Intracellular and luminal ion concentrations in sea turtle salt glands determined by x-ray microanalysis

Summary The lachrymal salt glands of hatchlings of the green sea turtle (Chelonia mydas) secrete a hyperosmotic (up to 2000 mosmol·kg^–1) NaCl solution. X-ray microanalysis of frozen-hydrated glands showed that during secretion intracellular Na⁺ concentration in the principal cells increased from 13 to 34 mmol·l^–1 of cell water, whilst Cl^– and K⁺ concentrations remained unchanged at 81 mmol·l^–1 and 160–174 mmol·l^–1, respectively. The high Cl^– concentration and the change in Na⁺ concentration are consistent with the prevailing paradigm for secretion by the structurally and functionally similar elasmobranch rectal gland. Concentrations of Na⁺, Cl^– and K⁺ in the lumina of secretory tubules of secreting (Na⁺ 122, Cl^– 167, K⁺ 38 mmol·l^–1) and non-secreting (Na⁺ 114, Cl^–1 174, K⁺ 44 mmol·l^–1) glands were similar and the fluid was calculated to be approximately isosmotic with blood. In the central canals Na⁺ and Cl^– concentrations were similar but K⁺ concentration was lower (11–15 mmol·l^–1). It is concluded that either a high transepithelial NaCl gradient in secretory tubules and central canals is very rapidly dissipated during the short time between gland excision and freezing, or that ductal modification of an initial isosmotic secretion occurs.     

Control of salt gland activity in the hatchling green sea turtle, Chelonia mydas

 We studied the control of salt gland secretion in hatchling Chelonia mydas. The threshold salt load to activate salt secretion was between 400 μmol NaCl 100 g bodymass (BM)⁻¹ and 600 μmol NaCl 100 g BM⁻¹, which caused an increase in plasma sodium concentration of 13% to 19%. Following a salt load of 2700 μmol NaCl 100 g BM⁻¹, salt gland secretion commenced in 12 ± 1.3 min and reached maximal secretory concentration within 2–7 min. Maximal secretory rate of a single gland averaged 415 μmol Na 100 g BM⁻¹ h⁻¹. Plasma sodium concentration and total osmotic concentration after salt loading were significantly higher than pretreatment values within 2 min. Adrenalin (25 μg kg BM⁻¹) and the cholinergic agonist methacholine (1 mg kg BM⁻¹) inhibited salt gland activity. Atropine (10 mg kg BM⁻¹) reversed methacholine inhibition and stimulated salt gland secretion when administered with a subthreshold salt load. Arginine vasotocin produced a transient reduction in sodium secretion by the active gland, while atrial natriuretic factor, vasoactive intestinal peptide and neuropeptide Y had no measurable effect on any aspect of salt gland secretion. Our results demonstrated that secretion of the salt gland in C. mydas can be modified by neural and hormonal chemicals in vivo and that the cholinergic and adrenergic stimulation of an exocrine gland do not appear to have the typical, antagonist actions on the chelonian salt gland. Accepted: 28 September 1999     

Initiation and ultrastructure of a reptilian fibroblast cell line obtained from cutaneous fibropapillomas of the green turtle,Chelonia mydas

Summary Two fibroblastic cell lines were established from explants of fibropapillomas of each of two different green turtles (Chelonia mydas). These cells, designated GTFP (Green Turtle Fibropapilloma), were subcultured approximately 30 times at 30°C in Eagle’s minimal essential media supplemented with 2 to 10% fetal bovine serum. The ultrastructural morphology of the cultured fibroblasts is described. The cells contained abundant rough endoplasmic reticulum, polyribosomes, and mitochondria; collagen fibrils were visible in the extracellular space. No viruslike particles or evidence of other pathogenic agents could be demonstrated by electron microscopy in any of the cultured cells examined. Supported in part by a grant from Sea World Research Institute, Hubbs Marine Research Center, San Diego, California 92109, and the Chelonia Institute, Arlington, Virginia 22209. Published as University of Florida, College of Veterinary Medicine, Journal Series no. 192.     

The duct system of the avian salt gland as a transporting epithelium: structure and morphometry in the duck Anas platyrhynchos

Summary The duct system of the nasal salt gland of the duck comprises central canals, secondary ducts and main ducts. The secondary and main ducts consist of a layer of columnar cells overlying a layer of small cuboidal cells. The columnar cells have complex intercellular spaces showing evidence of Na⁺ K⁺ -ATPase at the apical regions. Approximately 70% of surface area of the duct system is external to the gland. During adaptation to salt water the duct system increases in size as does the gland. Although the components of the gland of adapted ducks, including the duct system within the gland, increase in size compared with normal ducks, the percentage volume densities of the components remain similar in both categories of ducks, i.e. the duct system increases in size in proportion to the glandular tissue. The volume of the duct system external to the gland is six to seven times larger than the volume within the gland. Thus, if ductal modification of secreted fluid occurs, it will be most likely to take place in the ducts external to the gland.Total surface areas of the duct system were measured from serial sections of glands and ducts from one normal and one adapted duck. These were used to calculate possible flux rates of water and sodium across the duct epithelium, assuming the occurrence of either water reabsorption or sodium secretion. Although these flux rates are high it is shown that they are similar to calculated flux rates across the luminal surface of the secretory tubules.     

Salt gland blood flow in the hatchling green turtle, Chelonia mydas

Microsphere and morphometric techniques were used to investigate any circulatory changes that accompany secretion by the salt glands of hatchling Chelonia mydas. Salt glands were activated by a salt load of 27.0 mmol NaCl kg body mass (BM)⁻¹, resulting in a mean sodium secretion rate of 4.14 ± 0.11 mmol Na kg BM⁻¹ h⁻¹ for a single gland. Microsphere entrapment was approximately 160–180 times greater in the active salt gland than the inactive gland, inferring a similar change in blood flow through salt gland capillaries. The concentration of microspheres trapped in the salt gland was significantly correlated with the rate of tear production (ml kg BM⁻¹ h⁻¹) and the total rate of sodium secretion (mmol Na kg BM⁻¹ h⁻¹) but not with tear sodium concentration (mmol Na l⁻¹). Adrenaline (500 μg kg BM⁻¹) inhibited tear production within 2 min and reduced microsphere entrapment by approximately 95% compared with active glands. The volume of filled blood vessels increased from 0.03 ± 0.01% of secretory lobe volume in inactive salt gland sections to 0.70 ± 0.11% in active gland sections. The results demonstrate that capillary blood flow in the salt gland of C. mydas can regulate the activity of the gland as a whole. Accepted: 12 July 2000     

Secretory capacity of the lachrymal salt gland of hatchling sea turtles,Chelonia mydas

Summary The lachrymal salt glands ofChelonia mydas were functional when hatchlings emerged from the nest. Osmotic concentrations up to 720 mosmol kg^–1 were recorded in spontaneously produced tears (salt gland secretions). When injected with a Na⁺ load (1500–2700 mol (100 g)^–1) newly emerged hatchlings produced tears ranging in osmotic concentration from 1000–1900 mosmol kg^–1 with Na⁺ secretion rates from single glands of 200–475 mol (100 g·h)^–1. In these circumstances the rate of sodium excretion, via the salt glands, was equivalent to the sodium content of 0.2 to 0.5 ml of sea water per hour. Since the apparent drinking rate of hatchlings within the first two days of entering sea water was approximately 0.5 to 1.7 ml per day, the excretion of Na⁺ imbibed by drinking is well within the secretory capacity of the lachrymal salt glands.In feeding hatchlings extraordinarily high Na⁺ secretion rates were induced by Na⁺ loading. Hatchlings which were loaded with Na⁺ by injection (1500–5400 mol (100 g)^–1) produced tears having osmotic concentrations between 1500 and >2000 mosmol kg^–1. The Na⁺ secretion rates from single glands were 750–4185 mol (100 g·h)^–1 with extremely high short term rates of 10700 mol (100 g·h)^–1 (50 mol min^–1 for 28 g hatchlings).In terms of gland mass the highest long term secretion rate translates into 21 mmol of Na⁺ per gram of salt gland per hour and is the highest secretion rate yet recorded for a reptilian salt gland. This rate is almost three times the highest rate recorded for sea snakes (8 mmol g·h^–1) and is similar to rates commonly observed in avian salt glands (25 mmol g·h^–1).Secretion by the lachrymal salt glands was initiated by increased blood concentrations of Na⁺ or K⁺, K⁺ being as effective as Na⁺ but with the composition of the teras being virtually unchanged compared to tears from Na⁺ stimulated hatchlings. Preliminary experiments indicated that secretion was not initiated by increased Cl^– concentration in the blood or by increased volume or osmotic concentration of the blood.Abbreviation O.P. osmotic pressure     

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4°C with high affinity (K _a = 1.49 ± 0.09 × 10⁹ M⁻¹; 0.17 ± 0.02 × 10⁷ M⁻¹) and low binding capacity (B _max = 3.24 ± 0.84 × 10⁻⁵ M; 0.33 ± 0.06 × 10⁻⁴ M). The binding affinity and capacity of testosterone at 23 and 36°C, respectively were similar to those determined at 4°C. However, oestradiol showed no binding activity at 36°C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36°C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.     

Pulmonary function of the green sea turtle, Chelonia mydas

Lung volumes, oxygen uptake (VO2), end-tidal PO2, and PCO2, diffusing capacity of the lungs for CO (DLCO), pulmonary blood flow (QL) and respiratory frequency were measured in the green sea turtle (Chelonia mydas) (49-127 kg body wt). Mean lung volume (VL) determined from helium dilution was 57 ml/kg and physiological dead space volume (VD) was about 3.6 ml/kg. QL, determined from acetylene uptake during rebreathing, increased in proportion to VO2 with temperature. Therefore, constant O2 content difference was maintained between pulmonary arterial and venous blood. DLCO, measured using a rebreathing technique, was 0.04 ml X kg-1 X min-1 X Torr-1 at 25 degrees C. Several cardiopulmonary characteristics in C. mydas are advantageous to diving: large tidal volume relative to functional residual capacity promotes fast exchange of the alveolar gas when the turtle surfaces for breathing: and the concomitant rise of pulmonary blood flow and O2 uptake with temperature assures efficient O2 transport regardless of wide temperature variations encountered during migrations.     

Characterization of single nucleotide polymorphism markers for the green sea turtle (Chelonia mydas)

We present data on 29 new single nucleotide polymorphism assays for the green sea turtle, Chelonia mydas. DNA extracts from 39 green turtles were used for two methods of single nucleotide polymorphism discovery. The first approach employed an amplified fragment length polymorphism technique. The second technique screened a microsatellite library. Allele-specific amplification assays were developed for high-throughput single nucleotide polymorphism genotyping and tested on two Pacific C. mydas nesting populations. Observed heterozygosities ranged from 0 to 0.95 for a Hawaiian population and from 0 to 0.85 for a Galapagos population. Each of the populations had one locus out of Hardy-Weinberg equilibrium, SSCM2b and SSCM5 for Hawaii and Galapagos, respectively. No loci showed significant genotypic linkage disequilibrium across an expanded set of four Pacific nesting populations. However, two loci, SSCM4 and SSCM10b showed linkage disequilibrium across three populations indicating possible association.     

Phylogeography of the green turtle, Chelonia mydas, in the Southwest Indian Ocean

Patterns of mitochondrial DNA (mtDNA) variation were used to analyse the population genetic structure of southwestern Indian Ocean green turtle (Chelonia mydas) populations. Analysis of sequence variation over 396 bp of the mtDNA control region revealed seven haplotypes among 288 individuals from 10 nesting sites in the Southwest Indian Ocean. This is the first time that Atlantic Ocean haplotypes have been recorded among any Indo-Pacific nesting populations. Previous studies indicated that the Cape of Good Hope was a major biogeographical barrier between the Atlantic and Indian Oceans because evidence for gene flow in the last 1.5 million years has yet to emerge. This study, by sampling localities adjacent to this barrier, demonstrates that recent gene flow has occurred from the Atlantic Ocean into the Indian Ocean via the Cape of Good Hope. We also found compelling genetic evidence that green turtles nesting at the rookeries of the South Mozambique Channel (SMC) and those nesting in the North Mozambique Channel (NMC) belong to separate genetic stocks. Furthermore, the SMC could be subdivided in two different genetic stocks, one in Europa and the other one in Juan de Nova. We suggest that this particular genetic pattern along the Mozambique Channel is attributable to a recent colonization from the Atlantic Ocean and is maintained by oceanic conditions in the northern and southern Mozambique Channel that influence early stages in the green turtle life cycle.     

The effect of organochlorines and heavy metals on sex steroid-binding proteins in vitro in the plasma of nesting green turtles, Chelonia mydas

In this study on green turtles, Chelonia mydas, from Peninsular Malaysia, the effect of selected environmental toxicants was examined in vitro. Emphasis was placed on purported hormone-mimicking chemicals such as dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene, dieldrin, lead, zinc and copper. Five concentrations were used: high (1 mg/L), medium (10⁻¹ mg/L), low (10⁻² mg/L), very low (10⁻⁶ mg/L) and control (diluted carrier solvent but no toxicants). The results suggest that environmental pesticides and heavy metals may significantly alter the binding of steroids [i.e. testosterone (T) and oestradiol] to the plasma proteins in vitro. Competition studies showed that only Cu competed for binding sites with testosterone in the plasma collected from nesting C. mydas. Dieldrin and all heavy metals competed with oestradiol for binding sites. Furthermore, testosterone binding affinity was affected at various DDT concentrations and was hypothesised that DDT in vivo may act to inhibit steroid-protein interactions in nesting C. mydas. Although the precise molecular mechanism is yet to be described, DDT could have an effect upon the protein conformation thus affecting T binding (e.g. the T binding site on the steroid hormone binding protein molecule).     

Spatial distribution of turtle barnacles on the green sea turtle, Chelonia mydas

Distribution patterns of epibiotic barnacles on green sea turtles were investigated in waters neighboring Okinawa, Japan. A number of barnacle species were found to coexist on the turtles and were classified into three genera: Chelonibia, Platylepas and Stomatolepas. Attachment sites on the turtles varied among the barnacle species, suggesting that there is niche partitioning with respect to their microhabitat selection. Turtle bodies offer a “patchy” environment for barnacles, so we also analyzed coexistence patterns in the context of an aggregation model. Within each genus, individual barnacles showed a clumped distribution. The different genera do not have mutually exclusive distribution patterns, but instead occur on the same turtle to various degrees. However, when turtles were divided into two size classes, both the level of aggregation and the degree of interspecific overlap among the barnacles was significantly lower on large turtles. We suggest that obtaining basic information on turtle epibionts will shed light on the biology of wild turtles, which is still largely unknown.     

Nocturnal activity in the green sea turtle alters daily profiles of melatonin and corticosterone

In nature, green turtles (Chelonia mydas) can exhibit nocturnal activity in addition to their typically diurnal activity cycle. We examined whether nocturnal activity in captive and free-living green turtles altered daily plasma profiles of melatonin (MEL) and corticosterone (CORT). In captivity, diurnally active green turtles expressed distinct diel cycles in MEL and CORT; a nocturnal rise was observed in MEL and a diurnal rise was observed in CORT. However, when induced to perform both low- and high-intensity nocturnal activity, captive green turtles exhibited a significant decrease in MEL, compared to inactive controls. In contrast, plasma CORT increased significantly with nocturnal activity, and further, the relative increase in CORT was correlated with the intensity of the nocturnal behavior. In free-living green turtles that performed nocturnal activity including: nesting, mate searching, and feeding/swimming behaviors, plasma profiles in MEL and CORT exhibited relatively little, or no, daily fluctuation. Our findings demonstrate that nocturnal activity in green turtles is often associated with MEL and CORT profiles that resemble those measured during the day. We speculate that these conspicuous changes in MEL and CORT during nocturnal activity could either support or promote behaviors that enable acquisition of transient resources important to the survival and reproductive success of green turtles.     

Characterization of polymorphic microsatellite markers for the green turtle (Chelonia mydas)

We describe primers and polymerase chain reaction conditions to amplify 12 microsatellite loci from the green turtle (Chelonia mydas), including one dinucleotide, four trinucleotide and seven tetranucleotide loci. The primers were tested on 78 individuals from a Pacific population nesting in the Hawaiian Islands. The primer pairs developed in this study yielded an average of 8.33 alleles per locus (range of 3-15 alleles), an average observed heterozygosity of 0.668 (range 0.309-0.910), and an average polymorphic information content of 0.647 (range 0.287-0.894).     

Conservation Genetics of the East Pacific Green Turtle (Chelonia mydas) in Michoacan, Mexico

The main continental nesting rookeries of the east Pacific green turtle (EPGT), Chelonia mydas, on the Michoacan (Mexico) coast suffered drastic population declines following intense exploitation in the 1960s--1970s with annual abundance of nesting females plummeting from about 25,000 to an average of about 1400 between 1982 and 2001. Analyses of data from three nDNA microsatellite loci and 400 bp mtDNA control region sequences from a total of 123 nesting females sampled from four Michoacan rookeries found no evidence of population sub-structuring. The recent order of magnitude reduction in the population size shows no apparent impact on genetic diversity in either control region sequences (overall h = 0.48; pi = 0.0036) or microsatellite loci (overall Na = 20.8; Hexp = 0.895). Our estimates of annual effective female population size (Nef; from theta = 2Nemicron) of 1.9-2.3 x 10(3), in spite of being an order of magnitude below historical records, appear to be sufficient to allow recovery of this population without significant loss of genetic diversity. These findings highlight the importance of continued conservation to reverse the decline of this population before it becomes vulnerable to genetic erosion.     

A bloom of Lyngbya majuscula in Shoalwater Bay, Queensland, Australia: An important feeding ground for the green turtle (Chelonia mydas)

Lyngbya majuscula, a toxic cyanobacterium, was observed blooming during June–July (winter) 2002 in Shoalwater Bay, Queensland, Australia, an important feeding area for a large population of green turtles (Chelonia mydas). The bloom was mapped and extensive mats of L. majuscula were observed overgrowing seagrass beds along at least 18 km of coast, and covering a surface area of more than 11 km². Higher than average rainfall preceded the bloom and high water temperatures in the preceding summer may have contributed to the bloom. In bloom samples, lyngbyatoxin A (LA) was found to be present in low concentration (26 μg kg⁻¹_{(dry weight)}), but debromoaplysiatoxin (DAT) was not detected. The diet of 46 green turtles was assessed during the bloom and L. majuscula was found in 51% of the samples, however, overall it contributed only 2% of the animals’ diets. L. majuscula contribution to turtle diet was found to increase as the availability of the cyanobacterium increased. The bloom appeared to have no immediate impact on turtle body condition, however, the presence of a greater proportion of damaged seagrass leaves in diet in conjunction with decreases in plasma concentrations of sodium and glucose could suggest that the turtles may have been exposed to a substandard diet as a result of the bloom. This is the first confirmed report of L. majuscula blooming in winter in Shoalwater Bay, Queensland, Australia and demonstrates that turtles consume the toxic cyanobacterium in the wild, and that they are potentially exposed to tumour promoting compounds produced by this organism.     

The ontogeny of pulmonary surfactant secretion in the embryonic green sea turtle (Chelonia mydas).

Pulmonary surfactant, consisting predominantly of phosphatidylcholine (PC), is secreted from Type II cells into the lungs of all air-breathing vertebrates, where it functions to reduce surface tension. In mammals, glucocorticoids and thyroid hormones contribute to the maturation of the surfactant system. It is possible that phylogeny, lung structure, and the environment may influence the development of the surfactant system. Here, we investigate the ontogeny of PC secretion from cocultured Type II cells and fibroblasts in the sea turtle, Chelonia mydas, following 58, 62, and 73 d of incubation and after hatching. The influence of glucocorticoids and thyroid hormones on PC secretion was also examined. Basal PC secretion was lowest at day 58 (3%) and reached a maximal secretion rate of 10% posthatch. Dexamethasone (Dex) alone stimulated PC secretion only at day 58. Triiodothyronine (T(3)) stimulated PC secretion in cells isolated from days 58 and 73 embryos and from hatchling turtles. A combination of Dex and T(3) stimulated PC secretion at all time points.     

Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle,Chelonia mydas

Summary A continuous line of epithelioid cells was established from explant skin tissues of the green sea turtle,Chelonia mydas. These cells, designated GTS, have been subcultured more than 60 times in commercially available mammalian cell culture medium supplemented with 5% bovine calf serum. Of those temperatures tested, optimal growth was achieved at 30°C although replication occurred between 16 and 37°C. These cells may be held as monolayers at 8°C or stored frozen in growth medium containing 10% dimethylsulfoxide at −70 or −196°C. The modal number of 55 chromosomes per cell is in agreement with the heterogametic female diploid number of this species. The GTS line represents the first established culture of normal epithelioid skin cells to be reported for a poikilothermic species.     

Sex steroid binding proteins in the plasma of hatchling <Emphasis Type="Italic">Chelonia mydas</Emphasis>

Sex steroid binding proteins were identified in hatchling female and male Chelonia mydas by dialysis and steady-state gel electrophoresis when examined at 4 degrees C. A testosterone binding protein with high binding affinity (K (a) = 0.98 +/- 0.5 x 10(8) M(-1)) and low to moderate binding capacity (B (max) = 7.58 +/- 4.2 x 10(-5) M) was observed in male hatchlings. An oestradiol binding protein with high affinity (K (a) = 0.35 +/- 1.8 x 10(8) M(-1)) and low to moderate binding capacity (B (max) = 0.16 +/- 0.5 x 10(-4) M) was identified in female hatchlings. This study confirmed that sex steroid binding proteins (SSBPs) become inactivate in both sexes at 36 degrees C, the maximum body temperature of sea turtle hatchlings at emergence. The inactivation of SSBPs at this temperature indicates that sex steroid hormones circulate freely in the body of the green turtles and are biologically available in the blood plasma. This observation is consistent with female and male hatchling C. mydas having different physiological (hormonal) and developmental requirements around the time of emergence. Moreover, concurrently conducted competition studies showed that sex steroids including testosterone and oestradiol do compete for binding sites in both male and female C. mydas hatchling plasma. Competition also occurred between testosterone and dihydrotestosterone for binding sites in the male C. mydas plasma. However, competition studies in the plasma of female hatchling C. mydas demonstrate that oestrone does not compete with oestradiol for binding sites.     

Phylogeography and population structure of the Atlantic and Mediterranean green turtle Chelonia mydas: a mitochondrial DNA control region sequence assessment

Mitochondrial (mt) DNA sequences were analysed to resolve the phylogeography and population genetic structure of Atlantic and Mediterranean populations of green turtles ( Chelonia mydas ). Analysis of sequence variation over 487 base pairs of the control (D-loop) region identified 18 haplotypes among 147 individuals from nine nesting populations. Pairwise comparisons of haplotype frequencies distinguished most nesting colonies, indicating significant genetic differentiation among rookeries and a strong propensity for natal homing behaviour by nesting females. Comparison of control region sequence data to earlier restriction fragment length polymorphism (RFLP) data for the same individuals demonstrates approximately a sixfold higher substitution rate in the 5' end of the control region. The sequence data provide higher resolution both in terms of the number of mtDNA genotype variants and the phylogeographic relationships detected within the Atlantic region, and reveal a gene genealogy that distinguishes two groups of haplotypes corresponding to (i) the western Caribbean and Mediterranean, and (ii) eastern Caribbean, South Atlantic and West Africa. The data suggest that phylogeographic patterns in the Atlantic Ocean may be interpreted in terms of female nest site fidelity and episodic dispersal events. The distribution of mtDNA haplotypes within the region is thus explained by the geological and climatic alternations (glacial and interglacial) over the last million years.