Ediacaran metazoan reveals lophotrochozoan affinity and deepens root of Cambrian Explosion

Through exceptional preservation, we establish a phylogenetic connection between Ediacaran and Cambrian metazoans. We describe the first three-dimensional, pyritized soft tissue in Namacalathus from the Ediacaran Nama Group, Namibia, which follows the underlying form of a stalked, cup-shaped, calcitic skeleton, with six radially arranged lobes projecting into an apical opening and lateral lumens. A thick body wall and probable J-shaped gut are present within the cup, and the middle layer of the often-spinose skeleton and skeletal pores are selectively pyritized, supporting an organic-rich composition and tripartite construction with possible sensory punctae. These features suggest a total group lophotrochozoan affinity. These morphological data support molecular phylogenies and demonstrates that the origin of modern lophotrochozoan phyla, and their ability to biomineralize, had deep roots in the Ediacaran.

The Cuticles of (?) Thylacocephalan Arthropod from the Basal Choteč Event (Choteč Formation, Eifelian; Barrandian Area, Czech Republic)

Small fragments of phosphatic cuticle have been observed in dark limestone of the early Eifelian age (Choteč Formation) in the interval of the Basal Choteč Event. The cuticle is two-layered, primarily folded, with a chamber between outer and inner walls. Fragments likely represent small cuticle pieces from the margins of the carapace. The exterior of the cuticle is nearly smooth bearing irregular network of wrinkled polygons or shallow pits. Low conical mound-like to high thorn-like spines with annular structure extend from both outer and inner surface of cuticle. Wrinkled and folded bases of these spines indicate moderate flexibility of cuticle. Spines are hollow, the higher ones often with apical opening. The inner surface of carapace carries smaller spines or is nearly smooth. Chamber walls inside the cara-pace are with folds and other structures supporting stiffness of the cuticle. The internal walls of the cuticle are covered by polygonal bumps. These uniformly sized and shaped bumps are about 1 μm sized and likely represents imprints of the epithelial cells adjoined to the basal membranous layer of endocuticle.Biological affinity of cuticle fragments is unclear. They surely represent pieces of the arthropod cara-pace, the most probably a thylacocephalan. Associated fossils indicate a deeper marine environment. Bloom of prasinophytes, abundance of dacryoconarids and organophosphatic brachiopods, and striking rarity and diminutive size of other fauna indicate eutrophic conditions in a neritic sea, likely with hypoxic bottom water. Nectonic mode of life in open sea can be suggested for an animal bearing this cuticle.

Efficacy of four irrigation needles in cleaning the apical third of root canals

This study aimed to evaluate the influence of irrigation needle gauge and design, and the final root canal diameter on the apical cleaning efficacy. Twelve human mandibular incisors were used. At different stages of root canal widening (sizes 20, 30 and 40 K-files), root canals were filled with radiopaque contrast medium. Four different needles were evaluated: 23G with side opening, 22G with apical opening, 30G with side opening and 30G with apical opening. Irrigation was carried out with 2 mL distilled water. The same tooth was radiographed with a digital system several times to assess the four types of needle in those three stages of canal widening. Pre-irrigation (canals filled with contrast) and post-irrigation (canals with remaining contrast) images were submitted to digital subtraction using the Adobe Photoshop CS4 program. Pre-irrigation (filled with contrast) and subtracted (cleaned by irrigation) areas were outlined by a trained and blinded operator using the Image Tool 3.0 software. Their ratio was calculated to express the percentage of apical cleaning in each stage of canal widening (sizes 20, 30 and 40 K-files) with each of the four needles. Data obtained were subjected to one-way ANOVA and Tukey's tests. The 30G needles with side and apical opening promoted better apical cleaning at all stages of root canal widening (p<0.05). In conclusion, smaller diameter needles were more efficacious in cleaning the apical third of the root canals, regardless of their design.

Potassium excretion through ROMK potassium channel expressed in gill mitochondrion-rich cells of Mozambique tilapia

Despite recent progress in physiology of fish ion homeostasis, the mechanism of plasma K+ regulation has remained unclear. Using Mozambique tilapia, a euryhaline teleost, we demonstrated that gill mitochondrion-rich (MR) cells were responsible for K+ excretion, using a newly invented technique that insolubilized and visualized K+ excreted from the gills. For a better understanding of the molecular mechanism of K+ excretion in the gills, cDNA sequences of renal outer medullary K+ channel (ROMK), potassium large conductance Ca2+-activated channel, subfamily M (Maxi-K), K+-Cl− cotransporters (KCC1, KCC2, and KCC4) were identified in tilapia as the candidate molecules that are involved in K+ handling. Among the cloned candidate molecules, only ROMK showed marked upregulation of mRNA levels in response to high external K+ concentration. In addition, immunofluorescence microscopy revealed that ROMK was localized in the apical opening of gill MR cells, and that the immunosignals were most intense in the fish acclimated to the environment with high K+ concentration. To confirm K+ excretion via ROMK, K+ insolubilization-visualization technique was applied again in combination with K+ channel blockers. The K+ precipitation was prevented in the presence of Ba2+, indicating that ROMK has a pivotal role in K+ excretion. The present study is the first to demonstrate that the fish excrete K+ from the gill MR cells, and that ROMK expressed in the apical opening of the MR cells is a main molecular pathway responsible for K+ excretion.

Functional plasticity of mitochondrion-rich cells in the skin of euryhaline medaka larvae (Oryzias latipes) subjected to salinity changes

A noninvasive technique, the scanning ion-selective electrode technique (SIET) was applied to measure Na+ and Cl− transport by the yolk-sac skin and individual mitochondrion-rich cells (MRCs) in intact medaka larvae ( Oryzias latipes ). In seawater (SW)-acclimated larvae, significant outward Na+ and Cl− gradients were measured at the yolk-sac surface, indicating secretions of Na+ and Cl− from the yolk-sac skin. With Na+ pump immunostaining and microscopic observation, two groups of MRCs were identified on the yolk-sac skin of SW-larvae. These were single MRCs (s-MRCs), which do not have an accompanying accessory cell (AC), and multicellular complex MRCs (mc-MRCs), which usually consist of an MRC and an accompanying AC. The percentage of mc-MRC was ∼60% in 30 parts per thousand of SW, and it decreased with the decrease of external salinity. By serial SIET probing over the surface of the MRCs and adjacent keratinocytes (KCs), significant outward fluxes of Na+ and Cl− were detected at the apical opening (membrane) of mc-MRCs, whereas only outward Cl− flux, but not Na+ flux, was detected at s-MRCs. Treatment with 100 μM ouabain or bumetanide effectively blocked the Na+ and Cl− secretion. Following freshwater (FW) to SW transfer, Na+ and Cl− secretions by the yolk-sac skin were fully developed in 5 h and 2 h, respectively. In contrast, both Na+ and Cl− secretions downregulated rapidly after SW to FW transfer. Sequential probing at individual MRCs found that Na+ and Cl− secretions declined dramatically after SW to FW transfer and Na+/Cl− uptake was detected at the same s-MRCs and mc-MRCs after 5 h. This study provides evidence demonstrating that ACs are required for Na+ excretion and MRCs possess a functional plasticity in changing from a Na+/Cl−-secreting cell to a Na+/Cl−-absorbing cell.

Chloride transport in mitochondrion-rich cells of euryhaline tilapia (Oreochromis mossambicus) larvae

A noninvasive scanning ion-selective electrode technique (SIET) was applied to measure Cl− transport at individual mitochondrion-rich cells (MRCs) in the skin of euryhaline tilapia ( Oreochromis mossambicus) larvae. In seawater (SW)-acclimated larvae, outward Cl− gradients (20∼80 mM higher than the background) were measured at the surface, indicating a secretion of Cl− from the skin. By serial probing over the surface of MRCs and adjacent keratinocytes (KCs), a significant outward flux of Cl− was detected at the apical opening (membrane) of MRCs. Treatment with 100 μM ouabain or bumetanide inhibited the Cl− secretion by ∼75%. In freshwater (FW)-acclimated larvae, a lower level of outward Cl− gradients (0.2∼1 mM) was measured at the skin surface. Low-Cl− water (<0.005 mM) acclimation increased the apical Na+-Cl− cotransporter (NCC) immunoreactivity of MRCs in the larval skin. An inward flux of Cl− was detected when probing the exterior surface of a group of MRCs (convex-MRCs) that express the NCC. An NCC inhibitor (100 μM metolazone) reduced the flux by ∼90%. This study provides direct and convincing evidence for Cl− transport by MRCs of SW- and FW-acclimated euryhaline tilapia and the involvement of an apical NCC in Cl− uptake of MRCs of FW-acclimated fish.

Functional regulation of H+-ATPase-rich cells in zebrafish embryos acclimated to an acidic environment

It is important to maintain internal pH homeostasis in biological systems. In our previous studies, H+-ATPase-rich (HR) cells were found to be responsible for proton secretion in the skin of zebrafish embryos during development. In this study, zebrafish embryos were exposed to acidic and basic waters to investigate the regulation of HR cell acid secretion during pH disturbances. Our results showed that the function of HR cells on the skin of zebrafish embryos can be upregulated in pH 4 water not only by increasing the cell number but also by enlarging the acid-secreting function of single cells. We also identified an “alveolar-type” apical opening under scanning electron microscopy observations of the apical membrane of HR cells, and the density and size of the alveolar type of apical openings were also increased in pH 4 water. p63 and PCNA immunostaining results also showed that additional HR cells in pH 4 water may be differentiated not only from ionocyte precursor cells but also newly proliferating epithelial stem cells.

Glycogen phosphorylase in glycogen-rich cells is involved in the energy supply for ion regulation in fish gill epithelia

The molecular and cellular mechanisms behind glycogen metabolism and the energy metabolite translocation between mammal neurons and astrocytes have been well studied. A similar mechanism is proposed for rapid mobilization of local energy stores to support energy-dependent transepithelial ion transport in gills of the Mozambique tilapia ( Oreochromis mossambicus). A novel gill glycogen phosphorylase isoform (tGPGG), which catalyzes the initial degradation of glycogen, was identified in branchial epithelial cells of O. mossambicus. Double in situ hybridization and immunocytochemistry demonstrated that tGPGG mRNA and glycogen were colocalized in glycogen-rich cells (GRCs), which surround ionocytes (labeled with a Na+-K+-ATPase antiserum) in gill epithelia. Concanavalin-A (a marker for the apical membrane) labeling indicated that GRCs and mitochondria-rich cells share the same apical opening. Quantitative real-time PCR analyses showed that tGPGG mRNA expression levels specifically responded to environmental salinity changes. Indeed, the glycogen content, glycogen phosphorylase (GP) protein level and total activity, and the density of tGPGG-expressing cells (i.e., GRCs) in fish acclimated to seawater (SW) were significantly higher than those in freshwater controls. Short-term acclimation to SW caused an evident depletion in the glycogen content of GRCs. Taken altogether, tGPGG expression in GRCs is stimulated by hyperosmotic challenge, and this may catalyze initial glycogen degradation to provide the adjacent ionocytes with energy to carry out iono- and osmoregulatory functions.

Metanotal gland of the genus Eidmanacris (Grylloidea, Phalangopsidae): taxonomic importance

The present study compares the metanotal gland through scanning electron microscopy in five species of Eidmanacris: E. corumbatai Garcia, 1998, E. alboannulata (Piza, 1960), E. dissimilis Desutter-Grandcolas, 1995, E. larvaeformis (Chopard, 1938) and Eidmanacris sp. The general external configuration of the gland was determined by the presence of two median projections with apical opening and a cluster of bristles just above these projections. Although there is a general pattern for this gland, each species has its own pattern, which can be defined mainly by the arrangement of the bristles and the position of the median projections. Our results suggest the taxonomical importance of these structures, which should be better analyzed when describing species of the genus Eidmanacris. In addition, while observing the reproductive behavior of these species, we concluded that the release of this gland secretion is important for the success of mating.