concentrations may fluctuate more than those in fatty tissues following recent dietary exposure or lipid mobilization. Paired fat and blood samples were collected from 44 live, juvenile loggerhead sea turtles and 10 juvenile Kemp's ridley sea turtle carcasses. Organochlorines were analyzed using gas chromatography with electron capture detection and mass spectrometry. Lipid-normalized OC concentrations measured in the blood significantly correlated to levels found in the fat samples of both species. This result suggests that sea turtle blood is a suitable alternative to fatty tissues for measuring OCs because blood concentrations reasonably represent those observed in the paired fat samples. However, blood OC concentrations calculated on a wet-mass basis were significantly and inversely correlated to lipid content in the fat samples. Therefore, caution should be used when monitoring spatial or temporal trends, as OC levels may increase in the blood following mobilization of fat stores, such as during long migrations, breeding, or disease events.

Adipose Tissue↗

Regulation of hemoglobin function and whole blood oxygen affinity by carbon dioxide and pH in the loggerhead (Caretta caretta) and green sea turtle (Chelonia mydas mydas).

The oxygen affinity of suspensions of erythrocytes from juvenile and adult loggerhead (Caretta caretta) and green sea (Chelonia mydas mydas) turtles decreased markedly with increasing concentrations of carbon dioxide (0 to near 15%) or hydrogen ion (pH 7.6 to pH 7.2). The P50's were higher with increases in pCO2, particularly at pH near 7.4, than were the P50's with increases in hydrogen ion concentration at any given CO2 concentration. Solutions of hemoglobins from the juvenile loggerhead (8-9 mos.) and green sea (10 mos.) turtles responded to 2, 3-DPG, ATP, or inositol-P5 when added at molar ratios of phosphate to hemoglobin of 4:1 and 20:1 in 0% and 6.29% CO2 but showed no decrease in oxygen affinity at these two CO2 levels in the green turtle when the molar ratio of phosphate to hemoglobin was 0.4. These compounds had little effect on the P50 of these hemoglobins in 14.6% CO2. The P50 of the adult loggerhead turtle hemoglobin did not increase in the presence of organic phosphates beyond the effect induced by CO2 alone. The P50 of hemoglobin from the adult green sea turtle increased only slightly when the molar ratio of phosphate to hemoglobin was 20:1 and at 0 and 6% CO2 concentration; little effect was observed at 14.6% CO2. These data demonstrate that blood oxygen affinities and hemoglobin function in these two species of marine turtles are altered significantly by CO2 and to a lesser degree by pH. It is suggested that such alterations may be of significance in vivo during prolonged diving when there are dramatic rises in blood pCO2 and [H+] and profound decreases in pO2.

Animals↗

Hepatitis B virus (HBV) infections in turtles.

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.

Animals↗