The sandwich structure of keratinous layers controls the form and growth orientation of chicken rhinotheca.

The upper beak bone of birds is known to be overlain by the rhinotheca, which is composed of the horny sheath of keratinous layers. However, the details of the structure and growth pattern of the rhinotheca are yet to be understood. In this study, the microstructure of the rhinotheca from chicken specimens of different growth stages (ranging from 1 to ~ 80 days old) was analyzed using a combination of thin section and scanning electron microscopy observations, and small-angle X-ray scattering analysis. We found that the rhinotheca comprises three different layers - outer, intermediate, and inner layers - throughout its growth. The outer layer arises from the proximal portion of the beak bone and covers the dorsal surface of the rhinotheca, whereas the intermediate and inner layers originate in the distal portion of the beak bone and underlie the outer layer. This tri-layered structure of the rhinotheca was also observed in wild bird specimens (grey wagtail, king quail, and brown dipper). On the median plane, micro-layers making up the outer and inner layers are bedded nearly parallel to the rostral bone at the base. However, more distally positioned micro-layers of the outer layer are more anteverted distally. The micro-layers of the intermediate layer are bedded nearly perpendicular to those of the outer and inner layers on the median plane. The growth of micro-layers in the intermediate layer adds thickness to the rhinotheca, which causes the difference in profile between the beak bone and the rhinotheca in the distal portion of the beak. Moreover, the entire intermediate layer grows distally as new proximal micro-layers form. The outer layer is dragged distally by the intermediate layer as a result of its distal growth, for the three layers are closely packed to each other at their boundaries. Furthermore, the occurrence of the intermediate and inner layers in the distal portion of the rostral bone may be because the distal end of the beak is frequently used and worn, and the rhinotheca therefore needs to be replaced more frequently at the distal end. The rhinotheca structure described here will be an important and useful factor in the reconstruction of the beaks of birds in extinct taxa.

Microscopic and immunohistochemical study on the cornification of the developing beak in the turtle Emydura macquarii.

The development and cornification of the ramphoteca (beak) in turtles are not known. The microscopic aspects of beak formation have been analyzed in the pleurodirian turtle Emydura macquarii using histological, immunocytochemical and ultrastructural methods. At embryonic Stage 15 the maxillar beak is originated from discontinuous placodes (one frontal and two oral) formed in the epidermis above and below the mouth that later merge into the epidermis of the beak. The mandibular beak is formed by two lateral placodes. In the placodes, basal keratinocytes in contact with local mesenchymal condensations become columnar, and generate suprabasal cells forming 5-6 layers of embryonic epidermis at Stages 17-20 and a compact shedding alpha-layer at the base of the embryonic epidermis. These keratinocytes contain irregular or aggregated reticular bodies made of 30-40 nm thick strands of coarse filaments, mixed with tonofilaments and sparse lipid droplets. Beneath the shedding layer are present 3-4 layers of keratinocytes accumulating coarse filaments mixed with beta-corneous packets, and underneath spindle-shaped beta-cells differentiate where beta-corneous packets completely replace the reticulate bodies. Differently from scales where corneocytes partially merge, beak corneocytes remain separated but they are joined by numerous interlocking spines. The production of beta-cells in the thick corneous layer of the developing beak, like in claws, occurs before the differentiation of beta-cells in the body scutes. This indicates that a massive mesenchymal condensation triggers beta-differentiation before this process is later activated in most of body scutes of the carapace and plastron. J. Morphol. 277:1309-1319, 2016. © 2016 Wiley Periodicals, Inc.

Hierarchical multiscale structure-property relationships of the red-bellied woodpecker (Melanerpes carolinus) beak.

We experimentally studied beaks of the red-bellied woodpecker to elucidate the hierarchical multiscale structure-property relationships. At the macroscale, the beak comprises three structural layers: an outer rhamphotheca layer (keratin sheath), a middle foam layer and an inner bony layer. The area fraction of each layer changes along the length of the beak giving rise to a varying constitutive behaviour similar to functionally graded materials. At the microscale, the rhamphotheca comprises keratin scales that are placed in an overlapping pattern; the middle foam layer has a porous structure; and the bony layer has a big centre cavity. At the nanoscale, a wavy gap between the keratin scales similar to a suture line was evidenced in the rhamphotheca; the middle foam layer joins two dissimilar materials; and mineralized collagen fibres were revealed in the inner bony layer. The nano- and micro-indentation tests revealed that the hardness (associated with the strength, modulus and stiffness) of the rhamphotheca layer (approx. 470 MPa for nano and approx. 320 MPa for micro) was two to three times less than that of the bony layer (approx. 1200 MPa for nano and approx. 630 MPa for micro). When compared to other birds (chicken, finch and toucan), the woodpecker's beak has more elongated keratin scales that can slide over each other thus admitting dissipation via shearing; has much less porosity in the bony layer thus strengthening the beak and focusing the stress wave; and has a wavy suture that admits local shearing at the nanoscale. The analysis of the woodpeckers' beaks provides some understanding of biological structural materials' mechanisms for energy absorption.

Macroscopic, histologic, and ultrastructural lesions associated with avian keratin disorder in black-capped chickadees (Poecile atricapillus).

An epizootic of beak abnormalities (avian keratin disorder) was recently detected among wild birds in Alaska. Here we describe the gross, histologic, and ultrastructural features of the disease in 30 affected adult black-capped chickadees (Poecile atricapillus). Grossly, there was elongation of the rhamphotheca, with varying degrees of lateral deviation, crossing, and gapping between the upper and lower beak. Not uncommonly, the claws were overgrown, and there was alopecia, scaling, and crusting of the skin. The most prominent histopathologic features in the beak included epidermal hyperplasia, hyperkeratosis, and core-like intrusions of necrotic debris. In affected birds, particularly those with moderate to severe beak overgrowth, there was remodeling of premaxillary and mandibular bones and various dermal lesions. Lesions analogous to those found in beaks were present in affected claws, indicating that this disorder may target both of these similar tissues. Mild to moderate hyperkeratosis occurred in other keratinized tissues, including skin, feather follicles, and, occasionally, sinus epithelium, but typically only in the presence of microbes. We did not find consistent evidence of a bacterial, fungal, or viral etiology for the beak lesions. The changes observed in affected birds did not correspond with any known avian diseases, suggesting a potentially novel hyperkeratotic disorder in wild birds.

Microanatomy of passerine hard-cornified tissues: beak and claw structure of the black-capped chickadee (Poecile atricapillus).

The microanatomy of healthy beaks and claws in passerine birds has not been well described in the literature, despite the importance of these structures in avian life. Histological processing of hard-cornified tissues is notoriously challenging and only a few reports on effective techniques have been published. An emerging epizootic of beak deformities among wild birds in Alaska and the Pacific Northwest region of North America recently highlighted the need for additional baseline information about avian hard-cornified structures. In this study, we examine the beak and claw of the Black-capped Chickadee (Poecile atricapillus), a common North American passerine that is affected by what has been described as "avian keratin disorder." We use light and scanning electron microscopy and high-magnification radiography to document the healthy microanatomy of these tissues and identify features of functional importance. We also describe detailed methods for histological processing of avian hard-cornified structures and discuss the utility of special stains. Results from this study will assist in future research on the functional anatomy and pathology of hard-cornified structures and will provide a necessary reference for ongoing investigations of avian keratin disorder in Black-capped Chickadees and other wild passerine species.

Comparative study of the mechanical properties, micro-structure, and composition of the cranial and beak bones of the great spotted woodpecker and the lark bird.

Woodpeckers are well able to resist head injury during repeated high speed impacts at 6-7 m s⁻¹ with decelerations up to 1000 g. This study was designed to compare the mechanical properties, microstructures and compositions of cranial bone and beak bone of great spotted woodpecker (Dendrocopos major) and the Mongolian sky lark (Melanocorypha mongolica). Microstructures were observed using micro-computed tomography and scanning electron microscopy and their compositions were characterized by X-ray powder diffraction and Fourier-transform infrared spectroscopy. Under high stress, the cranial bone and the beak of the woodpecker exhibited distinctive mechanical features, which were associated with differences in micro-structure and composition, compared with those of the lark. Evolutionary optimization of bone micro-structure has enabled functional adaptation to the woodpecker's specific lifestyle. Its characteristic micro-structure efficiently avoids head impact injury and may provide potential clues to the prevention of brain injury using bio-inspired designs of shock-absorbing materials.

Fine structural dependence of ultraviolet reflections in the King Penguin beak horn.

The visual perception of many birds extends into the near-ultraviolet (UV) spectrum and ultraviolet is used by some to communicate. The beak horn of the King Penguin (Aptenodytes patagonicus) intensely reflects in the ultraviolet and this appears to be implicated in partner choice. In a preliminary study, we recently demonstrated that this ultraviolet reflectance has a structural basis, resulting from crystal-like photonic structures, capable of reflecting in the near-UV. The present study attempted to define the origin of the photonic elements that produce the UV reflectance and to better understand how the UV signal is optimized by their fine structure. Using light and electron microscopic analysis combined with new spectrophotometric data, we describe here in detail the fine structure of the entire King Penguin beak horn in addition to that of its photonic crystals. The data obtained reveal a one-dimensional structural periodicity within this tissue and demonstrate a direct relationship between its fine structure and its function. In addition, they suggest how the photonic structures are produced and how they are stabilized. The measured lattice dimensions of the photonic crystals, together with morphological data on its composition, permit predictions of the wavelength of reflected light. These correlate well with experimentally observed values. The way the UV signal is optimized by the fine structure of the beak tissue is discussed with regard to its putative biological role.

Ultraviolet reflecting photonic microstructures in the King Penguin beak.

King and emperor penguins (Aptenodytes patagonicus and Aptenodytes forsteri) are the only species of marine birds so far known to reflect ultraviolet (UV) light from their beaks. Unlike humans, most birds perceive UV light and several species communicate using the near UV spectrum. Indeed, UV reflectance in addition to the colour of songbird feathers has been recognized as an important signal when choosing a mate. The king penguin is endowed with several highly coloured ornaments, notably its beak horn and breast and auricular plumage, but only its beak reflects UV, a property considered to influence its sexual attraction. Because no avian UV-reflecting pigments have yet been identified, the origin of such reflections is probably structural. In an attempt to identify the structures that give rise to UV reflectance, we combined reflectance spectrophotometry and morphological analysis by both light and electron microscopy, after experimental removal of surface layers of the beak horn. Here, we characterize for the first time a multilayer reflector photonic microstructure that produces the UV reflections in the king penguin beak.

Ultrastructural analysis of a putative magnetoreceptor in the beak of homing pigeons.

With the use of different light and electron microscopic methods, we investigated the subcellular organization of afferent trigeminal terminals in the upper beak of the homing pigeon, Columba livia, which are about 5 microm in diameter and contain superparamagnetic magnetite (SPM) crystals. The SPM nanocrystals are assembled in clusters (diameter, approximately 1-2 microm). About 10 to 15 of these clusters occur inside one nerve terminal, arranged along the cell membrane. Each SPM cluster is embedded in a solid fibrous cup, open towards the cell surface, to which the cluster adheres by delicate fiber strands. In addition to the SPM clusters, a second inorganic iron compound has been identified: noncrystalline platelets of iron phosphate (about 500 nm wide and long and maximally 100 nm thick) that occur along a fibrous core of the terminal. The anatomic features suggested that these nerve endings could detect small intensity changes of the geomagnetic field. Such stimuli can induce deformations of the SPM clusters, which could be transduced into primary receptor potentials by mechanosensitive membrane receptor channels. The subepidermal fat cells surrounding the nerve endings prevent the inside from external mechanical stimuli. These structural findings corresponded to conclusions inferred from rock magnetic measurements, theoretical calculations, model experiments, and behavioral data, which also matched previous electrophysiologic recordings from migratory birds.

Avian pox in sanderlings from Florida.

Asian pox was diagnosed in three sanderlings (Calidris alba) on Sanibel Island, Florida (USA) in February 1997. All three cases had large tumor-like lesions which contributed significantly to their mortality. Poxvirus infection was confirmed by cytology, histopathology, and electron microscopy. This is the first report of poxvirus infection in sanderlings.

TGFbeta3 promotes transformation of chicken palate medial edge epithelium to mesenchyme in vitro.

Epithelial-mesenchymal transformation plays an important role in the disappearance of the midline line epithelial seam in rodent palate, leading to confluence of the palate. The aim of this study was to test the potential of the naturally cleft chicken palate to become confluent under the influence of growth factors, such as TGFbeta3, which are known to promote epithelial-mesenchymal transformation. After labeling medial edge epithelia with carboxyfluorescein, palatal shelves (E8-9) with or without beak were dissected and cultured on agar gels. TGFbeta1, TGFbeta2 or TGFbeta3 was added to the chemically defined medium. By 24 hours in culture, medial edge epithelia form adherent midline seams in all paired groups without intact beaks. After 72 hours, seams in the TGFbeta3 groups disappear and palates become confluent due to epithelial-mesenchymal transformation, while seams remain mainly epithelial in control, TGFbeta1 and TGFbeta2 groups. Epithelium-derived mesenchymal cells are identified by carboxyfluorescein fluorescence with confocal microscopy and by membrane-bound carboxyfluorescein isolation bodies with electron microscopy. Labeled fibroblasts completely replace the labeled epithelia of origin in TGFbeta3-treated palates without beaks. Single palates are unable to undergo transformation, and paired palatal shelves with intact beaks do not adhere or undergo transformation, even when treated with TGFbeta3. Thus, physical contact of medial edge epithelia and formation of the midline seam are necessary for epithelial-mesenchymal transformation to be triggered. We conclude that there may be no fundamental difference in developmental potential of the medial edge epithelium for transformation to mesenchyme among reptiles, birds and mammals. The bird differs from other amniotes in having developed a beak and associated craniofacial structures that seemingly keep palatal processes separated in vivo. Even control medial edge epithelia partly transform to mesenchyme if placed in close contact. However, exogenous TGFbeta3 is required to achieve complete confluence of the chicken palate.

Alterations in Msx 1 and Msx 2 expression correlate with inhibition of outgrowth of chick facial primordia induced by retinoic acid.

Spatially-restricted expression domains of Msx 1 and Msx 2 in the developing chick face suggest that they may play a role in epithelial-mesenchymal interactions governing outgrowth of facial primordia. Retinoid application to developing chick faces reproducibly inhibits upper beak outgrowth but the lower beak is unaffected. In the normal face, high levels of Msx gene transcripts in upper and lower beak primordia correlate with regions of outgrowth. Following retinoid treatment, Msx 1 and Msx 2 transcripts are rapidly down-regulated in upper beak primordia where outgrowth is inhibited, but remain largely unchanged in lower beak primordia, where outgrowth is unaffected. Decreases in gene expression precede retinoid-induced morphological changes in the upper beak, suggesting that Msx gene products are involved in mediating the effect of retinoids on facial development.

Receptors in the bill of the platypus.

1. Afferent responses were recorded from filaments of the trigeminal nerve in each of two platypuses (Ornithorhynchus anatinus) anaesthetized with alpha-chloralose. All receptive fields were located along the lateral border of the upper bill. Discrete receptive fields could be identified as belonging to two distinct classes of sensory receptor. 2. The most prominent response was an irregular resting discharge which could be increased or decreased by weak electric pulses. These receptors were insensitive to moderately strong mechanical stimulation, and it was concluded that they were electroreceptors. 3. Each electroreceptor had a single spot of maximum sensitivity on the bill surface. When the stimulating electrode over this spot was the cathode it excited the receptor for the duration of the stimulating pulse, using stimulus strengths as low as 20 mV. When it was the anode, it inhibited the discharge. Cathodal excitation was followed by rebound inhibition and anodal inhibition by rebound excitation. 4. Receptors responded to cathodal steps with an initial high-frequency burst of impulses, followed by a lower maintained rate of discharge. Rapidly changing pulses were similarly effective in exciting receptors, adding support to the claim that platypuses are able to detect moving prey by the electrical activity associated with muscle contraction. 5. The centres of the receptive fields of two electroreceptors were marked by the insertion of fine entomological pins. Histological examination established the presence of a large mucus-secreting gland at the marked spot. The epidermal duct of the gland contained an elaborate myelinated innervation, with morphologically distinct axon terminals that we identify as the electroreceptors. 6. As well as electroreceptors, the skin of the bill contained three kinds of mechanoreceptors: slow-adapting receptors, rapidly adapting, vibration-sensitive receptors and receptors with an intermediate adaptation rate. The slowly adapting receptors were characterized by their low threshold to mechanical stimuli, irregular discharge and significant dynamic sensitivity. Vibration receptors showed maintained responses to sinusoidal vibration of the skin up to 600 Hz. 7. These experiments confirm an earlier report that the platypus bill is an electrodetector organ. The presence of electroreceptors of a unique structure and supplied by the trigeminal nerve indicates that electroreception has evolved independently in monotremes. This in turn emphasizes that monotremes are a highly evolved group which split off from the main mammalian stem a long time ago.

The bill tip organ of the chicken (Gallus gallus var. domesticus).

At the tip of the lower beak of the chicken there were found 15-20 specialised dermal papillae containing large numbers of mechanoreceptors. The Merkel or Grandry corpuscles were situated distally in the papillae and at the papillae base was a collection of Herbst corpuscles. The apex of the papillae, under the scanning electron microscope, appeared as a row of shallow pits on the surface of the beak just inside the mouth. These papillae resemble similar structures seen in other birds and are probably necessary for fine tactile discrimination.

Analysis of upper beak defects in chicken embryos following with retinoic acid.

Implanting inert carriers soaked in retinoic acid into the anterior margin of the developing limb of chicken embryos leads to orofacial malformations as well as affecting pattern formation in the limb. Using anion-exchange beads as carriers, and soaking solutions of 1-10 mg/ml retinoic acid, almost 100% of the embryos have malformations of the face. The effects on the treated limbs range from symmetrical patterns of duplicated digits (maximum number of digits being four) to truncations in which no digits were formed at all. Typically, in the malformed faces the upper beak is completely absent, no nostrils are present and the front of the face forms a scalloped rim of tissue above the mouth. By reference to normal beak development, the seven bulges of tissue that make up the rim can be identified as derivatives of the masses of tissue that normally would fuse to form the upper beak. The roof of the mouth consists of three bulges of tissue flanked by widely separated palatal shelves. The defect can thus be classified as severe bilateral clefting of the primary palate. By examining the morphology of the faces of treated embryos, the origin of the defect can be traced to failure of the frontonasal mass to enlarge. Thus, the oronasal fissures are very wide and fusion across them to form the primary palate cannot occur. The way in which retinoic acid brings about the defect is discussed in relation to possible mechanisms involved in the production of cleft palate. The parallel is noted between the associated effects of retinoic acid on beak and limb morphogenesis and the chick mutation cpp, that also affects both face and limbs.

[Ultrastructural studies of the duck cere with special reference to Grandry and Herbst bodies]. / Ultrastrukturelle Untersuchungen an der Wachshaut von Enten unter besonderer Berücksichtigung der Grandryschen und Herbstschen Körper.

The results of the investigations into the cere of the domestic duck (Anas boscas domestica) and the mallard (Anas platyrhyncha) may be summarized as follows. The parts of ceroma are the epidermis and the corium. The epidermis is a laminated epithelium and the corium consists of fibrous connective tissues. The later one frequently contains mastocytes and pigment cells. There are many blood vessels, Schwann's cells, nerve fibres, Grandry's and Herbst's bodies. The sensory cells of Grandry's bodies are bodies with processes. They are characterized by the round nucleus, dense core vesicles, the small Golgi's complexes, the narrow endoplasmatic ducts and the cytoplasmatic fascicles. In the nerve fibre running between the sensory cells there are plenty of mitochondria but few clear vesicles and neurofilaments. The axolemma, cytolemma, and the empty space lying between these are sharply obvious. In Grandry's bodies with more than one sensory cells the nerve fibre ramifies. In the branches there are no mitochondria. These are replaced by clear vesicles. In Grandry's bodies with one sensory cell the axon ends in a disc growing narrow at its termination. Both of them are full of mitochondria. The terminal disc is characterized by the elliptical bodies and the thickenings of the membrane. Herbst's body consists of three parts. One of these is the inner knob, the other is the inner cavity, the third is the external capsule. The inner knob consists of two series of cells. The number of cells is ten in both rows each. From the cells, 20 to 50 lamina-like processes are originating. The laminar system of every cell is connected with the system of the cell before and behind it, as well as with the laminar system of the other side. In the nerve fibre running between the rows of cells mainly in its terminal part, some groups are formed by mitochondria, the dense-core vesicles, and the elliptical bodies. The inner cavity consists of laminae of different breadths, limited by cavities full of some meshwork. The laminae vary in form and extent. There are among them some ramifying ones, some forms terminating in a point and some of blunt ends. They are characterized by dense and long ribosomeseries, arranged close to one another. The external capsule consists of several parallel laminae, separated from one another by collagenous fibrin fascicles. The laminae are mostly narrow, their cytoplasm is spongoid, they are characterized by egg-shaped cysterns. There is no synaptic organization either in Grandry's or in Herbst's body. The junction between the membrane is to be regarded as a parallel contact in both places.