Establishing physiological blood parameters in the loggerhead sea turtle (Caretta caretta)

The loggerhead sea turtle (Caretta caretta), one of the three sea turtle species inhabiting the Mediterranean Sea, is an endangered species. However, although frequently treated in marine animal rescue centers, and the subject of several studies in literature, as yet, few studies have been conducted on large numbers of loggerhead sea turtles in order to establish physiological reference ranges that enable the identification of pathological values. The lack of studies on reference parameters probably depends on the fact that marine turtles treated in wildlife rescue centers are usually in a critical conditions, thus precluding the collection of data on healthy animals and compromising the reliability of any data obtained from them. The present biological study was therefore conducted in order to obtain a database from healthy animals in natural conditions. Serum biochemistry and serum protein electrophoresis were performed on blood samples obtained from 65 healthy adult loggerhead sea turtles captured and delivered to the Sea Turtle Rescue Center of Linosa (Italy); the blood samples were collected during the clinical examination of rescued animals. Laboratory analyses of serum samples were made in order to establish reference parameters, commonly required for laboratory diagnoses in mammals and diseased animals.

Tetranucleotide markers from the loggerhead sea turtle (Caretta caretta) and their cross-amplification in other marine turtle species

The loggerhead sea turtle (Caretta caretta) is a federally threatened species and listed as endangered by the World Conservation Union (IUCN). We describe primers and polymerase chain reaction (PCR) conditions to amplify 11 novel tetranucleotide microsatellite loci from the loggerhead sea turtle. We tested primers using samples from 22 females that nested at Melbourne Beach, Florida (USA). Primer pairs yielded an average of 11.2 alleles per locus (range of 4–24), an average observed heterozygosity of 0.83 (range 0.59–0.96), and an average polymorphic information content of 0.80 (range 0.62–0.94). We also demonstrate the utility of these primers, in addition to primers for 15 loci previously described, for amplifying microsatellite loci in four additional species representing the two extant marine turtle families: olive ridley (Lepidochelys olivacea), hawksbill (Eretmochelys imbricata), green turtle (Chelonia mydas), and leatherback (Dermochelys coriacea).     

Twelve new polymorphic microsatellite markers from the loggerhead sea turtle (Caretta caretta) and cross-species amplification on other marine turtle species

We describe 12 new polymorphic dinucleotide microsatellite loci and multiplex Polymerase Chain Reaction conditions from the loggerhead sea turtle Caretta caretta. Levels of polymorphism were assessed in 50 individuals from the nesting population of the Cape Verde Islands. Number of alleles ranged from 3 to 13 (average of 7.33) and the values of observed heterozygosities from 0.32 to 0.80 (average of 0.61). Cross-species amplification on three other marine turtles, Chelonia mydas, Eretmochelys imbricata and Dermochelys coriacea, revealed polymorphism and variability at eight, eleven and three loci, respectively.     

Unfolding of the loggerhead sea turtle (Caretta caretta) myoglobin: A (1)H-NMR and electronic absorbance study

The effect of urea concentration on the backbone solution structure of the cyanide derivative of ferric Caretta caretta myoglobin (at pH 5.4) is reported. By addition of urea, sequential and long-range nuclear Overhauser effects (NOEs) are gradually lost. By using the residual NOE constraints to build the molecular model, a picture of the unfolding pathway was obtained. When the urea concentration is raised to 2.2 M, helices A and B appear largely disordered; helices C, D, and F loose structural constraints at 3.0 M urea. At urea concentration >6 M, the protein appears to be fully unfolded, including the GH hairpin and helix E stabilizing the prosthetic group. Reversible and cooperative denaturation isotherms obtained by following NOE peaks are considerably different from those obtained by monitoring electronic absorption changes. The reversible and cooperative urea-dependent folding-unfolding process of C. caretta myoglobin follows the minimum three-state mechanism N long left and right arrow X long left and right arrow D, where X represents a disordered globin structure (occurring at approximately 4 M urea) that still binds the heme.     

Metabolic heating in Mediterranean loggerhead sea turtle clutches

Offspring sex ratio is an important demographic parameter and, given its determination by incubation temperature in sea turtles, might be a key factor for their conservation under climate warming. An appealing approach to estimate hatchling sex ratios is to measure sand temperatures at nest depth and deduce hatchling sex ratios from a beforehand-established relationship of hatchling sex ratio and sand temperature. Such estimates will only be accurate though if metabolic heat produced by the embryos is considered. Judging whether metabolic heating has a potential effect on hatchling sex ratios without actually measuring temperature within clutches would greatly facilitate monitoring protocols. We tested for a relationship between the amount of metabolic heating and the number of developed embryos as well as clutch size in the largest known loggerhead sea turtle (Caretta caretta) population of the Mediterranean on Zakynthos (Greece). Temperatures were measured within 20 nests as well as at a reference site in the sand at nest depth. Metabolic heating was detected, but only during the last third of the incubation period did nests heat up considerably (1.6 °C on average) above the temperature of the surrounding sand. During the middle third of incubation, when sex is determined, the amount of metabolic heating was negligible. The amount of metabolic heating during the last third of the incubation duration was significantly correlated to the number of offspring developed to at least about 75% of incubation duration. This factor explained nearly 50% of variation in metabolic heating. Metabolic heating was also significantly correlated to clutch size. Given that clutch size within the Mediterranean is largest in Zakynthos loggerheads, we conclude that metabolic heating can be ignored in the estimate of hatchling sex ratios in Mediterranean loggerhead populations. These results thus provide the basis for a feasible monitoring of hatchling sex ratios in the loggerhead sea turtle in the Mediterranean.     

Tetranucleotide microsatellites from the loggerhead sea turtle (Caretta caretta)

We describe primers and polymerase chain reaction conditions to amplify 15 tetranucleotide microsatellite loci from the loggerhead sea turtle (Caretta caretta). The primers were tested on 30 individuals that nested along the Georgia, USA coast. The primer pairs developed in this study yielded an average of 13.9 alleles per locus (range of 10–21), an average observed heterozygosity of 0.91 (range 0.79–1.00), and an average polymorphic information content of 0.88 (range 0.84–0.92).     

Invertebrate infestation on eggs and hatchlings of the loggerhead turtle, Caretta caretta, in Dalaman, Turkey

The damage caused by some invertebrates to the eggs and hatchlings of loggerhead turtles, Caretta caretta, was investigated during the summer of 2002 on Dalaman beach, Turkey. The specimens, identified to family or genus levels, from nine families representing seven orders were recorded as infesting nests of loggerhead turtles. The heaviest impacts on loggerhead turtle nests was made by Pimelia sp. (Tenebrionidae, Coleoptera). Twenty-four (36.3%) out of 66 intact loggerhead hatched nests were affected by these larvae. Larval damage by Pimelia sp. was recorded in 188 (10.6%) out of 1773 eggs, but only in two (0.28%) hatchlings. The results show that fewer insects were in the nest the further from vegetation and therefore the relocation of nests from the water's edge to further inland close to vegetation may increase the infestation rate of the eggs.     

Discovery of a regular nesting area of loggerhead turtle <Emphasis Type="Italic">Caretta caretta</Emphasis> in southern Italy: a new perspective for national conservation

Loggerhead turtle Caretta caretta nesting in Italy had been reported to be limited to the Pelagian Islands and only sporadically elsewhere. As presence of loggerhead turtle nests had occasionally been reported (1988–1999) along about 200 km of the Ionian coast of Calabria, we carried out a project to assess the actual state of the nesting population between 2000 and 2004. We divided the coastline in two sectors (A: 52 km, and B: 146 km) that were monitored from mid-June to end of July for a total of n = 174 monitoring days and 1,813.6 km patrolled on foot with different intensities (extensive versus intensive). In sector B, through extensive monitoring we did not find any emergence tracks, but in sector A by intensive survey (2002–2004: one survey/3.64 days) we detected 3–8 nests/year. In total, 25 nests (both observed and reported), were recorded in our study area, and an assessment of a total of 15–16 nests/year was suggested. These figures, within the national scenario depicted from the review of known nesting events in the last 40 years (88 records concerning more than 143–144 nests), show that loggerhead turtle nesting has been underestimated in Italy, due to inadequate monitoring protocols, and that nesting is more frequent than expected (at least 30–40 nests/year). Conservation strategies in Italy should then focus not only on the reduction of mortality at sea, but also include large-scale actions to preserve scattered (but regular) nesting events.     

Conservation implications of complex population structure: lessons from the loggerhead turtle (Caretta caretta)

Complex population structure can result from either sex-biased gene flow or population overlap during migrations. Loggerhead turtles (Caretta caretta) have both traits, providing an instructive case history for wildlife management. Based on surveys of maternally inherited mtDNA, pelagic post-hatchlings show no population structure across the northern Atlantic (phi(ST) < 0.001, P = 0.919), subadults in coastal habitat show low structure among locations (phi(ST) = 0.01, P < 0.005), and nesting colonies along the southeastern coast of the United States have strong structure (phi(ST) = 0.42, P < 0.001). Thus the level of population structure increases through progressive life history stages. In contrast, a survey of biparentally inherited microsatellite DNA shows no significant population structure: R(ST) < 0.001; F(ST) = 0.002 (P > 0.05) across the same nesting colonies. These results indicate that loggerhead females home faithfully to their natal nesting colony, but males provide an avenue of gene flow between regional nesting colonies, probably via opportunistic mating in migratory corridors. As a result, all breeding populations in the southeastern United States have similar levels of microsatellite diversity (H(E) = 0.70-0.89), whereas mtDNA haplotype diversity varies dramatically (h = 0.00-0.66). Under a conventional interpretation of the nuclear DNA data, the entire southeastern United States would be regarded as a single management unit, yet the mtDNA data indicate multiple isolated populations. This complex population structure mandates a different management strategy at each life stage. Perturbations to pelagic juveniles will have a diffuse impact on Atlantic nesting colonies, mortality of subadults will have a more focused impact on nearby breeding populations, and disturbances to adults will have pinpoint impact on corresponding breeding populations. These findings demonstrate that surveys of multiple life stages are desirable to resolve management units in migratory marine species.     

Molecular evidence of male-biased dispersal in loggerhead turtle juveniles

Serum testosterone levels and mtDNA haplotypes were obtained from 65 juvenile loggerhead turtles (Caretta caretta, L.) incidentally caught in the central Mediterranean. The group of specimens carrying a haplotype specific for the northwest Atlantic had higher testosterone levels, and so included more males, than the other one. Since primary sex ratios of northwest Atlantic colonies are strongly skewed towards females, results indicate a male bias among Atlantic turtles entering the Mediterranean. This demonstrates for the first time a sex-biased dispersal of specimens in the pelagic phase, an important factor to be considered in conservation programs.     

Post-nesting movements and submergence patterns of loggerhead marine turtles in the Mediterranean assessed by satellite tracking

Two female loggerhead turtles (Caretta caretta) were tracked, following nesting at Alagadi Beach (35°33′N, 33°47′E), Northern Cyprus, eastern Mediterranean for 60 and 82 days, respectively. The two individuals showed marked differences in their behaviour. Individual A was tracked to Syrian coastal waters, whereas individual B travelled around the coast of Northern Cyprus to a foraging site in the waters off the east coast of Northern Cyprus. Submergence durations varied markedly during different phases of the migration, suggesting coastal foraging/resting at certain stages en route with sustained directed travelling movements during initial coastal movements and open ocean crossing. Both turtles showed fidelity to foraging grounds for the duration of transmissions (Turtle A: 36 days; Turtle B: 58 days). In both cases, locations were centred in inshore waters although the two individuals exhibited quite different submergence patterns. Individual A carried out very short dives of typical duration <10 min, whereas Individual B carried out longer dives with typical duration >20 min. Diel differences in submergence duration at the foraging grounds suggested longer dives at night/early morning for both turtles. For Turtle A, there was a general reduction in submergence duration as the period of residence increased; a pattern that may have been related to increasing temperature experienced. The total distance travelled by the two turtles (320 and 227 km) was relatively short when compared to satellite tracking studies of conspecifics following nesting in South Africa and USA and tagging studies of nesting loggerhead turtles in Greece and Australia. It is hypothesized that short migratory distance may be correlated with both the small body size and the relatively high frequency of remigration in this population.     

Effect of maternal foraging habitat on offspring quality in the loggerhead sea turtle (Caretta caretta)

Exploring a trade‐off between quantity and quality of offspring allows differences in the fitness between alternative life histories to be accurately evaluated. We addressed the mechanism that maintains alternative life histories (small oceanic planktivores vs. large neritic benthivores) observed in a loggerhead sea turtle (Caretta caretta) population, which has been suggested to be environmental, based on the lack of genetic structure and a large difference in reproductive output. We examined whether maternal foraging habitat affects offspring quality, by measuring the morphology, emergence success, and righting response of hatchlings following incubation in a common open sand area over the whole nesting season at Yakushima Island, Japan, and by recording early growth and survival of offspring that were reared in a common environment at a Japanese aquarium. Furthermore, we tested whether sea turtles adjust egg size in response to temporal shifts of the incubation environment. There were no significant differences in any hatchling traits between oceanic and neritic foragers (which were classified by stable isotope ratios), except for clutches laid during the warmest period of the nesting season. There were also no significant differences in the growth and survival of offspring originating from the two foragers. The size of eggs from both foragers significantly increased as the season progressed, even though the rookery had heavy rainfall, negating the need to counteract heat‐related reduction in hatchling morphology. In comparison, the sizes of adult body and clutches from both foragers did not vary significantly. The results further support our previous suggestions that the size‐related foraging dichotomy exhibited by adult sea turtles does not have a genetic basis, but derives from phenotypic plasticity. Adjustment in reproductive investment may be associated with: (1) predation avoidance, (2) founder effect, and/or (3) annual variation in egg size.     

Abundance and distribution of the endangered loggerhead turtle in Spanish Mediterranean waters and the conservation implications

During 2 years (2001–2003), we performed seasonal aerial surveys in the central Spanish Mediterranean following the transect line methodology in order to determine the abundance and distribution patterns of loggerhead turtles Caretta caretta . We surveyed a total of 16 700 km, accounting for 770 turtle sightings. Loggerhead turtles were present with high abundance all year round. No seasonal differences in abundance were found, except in spring 2001, where the density of turtles was higher than in the other seasons. Our results show that the Western Mediterranean is not a 'summer' feeding area as proposed previously, as a high number of turtles are present throughout the year. The average surface density of turtles in the whole study area was 0.21 turtles km⁻² [95% confidence interval (CI): 0.17–0.25], and the mean abundance was 6653 turtles (95% CI: 5514–8027). The data relate to the number of turtles on the surface only, as diving turtles escape observation. Correcting our estimations of diving behaviour data in the area, the absolute abundance was 18 954 turtles (95% CI: 6679–53 786). Bearing in mind that around 25 000 loggerheads are caught per year in the Spanish Mediterranean, our results indicate that accidental captures seem to be a significant threat for this species, and conservation measures have to be implemented to avoid a non-sustainable situation.     

Genetic polymorphisms of Echinococcus tapeworms in China as determined by mitochondrial and nuclear DNA sequences

The genetic polymorphisms of Echinococcus spp. in the eastern Tibetan Plateau and the Xinjiang Uyghur Autonomous Region were evaluated by DNA sequencing analyses of genes for mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear elongation factor-1 alpha (ef1a). We collected 68 isolates of Echinococcus granulosus sensu stricto (s.s.) from Xinjiang and 113 isolates of E. granulosus s. s., 49 isolates of Echinococcus multilocularis and 34 isolates of Echinococcus shiquicus from the Tibetan Plateau. The results of molecular identification by mitochondrial and nuclear markers were identical, suggesting the infrequency of introgressive hybridization. A considerable intraspecific variation was detected in mitochondrial cox1 sequences. The parsimonious network of cox1 haplotypes showed star-like features in E. granulosus s. s. and E. multilocularis, but a divergent feature in E. shiquicus. The cox1 neutrality indexes computed by Tajima’s D and Fu’s Fs tests showed high negative values in E. granulosus s. s. and E. multilocularis, indicating significant deviations from neutrality. In contrast, the low positive values of both tests were obtained in E. shiquicus. These results suggest the following hypotheses: (i) recent founder effects arose in E. granulosus and E. multilocularis after introducing particular individuals into the endemic areas by anthropogenic movement or natural migration of host mammals, and (ii) the ancestor of E. shiquicus was segregated into the Tibetan Plateau by colonising alpine mammals and its mitochondrial locus has evolved without bottleneck effects.     

Significant genetic differentiation between the Yellow Sea and East China Sea populations of cocktail shrimp Trachypenaeus curvirostris revealed by the mitochondrial DNA COI gene

The cocktail shrimp Trachypenaeus curvirostris is an ecologically and economically important shrimp species in the Yellow Sea and East China Sea. However, there is no information about its population genetic structure. The population genetic structure and level of gene flow of T. curvirostris from the Yellow Sea and the East China Sea were studied with a 658-bp segment of mtDNA COI gene. In total, 85 individuals were collected from five locations and 13 haplotypes were obtained. The genetic variation of COI gene in five populations was moderate, giving an overall haplotype diversity of 0.6888 ± 0.0432 and nucleotide diversity of 0.0069 ± 0.0038. Conflicting to our expectation, significant genetic differentiation was detected in this species. The result revealed two genetically divergent lineages, displaying clear different geographical distributions in the studied area. The significant genetic differentiation between the Yellow Sea and East China Sea populations might be caused by the Yangtze River outflow. Mismatch distribution revealed that T. curvirostris had undergone population range expansion, possibly before 103,400–109,700 years ago in the last interglaciation, rejecting the sudden demographic expansion.     

Evidence for transoceanic migrations by loggerhead sea turtles in the southern Pacific Ocean

Post-hatchling loggerhead turtles (Caretta caretta) in the northern Pacific and northern Atlantic Oceans undertake transoceanic developmental migrations. Similar migratory behaviour is hypothesized in the South Pacific Ocean as post-hatchling loggerhead turtles are observed in Peruvian fisheries, yet no loggerhead rookeries occur along the coast of South America. This hypothesis was supported by analyses of the size-class distribution of 123 post-hatchling turtles in the South Pacific and genetic analysis of mtDNA haplotypes of 103 nesting females in the southwest Pacific, 19 post-hatchlings stranded on the southeastern Australian beaches and 22 post-hatchlings caught by Peruvian longline fisheries. Only two haplotypes (CCP1 93% and CCP5 7%) were observed across all samples, and there were no significant differences in haplotype frequencies between the southwest Pacific rookeries and the post-hatchlings. By contrast, the predominant CCP1 haplotype is rarely observed in North Pacific rookeries and haplotype frequencies were strongly differentiated between the two regions (F_st=0.82; p=<0.00001). These results suggest that post-hatchling loggerhead turtles emerging from the southwest Pacific rookeries are undertaking transoceanic migrations to the southeastern Pacific Ocean, thus emphasizing the need for a broader focus on juvenile mortality throughout the South Pacific to develop effective conservation strategies.     

Patterns of nuclear and mitochondrial DNA variation in Iberian populations of <Emphasis Type="Italic">Emys orbicularis</Emphasis> (Emydidae): conservation implications

The European pond turtle (Emys orbicularis) is threatened and in decline in several regions of its natural range, due to habitat loss combined with population fragmentation. In this work, we have focused our efforts on studying the genetic diversity and structure of Iberian populations with a fine-scale sampling (254 turtles in 10 populations) and a representation from North Africa and Balearic island populations. Using both nuclear and mitochondrial markers (seven microsatellites, ∼1048 bp nDNA and ∼1500 bp mtDNA) we have carried out phylogenetic and demographic analyses. Our results show low values of genetic diversity at the mitochondrial level although our microsatellite dataset revealed relatively high levels of genetic variability with a latitudinal genetic trend decreasing from southern to northern populations. A moderate degree of genetic differentiation was estimated for Iberian populations (genetic distances, F _ST values and clusters in the Bayesian analysis). The results in this study combining mtDNA and nDNA, provide the most comprehensive population genetic data for E. orbicularis in the Iberian Peninsula. Our results suggest that Iberian populations within the Iberian–Moroccan lineage should be considered as a single subspecies with five management units, and emphasize the importance of habitat management rather than population reinforcement (i.e. captive breeding and reintroduction) in this long-lived species.