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PubMed2004

Differentiation of the epidermis in turtle: an immunocytochemical, autoradiographic and electrophoretic analysis.

Proteins involved in the process of cornification of turtle epidermis are not well known. The present immunocytochemical, electrophoretic and autoradiographic study reports on the localization patterns and molecular weights of keratins, which are cornification proteins, and of tritiated histidine in turtle epidermis. Alpha-keratins with a molecular weight of 40-62 kDa are present in the epidermis. Beta-keratin is mainly detectable in the stratum corneum of the carapace and plastron, but is rarely present or even absent in the corneous layer of limb, tail and neck epidermis. After electrophoresis and immunoblotting with an antibody against chicken scale beta-keratin, bands at 15-17, 22-24, and 36-38 kDa appeared. This antibody recognized weaker bands at 38-40 and 58-60 kDa in the soft epidermis. After reduction and carboxymethylation of proteins extracted from carapace and plastron, but not of proteins from the soft epidermis, protein bands at 15-17 and 35-37 kDa were found when using the anti-beta 1-keratin antibody. Loricrin-, filaggrin-, sciellin-, and transglutaminase-like immunostaining was detectable only in the transitional and lowermost corneous layers of the soft epidermis. Vesicular bodies in the transitional layer were immunolabeled by the anti-loricrin antibody, and weakly by the anti-filaggrin and anti-transglutaminase antibodies. In immunoblots, the anti-loricrin antibody reacted with a major band at 50-54 kDa in both carapace-plastron and soft epidermis. The anti-sciellin antibody detected major bands at 38-40 and 50 kDa in hard epidermis, and at 50 and 54-56 kDa in soft epidermis. Filaggrin-like immunostained bands were observed at 50-55 and 62-64 kDa. This immunostaining was probably due to a common epitope in filaggrin and some keratins. Histidine was evenly incorporated in the epidermis, and the ultrastructural study showed random labeling, often associated with keratin bundles of alpha and beta-keratinocytes. Histidine-labeled protein bands were not found in the carapace-plastron. In the soft epidermis, weakly labeled bands at 15-20, 25, and 45-60 kDa were found occasionally. The latter bands probably represented neo-synthesized keratins as was also indicated by the ultrastructural autoradiographic analysis. In conclusion, our study suggests that proteins with epitopes that they have in common with cornification proteins of mammalian epidermis are also present in the epidermis of turtle.

Animals↗
PubMed2006

Anoxia tolerance in turtles: metabolic regulation and gene expression.

Freshwater turtles of the Trachemys and Chrysemys genera are champion facultative anaerobes able to survive for several months without oxygen during winter hibernation in cold water. They have been widely used as models to identify and understand the molecular mechanisms of natural anoxia tolerance and the molecular basis of the hypoxic/ischemic injuries that occur in oxygen-sensitive systems and underlie medical problems such as heart attack and stroke. Peter L. Lutz spent much of his career investigating turtle anaerobiosis with a particular focus on the mechanisms of brain ion homeostasis and neurotransmitter responses to anoxia exposure and the mechanisms that suppress brain ion channel function and neuronal excitability during anaerobiosis. Our interests intersected over the mechanisms of metabolic rate depression which is key to long term anoxia survival. Studies in my lab have shown that a key mechanism of metabolic arrest is reversible protein phosphorylation which provides coordinated suppression of the rates of multiple ATP-producing, ATP-utilizing and related cellular processes to allow organisms to enter a stable hypometabolic state. Anoxia tolerance is also supported by selective gene expression as revealed by recent studies using cDNA library and DNA array screening. New studies with both adult T. scripta elegans and hatchling C. picta marginata have identified prominent groups of genes that are up-regulated under anoxia in turtle organs, in several cases suggesting aspects of cell function and metabolic regulation that have not previously been associated with anaerobiosis. These groups of anoxia-responsive genes include mitochondrially-encoded subunits of electron transport chain proteins, iron storage proteins, antioxidant enzymes, serine protease inhibitors, transmembrane solute carriers, neurotransmitter receptors and transporters, and shock proteins.

Adaptation, Physiological↗
PubMed2006

Exercise warms adult leatherback turtles.

Leatherback sea turtles (Dermochelys coriacea) can maintain body temperature (T(B)) up to 18 degrees C above that of the surrounding sea water (T(W)) which allows leatherbacks to enter cold temperate waters and have the largest global range of any reptile. Using a cylindrical model of a leatherback we investigated the extent to which heat production through variation of swim speed could be used in a leatherback's thermal strategy. Drag force of a full scale cast of a leatherback was measured in a low velocity wind tunnel to obtain an estimate of the metabolic cost needed to offset drag. Heat released in the core of a turtle as a byproduct of the metabolic cost of locomotion is conducted from the core of the turtle to the surrounding water through its insulation layer. By keeping insulation thickness constant, we highlight the effectiveness of swim speed in maintaining T(B)-T(W). Our model, when tested against published data at a given T(W), showed a close correlation between predicted and measured swimming speed at a given T(B). We conclude that the ability to maintain a large T(B)-T(W) is an interplay between mass, insulation thickness and water temperature selection but behavioural control of swimming speed predominates.

Animals↗
PubMed2006

Nonadditive interactions between animal and plant diet items in an omnivorous freshwater turtle Trachemys scripta.

Nonadditive interactions occur when diet items interact with one another such that the net energy or nutrient gain from a mixed diet differs from that predicted by summing the gains from individual diet components. We quantified nonadditive effects between duckweed, Lemma valdiviana, and grass shrimp, Palaemontes paludosus, in the freshwater turtle Trachemys scripta. We fed turtles 100% duckweed, 100% shrimp, and two mixed diets containing 67% duckweed, 33% shrimp and 14% duckweed, 86% shrimp (dry matter basis). During each feeding trial, we measured intake, digestibility, and transit time of the diet, and upon conclusion, short-chain fatty acid concentrations in turtle digestive tracts. Digestibility was lower on the 67% duckweed diet, but higher on the 14% diet. These apparent nonadditive interactions may be due to differences in transit time of duckweed and shrimp. We believe this is the first evidence of two diet items producing opposing nonadditive effects when fed in different ratios.

Animals↗
PubMed2005

Androgen receptor in the oviduct of the turtle, Trachemys scripta.

Circulating androgens reach high concentrations in females of some reptiles and amphibians. We are testing the hypothesis that androgens can act directly in female reptilian reproductive tissues, via the androgen receptor. In this study, we sought to determine if androgen receptors are present in the oviduct of the turtle, Trachemys scripta, using radioligand-binding assays and immunological assays. An androgen-binding site was detected in turtle oviductal cytosol and oviductal nuclear extract by radioligand binding assay, using (3)H-dihydrotestosterone (DHT) as the ligand. This site was saturable (B(max)=11 pmol/g tissue), had a high affinity (10(-10) M), and showed specificity typical of androgen receptors (DHT>testosterone, progesterone>>estradiol, cortisol). Western blotting using an anti-androgen receptor antibody revealed a band of immunoreactivity in oviductal cytosol at approximately 115 kDa, and a more prominent band at 50 kDa, possibly indicating a truncated form of the androgen receptor. Immunohistochemistry revealed crossreactivity of the androgen receptor antibody against oviductal glandular cells but not against oviductal luminal epithelial or muscularis cells. The presence of androgen receptor in the turtle oviduct suggests that androgens have a role in female reproduction and that their action can be mediated directly by androgen receptor.

Animals↗
PubMed2003

Arsenic accumulation in livers of pinnipeds, seabirds and sea turtles: subcellular distribution and interaction between arsenobetaine and glycine betaine.

Concentrations of total arsenic and individual arsenic compounds were determined in liver samples of pinnipeds (northern fur seal Callorhinus ursinus and ringed seal Pusa hispida), seabirds (black-footed albatross Diomedea nigripes and black-tailed gull Larus crassirostris) and sea turtles (hawksbill turtle Eretmochelys imbricata and green turtle Chelonia mydas). Among these species, the black-footed albatross contained the highest hepatic arsenic concentration (5.8+/-3.7 microg/g wet mass). Arsenobetaine was the major arsenic species found in the liver of all these higher tropic marine animals. To investigate the cause of high accumulation of arsenobetaine, subcellular distribution of arsenic and relationship between arsenobetaine and glycine betaine concentrations were examined in the livers of these animals. There was no relationship between total arsenic concentration and its subcellular distribution in liver tissues. However, a significant negative correlation was found between arsenobetaine and glycine betaine concentrations in the liver of six species examined. This result may indicate that arsenobetaine is accumulated in these marine animals as an osmolyte along with glycine betaine, which is a predominant osmolyte in marine animals because the chemical structure and properties of arsenobetaine are similar to those of glycine betaine.

Animals↗
PubMed2004

The effects of vitamin E on antiacid stress ability in juvenile soft-shelled turtles (Pelodiscus sinensis).

We determined the effect of dietary supplementation with vitamin E (0-, 50-, 250-, 500-, 1000- and 5000-mg/kg diet for 4 weeks) on antistress ability in juvenile soft-shelled turtle (Pelodiscus sinensis). Half of the turtles per dose group were treated by acid stress for 24 h. The results showed that phagocytosis of blood cells in the control group significantly decreased after acid stress while the other five groups had no significant changes compared with those of before stress. Serum bacteriolytic activity in the control group and the group supplemented with 50-mg vitamin E/kg diet significantly decreased after acid stress. The other four groups showed no significant differences compared with those before stress. Serum bactericidal activities in all groups notably decreased after acid stress, but the difference of serum bactericidal activity in before and after stress had a decreased tendency from the control group to the highest dose group. Serum cortisol levels in the control group were significantly increased while the other five groups had no notable increases after acid stress. Liver vitamin E levels in all groups had no notable changes compared with those before stress but there was a tendency to decrease after acid stress. These results suggest that acid stress depress immune function and increase serum cortisol levels in turtles while vitamin E alleviate the adverse effects caused by acid stress.

Acids↗
PubMed2005

Polycyclic aromatic hydrocarbons affect survival and development of common snapping turtle (Chelydra serpentina) embryos and hatchlings.

Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds found in the John Heinz National Wildlife Refuge in Philadelphia, Pennsylvania. We assessed the impact of PAHs and crude oil on snapping turtle development and behavior by exposing snapping turtle eggs from the Refuge and from three clean reference sites to individual PAHs or a crude oil mixture at stage 9 of embryonic development. Exposure to PAHs had a significant effect on survival rates in embryos from one clean reference site, but not in embryos from the other sites. There was a positive linear relationship between level of exposure to PAHs and severity of deformities in embryos collected from two of the clean reference sites. Neither righting response nor upper temperature tolerance (critical thermal maximum, CTM) of snapping turtle hatchlings with no or minor deformities was significantly affected by exposure to PAHs.

Animals↗
PubMedDate not supplied

Effects of organochlorine compounds on cytochrome P450 aromatase activity in an immortal sea turtle cell line.

Many classes of environmental contaminants affect the reproductive function of animals through interactions with the endocrine system. The primary components affected by endocrine active compounds (EACs) are the steroid receptors and the enzymes responsible for steroidogenesis. This study sought to develop an in vitro model for assessing EAC effects in sea turtles by examining their ability to alter cytochrome P450 aromatase (CYP19) activity. Aromatase is the enzyme responsible for the conversion of testosterone to estradiol. This enzyme is critical in the sexual differentiation of reptiles which demonstrate temperature-dependent sex determination. An immortal testis cell line GST-TS from a green sea turtle was grown in culture at 30 degrees C in RPMI 1640 media. The cells were exposed to three known aromatase inducers; dexamethasone (Dex), 8Br-cyclic AMP, or human chronic gonadotropin (HCG) and one aromatase inhibitor 4-androstenol-dione (4-OHA). In addition, the GST-TS cells were exposed to 0.1-30 microM atrazine and 3-100 microM 4,4'-DDE. The inducing compounds that have been shown to increase aromatase activity in other systems failed to induce aromatase activity in the GST-TS cells, yet exposure to the inhibiting compound, 4-OHA, did result in a significant reduction. Atrazine (0.1, 1.0 and 10 microM) significantly induced aromatase activity following a 24 h exposure, and 4,4'-DDE inhibited the activity but only at cytotoxic concentrations (100 microM). Based on these results, this in vitro model can be useful in examining the endocrine effects of EACs in sea turtles.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2005

Distribution of facial motor neurons in the pond turtle Pseudemys scripta elegans.

A tract tracing study was performed to examine the localization of the facial nucleus in the brain stem of the pond turtle, Pseudemys scripta elegans. Neurobiotin and the fluorescent tracers alexa fluor 488 and 594 were used to retrogradely label neurons of the abducens or facial nerves. The results showed that the facial nucleus has two subnuclei, a medial group and a lateral group. Measurements of cell size revealed no significant differences between these populations. Double labeling studies showed that the medial cell group of the facial nucleus lies between the principal and accessory abducens nuclei in the pons, whereas the lateral group lies adjacent to the accessory abducens nucleus. The facial nucleus of pond turtles largely overlaps the rostrocaudal extent of the accessory abducens nucleus, but extends well beyond it into the medulla. These data elucidate the position and distribution of the facial nucleus in the brain stem of pond turtles and contribute to the body of comparative neuroanatomical literature on the distribution of the cranial nerve nuclei of reptiles.

Animals↗
PubMed2006

Computed tomography of the vertebral column and coelomic structures in the normal loggerhead sea turtle (Caretta caretta).

The aim of this study was to determine the normal computed tomography (CT) appearance of the vertebral column and coelomic structures of the loggerhead sea turtle (Caretta caretta) and to use three-dimensional (3D) and multiplanar reconstructions to indicate the position of each organ in relation to the vertebrae and carapace. Transverse sections of 1mm thickness were performed in seven clinically healthy and in five dead loggerhead sea turtles using multi-detector CT equipment. A computer workstation was used for multiplanar and 3D reconstructions. Dead turtles were frozen and sectioned in the transverse, dorsal and sagittal planes to compare the anatomical structures' appearance with CT images. Clinically relevant organs including the oesophagus, stomach, trachea, bronchi, lungs, liver, gallbladder, heart, spleen, kidneys and vertebral canal were identified in CT images. Computed tomography provides detailed information on the respiratory system and skeleton; the location of the coelomic structures with respect to the carapace and the vertebrae that is provided in this work will facilitate the use of other ancillary diagnostic techniques such as ultrasound, radiography and biopsy, thereby improving safety of access in surgical procedures.

Abdomen↗
PubMed2006

Salmonella in free living terrestrial and aquatic turtles.

Detection of Salmonella in pet turtles has been the focus of extensive research, but its incidence in free living turtles is not well known. The aim of this study was to evaluate the incidence of Salmonella in terrestrial and aquatic species of chelonians inhabiting a National Park in southwestern Spain. Individuals of the terrestrial tortoise Testudo graeca (n = 16) and the aquatic turtles Emys orbicularis (n = 26) and Mauremys leprosa (n = 50) were investigated. Maximum incidence of Salmonella was recorded in the terrestrial species (100%). In contrast, the incidence of infected animals was low in the aquatic species, 12% in M. leprosa and 15.4% in E. orbicularis. Five serotypes of Salmonella belonging to subspecies enterica (I) and salamae (II) were identified. All serotypes were found in the terrestrial species, and three in the aquatic ones, suggesting that wild terrestrial chelonians are important reservoirs of Salmonella in our study area. Cloacal transmission during mating is the most probable mode of transmission among individuals.

Animals↗
PubMed2005

Synergistic role for pituitary growth hormone in the regulation of hepatic estrogen and progesterone receptors and vitellogenesis in female freshwater turtles, Chrysemys picta.

Previous studies using the freshwater turtle Chrysemys picta have demonstrated that estradiol, progesterone (P), testosterone (T), and growth hormone (GH) regulate hepatic vitellogenin synthesis, suggesting a multihormonal regulation of vitellogenesis in the turtle. In this study we further investigated the interaction between estradiol-17beta (E) and growth hormone in the regulation of vitellogenin (vtg) in hypophysectomized post-reproductive female turtles (C. picta). Northern blot analysis was used to monitor the changes in vtg mRNA, ER mRNA, and PR mRNA expression; Western blot to determine changes in PR isoform expression and a homologous ELISA for measurement of plasma vtg. Compared to sham-operated controls, hypophysectomy did not reduce the hepatic levels of any parameters below the seasonal norm. Changes in these parameters in hypophysectomized animals after administration of GH alone, estrogen alone, or GH in combination with estrogen were well correlated. The effect of estrogen alone was greater than that of GH, and elevated all end-points analyzed. With the exception of plasma vitellogenin, the effect of GH plus estrogen was significantly greater than either hormone alone. In contrast to changes in ER mRNA, vtg mRNA, and vtg protein, the effect of estrogen and GH plus estrogen treatments on PRA mRNA and PRC mRNA, although significant, was relatively modest. However, changes in PRA and PRB protein were large (5- to 10-fold), and of similar magnitude to the changes in ER mRNA, vtg mRNA, and plasma vtg. Further, PRA and PRB protein levels appeared to be differentially affected. Thus, in sham and normal animals, only PRB was detected, and the levels were similar. After GH administration, PRB increased 4- to 5-fold, and PRA became detectable. Estrogen increased both isoforms of PR equally (approximately 6-fold), and some synergism was apparent when the two hormones were administered together, PR levels being the highest in this group (about an 8-fold increase). The results suggest that although estrogen is the primary regulatory factor involved in activation of vitellogenin synthesis, basal levels of all primary components of signaling pathways involved in vitellogenesis measured here may be maintained in the absence of either estrogen or GH, and that GH alone can activate transcription of some of these important transcription factors.

Animals↗
PubMed2000

cDNA cloning of turtle prion protein.

Cloning of the cDNA coding for the 270-residue turtle prion protein is reported. It represents the most remote example thus far described. The entire coding region is comprised in a single exon, while a large intron interrupts the 5' UTR. The common structural features of the known prion proteins are all conserved in turtle PrP, whose identity degree to mammalian and avian proteins is about 40 and 58%, respectively. The most intriguing feature, unique to the turtle prion, is the presence of an EF-hand Ca(2+) binding motif in the C-terminal half of the protein.

Amino Acid Sequence↗
PubMed2003

Seasonal variation in plasma catecholamines and adipose tissue lipolysis in adult female green sea turtles (Chelonia mydas).

We investigated three aspects of potential interrenal regulation of reproduction in female green sea turtles, Chelonia mydas. First, seasonal trends in plasma catecholamines were examined from female C. mydas at different stages of their reproductive cycles. Second, variation in catecholamine levels during a nesting season were analysed in relation to restraint time, and ecological variables such as nesting habitat, body size, and reproductive investment. Third, catecholamine and corticosterone (CORT) induced lipolysis was investigated with adipose tissue collected from gravid green turtles, using in vitro incubations. Plasma epinephrine (EPI) was lowest in non-vitellogenic (1.55 +/- 0.26 ng/ml) and post-breeding (1.57 +/- 0.22 ng/ml) females, and highest in courting females (2.87 +/- 0.28). Concentrations of norepinephrine (NE) and EPI were relatively constant throughout a nesting season, and not significantly related to restraint time, reproductive investment or nesting habitat. In vitro concentrations of CORT (>3 ng/ml) and NE (2 ng/ml) induced significant release of glycerol after 6h of incubation. Epinephrine tended to induce an antilipolytic affect at low concentrations (0.25 ng/ml) and a net lipolytic response at higher concentrations (>1 ng/ml). Our data suggest that EPI may play a role in regulating body condition during vitellogenesis, and maintaining energy stores during prolonged aphagia during courtship and nesting in female green sea turtles. Furthermore, we provide preliminary evidence that suggests that catecholamine production may be either down regulated or de-sensitised in gravid female C. mydas.

Adipose Tissue↗
PubMed2003

Lipids and lipid-transporting proteins in Chrysemys picta: role of gonadal steroids and growth hormone in intact and hypophysectomized turtles.

In the freshwater turtle, the homeostatic control of plasma lipids and lipid-transporting proteins may be coordinately regulated by ovarian steroids and pituitary hormones such as growth hormone (GH). In order to elucidate the role of these hormones in the regulation of vitellogenesis and ovarian growth, we have investigated lipid metabolic changes in normal male and female turtles, and in hypophysectomized females with and without GH injections, in response to combinations of exogenously administered gonadal steroids (estradiol (E2), progesterone (P), and testosterone (T)). Determinations of total plasma triglycerides, cholesterol, vitellogenin, and apoA-I were performed. We have demonstrated that E2 alone and in combination with P significantly increased plasma apoA-I and triglyceride levels in both intact female and male turtles. Testosterone administered alone to males had no effect on any of the parameters measured. In hypophysectomized females, plasma apoA-I, vitellogenin, and triglyceride levels were all significantly elevated in animals which received GH and E2, compared to controls (sham and hypox) and those which received GH alone.

Animals↗
PubMed1997

PCB concentrations of eggs and chorioallantoic membranes of loggerhead sea turtles (Caretta caretta) from the Cape Romain National Wildlife Refuge.

PCBs were found in eggs and chorioallantoic membranes (CAMs) of loggerhead sea turtles (Caretta caretta). Total PCB concentrations in CAMs were larger than concentrations in eggs. Total PCB concentrations in egg and CAM tissues were highly correlated (r2 = 0.782; p = 0.0001). Sums of PCB congeners within homologue groups were also correlated in the two tissues. Data from these turtle eggs indicate 1) PCB concentrations in CAMs represent PCB concentrations in whole eggs and 2) CAMs can be collected in a nonlethal manner to determine PCB concentrations in sea turtle hatchlings.

Allantoin↗
PubMed2001

Congener-specific profile and toxicity assessment of PCBs in green turtles (Chelonia mydas) from the Hawaiian Islands.

Chemical pollution may play a role in the etiology of fibropapillomatosis in green turtles (Chelonia mydas). In this preliminary study, polychlorinated biphenyls (PCBs) were measured in the livers and adipose fats of green turtles collected after they were stranded on Oahu Island, Hawaii in 1992-1993. Average concentrations of total PCBs were 45-58 ng/g dry weight and 73-665 ng/g in the liver and adipose tissues, respectively. Hexachlorobiphenyls were predominant homologues, PCBs 153 and 138 were dominant congeners in all the turtle tissues. Among the most toxic coplanar congeners, in the order of abundance, were PCB 77 > 126 > 169. Estimated toxic equivalents (TEQs) of PCBs to 2,3,7,8-tetrachlorodibenzo-p-dioxin were 8-15 pg/g in the livers and 13-48 pg/g in the adipose tissues. PCB 126 contributed 85-91% of the total TEQs.

Adipose Tissue↗