Dental structure and tooth attachment modes in the common fangtooth Anoplogaster cornuta (Valenciennes, 1833) (Actinopterygii; Trachichthyiformes; Anoplogastridae).

We studied the structure and attachment modes of the teeth of adult Anoplogaster cornuta using light- and scanning-electron microscopic techniques. All teeth were monocuspid, composed solely of orthodentin, and lacked a covering enameloid cap. Fourteen teeth were present in the oral jaws, with three teeth each on the left and right premaxilla and four teeth each on the left and right dentary. The anteriormost premaxillary and dentary teeth were considerably larger than the more posteriorly located ones. The oral jaw teeth were transparent, non-depressible and firmly ankylosed to their respective dentigerous bone by a largely anosteocytic bone of attachment. No evidence for replacement of the large oral jaw teeth was found in the analyzed adult specimens. The bone of attachment exhibited lower calcium and phosphorus concentrations and a higher Ca/P ratio than the orthodentin. The connection between dentinal tooth shaft and bone of attachment was stabilized by a collar of mineralized collagen fibers. In contrast to the oral jaw teeth, the pharyngeal teeth exhibited a ring-like fibrous attachment to their supporting bones. This mode of attachment provides the teeth with some lateral mobility and allows their depression relative to their supporting bones, which may facilitate intra-pharyngeal prey transport. In contrast, a firm ankylosis was observed in numerous small teeth located on the branchial arches. The function of these teeth is presumably to increase the tightness of the pharyngeal basket and thereby the retention of small prey items in a species living in a habitat with only sparse food supply. Our findings corroborate earlier statements on the tooth attachment modes of the oral jaw teeth of Anoplogaster cornuta, but provide new findings for the attachment modes of pharyngeal teeth in this species.

Quantitative aspects of the ruminating process in giraffes (Giraffa camelopardalis) fed with different diets.

Giraffes are ruminants feeding on fresh browse and twigs in the wild, but in zoos, their diet is mainly based on alfalfa hay, grains, and pellets occasionally supplemented by twigs and foliage. These diets, which differ in composition and digestibility, affect the behavior of the animals, tooth wear patterns, and chewing efficiency. We quantified several parameters of the rumination process in ten zoo housed giraffes of different sexes and ages fed either with alfalfa hay, fresh browse, or a combination of both. Chewing during rumination was highly ritualized and specimens showed an even distribution of chewing directions during this process, which prevents uneven tooth wear and use of chewing muscles. During rumination of alfalfa hay, chewing cycles of the giraffes took on average 49 s and included 54 jaw movements compared to 37 s and 42 jaw movements during rumination of browse, respectively. Single jaw movements (measured as basic chewing rates) were on average significantly slower during rumination of alfalfa hay (alfalfa: 1.10 chews per second, browse: 1.17 chews per second) and intercycle times between two chewing cycles took significantly longer (alfalfa: 7.77 s, browse: 7.46 s). Our results clearly indicate that several rumination parameters are influenced by the type of diet.

Gelsolin in Onychophora and Tardigrada with notes on its variability in the Ecdysozoa.

Rearrangements of the filamentous actin network involve a broad range of actin binding proteins. Among these, the gelsolin proteins sever actin filaments, cap their fast growing end and nucleate actin assembly in a calcium-dependent manner. Here, we focus on the gelsolin of the onychophoran Peripatoides novaezealandiae and the eutardigrade Hypsibius dujardini. From the cDNA of P. novaezealandiae we obtained the complete coding sequence with an open reading frame of 2178bp. It encodes a protein of 726 amino acids with a calculated molecular mass of 82,610.9Da and a pI of 5.57. This sequence is comprised of six segments (S1-S6). However, analysis of data from TardiBase reveals that the gelsolin of the eutardigrade Hypsibius dujardini has only three segments (S1-S3). The coding sequence consist of 1119bp for 373 amino acids with a calculated molecular mass of 42,440.95Da and a pI of 6.17. The Peripatoides and Hypsibius gelsolin revealed both conserved binding motifs for G-actin, F-actin and phosphatidylinositol 4,5-bisphosphate (PIP2), along with a full set of type-1 and type-2 Ca2+-binding sites which could result in the binding of eight and four calcium ions, respectively. Both gelsolin proteins lack a C-terminal latch-helix indicating a more rapid activation in the submicromolar Ca2+ range. We suggest that a gelsolin with three segments was present in the last common ancestor of the ecdysozoan clade Panarthropoda (Onychophora, Tardigrada, Arthropoda), primarily because the gelsolin of all non-Ecdysozoa studied so far (except Chordata) reveals this number of segments. Mapping of our molecular data onto a well-established phylogeny revealed that the number of gelsolin segments does not correlate with the phylogenetic lineage but rather with particular functional demands to alter the kinetics of actin polymerization.

Single-distance phase retrieval algorithm for Bragg Magnifier microscope.

We present an improved, single-distance phase retrieval algorithm applicable for holographic X-ray imaging of biological objects for an in-line germanium Bragg Magnifier Microscope (BMM). The proposed algorithm takes advantage of a modified shrink-wrap algorithm for phase objects, robust unwrapping algorithm as well as other reasonable constraints applied to the wavefield at the object and the detector plane. The performance of the algorithm is analyzed on phantom objects and the results are shown and discussed. We demonstrated the suitability of the algorithm for the phase retrieval on a more complex biological specimen Tardigrade, where we achieved successful phase retrieval from only a single hologram. The spatial resolution obtained by Fourier spectral power method for biological objects is ∼ 300 nm, the same value as obtained from the reconstructed test pattern. Our results achieved using the new algorithm confirmed the potential of BMM for in-vivo, dose-efficient single-shot imaging of biological objects.

Micromorphology of the elytral cuticle of beetles, with an emphasis on weevils (Coleoptera: Curculionoidea).

The elytral cuticle of 40 beetle species, comprising 14 weevils (Curculionoidea) and 26 representatives of other taxa, is examined. All weevils and 18 other species have an endocuticle with prominent macrofibers, which corresponds to a modified pseudo-orthogonal cuticle. Angles between successive layers of macrofibers range between 30° and 90°, but are constantly less than 60° in weevils. In all Curculionoidea, as well as in one buprestid and one erotylid species exo- and endocuticle are densely interlocked. In the weevil Sitophilus granarius, transmission electron microscopy revealed vertical microfibrils extending from the exocuticle between the macrofibers of the underlaying endocuticle. Vertical microfibrils connecting successive macrofiber layers of the endocuticle were observed in S. granarius and Trigonopterus nasutus. Distinct cuticular characters are traced on a beetle phylogeny: the angles between unidirectional endocuticle layers; the presence and the shape of endocuticular macrofibers; and the interlocking of exo- and endocuticle. While character traits seem to be more or less randomly distributed among Coleoptera, the Curculionoidea have a uniform groundplan: The "weevil-specific" combination of characters includes 1) interlocking of exo- and endocuticle, 2) an endocuticle with distinct ovoid macrofibers embedded in a matrix and 3) comparatively small angles between successive endocuticular layers. Thus, phylogenetic constraints appear equally important to functional factors in the construction of the weevil elytron.

Non-invasive measurement of frog skin reflectivity in high spatial resolution using a dual hyperspectral approach.

BACKGROUND: Most spectral data for the amphibian integument are limited to the visible spectrum of light and have been collected using point measurements with low spatial resolution. In the present study a dual camera setup consisting of two push broom hyperspectral imaging systems was employed, which produces reflectance images between 400 and 2500 nm with high spectral and spatial resolution and a high dynamic range. METHODOLOGY/PRINCIPAL FINDINGS: We briefly introduce the system and document the high efficiency of this technique analyzing exemplarily the spectral reflectivity of the integument of three arboreal anuran species (Litoria caerulea, Agalychnis callidryas and Hyla arborea), all of which appear green to the human eye. The imaging setup generates a high number of spectral bands within seconds and allows non-invasive characterization of spectral characteristics with relatively high working distance. Despite the comparatively uniform coloration, spectral reflectivity between 700 and 1100 nm differed markedly among the species. In contrast to H. arborea, L. caerulea and A. callidryas showed reflection in this range. For all three species, reflectivity above 1100 nm is primarily defined by water absorption. Furthermore, the high resolution allowed examining even small structures such as fingers and toes, which in A. callidryas showed an increased reflectivity in the near infrared part of the spectrum. CONCLUSION/SIGNIFICANCE: Hyperspectral imaging was found to be a very useful alternative technique combining the spectral resolution of spectrometric measurements with a higher spatial resolution. In addition, we used Digital Infrared/Red-Edge Photography as new simple method to roughly determine the near infrared reflectivity of frog specimens in field, where hyperspectral imaging is typically difficult.

EF-hand proteins and the regulation of actin-myosin interaction in the eutardigrade Hypsibius klebelsbergi (tardigrada).

Many tardigrade species resist harsh environmental conditions by entering anhydrobiosis or cryobiosis. Desiccation as well as freeze resistance probably leads to changes of the ionic balance that includes the intracellular calcium concentration. In order to search for protein modifications affecting the calcium homoeostasis, we studied the regulatory system controlling actin-myosin interaction of the eutardigrade Hypsibius klebelsbergi and identified full-length cDNA clones for troponin C (TnC, 824 bp), calmodulin (CaM, 1,407 bp), essential myosin light chain (eMLC, 1,015 bp), and regulatory myosin light chain (rMLC, 984 bp) from a cDNA library. All four proteins belong to the EF-hand superfamily typified by a calcium coordinating helix-loop-helix motif. Further, we cloned and obtained recombinant TnC and both MLCs. CaM and TnC revealed four and two potential calcium-binding domains, respectively. Gel mobility shift assays demonstrated calcium-induced conformational transition of TnC. From both MLCs, only the rMLC showed one potential N-terminal EF-hand domain. Additionally, sequence properties suggest phosphorylation of this myosin light chain. Based on our results, we suggest a dual-regulated system at least in somatic muscles for tardigrades with a calcium-dependent tropomyosin-troponin complex bound to the actin filaments and a phosphorylation of the rMLC turning on and off both actin and myosin. Our results indicate no special modifications of the molecular structure and function of the EF-hand proteins in tardigrades. Phylogenetic trees of 131 TnCs, 96 rMLCs, and 62 eMLCs indicate affinities to Ecdysozoa, but also to some other taxa suggesting that our results reflect the complex evolution of these proteins rather than phylogenetic relationships.

Morphology of skin incubation in Pipa carvalhoi (Anura: Pipidae).

South American Pipidae show a unique reproductive mode, in which the fertilized eggs develop in temporarily formed brood chambers of the dorsal skin after eggs have been deposited on the back of the female. We studied the skin incubation of Pipa carvalhoi using light microscopy and scanning electron microscopy. The skin consists of a stratified epithelium with a one-layered stratum corneum, and the dermis. The dermis of the dorsal skin of nonreproductive and reproductive females lacks a distinct stratum compactum, which is typical for most anuran skins. The entire dermis shows irregularly arranged collagen bundles like a stratum spongiosum. Before egg laying, the skin swells, primarily by thickening and further by loosening of the middle zone of the dermis. In the epidermis, large furrows develop that are the prospective sites of egg nidation. The epidermis, which forms a brood chamber around the developing egg becomes bi-layered and very thin and lacks a stratum corneum. Further, the dermis loosens and becomes heavily vascularized. Egg carrying females do not have mature oocytes in their ovaries indicating a slow down or interruption of egg maturation during this period. Similarities with the brood pouch of marsupial frogs are discussed.

Parotoid macroglands in toad (Rhinella jimi): their structure and functioning in passive defence.

When toads (Rhinella) are threatened they inflate their lungs and tilt the body towards the predator, exposing their parotoid macroglands. Venom discharge, however, needs a mechanical pressure onto the parotoids exerted by the bite of the predator. The structure of Rhinella jimi parotoids was described before and after manual compression onto the macroglands mimicking a predator attack. Parotoids are formed by honeycomb-like collagenous alveoli. Each alveolus contains a syncytial gland enveloped by a myoepithelium and is provided with a duct surrounded by differentiated glands. The epithelium lining the duct is very thick and practically obstructs the ductal lumen, leaving only a narrow slit in the centre. After mechanical compression the venom is expelled as a thin jet and the venom glands are entirely emptied. The force applied by a bite of a potential predator may increase alveolar pressure, forcing the venom to be expelled as a thin jet through the narrow ductal slit. We suggest that the mechanism for venom discharge within all bufonids is possibly similar to that described herein for Rhinella jimi and that parotoids should be considered as cutaneous organs separate from the rest of the skin specially evolved for an efficient passive defence.

Parotoid macroglands in toad (Rhinella jimi): Their structure and functioning in passive defence

When toads (Rhinella) are threatened they inflate their lungs and tilt the body towards the predator, exposing their parotoid macroglands. Venom discharge, however, needs a mechanical pressure onto the parotoids exerted by the bite of the predator. The structure of Rhinella jimi parotoids was described before and after manual compression onto the macroglands mimicking a predator attack. Parotoids are formed by honeycomb-like collagenous alveoli. Each alveolus contains a syncytial gland enveloped by a myoepithelium and is provided with a duct surrounded by differentiated glands. The epithelium lining the duct is very thick and practically obstructs the ductal lumen, leaving only a narrow slit in the centre. After mechanical compression the venom is expelled as a thin jet and the venom glands are entirely emptied. The force applied by a bite of a potential predator may increase alveolar pressure, forcing the venom to be expelled as a thin jet through the narrow ductal slit. We suggest that the mechanism for venom discharge within all bufonids is possibly similar to that described herein for Rhinella jimi and that parotoids should be considered as cutaneous organs separate from the rest of the skin specially evolved for an efficient passive defence.

Comments on the eyes of tardigrades.

A survey is given on the scarce information on the visual organs (eyes or ocelli) of Tardigrada. Many Eutardigrada and some Arthrotardigrada, namely the Echiniscidae, possess inverse pigment-cup ocelli, which are located in the outer lobe of the brain, and probably are of cerebral origin. Occurrence of such organs in tardigrades, suggested as being eyeless, has never been checked. Depending on the species, response to light (photokinesis) is negative, positive or indifferent, and may change during the ontogeny. The tardigrade eyes of the two eutardigrades examined up to now comprise a single pigment cup cell, one or two microvillous (rhabdomeric) sensory cells and ciliary sensory cell(s). In the eyes of the eutardigrade Milnesium tardigradum the cilia are differentiated in an outer branching segment and an inner (dendritic) segment. Because of the scarcity of information on the tardigrade eyes, their homology with the visual organs of other bilaterians is currently difficult to establish and further comparative studies are needed. Thus, the significance of these eyes for the evolution of arthropod visual systems is unclear yet.

Parental investment by skin feeding in a caecilian amphibian.

Although the initial growth and development of most multicellular animals depends on the provision of yolk, there are many varied contrivances by which animals provide additional or alternative investment in their offspring. Providing offspring with additional nutrition should be favoured by natural selection when the consequent increased fitness of the young offsets any corresponding reduction in fecundity. Alternative forms of nutrition may allow parents to delay and potentially redirect their investment. Here we report a remarkable form of parental care and mechanism of parent-offspring nutrient transfer in a caecilian amphibian. Boulengerula taitanus is a direct-developing, oviparous caecilian, the skin of which is transformed in brooding females to provide a rich supply of nutrients for the developing offspring. Young animals are equipped with a specialized dentition, which they use to peel and eat the outer layer of their mother's modified skin. This new form of parental care provides a plausible intermediate stage in the evolution of viviparity in caecilians. At independence, offspring of viviparous and of oviparous dermatotrophic caecilians are relatively large despite being provided with relatively little yolk. The specialized dentition of skin-feeding (dermatophagous) caecilians may constitute a preadaptation to the fetal feeding on the oviduct lining of viviparous caecilians.

Parental investment by skin feeding in a caecilian amphibian

Although the initial growth and development of most multi-cellular animals depends on the provision of yolk, there are many varied contrivances by which animals provide additional or alternative investment in their offspring 1. Providing offspring with additional nutrition should be favoured by natural selection when the consequent increased fitness of the young offsets any corresponding reduction in fecundity2. Alternative forms of nutrition may allow parents to delay and potentially redirect their investment. Here we report a remarkable form of parental care and mechanism of parent-offspring nutrient transfer in a caecilian amphibian. Boulengerula taitanus is a direct-developing, oviparous caecilian3, the skin of which is transformed in brooding females to provide a rich supply of nutrients for the developing offspring. Young animals are equipped with a specialized dentition, which they use to peel and eat the outer layer of their mother's modified skin. This new form of parental care provides a plausible intermediate stage in the evolution of viviparity in caecilians. At independence, offspring of viviparous and of oviparous dermatotrophic caecilians are relatively large despite being provided with relatively little yolk. The specialized dentition of skin-feeding (dermatophagous) caecilians may constitute a pre-adaptation to the fetal feeding on the oviduct lining of viviparous caecilians.

Notes on cranial ontogeny and delayed metamorphosis in the hynobiid salamander Ranodon sibiricus Kessler, 1866 (Urodela).

The skull of larvae, juveniles and adults of the rare and primitive hynobiid salamander Ranodon sibiricus was re-examined using transparencies and illustrated by new graphics. The earliest larva available for investigations already had the dominant bones. The maxillary, however, was still lacking. Previous descriptions regarding the appearance and growth of bones could be largely confirmed. The vomer, first seen as a relatively small obliquely arranged dentate bar in the 3.8 cm long larva, became larger during ontogeny, but did not change its position remarkably. The vomerine pars dentalis with only a single tooth line was straight in larvae and juveniles, but was slightly curved in adults allowing for distinction of an outer and inner portion. This feature is typical and more pronounced in most other hynobiids. The significance of the vomer and vomerine dentition for systematic and phylogenetic purposes and its changes during metamorphosis are briefly discussed. Two of the specimens examined showed delayed metamorphosis very likely caused by low temperatures. Here the temporal course of transformation was "stretched" and therefore some alterations, e.g. regression of the palatinal portion of the palatopterygoid, were shown more clearly. Continuous growth of some skull elements in these individuals suggested a relative independence from metamorphosis perhaps due to variable thyroid activity and/or independent changes in individual tissue sensitivities. It is suggested that remodelling of the mouth roof could be used for staging urodele ontogeny.

Genital structures in the entelegyne widow spider Latrodectus revivensis (Arachnida; Araneae; Theridiidae) indicate a low ability for cryptic female choice by sperm manipulation.

The female genital structures of the entelegyne spider Latrodectus revivensis are described using semithin sections and scanning electron microscopy. Apart from the tactile hairs overhanging the opening of the atrium, the contact zones of the female epigynum are devoid of any sensilla, indicating that the female does not discriminate in favor or against males due to their genital size or stimulation through copulatory courtship. The dumb-bell shape and the spatial separation of the entrance and the exit of the paired spermathecae suggest that they are functionally of the conduit type. Not described for other entelegyne spiders so far, the small fertilization ducts originating from the spermathecae of each side lead to a common fertilization duct that connects the spermathecae to the uterus externus. During oviposition, it is most likely that spermatozoa are indiscriminately sucked out of the spermathecal lumina by the low pressure produced by the contraction of the muscle extending from the epigynal plate to the common fertilization duct. As no greater amounts of secretion are produced by the female during oviposition, and no activated sperm are present within the female genital tract, the secretion produced by the spermathecal epithelium does not serve in displacement or (selective) activation of spermatozoa. These findings suggest that female L. revivensis are not able to exert cryptic female choice by selectively choosing spermatozoa of certain males.

Genomic organization of the cadmium-inducible tandem repeat 25-kDa metallothionein of the oligochaete worm Enchytraeus buchholzi.

The terrestric oligochaete worm Enchytraeus buchholzi survives in cadmium (Cd)-polluted environments by aid of its Cd-inducible 25 kDa cysteine-rich protein (CRP). Here, we analyze promoter and structure of the crp gene and compare its relationship to MT genes. The crp gene, approximately 12 kbp long, consists of 10 exons with exons 2 to 9 encoding eight almost identical repeats of predominantly 31 amino acids of the CRP. The introns of the crp gene contain various repetitive elements including retrotransposon-like sequences. The 683-bp promoter of the non-constitutive crp gene exhibits a much higher basal activity than the mouse MT-II promoter in HepG2 cells. Essential for crp promoter activity is the distal region (-683/-521) with a GC box and the proximal region (-308/-8) with the four MREa, b, c, d and AP-1, -2, -3 elements, whereas the central portion (-521/-309) with CAAT box, CRE and a XRE causes promoter repression. The TATA box-, MREc- and the AP-2, -3-containing region are required for high crp promoter activity. Our data support the view that the crp gene is a unique MT-gene and has evolved by exon duplications from a MT-like ancestral gene.

The dentition of European Speleomantes spp. (Urodela, Plethodontidae) with special regard to sexual dimorphism.

Dentigerous bones and dentition of juveniles and adults of several European Speleomantes-species were investigated using scanning electron microscopy. In the juveniles examined, all dentigerous bones bore bicuspid teeth. The adult males showed the well-known dentitional sexual dimorphism, i.e. monocuspid largely conical, elongated and strongly curved premaxillary teeth. However, teeth of the anterior portion of the maxillae and some teeth of the dentary were also clearly monocuspid; the remaining teeth had differently shaped apices considered to be different grades of bicuspidity. Vomerine teeth were exclusively bicuspid. Monocuspid, but smaller teeth were also found on the premaxilla of an adult female. The conical monocuspid tooth in adults is a modified metamorphosed tooth. Monocuspids, at least on the premaxillae, may be constantly present in males and development of monocuspids probably depends on differential sensitivities of the tooth producing tissue for androgen stimuli.

Cadmium resistance conferred to yeast by a non-metallothionein-encoding gene of the earthworm Enchytraeus.

The earthworm Enchytraeus is able to survive in cadmium (Cd)-polluted environments. Upon Cd exposure, the worms express a gene encoding the putative non-metallothionein 25-kDa cysteine-rich protein (CRP), which contains eight repeats with highly conserved cysteines in Cys-X-Cys and Cys-Cys arrangements exhibiting 36-53% identities to the 6-7-kDa metallothioneins of different organisms. Here, we demonstrate that the CRP protein confers a highly Cd-resistant phenotype to a Cd-hypersensitive yeast strain. Cd resistance increases with increasing numbers of expressed CRP repeats, but even one 3-kDa CRP repeat still mediates Cd resistance. Site-directed mutagenesis reveals that each single cysteine within a given repeat is important for Cd resistance, though to a different extent. However, replacement of other conserved amino acids such as Pro(136) and Asp(196) at the CRP repeat junctions does not affect Cd resistance. Our data indicate (i) that the non-metallothionein CRP protein is able to detoxify Cd and (ii) that this is dependent on the availability of sulfhydryl groups of the conserved cysteines.