Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea). VI. Anterior testicular ducts and their nomenclature

In this study, the anterior testicular ducts of the North American natricine snake Seminatrix pygaea are described using light and electron microscopy. From the seminiferous tubules, the rete testis passes into the epididymal sheath, a structure along the medial border of the testis heavily invested with collagen fibers. The rete testis consists of simple, nonciliated cuboidal epithelium (principal cells). The intratesticular ducts of the rete testis are narrow (50–70 μm) at their junction with the seminiferous tubules, widen (80–100 μm) as they extend extratesticularly, and divide into smaller branches as they anastomose with the next tubules, the ductuli efferentes. The ductuli efferentes are lined by simple cuboidal epithelium but possess nonciliated principal cells as well as ciliated cells. These are the only ducts in the male reproductive system with ciliated cells. The ductuli efferentes are narrow (25–45 μm), divide into numerous branches, and are highly convoluted. The ductus epididymis is the largest duct in diameter (240–330 μm), and the diameter widens and the epithelium thins posteriorly. The ductus epididymis is lined by nonciliated, columnar principal cells and basal cells. No regional differences in the ductus epididymis are apparent. Ultrastructural evidence suggests that all of the nonciliated principal cells in each of the anterior testicular ducts function in both absorption and secretion. Absorption occurs via small endocytic vesicles, some of which appear coated. Secretion is by a constitutive pathway in which small vesicles and a flocculent material are released via a merocrine process or through the formation of apocrine blebs. The secretory product is a glycoprotein. Overall, the characteristics of the anterior testicular ducts of this snake are concordant with those of other amniotes, and the traditional names used for snakes are changed to conform with those used for other sauropsids and mammals. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

The anatomy of the upper cretaceous snake Najash rionegrina Apesteguía & Zaher, 2006, and the evolution of limblessness in snakes

Najash rionegrina Apesteguía & Zaher, 2006 , a terrestrial fossil snake from the Upper Cretaceous of Argentina, represents the first known snake with a sacrum associated with robust, well‐developed hind limbs. Najash rionegrina documents an important gap in the evolutionary development towards limblessness, because its phylogenetic affinities suggest that it is the sister group of all modern snakes, including the limbed Tethyan snakes Pachyrhachis, Haasiophis, and Eupodophis. The latter three limbed marine fossil snakes are shown to be more derived morphologically, because they lack a sacrum, but have articulated lymphapophyses, and their appendicular skeleton is enclosed by the rib cage, as in modern snakes.     

Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians

Alibardi, L. 2011. Observations on the ultrastructure and distribution of chromatophores in the skin of chelonians. —Acta Zoologica (Stockholm) 00 :1–11. The cytology and distribution of chromatophores responsible for skin pigmentation in chelonians is analyzed. Epidermal melanocytes are involved in the formation of dark spots or stripes in growing shelled and non‐shelled skin. Melanocytes rest in the basal layer of the epidermis and transfer melanosomes into keratinocytes during epidermal growth. Dermal melanophores and other chromatophores instead remain in the dermis and form the gray background of the skin. When dermal melanophores condense, they give origin to the dense spots or stripes in areas where no epidermal melanocytes are present. In the latter case, the epidermis and the corneous layer are transparent and reveal the dermal distribution of melanophores and other chromatophores underneath. As a result of this basic process of distribution of pigment cells, the dark areas visible in scales can have a double origin (epidermal and dermal) or a single origin (epidermal or dermal). Xanthophores, lipophores, and a cell containing both pterinosomes and lipid droplets are sparse in the loose dermis while iridophores are rarely seen in the skin of chelonians analyzed in the present study. Xanthophores and lipophores contribute to form the pale, yellow or oranges hues present among the dark areas of the skin in turtles.     

Ultrastructural features of skin pigmentation in the lizard Heloderma suspectum with emphasis on xanto‐melanophores

The morphological origin of the dark and pink‐orange areas in the skin of the venomous lizard Heloderma suspectum is not known. Histology and electron microscopy show that dark‐grey areas of the skin derived from dermal chromatophores localized in specific areas present underneath the epidermis. A dynamic chromatophoric unit in the dermis is absent. In the darkest areas of the skin, the accumulation of melanosomes in cells of the beta‐layer contributes to increase the black intensity. In the orange‐pink areas, the superficial dermis contains xantophores storing numerous carotenoid vesicles, rare or absent lamellated pterinosomes and a variable number of melanosomes. These xanto‐melanophores predominate over the remaining chromatophores and form a continuous stratum underneath the epidermis. Beneath this lipoid‐rich stratum, iridophores are infrequent and do not form a continuous layer in the dermis. In the paler areas of the skin, melanophores are sparse in both superficial and deeper part of the dermis where irregularly oriented bundles of collagen fibrils are present. The prevalent xanto‐melanophores localized in the pink‐orange areas of the skin contribute to an effective sunlight protection in desert conditions in addition to the darker regions occupied by melanophores.     

Immunocytochemistry indicates that glycine‐rich beta‐proteins are present in the beta‐layer,while cysteine‐rich beta‐proteins are present in beta‐ and alpha‐layers of snake epidermis

Immunolocalization of glycine‐rich and cysteine–glycine‐medium‐rich beta‐proteins (Beta‐keratins) in snake epidermis indicates a different distribution between beta‐ and alpha‐layers. Acta Zoologica, Stockholm. The epidermis of snakes consists of hard beta‐keratin layers alternated with softer and pliable alpha‐keratin layers. Using Western blot, light and ultrastructural immunolocalization, we have analyzed the distribution of two specific beta‐proteins (formerly beta‐keratins) in the epidermis of snakes. The study indicates that the antibody HgG5, recognizing glycine‐rich beta‐proteins of 12–15 kDa, is poorly or not reactive with the beta‐layer of snake epidermis. This suggests that glycine‐rich proteins similar to those present in lizards are altered during maturation of the beta‐layer. Conversely, a glycine–cysteine‐medium‐rich beta‐protein (HgGC10) of 10–12 kDa is present in beta‐ and alpha‐layers, but it is reduced or disappears in precorneous and suprabasal cells destined to give rise to beta‐ and alpha‐cells. Together with the previous studies on reptilian epidermis, the present results suggest that beta‐proteins rich in glycine mainly accumulate on a scaffold of alpha‐keratin producing a resistant and hydrophobic beta‐layer. Conversely, beta‐proteins lower in glycine but higher in cysteine accumulate on alpha‐keratin filaments present in beta‐ and alpha‐layers producing resistant but more pliable layers.     

Epidermal differentiation during ontogeny and after hatching in the snake Liasis fuscus (Pythonidae,Serpentes, Reptilia), with emphasis on the formation of the shedding complex

Differentiation and localization of keratin in the epidermis during embryonic development and up to 3 months posthatching in the Australian water python, Liasis fuscus, was studied by ultrastructural and immunocytochemical methods. Scales arise from dome-like folds in the skin that produce tightly imbricating scales. The dermis of these scales is completely differentiated before any epidermal differentiation begins, with a loose dermis made of mesenchymal cells beneath the differentiating outer scale surface. At this stage (33) the embryo is still unpigmented and two layers of suprabasal cells contain abundant glycogen. At Stage 34 (beginning of pigmentation) the first layers of cells beneath the bilayered periderm (presumptive clear and oberhautchen layers) have not yet formed a shedding complex, within which prehatching shedding takes place. At Stage 35 the shedding complex, consisting of the clear and oberhautchen layers, is discernible. The clear layer contains a fine fibrous network that faces the underlying oberhautchen, where the spinulae initially contain a core of fibrous material and small beta-keratin packets. Differentiation continues at Stage 36 when the beta-layer forms and beta-keratin packets are deposited both on the fibrous core of the oberhautchen and within beta-cells. Mesos cells are produced from the germinal layer but remain undifferentiated. At Stage 37, before hatching, the beta-layer is compact, the mesos layer contains mesos granules, and cells of the alpha-layer are present but are not yet keratinized. They are still only partially differentiated a few hours after hatching, when a new shedding complex is forming underneath. Using antibodies against chick scale beta-keratin resolved at high magnification with immunofluorescent or immunogold conjugates, we offer the first molecular confirmation that in snakes only the oberhautchen component of the shedding complex and the underlying beta cells contain beta-keratin. Initially, there is little immunoreactivity in the small beta-packets of the oberhautchen, but it increases after fusion with the underlying cells to produce the syncytial beta layer. The beta-keratin packets coalesce with the tonofilaments, including those attached to desmosomes, which rapidly disappear in both oberhautchen and beta-cells as differentiation progresses. The labeling is low to absent in forming mesos-cells beneath the beta-layer. This study further supports the hypothesis that the shedding complex in lepidosaurian reptiles evolved after there was a segregation between alpha-keratogenic cells from beta-keratogenic cells during epidermal renewal.     

Microornamentation of leaf chameleons (Chamaeleonidae: Brookesia,Rhampholeon, and Rieppeleon)—with comments on the evolution of microstructures in the chamaeleonidae

Chameleons (Chamaeleonidae) feature many adaptations to their arboreal lifestyle, including zygodactylous feet, a prehensile tail, and epidermal microstructures. In arboreal tree chameleons, the substrate‐contacting site of the feet and tail is covered by microscopic hair‐like structures (setae) of 6–20 µm length. Their friction enhancing function has been shown in recent studies. Leaf chameleons and one representative of the tree chameleons (Chamaeleo namaquensis) secondarily have become ground‐dwelling. Because leaf chameleons are paraphyletic, one could expect that in the three leaf chameleon genera Brookesia, Rhampholeon, and Rieppeleon and the tree chameleon Ch. namaquensis, epidermis has adapted independently to terrestrial locomotion. Using scanning electron microscopy, we investigated the substrate‐contacting surfaces of the feet (subdigital) of 17 leaf chameleon species and five tree chameleon species that have not yet been examined. Additionally, surfaces not involved in locomotion, the flanks (dorsolateral), and scale interstices, were examined. Although the subdigital microstructures in leaf chameleons are more diverse than in tree chameleons, we found some features across the genera. The subdigital microornamentation of Rhampholeon spinosus consists of long thin setae and spines, comparable to those of tree chameleons. All other Rhampholeon species have spines or short but broad setae. Rh. spectrum had tooth‐like structures instead of setae. Subdigital scales of Brookesia have either thorns or conical scale‐tops in the center and feature honeycomb microstructures. In Rieppeleon, subdigital scales have a thorn. Scale surfaces are covered by honeycombs and short hair‐like structures (spines). As subdigital scales with a thorn in the center and honeycomb microstructures were also found in the terrestrial tree chameleon Ch. namaquensis, one can assume that this geometry is a convergent adaptation to terrestrial locomotion. Despite the great number of genus‐specific traits, the convergent evolution of honey‐comb structures in Brookesia, Rieppeleon, and Ch. namaquensis and the high variability of spines and setae in Rhampholeon suggests a rapid adaptation of subdigital microornamentation in Chamaeleonidae. J. Morphol. 276:167–184, 2015. © 2014 Wiley Periodicals, Inc.     

Ferulic acid in the treatment of post‐diabetes testicular damage: relevance to the down regulation of apoptosis correlates with antioxidant status via modulation of TGF‐β1, IL‐1β and Akt signalling

The aim of this study was to investigate the protective effect of ferulic acid at different doses (50 mg kg^?1 alternative day and 50 mg kg^?1 daily) on the streptozotocin (STZ)‐induced post‐diabetes rat testicular damage. Diabetes was induced by a single intraperitoneal injection of STZ (50 mg/kg). Rats treated with ferulic acid were given once a day orally for 10 weeks, starting 3 days after STZ injection. Testis tissue and blood samples were collected for investigating biochemical analysis, antioxidant status, sperm parameters, and histopathological, immunohistochemical and apoptotic studies. Treatment with ferulic acid to diabetic rats significantly improved the body weight, testis weight, serum insulin level, serum testosterone level and sperm parameters (viability, motility and count). Histopathological study also revealed that ferulic acid‐treated diabetic rats showed an improved histological appearance. Our data indicated that significant reduction in the activity of apoptosis by using terminal deoxyuridine triphosphate nick end‐labelling and reduced expression of transforming growth factor‐β1 and interleukin‐1β in the testis tissue of ferulic acid‐treated diabetic rats. Conversely, it was also revealed that ferulic acid‐treated diabetic rats markedly enhanced the serine/threonine protein kinase protein expression in the testis tissue. Our result suggests that ferulic acid inhibits testicular damage in diabetic rats by declining oxidative stress. Copyright © 2013 John Wiley & Sons, Ltd.     

Derived sperm morphology in the interstitial sea cucumber Rhabdomolgus ruber,with observations on oogenesis and spawning behavior

Some life history features of the interstitial sea cucumber Rhabdomolgus ruber are described from intertidal specimens collected from the northern coast of Maine. Histological studies suggest that the population consists of hermaphrodites with gametogenesis being initiated in April and reproduction beginning in May and continuing through the summer months. Sexually mature adults possess a single, blind‐ended gonadal tubule that functions as an ovotestis by producing both eggs and sperm. The ovotestis wall consists of an outer peritoneum composed of flagellated epithelial cells and muscles; an inner germinal epithelium of germ and somatic cells; and a middle connective tissue (hemal) compartment bounded by the basal laminas of the peritoneum and germinal epithelium. During the reproductive season, the gonadal tubule contains all stages of oocyte development. Vitellogenesis appears to involve the biosynthetic activities of the Golgi complex and rough endoplasmic reticulum. A few specimens had transitional ovotestes with mature sperm in the gonad lumen and asynchronously developing oocytes and a small number of spermatocytes within the germinal epithelium. The mature spermatozoon is an ent‐aquasperm with ultrastructural features significantly different from those described from other echinoderm classes including a highly elongated acrosome, a large periacrosomal region between the acrosome and nucleus, numerous unfused mitochondria in the midpiece, and a cytoplasmic sleeve or collar extending posteriorly along the proximal portion of the flagellum. The sperm head reaches 11.5 μm in length (combined midpiece, nucleus, periacrosomal region, acrosome), making it the longest yet reported from the Holothuroidea and among the longest in the Echinodermata. Some elements of this derived morphology could be attributed to fertilization biology, but others may have phylogenetic significance. Spawning behavior was observed in which two individuals appeared to pseudocopulate by intertwining their oral tentacles for several minutes before one of them abruptly secreted an egg mass containing three eggs.     

Short‐term heat stress impairs testicular functions in the American oyster,Crassostrea virginica: Molecular mechanisms and induction of oxidative stress and apoptosis in spermatogenic cells

Global temperature is increasing due to anthropogenic activities. Abnormal temperature has devastating effects on growth, reproduction, and development of aquatic organisms. In this study, we examined the effects of short‐term exposure to elevated temperatures (28 and 32°C for 1 week) on testicular functions, heat shock protein‐70 (HSP70), dinitrophenyl protein (DNP, a biomarker of reactive oxygen species [ROS]), and nitrotyrosine protein (NTP, an indicator of reactive nitrogen species [RNS]) expressions, protein carbonyl (PC, a measure of ROS) contents, nitrates/nitrites (NOx, a metabolite of nitric oxide) levels, extrapallial fluid (EPF) conditions, and cellular apoptosis in American oyster (Crassostrea virginica). Higher temperatures significantly decreased (~26%) sperm production in oysters compared with controls (24°C). HSP70, NTP, and DNP expressions were increased after heat exposure, consistent with increased EPF pH, and cellular apoptosis. The enhanced apoptosis in spermatogenic cells is associated with increased caspase 3/7 activity, PC contents, and NOx levels in testicular tissues. Together these results suggest that elevated temperature drastically increases oxidative stress and cellular apoptosis which in turn leads to decreased testicular functions in oysters. To the best of our knowledge, this study reports the first findings on the impacts of elevated temperatures on testicular functions in oysters.     

The ontogeny of the shell in side‐necked turtles,with emphasis on the homologies of costal and neural bones

Although we are starting to understand the molecular basis of shell development based on the study of cryptodires, basic comparative ontogenetic data for the other major clade of living turtle, the pleurodires, are largely missing. Herein, the developmental and phylogenetic relation between the bony shell and endoskeleton of Pleurodira are examined by studying histological serial sections of nine specimens of three different species, including an ontogenetic series of Emydura subglobosa. Emphasis is given to the portion of the carapace in which ribs and vertebral spinous processes become part of the carapace. Central questions are how neurals and costals are formed in pleurodiran turtles, whether costals and neurals are of endoskeletal or exoskeletal origin, and what ontogenetic factors relate to neural reduction of some Pleurodira. The neurals and costals do not develop as independent ossification centers, but they are initial outgrowths of the periosteal collar of endoskeletal ribs and neural arches. Slightly later in development, the ossification of both shell elements continues without a distinct periosteum but by metaplastically ossifying precondensed soft‐tissue integumentary structures. Through ontogeny, ribs of the turtles studied are closely associated with the hypaxial intercostalis musculature while epaxial interspinalis musculature connects the neural arches. We here propose an alternative structural hypothesis for the neural reduction and, ultimately, the complete loss of the neural series. The complete reduction of neurals in Emydura spp. may be linked to heterochrony, accompanied by a restricted influence of epaxial musculature and epidermal–dermal interaction in shell bone formation. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Microanatomy of the male and female reproductive tracts in the long‐tailed butterfly ray Gymnura poecilura,an elasmobranch with unusual characteristics

The anatomy of the male and female reproductive systems was investigated in the long‐tailed butterfly ray Gymnura poecilura using gross observation and light microscopy. The testes are highly asymmetrical, to the extent that only the left testis is functional and the right testis is completely absent. Both of the male genital ducts are present and symmetrical, although spermatozoa only occur in the left duct. The genital ducts are straight and unconvoluted, with regular incomplete internal partitions throughout. Females do not possess a right ovary, nor do the oviducal glands exhibit distinct club and papillary zones, and the baffle zone lacks baffle plates. In all sections of the gland, the tubules display different secretory activities depending on the proximity to the gland lumen. The gland produces a thin egg membrane that encases each egg individually, while the endometrium is formed into trophonemata.     

Multiple phases of mg‐calcite in crustose coralline algae suggest caution for temperature proxy and ocean acidification assessment: lessons from the ultrastructure and biomineralization in Phymatolithon (Rhodophyta,Corallinales)1

Magnesium content, strongly correlated with temperature, has been developed as a climate archive for the late Holocene without considering anatomical controls on Mg content. In this paper, we explore the ultrastructure and cellular scale Mg‐content variations within four species of North Atlantic crust‐forming Phymatolithon. The cell wall has radial grains of Mg‐calcite, whereas the interfilament (middle lamella) has grains aligned parallel to the filament axis. The proportion of interfilament and cell wall carbonate varies by tissue and species. Three distinct primary phases of Mg‐calcite were identified: interfilament Mg‐calcite (mean 8.9 mol% MgCO₃), perithallial cell walls Mg‐calcite (mean 13.4 mol% MgCO₃), and hypothallium Mg‐calcite (mean 17.1 mol% MgCO₃). Magnesium content for the bulk crust, an average of all phases present, showed a strongly correlated (R² = 0.975) increase of 0.31 mol% MgCO₃ per °C. Of concern for climate reconstructions is the potential for false warming signals from undetected postgrazing wound repair carbonate that is substantially enriched in Mg, unrelated to temperature. Within a single crust, Mg‐content of component carbonates ranged from 8 to 20 mol% MgCO₃, representing theoretical thermodynamic stabilities from aragonite‐equivalent to unstable higher‐Mg‐calcite. It is unlikely that existing current predictions of ocean acidification impact on coralline algae, based on saturation states calculated using average Mg contents, provide an environmentally relevant estimate.     

Nonparametric inference for the cumulative incidence function of a competing risk,with an emphasis on confidence bands in the presence of left‐truncation

Motivated by a study on pregnancy outcome, a computationally simple resampling procedure for nonparametric analysis of the cumulative incidence function of a competing risk is investigated for left‐truncated data. We also modify the original procedure to producing the more desirable Greenwood‐type variance estimates. These approaches are used to construct simultaneous confidence bands of the cumulative incidence functions which is otherwise hampered by the complicated nature of the covariance process. Simulation results and a real data example are provided.     

Species‐specific preferences of German recreational anglers for freshwater fishing experiences,with emphasis on the intrinsic utilities of fish stocking and wild fishes

To answer the question, whether anglers have an intrinsic preference for stocking or a preference for catch outcomes (e.g. catch rates) believed to be maintained by stocking, a discrete choice experiment was conducted among a sample of anglers (n = 1335) in Lower Saxony, Germany. After controlling for catch aspects of the fishing experience, no significant influence of two stocking attributes (stocking frequency and composition of the catch in terms of wild v. hatchery fishes) on the utility gained from fishing was found for any of the freshwater species that were studied. It was concluded that the previously documented large appreciation of fish stocking by anglers may be indicative of an underlying preference for sufficiently high catches rather than reflect an intrinsic preference for stocking or the catching of wild fishes per se.     

Confusing concepts in Lateglacial stratigraphy and geochronology: origin, consequences, conclusions (with special emphasis on the type locality Bøllingsø)

A great confusion currently exists on the stratigraphic and geochronologic subdivision of the Weichselian Lateglacial. This paper unravels the development of this confusion, which is interpreted to be mainly due to a combination of wrongly used stratigraphical concepts, incorrect interpretations, and erroneous correlation between various sites and records. Special emphasis is laid on the palynological methods of Johs. Iversen and on the type locality Bøllingsø, from which the original (partly previously unpublished) data are presented and evaluated. It is concluded that almost all stratigraphic schemes and terms have lost their original value.     

A re‐examination of the molecular phylogeny and biogeography of the genus Schizothorax (Teleostei: Cyprinidae) through enhanced sampling,with emphasis on the species in the Yunnan–Guizhou Plateau,China

The mitochondrial DNA cytochrome b sequences of 36 Schizothorax species from 51 localities in the Yunnan–Guizhou Plateau (YGP) and its adjacent areas were analysed. Maximum parsimony, Maximum likelihood and Bayesian phylogenetic analyses were performed to examine the relationships of Schizothorax species. A hypothesis of the phylogenetic relationships of the species is given. A relaxed molecular clock based on Bayesian evolutionary analysis was used to tentatively calculate the divergence times of Schizothorax. Samples from the YGP were tentatively grouped into three geographically distributed clades: the Tsangpo‐Irrawaddy, the Mekong‐Salween and the Trans‐Jinsha River (including Jinsha, Red, Nanpan and Beipan Rivers). Calibration of the molecular clock revealed that two geological periods, the late Miocene about 10 million years before present (Myr BP) and the Pliocene (4.0 Myr BP), were important times in the vicariant speciation of Schizothorax. The phylogenetic history of the species is congruent with events caused by the uplift of the Tibetan Plateau and the YGP. The divergence of Schizothorax species in YGP began in the Pliocene. Our phylogenetic trees did not support the hypothesis that the paleo Jinsha River was drained through the Yangtze River‐Jianchuan Lake‐Erhai Lake to the Red River. Schizothorax in the Beipan River were derived from the Jinsha River.