Many studies have yet been conducted on suction feeding in aquatic salamander species. Within the Salamandridae, the crested newt Triturus dobrogicus (Kiritzescu, 1903), occurring from the Austrian Danube floodplains to the Danube Delta, was not subject of investigations so far. The present study examines the kinematics of aquatic suction feeding in this species by means of high-speed videography. Recordings of five individuals of different size and sex while feeding on bloodworms were conducted, in order to identify potential discrepancies among individuals and sizes. Five coordinate points were digitized from recordings of prey capture and twelve time- and velocity-determined variables were evaluated. All specimens follow a typical inertial suction feeding process, where rapid hyoid depression expands the buccal cavity. Generated negative pressure within the buccal cavity causes influx of water along with the prey item into the mouth. Results demonstrate higher distance values and angles for gape in individuals with smaller size. In addition, hyoid depression is maximized in smaller individuals. While Triturus dobrogicus resembles a typical inertial suction feeder in its functional morphology, intraspecific differences could be found regarding the correlation of different feeding patterns and body size.

Dual-energy computed tomography (DECT) uses two different x-ray energy spectra in order to differentiate between tissues, materials or elements in a single sample or patient. DECT is becoming increasingly popular in clinical imaging and preclinical in vivo imaging of small animal models, but there have been only very few reports on ex vivo DECT of biological samples at microscopic resolutions. The present study has three main aims. First, we explore the potential of microscopic DECT (microDECT) for delivering isotropic multichannel 3D images of fixed biological samples with standard commercial laboratory-based microCT setups at spatial resolutions reaching below 10 math formulam. Second, we aim for retaining the maximum image resolution and quality during the material decomposition. Third, we want to test the suitability for microDECT imaging of different contrast agents currently used for ex vivo staining of biological samples. To address these aims, we used microCT scans of four different samples stained with x-ray dense contrast agents. MicroDECT scans were acquired with five different commercial microCT scanners from four companies. We present a detailed description of the microDECT workflow, including sample preparation, image acquisition, image processing and postreconstruction material decomposition, which may serve as practical guide for applying microDECT. The MATLAB script (The Mathworks Inc., Natick, MA, USA) used for material decomposition (including a graphical user interface) is provided as a supplement to this paper (https://github.com/microDECT/DECTDec). In general, the presented microDECT workflow yielded satisfactory results for all tested specimens. Original scan resolutions have been mostly retained in the separate material fractions after basis material decomposition. In addition to decomposition of mineralized tissues (inherent sample contrast) and stained soft tissues, we present a case of double labelling of different soft tissues with subsequent material decomposition. We conclude that, in contrast to in vivo DECT examinations, small ex vivo specimens offer some clear advantages regarding technical parameters of the microCT setup and the use of contrast agents. These include a higher flexibility in source peak voltages and x-ray filters, a lower degree of beam hardening due to small sample size, the lack of restriction to nontoxic contrast agents and the lack of a limit in exposure time and radiation dose. We argue that microDECT, because of its flexibility combined with already established contrast agents and the vast number of currently unexploited stains, will in future represent an important technique for various applications in biological research.

In tetrapods, the ability to take up food on land is based on certain morphological requirements of the oropharynx in general and the feeding apparatus in particular. Recent paleoecological studies implay that tortoises (Testudinidae) developed terrestrial forms convergent to other amniotes and they had to meet the morphological oropharyngeal requironments independendently. As only limited information was available to date on the oropharyngeal morphology in testudinids, this study provides a detailed analysis of the oropharynx in Manouria emys emys with special emphasis on form and function of the tongue. Even if Manouria is considered a very basal member of the only terrestrial turtle clade and it was hypothesized it could have retained some features reflecting the aquatic ancestor, Manouria shows oropharyngeal characteristics found in “higher” testudinids. Accordingly, the oropharyngeal cavity in Manouria is richly structured and the glands are large and complexly organized. The tongue is large and fleshy and bears numerous slender papillae which are not supplied by lingual muscles. The hyolingual skeleton is mainly cartilaginous and the enlarged anterior elements support the tongue and provide insertion sides for the well developed lingual muscles which show striking differences to other reptiles. We conclude that the morpho-functional design of the oropharynx in Manouria shows clear differences to semiaquatic and aquatic turtles, as well as other reptiles and amniotes in general.

The Indochinese box turtle Cuora galbinifrons is regarded as a purely terrestrial species, but our results demonstrate that it can feed both on land and in water. The inverse relationship between the relative development of the hyoid apparatus and the tongue found in the most investigated chelonians is not valid in the Indochinese box turtle. Our morphological analysis of the feeding apparatus reveals that the palate shape and the design of the tongue are consistent with terrestrial feeders, but the construction of the hyoid complex is more characteristic of aquatic feeders. Previous studies have demonstrated that tongue enlargement negatively impacts the capacity of the turtles to suction feed. The present study focuses on the aquatic intraoral prey transport kinematic patterns. Our analysis is based on high-speed Wlms with 250 fr/s and high-speed cineradiography with 50 fr/s. The aquatic intraoral food transport mechanisms diVer depending on prey size: small items are transported predominantly by “inertial suction”, whereas larger items are moved by the tongue - normally a clear terrestrial strategy. As the genus Cuora is ancestrally aquatic, the use of lingual food transport in the aquatic environment is presumably an aberrant modus typical only for the most terrestrial among the Asian box turtles.

In tetrapods, the oropharyngeal cavity and its anatomical structures are mainly, but not exclusively, responsible for the uptake and intraoral transport of food. In this study, we provide structural evidence for a second function of the oropharynx in the North American common musk turtle, Sternotherus odoratus, Kinosternidae: aquatic gas exchange. Using high-speed video, we demonstrate that S. odoratus can grasp food on land by its jaws, but is afterward incapable of lingual based intraoral transport; food is always lost during such an attempt. Scanning electron microscopy and light microscopy reveal that the reason for this is a poorly developed tongue. Although small, the tongue bears a variety of lobe-like papillae, which might be misinterpreted as an adaptation for terrestrial food uptake. Similar papillae also cover most of the oropharynx. They are highly vascularized as shown by light microscopy and may play an important role in aquatic gas exchange. The vascularization of the oropharyngeal papillae in S. odoratus is then compared with that in Emys orbicularis, an aquatic emydid with similar ecology but lacking the ability of underwater respiration. Oropharyngeal papillae responsible for aquatic respiration are also found in soft-shelled turtles (Trionychidae), the putative sister group of the kinosternids. This trait could therefore represent a shared, ancestral character of both groups involving advantages in the aquatic environment they inhabit.

The feeding apparatus of the fringed turtle Chelus fimbriatus (Schneider, 1783) was studied to elucidate the feeding mechanics of an aquatic feeding specialist that has never been investigated in detail before, regarding gross morphology. The skull and hyoid apparatus as well as associated musculature were examined by computer tomography and dissection; the tongue was examined by scanning electron microscopy. The flat skull, the possibility to enormously depress the mandible combined with a cheek-like development, the large, ossified hyoid apparatus, and a well-distensible esophagus enable the turtle to produce an enormous suction force the prey is inhaled with. The jaw adductors are poorly developed in relation to other turtles and thus help keep the skull shape flat; nevertheless, they are able to generate high velocities and exhibit some new performance lines. The hyoid musculature is as well-developed as the hyoid apparatus itself, promoting the high depression velocity that is necessary for good feeding performance. The tongue is nearly reduced and lacks dorsal morphological differentiations. Taking all the morphological features into account, C. fimbriatus is an extremely well-adapted turtle making this species a very interesting object of investigation.

For a period of one year we injected a solution of stream water enriched with glucose and inorganic nitrogen and phosphorus at two experimental sites into the hyporheic sediments of the Oberer Seebach, Austria. The biofilm reacted with a quantitative increase after two weeks. The hyporheic invertebrates were sampled with the Cage Pipe Trap method, where the number of trapped animals is determined by the spatial density and the activity of the invertebrates. Within two and six weeks, the hyporheic invertebrates exhibited a reaction indicating an utilization of the new food resources. Over a longer period of one year, three different reaction patterns appeared. The number of nematods and ostracods increased extensively, presumably caused by the modification of the spatial structure of the environment due to biofilm growth. The number of the small sized invertebrates decreased, reflecting the reduced feeding effort. And the number of the large insect larvae increased indicating that these group is mainly limited by space. The hyporheic zone is described as a 'self-cleaning DOC filter', an attribute that is particularly assigned to the ecotone between the riparian soil zone and the stream hyporheic zone.

This study employed light microscopic (LM), scanning electron microscopic (SEM), and transmission electron microscopic (TEM) methods to provide detailed morphological information on the histological and ultrastructural features of the dorsal tongue epithelium of Rhinoclemmys pulcherrima incisa.
SEM revealed columnar papillae laterally, as well as papillae, which tend to have a ridge-like appearance in the center of the tongue. LM and TEM showed three different zones of lingual epithelium: a stratified apical area with serous cells at the top of the papillae, a stratified lateral area with both serous and mucus cells, and an unstratified glandular area consisting of distinct glandular ducts with mucus cells.
Comparison with morphological data from other turtles shows that the lingual epithelial structure in R. p. incisa is in accordance with that observed for other generalized omnivores which prefer a terrestrial lifestyle, thus matching the ecological information about this species.

The marine Stilbonematinae (Nematoda) are known for their highly specific mutualistic association with thiotrophic ectosymbiotic bacteria. The mechanism mediating recognition and binding between symbionts and host was studied in 5 host species. When incubated with D-mannose and L-rhamnose the symbionts detached in 2 species, Laxus cosmopolitus and L. oneistus, most likely due to competitive interactions with sugars involved in the binding mechanism; 3 other species, Stilbonema maium, Eubostrichus topiarius and E. dianae, did not lose their bacteria during any tested sugar incubations. Incubations with lectins binding specifically to D-glucose/D-mannose (ConA, concanavalin agglutinin) and to D-mannose (NPA, Narcissus pseudonarcissus agglutinin) respectively, both in vivo and on ultrathin sections, confirmed that accessible D-mannose is located on the symbionts of L. cosmopolitus, but not on the host’s surface. Our results showed an involvement of D-mannose and L-rhamnose residues of the bacterial surface in the attachment mechanism. We hypothesize that the recognition and binding of the environmentally transmitted symbionts in the 2 Laxus species, which harbor only 1 phylotype of symbiotic γ-proteobacterium each, is most probably mediated through a yet unknown mannose/rhamnose-specific lectin of host origin.

Using light as well as transmission and scanning electron microscopical methods, the three-dimensional structure of the tongue surface and the composition of the dorsal lingual epithelium of three chelonian species was observed. Subjects of examinations were Chelus fimbriatus, Pelusios castaneus, and Rhinoclemmys pulcherrima incisa, three species adapted to life in aquatic or terrestrial environment to a different extent.
Differences between these three species concern the form of the dorsal tongue morphology as well as the presence of different areas of the epithelium covering the lingual papillae and in the abundance of its two types of secretory cells: in Chelus fimbriatus, the species most adapted to life in water, the tongue is very reduced, and dorsal differentiations are absent. Parts of the lingual epithelium being rich in mucus cells - lingual glands sensu lato - cannot be expected. This has to be interpreted as the best possible adaptation of this carnivorous species to suction feeding, in which a large tongue with lingual papillae would prove to be a hindrance. On the contrary, the generalistic aquatic species Pelusios castaneus shows a functionable tongue with moderate lingual papillae. A differentiation within the dorsal lingual epithelium into an apical area at the oral-sided surface of the papilla and the lateral area at the papilla's lateral sides can be observed; mucus cells are dispersed over the whole lingual epithelium, but concentrate in lateral areas. In a hypothetical sequence of the species observed to date from aquatic to terrestrial, this distribution can be seen as it is the beginning of the formation of lingual glands sensu stricto, and corresponds with the ecology of this omnivorous and aquatic species as well as with its feeding kinematics. Rhinoclemmys pulcherrima incisa, a terrestrial species, has in comparison with similar species a less movable tongue and less prominent lingual papillae at the tongue surface. However, it shows well developed lingual glands in the form of distinct glandular ducts (lingual glands sensu stricto). The preferred habitat land as well as the grasping of the food with the jaws correlates with this morphology.
The ultrastructural composition of the lingual epithelium and its cells fit well into other data from turtles gained to date. Principally, four cell layers have to be distinguished: the basal layer just above the basal membrane, the deep and the superficial intermediate layer, and the surface layer. Deviations from this can be found in parts of the epithelium being rich in mucus cells, where the intermediate layers tend to be reduced or where the stratification is distorted. The process of maturation of both the essential cell types, the granula cells and the mucus cells, and of their secretory granules, is in all observed species comparable and is in agree with data from literature.

The ultrastructure of the dorsal lingual epithelium of the semi-aquatic West African mud turtle, Pelusios castaneus, is described. Our goal is to give additional information to previous studies of this species such as feeding pattern analysis and gross morphology. Tissue specimens were fixed in modified Karnovsky solution followed by osmium tetroxide, embedded in epoxy resin and observed using light and transmission electron microscopy. The dorsal tongue surface is covered with moderate papillae, which are coated by a stratified epithelium overlying a connective tissue core. Two epithelial regions can be differentiated, although differences are not very obvious: the apical area, where granular cells are more abundant than mucus cells, and the lateral area, where cell distribution is opposite. Within the epithelium, different layers are discernable on the basis of the cells’ organelles, corresponding with a process of cell maturation and formation of different granules. These results together with data of previous studies of this species show that the ultrastructure of the lingual epithelium is similar to other turtles adapted to semi-aquatic environments; functional and morphological data indicate a generalist, being well but not highly adapted to feeding in an aquatic environment.

Feeding mechanics of vertebrates depend on physical constraints of the surrounding media, water or air. Such functions are inseparably combined with form. The aim of this study is to show this linkage for the pleurodiran freshwater turtle Pelusios castaneus, and, additionally, to point out the major functional and biomechanical distinctions between aquatic and terrestrial feeding turtles as well as several intermediate forms. Gross morphological investigations of skull, hyoid, tongue and connected musculature, as well as scanning electron microscopy of the tongue surface show typical features of an aquatic feeder: e.g., strong developed hyoid apparatus versus a small tongue with only moderate papillae; massive jaw and hyoid musculature. Additionally, the special function of the esophagus during feeding is investigated to elucidate the problems of a bidirectional feeder. The esophagus is highly distensible in order to store the excess water sucked in during feeding until the prey is fixed by the jaws. The distension is probably achieved by a coincidence of active (branchial horn) as well as passive (water) components.
P. castaneus is a feeding generalist, and is well adapted to the aquatic medium, in terms of its functional as well as morphological features.

Background: Turtles are adapted to different environments, such as freshwater, marine and terrestrial habitats. Examination of histological and ultrastructural features of the dorsal lingual epithelium of the red-eared turtle, Trachemys scripta elegans, and comparison of the results with those of other turtles should elucidate the relationship between morphology of tongues as well as fine structure of lingual epithelia and chelonian feeding mechanisms.
Methods: Light microscopical (LM), scanning (SEM) and transmission (TEM) electron microscopical methods were used.
Results: SEM revealed a distribution of lingual papillae all over the dorsal tongue surface. Single epithelial cells can be discerned, with short microvilli on their surface. LM studies show differences within the stratified epithelium between the lateral and the apical side of the papillae. In TEM, these differences become more obvious: while the basal and deep intermediate layer is similar in both sides of the papillae, mucus granules begin to form at the edge of the superficial intermediate layer at the lateral side. Cells containing fine secretory granules are visible there, too. On the other hand, at the apical side, only fine-granule-containing cells are visible.
Conclusions: This study indicates, that the histology and ultrastructure of the lingual epithelium of Trachemys scripta elegans is similar to that of other turtles adapted to freshwater environments, but differs from turtles living in marine or terrestrial habits. These differences can be explained in terms of the adaptation of turtles to their particular life circumstances.

Using light and electron microscopic methods, the epithelium covering the dorsal side of the tongue in two chelonian species was observed. Subjects of the examination have been Platemys pallidipectoris Freiberg 1945 and Trachemys scripta (Schoepf, 1792) elegans (Wied, 1839), two species adepted to life in an aquatic environment in a different extent.
Between the two species differences in the dorsal tongue morphology are present. The surface of the tongue of P. pallidipectoris is almost flat; on the contrary, on the tongue of T. s. elegans papillae are clearly formed. This is in agreement with other findings which are found out to date concerning the different feeding mechanisms of water living, semi-terrestrial or completely terrestrial living vertebrates: a small tongue is better in species using suction-feeding as main mechanism for feeding. The rush of water, which occurs when the floor of the mouth is suddenly lowered, sucks the food into the oral cavity. The semi-aquatic species T. s. elegans is able to feed on land too, but therefore a more moveable tongue with mucus cells for manipulation and for making slippery of the food is necessary. The microanatomical and ultrastructural structure of the tongue epithelium and its cells is in a general way the same as observed in other vertebrates. Four layers are distinguishable: the basal layer with the basement membrane, the deep intermediate and the superficial layer and the surface layer. In T. s. elegans, one can distinguish between a mainly mucus secreting area on the lateral side of the papillae and an apical area without mucus cells. In P. pallidipectoris, on the other side, only one type of secreting cells is visible in the tongue epithelium.

Scanning electron microscopy reveals that the flat tongue of Platemys pallidipectoris has shallow grooves, and no lingual papillae. The surface of the tongue is covered with dome-shaped bulges, each corresponding to a single cell. Short microvilli are distributed over the cell surface. Light microscopy shows a stratified cuboidal epithelium with an underlying strong connective tissue. Transmission electron microscopy indicates four layers. The basal cells of the epithelium are electron translucent, have a large central nucleus and a cytoplasm with keratin tonofilaments. Plasma cells with abundant rough endoplasmic reticulum and mitochondria occur in the basal layer. Production of secretory granules begins in the more electron-dense intermediate layers and increases as the cells move toward the surface. The membranes of the cells of the deep intermediate layer form processes that project into relatively wide intercellular spaces. In the superficial intermediate layer, the cytoplasm of the cells contains numerous fine granules; these increase in number but not in size in more distal layers. The cells of the surface layer are electron translucent with a round nucleus. Contents of their fine granules are secreted into the oral cavity.

Within this study, several different turtle species were examined by means of light microscopy and scanning electron microscopy with respect to its dorsal tongue morphology. Results were set in relation with the species' life circumstances. 
It could be shown that dorsal tongue morphology ranges from a very small, functional absent tongue in highly aquatic species (Chelus fimbriatus) via a very flat tongue topography without lingual glands (Acanthochelys pallidipectoris) to gradually higher and ridge-like papillae with beginning formation of glandular structures in semi-aquatic (Pelusios castaneus) and intermediate species (Trachemys scripta elegans). In semi-terrestrial species (Cuora amboinensis), already well-developed glandular regions can be found. The other endpoint of variety is resembled by very high and columnar papillae with distinct mucus glands in terrestrial (Rhinoclemmys pulcherrima) or highly terrestrial animals (Testudo hermanni).
Thus, tongue morphology correlates to a high degree with the species' life circumstances and with the feeding patterns primarily used by the animals: Highly aquatic species, using suction as the main mechanism for food uptake, try to reduce their tongue or at least keep it very small to prevent hindrance of the water flow during feeding process. The more a turtle species occupies terrestrial habitats, the more it uses terrestrial feeding patterns such as lingual feeding, where a well developed tongue morphology and lingual glands are necessary for food uptake and bolus formation. [see the whole poster as *.gif! (980kb)]

Among turtles, generally two modes of feeding have been recognized: A terrestrial feeding pattern, where the animal uses its tongue for manipulation of food, and an aquatic suction feeding mechanism, where water acts as a hydraulic tongue. Whereas in the terrestrial mode a prominent tongue is useful, in the latter type a small tongue is necessary in order to prevent hindrance of the rush of water into the mouth. In the present study, tissue samples were fixed and afterwards prepared for scanning electron microscopy. The tongue of Pelusios castaneus is somewhat triangular in shape with a round apex. At low magnification, ridge-like and irregularly shaped structures are visible, which seem to correspond to very moderate papillae. At higher magnification, dome shaped bulges are visible. Each of these bulges coincidents with a single cell of the surface layer of the lingual epithelium. Between these cells cell borders are clearly visible and short microvilli are widely distributed on the free surface side of each cell. This discreet morphology of the dorsal tongue surface reflects on the one hand an aquatic feeding pattern, but on the other hand indicates that this species tends to feed on more immobile prey than other pleurodiran species.