Turtle skull development unveils a molecular basis for amniote cranial diversity.

Amniote skulls display diverse architectural patterns including remarkable variations in the number of temporal arches surrounding the upper and lower temporal fenestrae. However, the cellular and molecular basis underlying this diversification remains elusive. Turtles are a useful model to understand skull diversity due to the presence of secondarily closed temporal fenestrae and different extents of temporal emarginations (marginal reduction of dermal bones). Here, we analyzed embryos of three turtle species with varying degrees of temporal emargination and identified shared widespread coexpression of upstream osteogenic genes Msx2 and Runx2 and species-specific expression of more downstream osteogenic genes Sp7 and Sparc in the head. Further analysis of representative amniote embryos revealed differential expression patterns of osteogenic genes in the temporal region, suggesting that the spatiotemporal regulation of Msx2, Runx2, and Sp7 distinguishes the temporal skull morphology among amniotes. Moreover, the presence of Msx2- and/or Runx2-positive temporal mesenchyme with osteogenic potential may have contributed to their extremely diverse cranial morphology in reptiles.

Histological features and Gαolf expression patterns in the nasal cavity of sea turtles.

Sea turtles use olfaction to detect volatile and water-soluble substances. The nasal cavity of green turtles (Chelonia mydas) comprises morphologically defined the anterodorsal, anteroventral, and posterodorsal diverticula, as well as a single posteroventral fossa. Here, we detailed the histological features of the nasal cavity of a mature female green turtle. The posterodorsal diverticulum contained spongy-like venous sinuses and a wave-shaped sensory epithelium that favored ventilation. Secretory structures that were significant in sensory and non-sensory epithelia were probably involved in protection against seawater. These findings suggested that green turtles efficiently intake airborne substances and dissolve water-soluble substances in mucous, while suppressing the effects of salts. In addition, positive staining of Gαs/olf that couples with olfactory, but not vomeronasal, receptors was predominant in all three types of sensory epithelium in the nasal cavity. Both of airborne and water-soluble odorants seemed to be detected in cells expressing Gαolf and olfactory receptors.

Histological findings of sperm storage in green turtle (Chelonia mydas) oviduct.

Green turtles (Chelonia mydas) are seasonal breeders with a time lag between mating and nesting periods. We therefore investigated whether female turtles store sperm like some other animals by histologically and ultrastructurally analyzing oviducts collected from three mature female free-ranging green turtles during the breeding season in the Ogasawara Islands, Japan. The oviduct comprised an infundibulum, magnum, isthmus, uterus, and vagina. Sperm was found in the isthmus of all turtles examined. Some spermatozoa were found in the duct and acini of glands in the isthmus of two turtles with oviducts containing eggs, and a few were also located in the transition area between the uterus and vagina of one of the turtles. On the other hand, we also found abundant spermatozoa on the luminal surface of the isthmus of one turtle captured during mating. In most reptiles, fertilization occurs in the infundibulum or albumen region, and thus the isthmus near those areas might be suitable for storing sperm in female turtles.

Detection and molecular characteristics of Pyelosomum cochlear (Digenea: Pronocephalidae) in the urinary bladder of the green sea turtle (Chelonia mydas) in the Northwest Pacific Ocean.

The genus Pyelosomum consists of parasitic flukes occurring primarily in marine turtles; Pyelosomum cochlear Looss 1899 is the only species of this genus that parasitizes the urinary bladder. In this study, we detected flukes in the urinary bladders of 20 of 88 green sea turtles (Chelonia mydas) harvested in the Ogasawara Islands, in the Northwest Pacific Ocean. We identified the flukes as P. cochlear based on detailed morphological observations and comparisons of morphometric measurements of the species reported previously. Nucleotide sequences of nuclear ribosomal 18S and 28S regions and the mitochondrial cytochrome c oxidase subunit 1 (COI) region were determined for the flukes. The 18S and 28S phylogenetic trees revealed that the species of the superfamily Pronocephaloidea, including P. cochlear, constituted a single clade, but the species of the family Pronocephalidae did not constitute a single taxon. These findings suggest that Pronocephalidae is a paraphyletic group. The COI sequences of P. cochlear exhibited high genetic diversity, suggesting that they would be useful markers to understand the genetic structure of the parasite and its evolutionary relationship with the host turtle populations. This is the first study to provide the nucleotide sequences of Pyelosomum species; these data will be available for further molecular studies of this genus and its related taxa.

Detection and molecular characteristics of Rhytidodoides sp. (Digenea: Rhytidodidae) from the gall bladder of green sea turtles (Chelonia mydas) in the Ogasawara Islands, Japan.

Trematodes of the genus Rhytidodoides are parasitic in marine turtles. Of the already known species, Rhytidodoides similis Price, 1939, occurs especially in the gall bladder. In this study, we surveyed 73 green sea turtles (Chelonia mydas) in the Ogasawara Islands, Japan, and detected Rhytidodoides sp. from the gall bladders of 18 turtles. A detailed morphological analysis revealed that the forebody of Rhytidodoides sp. differed slightly in shape from that of R. similis. There has been no information on DNA sequences of the family Rhytidodidae. A molecular phylogeny based on 28S rDNA sequences of Rhytidodoides sp. and related taxa suggested that the Rhytidodidae is sister to the other families of Echinostomatoidea. The intraspecific diversity of Rhytidodoides sp. was examined by using DNA sequences of mitochondrial cytochrome c oxidase subunit 1 gene (COI). The population genetic features of the COI haplotypes demonstrated that Rhytidodoides sp. is highly diverse in the Ogasawara Islands. The DNA sequences determined in this study will contribute to the species identification of congeners and the taxonomic reconsideration of the Echinostomatoidea.

Morphological features of the nasal cavities of hawksbill, olive ridley, and black sea turtles: Comparative studies with green, loggerhead and leatherback sea turtles.

We analyzed the internal structure of the nasal cavities of hawksbill, olive ridley and black sea turtles from computed tomography images. The nasal cavities of all three species consisted of a vestibule, nasopharyngeal duct and cavum nasi proprium that included anterodorsal, posterodorsal and anteroventral diverticula, and a small posteroventral salience formed by a fossa of the wall. These findings were similar to those of green and loggerhead sea turtles (Cheloniidae), but differed from those of leatherback sea turtles (Dermochelyidae). Compared to the Cheloniidae species, the nasal cavity in leatherback sea turtles was relatively shorter, wider and larger in volume. Those structural features of the nasal cavity of leatherback sea turtles might help to suppress heat dissipation and reduce water pressure within the nasal cavity in cold and deep waters.

Sensitivity of turtles to anticoagulant rodenticides: Risk assessment for green sea turtles (Chelonia mydas) in the Ogasawara Islands and comparison of warfarin sensitivity among turtle species.

Although anticoagulant rodenticides (ARs) are effectively used for the control of invasive rodents, nontarget species are also frequently exposed to ARs and secondary poisonings occur widely. However, little data is available on the effects of ARs, especially on marine organisms. To evaluate the effects of ARs on marine wildlife, we chose green sea turtles (Chelonia mydas), which are one of the most common marine organisms around the Ogasawara islands, as our primary study species. The sensitivity of these turtles to ARs was assessed using both in vivo and in vitro approaches. We administered 4 mg/kg of warfarin sodium either orally or intravenously to juvenile green sea turtles. The turtles exhibited slow pharmacokinetics, and prolongation of prothrombin time (PT) was observed only with intravenous warfarin administration. We also conducted an in vitro investigation using liver microsomes from green sea turtles, and two other turtle species (softshell turtle and red-eared slider) and rats. The cytochrome P450 metabolic activity in the liver of green sea turtles was lower than in rats. Additionally, vitamin K epoxide reductase (VKOR), which is the target enzyme of ARs, was inhibited by warfarin in the turtles at lower concentration levels than in rats. These data indicate that turtles may be more sensitive to ARs than rats. We expect that these findings will be helpful for sea turtle conservation following accidental AR-broadcast incidents.

The nasal cavity in sea turtles: adaptation to olfaction and seawater flow.

The nasal cavity of tetrapods has become phylogenetically adapted to the environment in terms of function, respiration, and olfaction. In addition, the nasal cavity of sea turtles plays an important role in seawater flow and water olfaction, unlike that of terrestrial species. Here, we describe the functional, morphological, and histological characteristics of the nasal cavity, and the odorant receptors encoded in the genome of sea turtles. The nasal cavity of sea turtles is well-suited to its complicated functions, and it significantly differs from those of other animals, including terrestrial and semi-aquatic turtles.

Computed tomographic analysis of internal structures within the nasal cavities of green, loggerhead and leatherback sea turtles.

The morphology of the tetrapod nasal cavity has adapted to the environment in terms of olfaction and respiration. Reports indicate that the internal structure of the nasal cavity of green sea turtles is more complex than that of turtles in general, but whether or not it is similar among sea turtle species remains unknown. The present study aimed to define the internal structures of the nasal cavity of green (Chelonian mydas), loggerhead (Caretta caretta) and leatherback (Dermochelys coriacea) sea turtles using computed tomography. The nasal cavity of green and loggerhead sea turtles contained anterodorsal, anteroventral, posterodorsal diverticula and a posteroventral excavation in the middle. In contrast, the nasal cavity of leatherback sea turtles had more complicated dorsal region comprising anterodorsal and posterodorsal diverticula, and two excavations between the nostril and anterodorsal diverticulum, but no distinct structures at the ventral region. The airway in the nasal cavity was shorter and thicker in the leatherback, than in the green and loggerhead turtles. These species differences might reflect ecological variety and different evolutionary strategies.

Investigating the effects of nest shading on the green turtle (Chelonia mydas) hatchling phenotype in the Ogasawara islands using a field-based split clutch experiment.

The Ogasawara Islands are an important rookery for the green turtle (Chelonia mydas) in the North Pacific. Green turtles possess temperature-dependent sex determination, and warmer incubation temperatures produce more females than males. Therefore, conservation practices such as nest shading may be required for this population to mitigate the effect of global warming on their sex ratio. To consider the application of such conservation practices in the Ogasawara population, it is fundamental to understand how artificially modified nest environments will affect green turtle hatchling phenotypes that influence their fitness. Here, we investigated the effects of nest shading on green turtle hatchling phenotypes in the Ogasawara population by using a split clutch experiment equally separating the clutch, relocating each half-clutch into an outdoor hatchery area either with or without shading, and observing the subsequent hatchling phenotype. Our results showed that the shading treatment produced hatchlings with a better self-righting response and a larger carapace size. Additionally, the shading treatment mostly reduced the production of hatchlings with a nonmodal scute pattern and produced hatchlings with a smaller unabsorbed yolk sac, which may be associated with their residual yolk mass. These results suggest that conservation practices such as shading could alter not only the sex ratio but also the hatchling phenotype that influences their fitness. Hence, our results suggest that applications of such conservation strategies must be carefully considered.

Main airway throughout the nasal cavity of green sea turtles is lined by keratinized stratified squamous epithelium.

Structural and histological features of the nasal cavity of sea turtles are largely different from those of other Testudines species. The sea turtle nasal cavity is a pair of tubular structures with three diverticula and an excavation in the center, and three types of sensory epithelium are present in these four significant structures. To more clarify the adaptation of the nasal cavity to marine life style in sea turtles, non-sensory epithelium in the nasal cavity of green sea turtles (Chelonia mydas) were histologically determined from nostril to choanae in this study. Unlike many other animals including terrestrial turtles, the vestibular area and nasopharyngeal duct were all lined by keratinized stratified squamous epithelium. In the main nasal cavity, the margins of each sensory epithelium turned into respiratory epithelium with goblet cells, followed by keratinized stratified squamous epithelium. Keratinized epithelium appears more appropriate in sea turtle upper airway to protect against osmotic pressure when they release seawater through the nostrils, and thus this histological feature of upper airway might reflect adaptation to marine life style.

Behavioral effects of scents from male mature Rathke glands on juvenile green sea turtles (Chelonia mydas).

Sea turtles can detect airborne and waterborne odors, but whether they recognize scents from the same species and if so, how they affect their behavior remains unknown. The present study evaluated the behavioral effects of odorants on juvenile green sea turtles (Chelonia mydas). The odorants were derived from Rathke glands (external scent glands) of mature male green sea turtles, and from two types of food. The activity of the juveniles increased when exposed to food scents, and significantly decreased compared with controls when exposed to scents from Rathke glands. These findings indicated that scents from the same species affect behavior, and that chemical communication via olfaction has important outcomes for sea turtles.

Infection by and Molecular Features of Learedius learedi (Digenea: Schistosomatoidea) in Green Sea Turtles (Chelonia mydas) on the Ogasawara Islands, Japan.

Learedius learedi Price, 1934 , is a blood fluke found in sea turtles, and the adult fluke parasitizes the cardiovascular system of the host. In this study we surveyed 46 green sea turtles, Chelonia mydas, on the Ogasawara Islands, Japan, and blood flukes were detected in the heart and blood vessels of 26 turtles. The flukes were identified as L. learedi based on a detailed morphological description. In addition, molecular identification and characterization of the parasite were performed. The nucleotide sequences of nuclear internal transcribed spacer 2 (ITS2) regions were almost identical to those of L. learedi reported previously, but not to those of Hapalotrema spp., which is the closest related genus. The nucleotide sequences of the 28S ribosomal DNA region formed a single clade with those of the reference L. learedi in the phylogenetic tree, but not with those of Hapalotrema spp. Therefore, the nucleotide sequences of ITS2 and 28S are robust markers for distinguishing L. learedi from other species. The nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (COI) region were analyzed to evaluate the genetic variations in L. learedi. The COI haplotypes revealed the extremely high genetic diversity of the species as well as the host turtles on the Ogasawara Islands. The haplotype frequency in the mitochondrial DNA of the green sea turtles on the Ogasawara Islands is known to be significantly different from those in other Pacific rookeries. Although the number of analyzed flukes is small in this study, no haplotype was close to that in other areas; on the basis of the data, we hypothesized that L. learedi differentiated along with the host turtles on the Ogasawara Islands.

Nasal Cavity of Green Sea Turtles Contains 3 Independent Sensory Epithelia.

The morphological and histological features of the nasal cavity are diverse among animal species, and the nasal cavities of terrestrial and semiaquatic turtles possess 2 regions lined with each different type of sensory epithelium. Sea turtles can inhale both of volatile and water-soluble odorants with high sensitivity, but details of the architectural features and the distribution of the sensory epithelia within the sea turtle nasal cavity remain uncertain. The present study analyzed the nasal cavity of green sea turtles using morphological, computed tomographic, and histological methods. We found that the middle region of the sea turtle nasal cavity is divided into anterodorsal, anteroventral, and posterodorsal diverticula and a posteroventral excavation by connective tissue containing cartilages. The posterodorsal diverticulum was lined with a thin sensory epithelium, and the anterodorsal and anteroventral diverticula were occupied by a single thick sensory epithelium. In addition, a relatively small area on the posteroventral excavation was covered by independent sensory epithelium that differed from other 2 types of epithelia, and a single thin bundle derived from the posteroventral excavation comprised the most medial nerve that joins the anterior end of the olfactory nerve tract. These findings suggested that the posteroventral excavation identified herein transfers stimuli through an independent circuit and plays different roles when odorants arise from other nasal regions.

Structure and functions of the placenta in common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales.

The structure and functions of placentas were examined in 3 species of rorqual whales, common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales, with the aim of confirming the structural characteristics of the chorion, including the presence of the areolar part, and clarifying steroidogenic activities and fetomaternal interactions in the placentas of these whales. Placentas were collected from the second phase of the Japanese Whale Research Program under Special Permit in the North Pacific (JARPN II). Histological and ultrastructural examinations revealed that these whale placentas were epitheliochorial placentas with the interdigitation of chorionic villi lined by monolayer uninucleate cells (trophoblast cells) and endometrial crypts as well as folded placentation by fold-like chorionic villi. Moreover, well-developed pouch-like areolae were observed in the placentas, and active absorption was suggested in the chorionic epithelial cells of the areolar part (areolar trophoblast cells). Berlin blue staining showed the presence of ferric ions (Fe(3+)) in the uterine glandular epithelial cells and within the stroma of chorionic villi in the areolar part. An immunohistochemical examination revealed tartrate-resistant acid phosphatase (TRAP; known as uteroferrin in uteri) in the cytoplasm of glandular cells and areolar trophoblast cells. This result suggested that, in cetaceans, uteroferrin is used to supply iron to the fetus. Furthermore, immunoreactivity for P450scc and P450arom was detected in trophoblast cells, but not in areolar trophoblast cells, suggesting that trophoblast cells synthesize estrogen in whale placentas. Therefore, we herein immunohistochemically revealed the localization of aromatase and uteroferrin in cetacean placentas during pregnancy for the first time.