High and variable mortality of leatherback turtles reveal possible anthropogenic impacts.

The number of nesting leatherback turtles (Dermochelys coriacea) in the eastern Pacific Ocean has declined dramatically since the late 1980s. This decline has been attributed to egg poaching and interactions with fisheries. However, it is not clear how much of the decline should also be ascribed to variability in the physical characteristics of the ocean. We used data on individually marked turtles that nest at Playa Grande, Costa Rica, to address whether climatic variability affects survival and inter-breeding interval. Because some turtles might nest undetected, we used capture-recapture models to model survival probability accounting for a detection failure. In addition, as the probability of reproduction is constrained by past nesting events, we formulated a new parameterization to estimate inter-breeding intervals and contrast hypotheses on the role of climatic covariates on reproductive frequency. Average annual survival for the period 1993-2011 was low (0.78) and varied over time ranging from 0.49 to 0.99 with a negative temporal trend mainly due to the high mortality values registered after 2004. Survival probability was not associated with the Multivariate ENSO Index of the South Pacific Ocean (MEI) but this index explained 24% of the temporal variability in the reproductive frequency. The probability of a turtle to permanently leave after the first encounter was 26%. This high proportion of transients might be associated with a high mortality cost of the first reproduction or with a long-distance nesting dispersal after the first nesting season. Although current data do not allow separating these two hypotheses, low encounter rate at other locations and high investment in reproduction, supports the first hypothesis. The low and variable annual survival probability has largely contributed to the decline of this leatherback population. The lack of correlation between survival probability and the most important climatic driver of oceanic processes in the Pacific discards a climate-related decline and point to anthropogenic sources of mortality as the main causes responsible for the observed population decline.

Survival and behavior of freshwater turtles after rehabilitation from an oil spill.

An oil spill in February 2000 at the John Heinz National Wildlife Refuge in southeastern Pennsylvania affected four species of freshwater turtles including painted turtles (Chrysemys picta), snapping turtles (Chelydra serpentina), red-eared slider turtles (Trachemys scripta), and red-bellied turtles (Pseudemys rubriventris). In the summer and fall of 2000, there were no differences in survival, home range, and temperature preference of 16 oil exposed/rehabilitated (OER) turtles, 18 possibly exposed (PE) turtles, and 32 non-exposed (NE) turtles as measured with temperature sensitive radio transmitters. Post-release mortality or transmitter loss was not correlated to oil exposure (OER = 25%, PE = 22%, NE = 31%). There were no statistically significant differences in home range minimum convex polygon area, (0.28

The leatherback turtle, Dermochelys coriacea, exhibits both polyandry and polygyny.

The leatherback turtle (Dermochelys coriacea) is an endangered species, and world-wide populations are declining. To understand better the mating structure of this pelagic and fragile species, we investigated paternity in nearly 1000 hatchlings from Playa Grande in Parque Marino Nacional Las Baulas, Costa Rica. We collected DNA samples from 36 adult female leatherbacks and assessed allele frequency distributions for three microsatellite loci. For 20 of these 36 females, we examined DNA from hatchlings representing multiple clutches, and in some cases assessed up to four successive clutches from the same female. We inferred paternal alleles by comparing maternal and hatchling genotypes. We could not reject the null hypothesis of single paternity in 12 of 20 families (31 of 50 clutches), but we did reject the null hypothesis in two families (eight of 50 clutches). In the remaining six families, the null hypothesis could not be accepted or rejected with certainty because the number of hatchlings exhibiting extra nonmaternal alleles was small, and could thus be a result of mutation or sample error. Successive clutches laid by the same female had the same paternal allelic contribution, indicating sperm storage or possibly monogamy. None of 20 females shared the same three-locus genotype whereas there were two instances of shared genotypes among 17 inferred paternal three-locus genotypes. We conclude that both polyandry and polygyny are part of the mating structure of this leatherback sea turtle population.

The effects of nest environment on calcium mobilization by leatherback turtle embryos (Dermochelys coriacea) during development.

We investigated the effect of sand moisture content and sand temperature on developmental success and the mobilization of calcium during development using laboratory incubated eggs (n=251) collected from leatherbacks nesting at Parque Nacional Marino Las Baulas, Costa Rica. Calcium concentrations of egg components [eggshell, yolk plus albumen (Y+A) and embryo] changed significantly through incubation for both viable and undeveloped eggs. In developed eggs, eggshell calcium content decreased 42.9% by day 60 of incubation. The Y+A calcium decreased by 20.8% until the last quarter of incubation, and then increased to 0.99% above initial Y+A calcium concentrations just prior to hatching. In undeveloped eggs, eggshell calcium content decreased by 25.7%, with the rate of decrease slowing significantly beyond day 30 of incubation. In contrast, Y+A calcium increased steadily through the 60-day incubation period. Embryos incorporated a higher proportion of calcium when incubated at a lower sand moisture content (5% H(2)O>12% H(2)O) and at lower sand temperatures (28.5 degrees C, 29.5 degrees C>31.0 degrees C). The total wet mass of freshly oviposited eggs was negatively correlated with calcium concentration per gram of eggshell (r=-0.569; P<0.001). Thus, each yolked egg, regardless of initial wet mass, had an average of 1.23 g (+/-0.43 g) of calcium per egg (Mean egg mass: 76.24+/-1.21 g). Both developmental success (24.1%) and hatching success (7.4%) of laboratory-incubated eggs were dependent to a greater extent on temperature than on moisture, with an increase in mortality as sand temperature increased. For natural nests on Playa Grande, developmental success (37.4%) and hatching success (19.8%) were similar in magnitude to the results obtained from the laboratory. The recent ENSO (El Niño Southern Oscillation) event and increased tidal activity may be responsible for the high embryonic mortality measured during the 1997-1998 nesting season.

Changes in gonadal and adrenal steroid levels in the leatherback sea turtle (Dermochelys coriacea) during the nesting cycle.

The reproductive endocrinology of nesting leatherback turtles (Dermochelys coriacea) was studied during the 1996-1997 and 1997-1998 nesting seasons at Los Baulas National Park, Playa Grande, Costa Rica. Blood samples were collected from nesting females throughout the season. Females were observed to nest up to 10 times during the nesting season. Plasma steroids were measured by radioimmunoassay and total plasma calcium was measured by flame atomic absorption spectrophotometry. Plasma testosterone and plasma estradiol levels declined throughout the nesting cycle of the female. Testosterone declined from a mean of 10.18 +/- 0.77 ng/ml at the beginning of the nesting cycle to 1.73 +/- 0.34 ng/ml at the end of the nesting cycle. Estradiol declined in a similar manner, ranging from a mean of 190.95 +/- 16.80 pg/ml at the beginning of the nesting cycle to 76.52 +/- 12.66 pg/ml at the end of the nesting cycle. Plasma progesterone and total calcium levels were relatively constant throughout the nesting cycle. Lack of fluctuation of total calcium levels, ranging from a mean high of 97.46 +/- 11.37 microg/ml to a mean low of 64.85 +/- 11.20 microg/ml, further suggests that vitellogenesis is complete prior to the arrival of the female at the nesting beach. Clutch size (both yolked and yolkless eggs) did not vary over the course of the nesting cycle.

Osmoregulation of the Nile crocodile, Crocodylus niloticus, in Lake St. Lucia, Kwazulu/Natal, South Africa.

Nile crocodiles of three age classes, hatched in captivity and reared in fresh water, when exposed acutely to water of 17 and 35 ppt NaCl, suffered marked dehydration, were lethargic, ceased to feed and lost mass. When exposed to gradually increasing salinities (3-35 ppt), with a short acclimation period at each salinity, crocodiles survived, continued to feed and increased in mass and size. All age classes had a relatively constant plasma osmolality across the salinity spectrum. Cloacal urine osmolality varied throughout the acclimation experiment, but did not increase with increasing salinity. No significant increase was found in plasma concentrations of any of the osmolytes. There was a trend of decreasing cloacal urine [Na(+)] and [Cl(-)] and increasing cloacal urine [K(+)] with increased salinity, indicating that urine was not an important route for Na(+) and Cl(-) excretion. Crocodiles exposed to saline conditions maintained relatively constant plasma uric acid concentrations, but urinary uric acid concentrations increased markedly with increasing salinities. This suggests that uric acid is the main constituent of nitrogenous waste excretion in saline exposed Nile crocodiles. As in Crocodylus porosus, C.niloticus has the physiological ability to survive and thrive in periodically hyper-osmotic environments. However, its euryhalinity is restricted, in that acute exposure to sea water leads to dehydration, but with an acclimation period at lower salinities, it survives and thrives in sea water.

Effects of maternal identity and incubation temperature on snapping turtle (Chelydra serpentina) metabolism.

Individual variation in physiological traits may have important consequences for offspring survivorship and adult fitness. Variance in offspring phenotypes is due to interindividual differences in genotype, environment, and/or maternal effects. This study examined the contributions of incubation environment, maternal effects, and clutch identity to individual variation in metabolic rates in the common snapping turtle, Chelydra serpentina. We measured standard metabolic rate, as determined by oxygen consumption, for 246 individuals representing 24 clutches at 15 degrees and 25 degrees C, and we measured standard metabolic rates additionally for 34 individuals at 20 degrees and 30 degrees C. Standard metabolic rate for 34 snapping turtles measured at 15 degrees, 20 degrees, 25 degrees, and 30 degrees C increased with increasing temperature. Mean standard metabolic rate for 246 individuals was 0.247 microL O(2) min(-1) g(-1) at 15 degrees C and 0.919 microL O(2) min(-1) g(-1) at 25 degrees C. At 15 degrees C, mass at hatching, individual mass, and egg mass had no significant effects on metabolic rate, but at 25 degrees C, mass at hatching, individual mass, and egg mass did have significant effects on metabolic rate. Incubation temperature had no significant effect on metabolic rate at 15 degrees, but it did have a significant effect at 25 degrees C. Clutch identity had a significant effect on metabolic rate at both 15 degrees and 25 degrees C. Interindividual variation in standard metabolic rate due to incubation temperature, and especially clutch identity, could have large effects on energy budgets. Results suggest that there were both environmental and genetic effects on standard metabolic rate.

Thermal independence of muscle tissue metabolism in the leatherback turtle, Dermochelys coriacea.

Metabolic rates of animal tissues typically increase with increasing temperature and thermoregulatory control in an animal is a regional or whole body process. Here we report that metabolic rates of isolated leatherback turtle (Dermochelys coriacea) pectoralis muscle are independent of temperature from 5-38 degrees C (Q10 = 1). Conversely, metabolic rates of green turtle (Chelonia mydas) pectoralis muscle exhibit a typical vertebrate response and increase with increasing temperature (Q10 = 1.3-3.0). Leatherbacks traverse oceanic waters with dramatic temperature differences during their migrations from sub-polar to equatorial regions. The metabolic stability of leatherback muscle effectively uncouples resting muscle metabolism from thermal constraints typical of other vertebrate tissues. Unique muscle physiology of leatherbacks has important implications for understanding vertebrate muscle function, and is another strong argument for preservation of this endangered species.

Sequence and expression analysis of WT1 and Sox9 in the red-eared slider turtle, Trachemys scripta.

Temperature-dependent sex-determination (TSD) is a phenomenon that has been characterized at the ecological, morphological, and endocrinological levels in some reptilian species. We have begun to investigate TSD at the level of molecular development by cloning, sequencing, and analyzing the expression of two genes, WT1 and Sox9, in the red-eared slider turtle Trachemys scripta. We obtained almost full-length cDNA clones for WT1 and Sox9 that were greater than 73% identical to the human homologues at the nucleotide level. WT1 was expressed in urogenital tissue at all developmental stages examined (Yntema stages 12-20) at incubation temperatures that produce males (26 degrees C) or females (32 degrees C). Sox9 was also expressed throughout these same stages, but some differences were observed. At both 26 degrees C and 32 degrees C Sox9 was expressed in the mesonephroi and the undifferentiated gonads until Yntema stage 20, when only the gonad from the 26 degrees C embryos expressed a high level. In addition, there were two transcripts of Sox9 at all stages, but the relative proportion of the two transcripts differed at the two temperatures. Although the similarities in gene expression between a TSD species and other species with genotypically determined sex probably reflect the common features of organogenesis, differences may illustrate unique mechanisms for TSD.

Molecular mechanisms of TSD in reptiles: a search for the magic bullet.

Significant progress has been made in understanding mechanisms of genetic sex determination. The ZFY gene encodes a zinc finger protein but is not the primary signal in sex determination. The SRY gene is the testis determining gene in man, mouse, rabbit, and probably marsupial mouse and wallaby. Temperature dependent sex determination probably involves a modification of development of the indifferent gonad due to differential expression of one or more specific DNA sequences whose behavior is controlled by some temperature sensitive process or to differential action of a gene product such as a protein. There are ZFY and SRY-like genes in reptiles. We cloned and sequenced a portion of the ZFY gene (Zft) from snapping turtle (Chelydra serpentina) that is found in both sexes. We cloned and sequenced portions of SRY-like genes (Sra for SRY-related-autosomal) from snapping turtle. Similar genes are found in alligator (Alligator mississippiensis) and lizards. Cladistic analysis suggests that there are two or three major families of SRY-like genes in vertebrates in addition to sex specific SRY genes located on the Y chromosome of eutherian and marsupial mammals. When placed on a phylogenetic tree these data indicate that Sras were present in early tetrapods. Sequestering of the SRY gene on the Y chromosome probably happened only once and this may have been the defining moment that set the mammalian line of Therapsid reptiles apart from other reptilian groups.

Sequence analysis of the ZFY and Sox genes in the turtle, Chelydra serpentina.

We have sequenced regions of the ZFY and Sox genes in the turtle Chelydra serpentina, a reptile with temperature-dependent sex determination. The ZFY gene in mammals encodes a transcription factor with multiple zinc fingers that may be involved in spermatogenesis as well as other processes. The turtle homologue, Zft, is 92% identical to the ZFY gene at the nucleotide and amino acid levels in the region of zinc fingers 7-12. There are several Sox genes in the turtle that are only 57-70% identical at the nucleotide level and about 55% identical at the amino acid level to the human sex-determining SRY gene. However, the turtle Sox genes, termed TSox, have the conserved motif called the HMG-box (for high mobility group DNA-binding protein) that defines a probable DNA-binding region, and thus are in the same gene family as the Sox genes of other organisms from Drosophila to man. One TSox sequence is identical at the amino acid level to a sequence found in birds, and is 98% identical to a sequence encoded autosomally in mouse and in man. The extent of sequence conservation among the Sox genes suggests that some of their functions may be conserved. Phylogenetic analysis of available Sox sequences including SRY (Sry) sequences suggests that there was a high degree of divergence between any possible immediate common ancestor of the turtle Sox sequences and the SRY (Sry) sequences.

Heat shock protein induction and induced thermal tolerance are independent in adult salamanders.

Ectothermic vertebrates become thermally tolerant (heat hardened) after exposure to heat shock. Eukaryotic cells show a similar response. Cellular thermal tolerance is correlated with the induction of heat shock proteins (hsps). We have investigated the relationship between heat hardening in salamanders and the induction of hsps in the tissues of these organisms. Although the synthesis of hsps can be induced in these animals by sublethal heat shocks, conditions required for hsp induction and heat hardening often do not coincide. We conclude that induced thermal tolerance in adult salamanders is independent of hsp induction in their tissues.

Avian eggs: barriers to the exchange of heat and mass.

Measured boundary-layer conductance to heat exchange for bird eggs varies with egg mass to the 0.53 power. Calculations based on the Nusselt-Reynolds relationship for a sphere and the thermal properties of air indicate that the conductance of the boundary layer to heat and to mass at any wind speed other than still air should scale with mass to the 0.53 power. Although the boundary layer contributes little to the total barrier to mass flux between bird eggs and their environment, we show that it is the major barrier to the exchange of heat. From these observations we infer that birds incubating eggs in natural nests can alter only the gradient affecting mass flux between their eggs and the environment while having the capability to change both the gradient and conductance affecting heat flux.

Temperature-dependent sex determination: current practices threaten conservation of sea turtles.

Temperature determines the sex of hatchling green turtles (Chelonia mydas) produced from eggs incubated in a beach hatchery under different temperature regimens. Cola and cool nests (less than 28 degrees C) produced almost no females (0 to 10 percent) and warm, thermostable nests (greater than 29.5 degrees C) produced almost all females (95 to 100 percent). A few intersex hatchlings were produced at lower temperatures. Since little concern is given to temperatures at which sea turtle eggs are incubated in artificial hatcheries, present conservation practices may be producing all male, all female, or even intersex hatchlings.