The laterophysic connection and swim bladder of butterflyfishes in the genus Chaetodon (Perciformes: Chaetodontidae)

The laterophysic connection (LC) is an association between bilaterally paired, anterior swim bladder extensions (horns) and medial openings in the supracleithral lateral line canals that diagnoses butterflyfishes in the genus Chaetodon. It has been hypothesized that the LC makes the lateral line system sensitive to sound pressure stimuli that are transmitted by the swim bladder horns and converted to fluid flow into the lateral line system via a laterophysic tympanum. The purpose of this study was to define variation in the morphology of the LC, swim bladder and swim bladder horns among 41 Chaetodon species from all 11 Chaetodon subgenera and a species from each of four non-Chaetodon genera using gross dissection, histological analysis as well as 2D or 3D CT (computed tomographic) imaging of live, anesthetized fishes. Our results demonstrate that the lateral line system appears rather unspecialized with well-ossified narrow canals in all species examined. Two LC types (direct and indirect), defined by whether or not the paired anterior swim bladder horns are in direct contact with a medial opening in the supracleithral lateral line canal, are found among species examined. Two variants on a direct LC and four variants of an indirect LC are defined by combinations of soft tissue anatomy (horn length [long/short] and width [wide/narrow], number of swim bladder chambers [one/two], and presence/absence of mucoid connective tissue in the medial opening in the supracleithrum). The combination of features defining each LC variant is predicted to have functional consequences for the bioacoustics of the system. These findings are consistent with the recent discovery that Chaetodon produce sounds during social interactions. The data presented here provide the comparative morphological context for the functional analysis of this novel swim bladder-lateral line connection.     

The structure of the canary's incubation patch

The gross morphology, histology and ultrastructure of the canary's incubation patch and the ventral apterium from which it arises are described. The apterium is vascularized by pectoral, external mammary, incubation, and prepubic arteries. It is innervated by cutaneous branches of spinal nerves. It has a surface area of 6 cm². Its epidermis is a stratified squamous epithelium with basal, intermediate, transitional and cornified layers. Cells in the stratum germinativum contain a normal array of organelles, but are characterized by tonofilaments, desmosomes and interdigitating surfaces. Cellular organelles disappear in the stratum transitivum and are replaced by large vacuoles and keratohyalin bands. Nonmyelinated nerve fibers are abundant in the stratum germinativum. The dermis consists of (1) an avascular layer of dense collagen subjacent to the epidermis and containing many nonmyelinated nerves, and (2) an underlying layer of areolar connective tissue containing blood vessels, lamellar corpuscles and nerves. A layer of coarse elastic fibers, reinforced by collagen and smooth muscle, separates the dermis from subcutaneous tissue. In contrast to the ventral apterium, the incubation patch is featherless and visibly hypervascular and edematous. Its epidermis is both hypertrophic and hyperplastic. Large spaces separate cells in the stratum germinativum. The visible hypervascularity is due to hyperemia and increased number and size of blood vessels in the dermis. Visible edema is due to the accumulation of fluid interstitially. Although no histological differences exist among various regions of the ventral apterium, such differences are present in the incubation patch.     

Skin structure and cornification proteins in the soft-shelled turtle Trionyx spiniferus

In contrast to most chelonians, the fully aquatic soft-shelled turtles have a smooth, unscaled, and pliable shell. The skin of the shell, tail, limbs, and neck of juveniles of Trionyx spiniferus has been studied by ultrastructural, immunocytochemical, and immunoblotting methods. The epidermis of the carapace and plastron has a thick corneous layer composed of alpha-corneocytes surrounded by a cornified cell envelope. The softer epidermis is similar to that of the shell but the epidermis and corneous layer are much thinner. Pre-corneous cells in both soft and shell epidermis are rich in vesicles produced in the Golgi apparatus and smooth endoplasmic vesicles, and contain numerous dense-core mucus-like and vesicular (lamellar) bodies. Secreted material is present among corneocytes where it probably forms an extensive intercellular lipid-mucus waterproof barrier. The dermis is very thick and composed of several layers of collagen bundles that form a plywood-patterned dermis. This dermis constitutes a strong mechanical barrier that compensates for the low content in beta-keratin, and lack of cornified scutes and dermal bones. The growth of the shell mainly occurs along the lateral margins. Immunocytochemistry reveals the presence of some beta-keratin in soft and shell epidermis, and this is confirmed by immunoblotting where bands at 18 and 32-35 kDa are present. Other proteins of the cornified cell envelope (loricrin and sciellin) or associated to lipid trafficking (caveolin-1) are also detected by immunoblotting. Loricrin positive bands at 24 and 57 kDa are present while bands cross-reactive for sciellin are seen at 45 and 53 kDa. Caveolin-1 positive bands are seen at 21-22 kDa. The presence of these proteins indicates that the epidermis is both coriaceous and waterproof. These results suggest that the shell of Trionyx is tough enough to be as mechanically efficient as the hard shell of the other turtles. At the same time, a soft shell is lighter, its shape is more easily controlled by muscles, and it allows a more controlled closure and retraction of limbs and neck inside the shell. Thus, the shell makes the animal more streamlined for swimming.     

Effects of induced ovulation by pregnant mare's serum and human chorionic gonadotropin on the sex ratio of mouse fetuses

The sex ratio of the fetuses from mice treated with pregnant mare's serum (PMS)/human chorionic gonadotropin (HCG) to induce ovulation did not differ appreciably from that of spontaneously ovulated controls. Rather, an intriguing observation was that the sex ratio in the right uterine horns tended to be lower than that in the left horns in both spontaneously ovulated controls and PMS/HCG-treated groups. We speculate on its possible relation to the observed right-left asymmetry of horn sizes (number of fetuses in the horn).     

The effect on fetal development and utero-placental blood flow of ligating a uterine artery in the rat near term

The uterine artery of one horn of 13 rats was ligated on day 18 of gestation; the remaining horn was used as a control. The effect, four days later, on blood flow to the reproductive tract, was measured with radioactive microspheres and compared to the effect on fetal and placental weights. Fetal survival in the ligated horns, 41 percent, was significantly lower (P less than 0.05) than that in the control horns, 98 percent. Fetal and placental weights of the survivors in the ligated horns, 3.159 +/- 0.133 g (SE) and 450 +/- 18 mg respectively, were similarly lower than those in the control horns, 3.814 +/- 0.111 g and 529 +/- 27 mg respectively. Maternal placental blood flow closely reflected the weight of tissue being supplied and was similar in the ligated and control horns, 129 +/- 21 and 130 +/- 18 ml.min(-1). 100g(-1), respectively. Myometrial blood flow was again similar in the ligated and control horns, 34 +/- 5 and 37 +/- 4 ml.min(-1). 100 g(-1), respectively, and in the ovarian, middle and cervical sections of each horn. These results are compatible with the view that ligation causes only a temporary reduction in uterine blood flow which permanently checks placental and fetal, or placental thus fetal, growth. Blood flow then returns to normal levels compatible with the reduced weights of tissues being supplied.     

ARTIFICIAL SELECTION ON HORN LENGTH-BODY SIZE ALLOMETRY IN THE HORNED BEETLE ONTHOPHAGUS ACUMINATUS (COLEOPTERA: SCARABAEIDAE)

Males of the horned beetle Onthophagus acuminatus Har. (Coleoptera: Scarabaeidae) exhibit horn length dimorphism due to a sigmoidal allometric relationship between horn length and body size: the steep slope of the allometry around the inflection of the sigmoid curve separates males into two groups; those larger than this inflection possess long horns, and those smaller than this inflection have short horns or lack horns. I examined the genetic basis of the allometric relationship between horn length and body size by selecting males that produced unusually long horns, and males that produced unusually short horns, for their respective body sizes. After seven generations of selection, lines selected for relatively long horns had significantly longer horn lengths for a given body size than lines selected for relatively short horns, indicating a heritable component to variation in the allometry. The sigmoidal shape of the allometry was not affected by this selection regime. Rather, selected lines differed in the position of the allometry along the body size axis. One consequence of lateral shifts in this allometric relationship was that the body size separating horned from hornless males (the point of inflection of the sigmoid curve) differed between selection lines: lines in which males were selected for relatively long horns began horn production at smaller body sizes than lines selected for relatively short horns. These results suggest that populations can evolve in response to selection on male horn length through modification of the growth relationship between horn length and body size.     

Structure of the epidermis of Australian Merino sheep over a 12-month period

Light-microscopic examination of frozen sections of skin taken from the dorsal thoraco-lumbar region of Australian Merino sheep in winter revealed that the thickness of the epidermis plus a sudanophilic layer was 24.9 micron in the interfollicular region. The uncornified epidermis (10.9 micron) was separated from the sudanophilic layer (14.0 micron) by a thin stratum corneum. It was concluded that the bulk of the sudanophilic layer was emulsified sebum in which was embedded a disorganized collection of desquamated cornified cells. Although large variances were observed in the thickness of the uncornified epidermis and of the sudanophilic layers between sheep and both within the between blocks of tissue obtained from individual sheep, there were no strong seasonal effects on either epidermal structure or layer thickness over a 12-month period. These results suggest that the Australian Merino differs from Finnish Landrace X Dorset Horn ewes, which are reported to possess, at least in winter, a thicker uncornified epidermis and a thicker stratum corneum that could be divided into two zones and was uniformly permeated by lipid.     

The enigmatic new Indochinese bovid, Pseudonovibos spiralis: an extraordinary forgery

In 1993, the discovery of several pairs of lyre-shaped horns with corrugations was recorded in Vietnam and regarded as evidence for a new, large bovid, later referred to as Pseudonovibos spiralis Peter & Feiler, 1994. Despite numerous field investigations made since 1994 in Vietnam and Cambodia, no new anatomical evidence has ever been found, except for two frontlets with their horn cores and horn sheaths. A new investigation has been carried out on six frontlets and based on a histological analysis of the keratin. It shows that the horns of P. spiralis are merely a skillful forgery made by carving and distorting ordinary cow horns. The horns of Pseudonovibos spiralis were, in some cases, positioned back onto the corresponding horn cores of domestic cow frontlets. The name Pseudonovibos spiralis is thus a junior synonym of Bos taurus, yet it remains valid and available.     

When ontogeny reveals what phylogeny hides: gain and loss of horns during development and evolution of horned beetles

How ecological, developmental and genetic mechanisms interact in the genesis and subsequent diversification of morphological novelties is unknown for the vast majority of traits and organisms. Here we explore the ecological, developmental, and genetic underpinnings of a class of traits that is both novel and highly diverse: beetle horns. Specifically, we focus on the origin and diversification of a particular horn type, those protruding from the pronotum, in the genus Onthophagus, a particularly speciose and morphologically diverse genus of horned beetles. We begin by documenting immature development of nine Onthophagus species and show that all of these species express pronotal horns in a developmentally transient fashion in at least one or both sexes. Similar to species that retain their horns to adulthood, transient horns grow during late larval development and are clearly visible in pupae. However, unlike species that express horns as adults, transient horns are resorbed during pupal development. In a large number of species this mechanisms allows fully horned pupae to molt into entirely hornless adults. Consequently, far more Onthophagus species appear to possess the ability to develop pronotal horns than is indicated by their adult phenotypes. We use our data to expand a recent phylogeny of the genus Onthophagus to explore how the widespread existence of developmentally transient horns alters our understanding of the origin and dynamics of morphological innovation and diversification in this genus. We find that including transient horn development into the phylogeny dramatically reduces the number of independent origins required to explain extant diversity patters and suggest that pronotal horns may have originated only a few times, or possibly only once, during early Onthophagus evolution. We then propose a new and previously undescribed function for pronotal horns during immature development. We provide histological as well as experimental data that illustrate that pronotal horns are crucial for successful ecdysis of the larval head capsule during the larval-to-pupal molt, and that this molting function appears to be unique to the genus Onthophagus and absent in the other scarabaeine genera. We discuss how this additional function may help explain the existence and maintenance of developmentally transient horns, and how at least some horn types of adult beetles may have evolved as exaptations from pupal structures originally evolved to perform an unrelated function.     

Specific distribution of barrier-relevant ceramides in the emerging epidermis and the periderm/subperiderm during chicken embryogenesis

During mammalian embryogenesis the emerging epidermis is temporarily covered by an epithelial monolayer, the periderm. In chicken, a second epithelial layer, the subperiderm, located underneath the periderm develops in later embryogenesis. Together the periderm and the subperiderm are referred to as the PSP unit. The cells of the PSP unit are tightly connected by tight junctions (TJ), thereby providing the embryo with an impermeable bilayered diffusion barrier. The emerging epidermis assumes its barrier function by cornification beginning at embryonic day 17 (E17) before at E18 the PSP unit undergoes desquamation. Lipid analysis of both epithelia after their mechanical separation revealed a dramatic increase to about 100-fold values of barrier-relevant ceramides, i.e. those known to essentially contribute to the diffusion barrier of the cornified envelope, in the emerging epidermis between E17 and E19. In contrast, the content of barrier-relevant ceramides in the PSP unit remained at constantly low levels throughout embryogenesis. These data strongly argue in favour of different mechanisms for the barrier function of the two epithelia. TJ in the PSP unit provide the main diffusion barrier protecting the embryo until beginning of desquamation at E18. At this developmental stage the content of cornified envelope-specific ceramides is substantially elevated, thus enabling the epidermis to fulfil its function as the major diffusion barrier after desquamation of the PSP unit. The observation that barrier-relevant ceramides are formed prior to desquamation of the PSP unit points to a precisely regulated sequence in that desquamation does not occur until the lipid-based barrier of the cornified envelope is completed and suggests in addition that these lipids might be essential regulators of the interaction between the PSP unit and the emerging epidermis.     

Embryonic keratinization in vertebrates in relation to land colonization

The embryogenesis and cytology of the epidermis in different vertebrates is variable in relation to the formation of a stratum corneum of different complexity. The latter process was essential for land colonization during vertebrate evolution and produced an efficient barrier in amniotes. Keratinocytes are made of cross‐linked keratins associated with specific proteins and lipids that are produced at advanced stages of embryogenesis when the epidermis becomes stratified. In these stages the epidermis changes from an aquatic to a terrestrial type, preadapted in preparation for the impact with the dry terrestrial environment that occurs at hatching or parturition. The epidermal barrier against water‐loss, mechanical and chemical stress, and microbe penetration is completely formed shortly before birth. Beneath the outer periderm, variably stratified embryonic layers containing glycine‐rich alpha‐keratins are formed in preparation for adult life. The following layers of the epidermis produce proteins for the formation of the cornified cell membrane and of the cornified material present in keratinocytes of the adult epidermis in reptiles, birds and mammals. The general features of the process of soft cornification in the embryonic epidermis of vertebrates are presented. Cornification in developing scales in reptiles, avian feathers and mammalian hairs is mainly related to the evolution of keratin‐associated proteins. The latter proteins form the resistant matrix of hard skin derivatives such as claws, beaks, nails and horns.     

Juvenile hormone mediates sexual dimorphism in horned beetles

The causes and consequences of sexual dimorphism are major themes in biology. Here we explore the endocrine regulation of sexual dimorphism in horned beetles. Specifically, we explore the role of juvenile hormone (JH) in regulating horn expression in females of two species with regular sexual dimorphism for pronotal horns (females have much shorter horns than males) and a third species with a rare reversed sexual dimorphism for both pronotal and head horns (females have much larger horns in both body regions compared with males). Applications of the JH analog methoprene caused females of the two more typical species to grow significantly shorter pronotal horns than control females, whereas no consistent effect on pronotal horn development was detected in the third, sex-reversed species. Instead, females in this species showed an unexpected and significant increase in head horn expression in response to methoprene treatment. Lastly, horn shape was also affected in females of one of the regularly sexually dimorphic species, but in the opposite direction than horn length. Although methoprene exerted a feminizing effect on female horn length in this species, it significantly masculinized horn shape by inducing a peculiar shape change observed naturally only in males. Our results suggest that JH influences both overall size and shape of female horns, but does so flexibly and as a function of species, sex and horn location. We use our results to review current models on the role of endocrine mechanisms in development and evolution of horned beetle diversity.     

Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in Ichthyosis vulgaris

Filaggrin is a histidine-rich, basic protein whose name was first proposed based on its ability to aggregate intermediate filaments in vitro. Based on this in vitro observation, it has generally been assumed that filaggrin functions in vivo as a matrix protein which causes keratin filaments to become densely packed in the terminally differentiated cornified cells. Inconsistent with this view however, is the well-known observation that keratin aggregation appears to proceed normally in the affected epidermis of ichthyosis vulgaris patients despite a greatly reduced quantity of filaggrin. To address this issue, we used immuno-electron microscopy to localize filaggrin and its cross-reactive precursor, profilaggrin, in human and mouse epidermis, as well as in ichthyosis vulgaris epidermis. We found that the localization of filaggrin in lower cornified cells correlates precisely with the formation of aggregated keratin filaments, and the disappearance of filaggrin in upper cornified cells correlates precisely with the loosening of keratin filaments. Furthermore, we showed that, even in ichthyosis vulgaris, small amounts of filaggrin/profilaggrin are present as electron-dense deposits associated with keratin filaments in the granular cells, and that the localization of this small amount of antigen again correlates with the aggregation state of keratin filaments. These data strongly suggest that filaggrin is indeed involved in filament aggregation in vivo.     

The effect of bone-marrow transplantation on decidua formation in pseudopregnant rats

A main cause of failed pregnancy is insolvency of the decidual reaction of endometrial cells. It is believed that bone-marrow cells (BMCs) are a partial source of decidual cells in endometrial tissue. In the present work, the effect is studied of transplantation of BMCs on the desidualization processes using the model of pseudopregnancy in rats. BMCs were flushed from rat femurs and tibias. On the fifth day of pseudopregnancy, a suspension of single BMCs was injected into one of the uterine horns. PBS injection into the contralateral horn without cells served as control. Rats were sacrificed on the 11th day of pseudopregnancy. The diameter in the meso-antimesometral direction in the experimental uterine horn increased by 1.5–2 times compared to the control horn. The weight of decidual tissue in the experimental horn was three times greater than the weight of the control horn. The presence of transplanted BMCs in decidual tissue was documented by preliminary double-staining of BMCs with membrane dye PKH26 Red and nuclear dye Hoechst 33342. Histological analysis of decidua sections after transplantation did not reveal any alterations in cell differentiation or tissue structure. We concluded that transplantation of BMCs stimulated decidualization in animals.     

Dimorphism of both head and prothoracic horn morphologies in male Copris acutidens (Coleoptera: Scarabaeoidea)

Allometric scaling of male horn morphologies of Copris acutidens, including the head and prothoracic horns, was analyzed. The analyses of scaling relationships indicated a discontinuous increase in head horn length and prothoracic horn height but a linear increase in prothoracic horn length relative to body size. The different results for length and height of the prothoracic horn suggest that the height is functionally more important for ensuring mates in the nests. Furthermore, both the head and prothoracic horns were dimorphic, and this characteristic was not found in the scarabaeoid beetle Onthophagus. Similarity in the switch point values for the head and prothoracic horns suggests that the dimorphism of the two kinds of horns may result from the same developmental threshold mechanism.     

Linear and threshold-dependent expression of secondary sexual traits in the same individual: insights from a horned beetle (Coleoptera: Scarabaeidae)

Elaborate horns or horn‐like structures in male scarab beetles commonly scale with body size either (a) in a linear fashion with horn size increasing relatively faster than body size or (b) in a threshold‐dependent, sigmoid fashion; that is, males smaller than a certain critical body size develop no or only rudimentary horns, whereas males larger than the threshold size express fully developed horns. The development of linear vs. sigmoid scaling relationships is thought to require fundamentally different regulatory mechanisms. Here we show that such disparate regulatory mechanisms may co‐occur in the same individual. Large males of the south‐east Asian Onthophagus (Proagoderus) watanabei (Ochi & Kon) (Scarabaeidae, Onthophagini) develop a pair of long, curved head horns as well as a single thoracic horn. We show that unlike paired head horns in a large number of Onthophagus species, in O. watanabei the relationship between head horns and body size is best explained by a linear model. Large males develop disproportionately longer horns than small males, but the difference in relative horn sizes across the range of body sizes is small compared to other Onthophagus species. However, the scaling relationship between the thoracic horn and body size is best explained by a strongly sigmoid model. Only males above a certain body size threshold express a thoracic horn and males smaller than this threshold express no horn at all. We found a significant positive correlation between head and thoracic horn length residuals, contrary to what would be expected if a resource allocation tradeoff during larval development would influence the length of both horn types. Our results suggest that the scaling relationship between body size and horn length, and the developmental regulation underlying these scaling relationships, may be quite different for different horns, even though these horns may develop in the same individual. We discuss our results in the context of the developmental biology of secondary sexual traits in beetles. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 473–480.     

Immuno-cross reactivity of transglutaminase and cornification marker proteins in the epidermis of vertebrates suggests common processes of soft cornification across species

In differentiating mammalian keratinocytes proteins are linked to the plasma membrane by epidermal transglutaminases through N-epsilon-(gamma-glutamyl)-lysine isopeptide bonds to form the cornified cell envelope. The presence of transglutaminases and their protein substrates in the epidermis of nonmammalian vertebrates is not known. The present study analyses the presence and localization of the above proteins in the epidermis using immuno-cross reactivity across different classes of amniotes. After immunoblotting, some protein bands appear labelled for loricrin, sciellin, and transglutaminase in most species. These proteins are scarce to absent in the epidermis of aquatic species (goldfish and newt) where a stratum corneum is absent or very thin. The molecular weight of transglutaminase immunoreactive bands generally varies between 40 to 62 kDa, with the most represented bands at 52-57 kDa in most species. The more intense loricrin- and sciellin-immunoreactive bands are seen at 50-55-62 kDa, but are weak or absent in aquatic vertebrates. Loricrine-like immunoreactivity is present in the epidermis where alpha-(soft)-keratinization occurs. Isopeptide bonds are mainly associated to bands in the range of 50-62 kDa. In vertebrates where hard-keratin is expressed (the beta-keratin corneous layer of sauropsids and in feathers) or in hair cortex of mammals, no loricrin-like, transglutaminase-, and isopeptide-bond-immunoreactivities are seen. Immunoblotting however shows loricrin-, sciellin-, and trasnsglutaminase-positive bands in the corneous layers containing beta-keratin. Histologically, the epidermis of most amniotes shows variable transglutaminase immunoreactivity, but isopeptide-bond and sciellin immunoreactivities are weak or undetactable in most species. The limitations of immunohistochemical methods are discussed and compared with results from immunoblotting. In reptilian epidermis transglutaminase is mainly localized in 0.15-0.3 microm dense granules or diffuse in transitional alpha-keratogenic cells. In beta-keratogenic cells few small dense granules show a weak immunolabeling. Transglutaminase is present in nuclei of terminal differentiating alpha- and beta-keratinocytes, as in those of mature inner and outer root sheath. The present study suggests that keratinization based on loricrin, sciellin and transglutaminase was probably present in the stratum corneoum of basic amniotes in the Carboniferous. These proteins were mainly maintained in alpha-keratogenic layers of amniotes but decreased in beta-keratogenic layers of sauropsids (reptiles and birds). The study suggests that similar proteins for the formation of the cornified cell envelope are present in alpha-keratinocytes across vertebrates but not in beta-keratinocytes.     

Sexually dimorphic male horns and their use in agonistic behaviors in the horn-headed cricket <Emphasis Type="Italic">Loxoblemmus doenitzi</Emphasis> (Orthoptera: Gryllidae)

Sexual dimorphism, the difference between the sexes in secondary sexual characters, is in general driven by processes of sexual selection. The horn-headed cricket, Loxoblemmus doenitzi, exhibits sexual dimorphism in head shape. Males have flat heads and triangular horns on both sides of their heads, whereas females have rounded heads and no horns. We hypothesized that male horns have evolved due to intra-sexual selection, in which males use these horns as weapons in aggressive interactions. We tested two predictions of this hypothesis by conducting agonistic trials with field-caught males of L. doenitzi: (1) the horns should be used in agonistic interactions between males, and (2) the asymmetry in horn size or horn use may determine contest outcome. Horn length was significantly correlated with thorax length and hind femur length. During agonistic interactions, males aggressively used their horns by beating the opponent’s horns with their own or by poking the opponent’s body. However, logistic regression analysis revealed that neither horn length nor horn use were significant factors for contest outcome. Instead, body size was significant for determining contest outcome. We discuss possible scenarios for evolution of male horns in L. doenitzi.