Ultrastructure of poison glands of South American frogs: a comparison between Physalaemus albonotatus and Leptodactylus chaquensis (Anura: Leptodactylidae)

Serous (poison) cutaneous glands of the leptodactylid species Physalaemus albonotatus and Leptodactylus chaquensis were compared using light and transmission electron microscopy. Glands in the two species share structural traits common in anurans, including the peripheral contractile sheath (myoepithelium) and the syncytial secretory unit that produces, stores, and modifies the poison. At the ultrastructural level, early steps of poison production are also similar and fit the usual path of proteosynthesis, involving rough endoplasmic reticulum (RER) and Golgi stacks (dictyosomes) in the peripheral syncytial cytoplasm. However, several differences are obvious during the maturational processes that lead post-Golgian products to their ultimate ultrastructural traits. In P. albonotatus, the dense product released from the dictyosomes acquires a thick repeating substructure, which, however, becomes looser in the inner portion of the syncytium. In L. chaquensis, serous maturation involves gradual condensation, and opaque, somewhat "vacuolized" granules are formed. These different maturational paths expressed during poison manufacturing in the two species agree with the polyphyletic origin of the family Leptodactylidae. On the other hand, data collected for P. albonotatus fit previous findings from P. biligonigerus and stress the view that poisons produced by congeneric species share similar (or identical) ultrastructural features.     

Three types of cutaneous glands in the skin of the salamandrid Pleurodeles waltl. A histological and ultrastructural study

Histological and ultrastructural investigations revealed three different multicellular skin gland types in the salamandrid Pleurodeles waltl. The mucous glands are small, with one layer of secretory cells surrounding a central lumen; they produce the viscous and slippery mucus film that has various functions in amphibians. The serous glands can be divided based on their histological and ultrastructural characters into the granular gland Type I (GGI) and the granular gland Type II (GGII). The first type (GGI) is moderately sized and distributed throughout the body surface, with higher concentrations in the parotoid and back regions. In contrast, the second type (GGII) is very large (for Pleurodeles) and was found only in the tail, with highest concentration in the tail dorsum. Both granular gland types contain mainly proteinaceous materials but differ in their morphological features including size, shape, cellular organization and vesicle distribution, vesicle size and vesicle shape. Both GGI and GGII are especially concentrated in body parts that are presented to an attacking predator and are hypothesized to produce repellent to poisonous substances to thwart potential aggressors. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Granular cutaneous glands in the frog Physalaemus biligonigerus (Anura, Leptodactylidae): comparison between ordinary serous and 'inguinal' glands

Beside the ordinary granular (or serous) glands, the skin of the leptodactylid frog Physalaemus biligonigerus possesses peculiar clusters of large granular units, the 'inguinal' glands, located in the dorsolateral areas of the pelvic girdle. Both gland types store their specific products within the syncytial cytoplasm of the secretory unit. These secretory materials consist of spheroidal or ellipsoidal bodies (granules) with a repeating substructure. The subcellular features of the immature products of the ordinary serous and inguinal glands are identical. However, these products undergo divergent maturative processes, leading to fluidation on the one hand and condensation on the other. Secretory release into the small gland lumen was observed in both cases, involving merocrine mechanisms. On the basis of the analysis of cutaneous serous gland polymorphism in anurans, the inguinal units in P. biligonigerus do not appear to be an independent line. Rather, these large units belong to the ordinary serous type and represent a gland population specialized in the storage of remarkable amounts of product used in chemical defence of the skin.     

Serous cutaneous glands in the South American horned frog Ceratophrys ornata (Leptodactylformes, Chthonobatrachia, Ceratophrydae): ultrastructural expression of poison biosynthesis and maturation

Serous cutaneous glands are described in newly metamorphosed and juvenile specimens of the horned frog Ceratophrys ornata using light and transmission electron microscopy. We report patterns of biosynthesis and maturation of the specific product of the gland secretory unit. The syncytial, secretory compartment possesses a complex of endoplasmic reticulum (predominantly smooth endoplasmic reticulum after metamorphosis) and Golgi stacks. The serous product is weak in density and is contained in vesicles involved in repeating merging processes. During this maturation activity, secondary lysosomes are observed, which derive from autophagic processes (crinophagy) involving the secretory materials. Ceratophrys ornata, a species representative of the type genus of the family Ceratophrydae, belongs to the heterogeneous group of anurans that, possibly as the result of convergence, all produce cutaneous poisons consisting of vesicles or faint density granules.     

Serous cutaneous glands in the paludiculine frog Physalaemus biligonigerus (Anura, Leptodactylidae): patterns of cytodifferentiation and secretory activity in premetamorphic specimens

The development of serous cutaneous glands is described in tadpoles of the leptodactylid frog Physalaemus biligonigerus , with attention to the cytodifferentiation processes of the secretory unit (adenomere). Secretory differentiation causes the adenomere to assume the structure of a syncytium and triggers biosynthesis of the serous product (poison), consisting of granules with peculiar repeating substructure. The secretory granules resemble glomerular-like aggregates of randomly orientated tubular subunits. This complex substructure derives from the rearrangement of the dense, compact material released by the Golgi apparatus in the form of small, membrane-bounded particles. Both early and advanced features of P. biligonigerus poison closely resemble those found during serous gland development in the Italian treefrog Hyla intermedia . The similar aspects described during serous biosynthesis in these leptodactylid and hylid frogs suggest they are phylogenetically informative. On the other hand, the peculiar features of their poisons may reflect the differential patternings of their secretory repertories along the divergent phylogenetic paths, which led these frogs to be currently included in different families.     

Development of integument and cutaneous glands in larval,juvenile and adult toads (Rhinella granulosa): a morphological and morphometric study

In this study, the development of integument and cutaneous glands in the toad Rhinella granulosa (Bufonidae) at different larval stages and in postmetamorphic and adult forms was examined. The analyses were conducted using histological, ultrastructural and morphometric methods. The results showed that cellular aggregations of precursor epidermal glands start to appear in stage 31 of (Herpetologica, 16, 1960 and 183) and then proliferate and invade the dermis. After stage 41, granular and mucous glands are very similar to those found in adults. The granular glands are syncytial and are surrounded by a distinct layer of myoepithelial cells. In the region of parotoid macroglands, the granular glands accumulate and their alveoli progressively increase until they reach adult size. An analysis by scanning electron microscopy showed the inner distribution of the syncytial nuclei and the myoepithelial cells. The morphological changes observed in the integument of tadpoles are associated with the gradual adaptation to terrestrial environments by preparing the individual for future chemical defence against predators and micro‐organisms.     

Oogenesis in Actinoposthia beklemischevi (Platyhelminthes, Acoela): an ultrastructural and cytochemical study

The oogenesis of the acoel Actinoposthia beklemischevi can be divided into a previtellogenic and a vitellogenic stage. Maturing oocytes are surrounded by accessory cells (a.c.) that produce electrondense granules, the content of which is released into the space between the oocyte and a.c. and gives rise to a thin primary egg envelope. The a.c. may also contribute to yolk synthesis by transferring low molecular weight precursors to the oocyte. Two types of inclusion are produced in maturing oocytes. Type I inclusions are small, roundish granules produced by the Golgi complex. They have a proteinaceous non-polyphenolic content which is discharged in the intercellular space and produce a thicker secondary egg envelope. Type I inclusions represent eggshell-forming granules (EFGs). Type II inclusions are variably sized globules progressively changing their shape from round to crescent. They appear to be produced by the ER, contain glycoproteins and remain scattered throughout the cytoplasm in large oocytes. Type II inclusions represent yolk. The main features of oogenesis in Actinoposthia are: (a) EFGs have a non-polyphenolic composition; (b) the egg envelope has a double origin and is not sclerotinized; (c) yolk production appears to be autosynthetic. The present ultrastructural findings are compared with those from other Acoelomorpha and Turbellaria.     

Spermiogenesis and spermatozoa in Acanthodasys aculeatus (Gastrotricha, Macrodasyida): an ultrastructural study

The spermatogenesis, the spermiogenetic process and the structure of the mature spermatozoon of Acanthodasys aculeatus (Gastrotricha, Macrodasyida) are described from an ultrastructural point of view. Several spermatogonia in mitotic divisions were seen, proving that euthely of gastrotrichs does not concern gonads. Spermiogenesis is characterized by the early formation of both the acrosome and the axoneme, by the subsequent appearances of a perinuclear helix and of a complex axial tubular structure in the acrosome and by the late development of the peraxonemal striated cylinder. The mature spermatozoon is filiform, and composed of a spiralized acrosome, a helical nuclear–mitochondrial complex and a long flagellum. The acrosome contains an axial tubular structure and the spring-shaped nucleus delimits a single, long mitochondrion. A perinuclear helix formed by the pro-acrosome surrounds the nuclear–mitochondrial complex extending for its whole length. A monolayered, obliquely striated cylinder encloses the 9 × 2 + 2 axoneme; its terminal part is empty because of the shortness of the axoneme.     

Previtellogenic development and vitellogenin synthesis in the fat body of a mosquito: an ultrastructural and immunocytochemical study

We describe two phases, previtellogenic and vitellogenic, in the activity of the trophocytes in the fat body of the mosquito Aedes aegypti. The previtellogenic phase, leading to trophocyte competence to synthesize vitellogenin (Vg), occurred during the first 3 days after eclosion. This phase was characterized by enlargement and activation of the nucleoli, proliferation of ribosomes and rough endoplasmic reticulum (RER), development of Golgi complexes, and extensive invaginations of the plasma membrane. During the vitellogenic phase, initiated by a blood meal, Vg was first detected, by immunofluorescence, 1 hr after feeding. The intensity of the immunoreaction increased for the next 24 hr, was declining at 30 hr, and had disappeared by 48 hr. Vg synthesis was characterized ultrastructurally by the enlargement of the RER and the formation of dense secretion granules in Golgi complexes. These secretion granules were two to three times larger at the peak of Vg synthesis than at the beginning. The granules discharged their contents by exocytosis. Two electron microscopical immunocytochemical methods, immunoferritin and peroxidase-antiperoxidase, confirmed this pathway of Vg processing. For the first 12 hr after feeding. Vg synthetic organelles proliferated and the active nucleoli were multilobed; thereafter, while Vg synthesis continued, the nucleoli began to regress into compact bodies. Termination of Vg synthesis was marked by autophagical degradation of Vg synthetic and processing organelles.     

Aberrant spermatozoa and spermatids,and observations on phagocytosis,in Sparganophilus tamesis (Sparganophilidae,Oligochaeta, Annelida): an ultrastructural study

In Sparganophilus tamesis replication, misalignment and misplacement phenomena occur in aberrant spermatids and spermatozoa, and giant nuclei are recorded in some sperm.

Sperm components replicate independently of each other, but midpiece replication was not observed. Aberrancy was commonest and showed greatest variation in the acrosomal region, and it is suggested that double acrosome tubes may share a common precursor primary vesicle.

Phagocytosis by coelomocytes and by cytophores occurs in the seminal vesicles. The spermathecae were empty and the possibility that the population is parthenogenetic is discussed.     

A new role for antennation in paper wasps (Hymenoptera,Vespidae): antennal courtship and sex dimorphic glands in antennomeres

Summary. Males of Polistes dominulus perform antennal vibrations and grasping of female antennae during pre-copulatory and copulatory phases. Male antennation plays a relevant role in mating success. In several antennomeres of males of P. dominulus and Vespa crabro, a further species in which we observed male antennation, we found secretory cells of class 1 and 3 associated to the same release site. In P. dominulus, class 3 cells with ampulla-like reservoirs and class 1 cells release their secretion through hairless multiporous areas. In V. crabro, tyloid-like structures are associated with large apodemes. The as yet unidentified secretion of these glands may act as a contact or low-volatile sex pheromone during courtship behaviour.Received 4 December 2003; revised 16 August 2004; accepted 7 September 2004.     

Synthesis of interkeratin matrix in differentiating lizard epidermis: an ultrastructural autoradiographic study after injection of tritiated proline and histidine

During epidermal differentiation in mammals, keratins and keratin-associated matrix proteins rich in histidine are synthesized to produce a corneous layer. Little is known about interkeratin proteins in nonmammalian vertebrates, especially in reptiles. Using ultrastructural autoradiography after injection of tritiated proline or histidine, the cytological process of synthesis of beta-keratin and interkeratin material was studied during differentiation of the epidermis of lizards. Proline is mainly incorporated in newly synthesized beta-keratin in beta-cells, and less in oberhautchen cells. Labeling is mainly seen among ribosomes within 30 min postinjection and appears in beta-keratin packets or long filaments 1-3 h later. Beta-keratin appears as an electron-pale matrix material that completely replaces alpha-keratin filaments in cells of the beta-layer. Tritiated histidine is mainly incorporated into keratohyalin-like granules of the clear layer, in dense keratin bundles of the oberhautchen layer, and also in dense keratin filaments of the alpha and lacunar layer. The detailed ultrastructural study shows that histidine-labeling is localized over a dense amorphous material associated with keratin filaments or in keratohyalin-like granules. Large keratohyalin-like granules take up labeled material at 5-22 h postinjection of tritiated histidine. This suggests that histidine is utilized for the synthesis of keratins and keratin-associated matrix material in alpha-keratinizing cells and in oberhautchen cells. As oberhautchen cells fuse with subjacent beta-cells to form a syncytium, two changes occur : incorporation of tritiated histidine, but uptake of proline increases. The incorporation of tritiated histidine in oberhautchen cells lowers after merging with cells of the beta-layer, whereas instead proline uptake increases. In beta-cells histidine-labeling is lower and randomly distributed over the cytoplasm and beta-keratin filaments. Thus, change in histidine uptake somehow indicates the transition from alpha- to beta-keratogenesis. This study indicates that a functional stratum corneum in the epidermis of amniotes originates only after the association of matrix and corneous cell envelope proteins with the original keratin scaffold of keratinocytes.     

The haemocytes of the salp Thalia democratica (Tunicata,Thaliacea): an ultrastructural and histochemical study in the oozoid

In comparative immunology and evolution of the chordate immune system, tunicates hold an important phylogenetic position as sister group of vertebrates. However, knowledge of the tunicate immune system is limited to the class Ascidiacea, in which some species are now considered model organisms. In the class Thaliacea, represented by fragile pelagic species, the few studies on their haemocytes go back to several decades ago and do not consider comparative aspects with ascidian haemocytes. In this study, we identified various haemocyte types and their distribution in the common salp Thalia democratica by comparative observations under light and electron microscopy and by histochemical, histoenzymatic and immunohistochemical techniques. By comparing specialisations with those of ascidian haemocytes, we detected an undifferentiated cell type (lymphocyte‐like cell) and three categories with four cell types, that is, (i) phagocytic line (hyaline amoebocyte and amoebocyte with large vacuoles), (ii) mast cell‐like line (granular cell) and (iii) storage cells (nephrocyte). Both phagocytes and granular cells appear to migrate in the tunic. Phagocytes adhere to the tunic which internally covers the oral siphon, where they probably function as sentinel cells of the pharynx. Results show the variety of haemolymph cells in the salp similar to phlebobranch ascidians.     

A new method to model change in cutaneous blood flow due to mechanical skin irritation part I: comparison between experimental and numerical data

Mechanical skin irritation creates vasodilation. Vasodilation of vascular networks induces increase in blood volume and blood velocity. Both can be measured by Laser Doppler Velocimetry. We propose in this article a method permitting comparison between experimental and numerical results. Experimental data was obtained by Laser Doppler Velocimetry. Numerical results were obtained by a continuous model of the vascular network. The model consists of three layers. First and last layer are described by anisotropic and heterogeneous porous media. They represent the irrigation and the drainage of the vascular system. The intermediate layer is described by a lumped parameter model that does not permit horizontal fluxes. All vessels are compliant. The permeabilities depend on the volumes of the specific layer. Skin irritation is modeled by a change in compliance of small arterial blood vessels. The comparison between experimental and numerical data is based on the model proposed by Bonner and Nossal [1981. Model for laser Doppler measurements of blood flow. Appl. Opt. 20, 2097-2107]. The model describes the Doppler frequency spectrum S(omega) as a function of the optical phenomena creating the frequency shift. The comparison is based on the model of the first moment M1 approximately integralomegaS(omega)domega. The variables of the first moment can be determined by results of the numerical model. We have shown, that it exists a linear relation between the change in compliance and the following increase in first moment. Using this linear relation experimental and numerical data can be compared.     

Functional significance of the male brood pouch in the reproductive strategies of pipefishes and seahorses: a morphological and ultrastructural comparative study on three anatomically different pouches

The morphological organization of the male brood pouch skin of three different species of syngnathids ( Nerophis ophidion, Syngnathus abaster and Hippocampus hippocampus ), investigated using light and electron microscopy, showed that each pouch had a skin with a different ultrastructure. This reflected different relationships between the paternal body and the developing embryos. In N. ophidion , the bilayered epidermis of the pouch consisted mainly of pavement cells (filament-containing cells) typical of fish skin. In S. abaster , pavement cells were interspersed with many mitochondria-rich cells. These cells varied in number during the different functional stages of the pouch and died by apoptosis after the breeding period. Modified secretory 'flame cone cells' rich in vesicles and granules characterized the epidermis of H. hippocampus . Although there were specific differences, the vascularized dermis was the only feature common to all three types of pouch. These findings suggest that the brood pouch in Syngnathidae has different functions, which may be related to the different reproductive strategies and ecology of each species.     

基于协整理论的经济增长与生态环境变化关系分析——以重庆市渝东南地区为例

许多研究文献忽略了经济增长和生态环境质量的双向作用机制,从而导致了变量之间的内生性偏差。与以往研究相比,主要在以下几方面进行分析:1)采用脉冲响应函数来分别考察生态环境与经济增长这两类变量之间的动态冲击反应。2)运用预测方差分解技术来考察经济增长与生态环境变化在解释因素变动时的相对重要性。结论:经济增长与生态环境之间存在长期的均衡关系,非平稳序列的经济增长指标、生态环境因子经过一阶差分后变得平稳,均为一阶单整,存在协整统计关系。从长期来看,人均GDP与废气排放量、固废排放量呈现负相关,而与矿产开采总量、化肥使用强度呈现正相关。其高度正相关性实证了该地区主要靠资源开发拉动经济增长。脉冲响应分析表明,快速经济增长是影响生态环境质量的重要原因,生态环境质量变化对经济增长存在着反作用力。就响应效果而言,生态环境对经济增长的负向响应表明生态环境退化将导致人们对生态环境质量需求偏好的改变,从而对经济增长进程的变化产生外在压力,但生态环境对经济增长的反馈效应具有滞后性。     

Investigation of the effects of vitamin D treatment on the ovarian AMH receptors in a polycystic ovary syndrome experimental model: an ultrastructural and immunohistochemical study

The aim of this study was to demonstrate the effects of vitamin D treatment on ultrastructural changes and AMHR2 expression in the ovary in PCOS rat model. A total of 24 female prepubertal rats were divided into 3 groups. In group 1, sesame oil was injected and used as control group. In group 2, PCOS was created by the injection of 6 mg/kg/day DHEA. In group 3, PCOS was created and 120 ng/100 g 1,25 (OH)₂D3 treatment was performed. At the end of the 28^th day, the blood samples were collected. The ovarian tissues were obtained for electron microscopic and immunohistochemical examinations.Serum AMH, testosterone, FSH, LH levels and LH/FSH ratios were higher in the PCOS group compared to the control group and decreased in the treatment group compared to the PCOS group. AMHR2 expression was increased in atretic and premature luteinizate antral follicles in the PCOS group compared to the control group, and decreased in the treatment group compared to the PCOS group. PCOS group electron micrographs showed degenerative changes in developing follicles, cystic follicles characterised with granulosa cell layer attenuation and thickening of the theca cell layer, and lipid accumulation in the interstitial cells. Structural changes observed in the PCOS group were improved with vitamin D treatment.As a result, there is an interaction between PCOS, AMH serum levels and AMHR2 in the ovarian follicles. Vitamin D has a positive effect on hormonal and structural changes in the PCOS group. We concluded that vitamin D supplementation may be beneficial in PCOS patients.     

Impact of temperature on the relationship between respiration and nitrogen concentration in roots: an analysis of scaling relationships, Q10 values and thermal acclimation ratios

* The impact of nitrogen (N) supply on the temperature response of root respiratory O(2) uptake (R) was assessed in several herbaceous species grown in solution culture. Warm-grown (25 : 20 degrees C, day:night) plants differing in root N concentration were shifted to 13 : 8 degrees C for 7 d to cold-acclimate. * Log-log plots of root R vs root N concentration both showed that R increased with increasing tissue N concentration, irrespective of the growth temperature. Although the regression slopes of the log-log plots did not differ between the warm-grown and cold-acclimated plants, cold-acclimated plants did exhibit a higher y-axis intercept than their warm-grown counterparts. This suggests that cold acclimation of root R is not entirely dependent on cold-induced increases in tissue N concentration and that scaling relationships (i.e. regression equations fitted to the log-log plots) between root R and N concentration are not fixed. * No systematic differences were found in the short-term Q(10) (proportional change in R per 10 degrees C change in temperature), or degree of cold acclimation (as measured by the proportional difference between warm- and cold-acclimated roots) among roots differing in root N concentration. The temperature response of root R is therefore insensitive to tissue N concentration. * The insensitivity of Q(10) values and acclimation to tissue N concentration raises the possibility that root R and its temperature sensitivity can be predicted for a range of N supply scenarios.     

Pneumocystis oryctolagi sp. nov., an uncultured fungus causing pneumonia in rabbits at weaning: review of current knowledge, and description of a new taxon on genotypic, phylogenetic and phenotypic bases

The genus Pneumocystis comprises noncultivable, highly diversified fungal pathogens dwelling in the lungs of mammals. The genus includes numerous host-species-specific species that are able to induce severe pneumonitis, especially in severely immunocompromised hosts. Pneumocystis organisms attach specifically to type-1 epithelial alveolar cells, showing a high level of subtle and efficient adaptation to the alveolar microenvironment. Pneumocystis species show little difference at the light microscopy level but DNA sequences of Pneumocystis from humans, other primates, rodents, rabbits, insectivores and other mammals present a host-species-related marked divergence. Consistently, selective infectivity could be proven by cross-infection experiments. Furthermore, phylogeny among primate Pneumocystis species was correlated with the phylogeny of their hosts. This observation suggested that cophylogeny could explain both the current distribution of pathogens in their hosts and the speciation. Thus, molecular, ultrastructural and biological differences among organisms from different mammals strengthen the view of multiple species existing within the genus Pneumocystis. The following species were subsequently described: Pneumocystis jirovecii in humans, Pneumocystis carinii and Pneumocystis wakefieldiae in rats, and Pneumocystis murina in mice. The present work focuses on Pneumocystis oryctolagi sp. nov. from Old-World rabbits. This new species has been described on the basis of both biological and phylogenetic species concepts.     

The interplay between habitat availability and population differentiation: a case study on genetic and morphological structure in an inland wader (Charadriiformes)

Long‐distance migrant waders breeding in the Arctic often have globally structured populations, largely because they were isolated in glacial or interstadial refugia or were restricted to fragmented coastal wetlands in winter. Conversely, inland species using continentally distributed wetlands appear to be less structured (more often panmictic), presumably because they are less likely to have been isolated by multiple refugia or by current events. We analyzed genetic variation in a widely distributed inland species, the ruff (Philomachus pugnax), sampled from seven Eurasian breeding localities, and from migration routes and wintering areas in Europe and Africa. One mitochondrial marker (N = 118) and eight nuclear microsatellites (N = 170) showed: (1) high genetic variation; (2) large genetic distances among mitochondrial (private) haplotypes within breeding populations; (3) the absence of a signature of isolation‐by‐distance; and (4) a distribution of private microsatellite alleles indicating dispersal between Scandinavia and Siberia but not between western and eastern Siberia. These results were consistent with a large refugial population during the Last Glacial Maximum, and postglacial long range expansions spreading ancestral polymorphisms, and not with a stepping‐stone model of gene flow. The divergence between breeding populations in Europe and Siberia was dated to approximately 12 000 years ago. Although genetic population structure is presently statistically non‐existent, support for evolving population structure came from analyses of geographical variation in two relevant phenotypic traits: wing length and the timing of migration. Analysis of 6077 individuals sampled on migration in 2002–08 revealed that, in each year, shorter‐winged birds migrated through significantly later than longer‐winged birds. The late‐passing birds were associated with more westerly breeding localities. In conclusion, the lack of genetic structuring in ruffs (and other inland species we examined) contrasts with strong structuring in many coastal species. This suggests that the ability to use more widely available inland habitat influences the evolution of genetic structure and the maintenance of genetic variation in waders. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 641–656.