New insights to discriminate between Sympterygia acuta Garman 1877 and Sympterygia bonapartii Müller & Henle, 1841 (Rajidae) of the Southwest Atlantic Ocean: on the use of geometric morphometrics and spinulation patterns

Geometric‐morphometric and spinulation pattern approaches were employed to assess the discrimination of two species of Sympterygia skates in the Southwest Atlantic: Sympterygia acuta Garman 1877 and Sympterygia bonapartii Müller & Henle, 1841. Two types of variables were employed: linear morphometrics measurements (LMMs), and interlandmark distances (IlDs). The Principal Component Analysis (PCA) and the discriminant analysis (DA) do not overlap between the species. Morphometrically, S. bonapartii is characterized by a wider disc, larger pelvic fins, larger eyes, a greater distance between nostrils and between the first pair of gill slits, a greater interorbital distance between spiracles, and a wider mouth. On the other hand, S. acuta has a larger disc due to the snout size. Regarding the allometric coefficient analysis performed on the IlDs, S. bonapartii showed a positive allometry for the variables that comprise the Box‐Truss 3 (Ventral protocol), which can be related to the mouth width and the first gill slits. This growth type is consistent with those observed in the Box‐Truss 3 (Outline protocol), which is related to the disc width. It has been interpreted that both structures could develop together. In opposition, S. acuta showed isometric growth for the above‐mentioned variables; on the other hand, S. acuta showed positive allometry for all variables that defined the snout in the three protocols, indicating that the snout has a distinctive growth relative to the size through ontogeny. S. bonapartii, however, showed an isometric snout growth. The remaining variables that defined the morphogeometrical protocols displayed the same type of growth. Regarding spinulation, the thorns and dermal denticles proved to be useful to discriminate both species. Thorns of the caudal region were large and oval with smooth edges in S. bonapartii, and oval with lobed edges in S. acuta. Dermal denticles in S. acuta also presented two elongated ridges with a third small ridge between them, whereas those of S. bonapartii presented three elongated ridges of the same size.     

Dysregulation of inter‐photoreceptor retinoid‐binding protein (IRBP) after induced Müller cell disruption

Reduced expression of a ~150 kDa protein was unexpectedly observed while investigating Norrin protein in a transgenic murine model in which Müller cells can be selectively and inducibly disrupted. Isolation of this unknown protein via ion exchange and hydrophobic interaction chromatography followed by Tandem mass spectrometry identified it as Inter‐photoreceptor retinoid‐binding protein (IRBP). Significantly reduced IRBP mRNA expression was observed at the early and late stages after Müller cell disruption. IRBP protein expression was also consistently reduced to 5.7% of the control level as early as 1 week after Müller cell disruption. This down‐regulation of IRBP was accompanied by focal hyperfluorescent dots and cytotoxic N‐retinylidene‐N‐retinylethanolamine (A2E) accumulation. In vitro treatment of cone photoreceptor cell lines with conditioned medium collected from stressed Müller cells suggested that Müller cells regulated photoreceptors expression of IRBP via secreted factor(s). In vivo studies suggested that one of these secreted factors was tumour necrosis factor alpha (TNFα). These findings suggest that dysregulation of IRBP expression caused by Müller cell dysfunction may be an important early event in photoreceptor degeneration in some retinal diseases.

     

Otx2 enhances transdifferentiation of Müller cells‐derived retinal stem cells into photoreceptor‐like cells

Retinal Müller glial cells have the potential of neurogenic retinal progenitor cells, and could reprogram into retinal‐specific cell types such as photoreceptor cells. How to promote the differentiation of Müller cells into photoreceptor cells represents a promising therapy strategy for retinal degeneration diseases. This study aimed to enhance the transdifferentiation of rat Müller cells‐derived retinal stem cells (MC‐RSCs) into photoreceptor‐like cells and explore the signalling mechanism. We dedifferentiated rat Müller cells into MC‐RSCs which were infected with Otx2 overexpression lentivirus or control. The positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus was significantly higher compared to control. Furthermore, pre‐treatment with Crx siRNA, Nrl siRNA, or GSK‐3 inhibitor SB‐216763 reduced the positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus. Finally, Otx2 induced photoreceptor precursor cells were injected into subretinal space of N‐methyl‐N‐nitrosourea induced rat model of retinal degeneration and partially recovered retinal degeneration in the rats. In conclusion, Otx2 enhances transdifferentiation of MC‐RSCs into photoreceptor‐like cells and this is associated with the inhibition of Wnt signalling. Otx2 is a potential target for gene therapy of retinal degenerative diseases.     

Murine dopaminergic Müller cells restore motor function in a model of Parkinson's disease

Müller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor‐related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller‐derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinson's disease, in which mouse Müller cells with the dopaminergic phenotype were transplanted into the striatum of hemi‐parkinsonian mice generated by unilateral injection of 6‐hydroxydopamine. These cells fully decreased the apomorphine‐induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb‐use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi‐parkinsonian mice confirmed by measurement of striatal dopamine after Müller cell grafting.

     

A new species of the genusDasyatis (Elasmobranchii: Dasyatididae) from southern Japan and lectotype designation ofD. zugei

Dasyatis acutirostra sp. nov. is described on the basis of 21 specimens from the East China Sea and southern part of Japanese waters. The new species is characterized by having small eyeball (2.4–3.8% of disc width), long snout (36.9–43.9% of disc width), 129–135 pectoral radiais, 23–28 pelvic radiais, 121–139 prespine separate vertebral centra and 22–24 intestinal valve turns, the absence of oral papillae, and the presence of ventral tail fold. Lectotype and paralectotypes were designated forD. zugei (Müller et Henle, 1841). Taxonomic confusion amongD. zugei and its allied species was worked out andD. cheni Teng, 1962 was synonymized withD. zugei.     

Nerve growth factor inhibits osmotic swelling of rat retinal glial (Müller) and bipolar cells by inducing glial cytokine release

Osmotic swelling of neurons and glial cells contributes to the development of retinal edema and neurodegeneration. We show that nerve growth factor (NGF) inhibits the swelling of glial (Müller) and bipolar cells in rat retinal slices induced by barium‐containing hypoosmotic solution. NGF also reduced Müller and bipolar cell swelling in the post‐ischemic retina. On the other hand, NGF prevented the swelling of freshly isolated Müller cells, but not of isolated bipolar cells, suggesting that NGF induces a release of factors from Müller cells that inhibit bipolar cell swelling in retinal slices. The inhibitory effect of NGF on Müller cell swelling was mediated by activation of TrkA (the receptor tyrosine kinase A), but not p75^NTR, and was prevented by blockers of metabotropic glutamate, P2Y₁, adenosine A₁, and fibroblast growth factor receptors. Basic fibroblast growth factor fully inhibited the swelling of freshly isolated Müller cells, but only partially the swelling of isolated bipolar cells. In addition, glial cell line‐derived neurotrophic factor and transforming growth factor‐β1, but not epidermal growth factor and platelet‐derived growth factor, reduced the swelling of bipolar cells. Both Müller and bipolar cells displayed TrkA immunoreactivity, while Müller cells were also immunostained for p75^NTR and NGF. The data suggest that the neuroprotective effect of NGF in the retina is in part mediated by prevention of the cytotoxic glial and bipolar cell swelling.

     

Reproductive biology of the Brazilian guitarfish,Pseudobatos horkelii (Müller & Henle, 1841) from southeastern Brazil,western South Atlantic

This study provides information on the seasonal occurrence, length‐width relationship, sexual development and size at first maturity of the critically endangered Brazilian guitarfish Pseudobatos horkelii in southeastern Brazil. Samples were obtained from September 2007 to August 2009 from commercial fisheries off São Paulo State coast at depths of 10–50 m, with net mesh sizes of 150 mm in the main net and 100 mm in the cod end. A total of 143 specimens (71 males, 72 females) were analyzed. Total length (TL), disc width (DW), sex and reproductive variables were recorded for each individual. Pseudobatos horkelii presents sexual dimorphism in length‐width relationships, with females attaining larger TL and DW. Females also mature at a larger TL, at 81.1% of the maximum TL, while males mature at 67.8% of the maximum TL. No specimens were captured in winter, and only a small sample size was observed in autumn. Males captured in spring and summer presented semen in the seminal vesicle, indicating that this species may reproduce in the warmer months, as already observed for other Rhinobatidae as well as for P. horkelii in southern Brazil.     

Hydrogen peroxide modulates energy metabolism and oxidative stress in cultures of permanent human Müller cells MIO‐M1

In this study the influence of hydrogen peroxide (H₂O₂) on the redox state, NADH protein binding, and mitochondrial membrane potential in Müller cells is investigated. Cultures of permanent human Müller cells MIO‐M1 were exposed to H₂O₂ in 75 µM and 150 µM concentration for two hours. Fluorescence emission spectra and lifetimes were measured by two‐photon microscopy (excitation wavelength: 740 nm) at the mitochondria which were identified in the microscopic images by their fluorescence properties (spectra and intensity). Two hours of H₂O₂ exposure did not impair viability of MIO‐M1 cells in culture. Whereas the ratio of flavine‐ to NADH fluorescence intensity did not change under either H₂O₂ concentration, the mean lifetime was significantly different between controls, not exposed to H₂O₂, and the 150 µM H₂O₂ exposure (972 ± 63 ps vs. 1152 ± 64 ps, p = 0.014). One hour after cessation of the H₂O₂ exposure, the value retuned to that of the control (983 ± 36 ps). A hyperpolarization of the mitochondrial membrane under 150 µM H₂O₂ was found. These findings suggest a shift form free to protein‐bound NADH in mitochondria as well as a hyperpolarization of their inner membrane which could be related to an impairment of Müller cell function despite their preserved viability.

Exposure of human Müller cells to hydrogen peroxide for two hours results in a reversible change of protein binding of mitochondrial NADH upon unchanged redox ratio. The mitochondrial membrane potential is increased during exposure.     

Wnt/β‐catenin‐signaling and the formation of Müller glia‐derived progenitors in the chick retina

Müller glia can be stimulated to de‐differentiate, proliferate, and form Müller glia‐derived progenitor cells (MGPCs) that are capable of producing retinal neurons. The signaling pathways that influence the de‐differentiation of mature Müller glia and proliferation of MGPCs may include the Wnt‐pathway. The purpose of this study was to investigate how Wnt‐signaling influences the formation of MGPCs in the chick retina in vivo. In NMDA‐damaged retinas where MGPCs are known to form, we find dynamic changes in retinal levels of potential readouts of Wnt‐signaling, including dkk1, dkk3, axin2, c‐myc, tcf‐1, and cd44. We find accumulations of nuclear β‐catenin in MGPCs that peaks at 3 days and rapidly declines by 5 days after NMDA‐treatment. Inhibition of Wnt‐signaling with XAV939 in damaged retinas suppressed the formation of MGPCs, increased expression of ascl1a and decreased hes5, but had no effect upon the differentiation of progeny produced by MGPCs. Activation of Wnt‐signaling, with GSK3β‐inhibitors, in the absence of retinal damage, failed to stimulate the formation of MGPCs, whereas activation of Wnt‐signaling in damaged retinas stimulated the formation of MGPCs. In the absence of retinal damage, FGF2/MAPK‐signaling stimulated the formation of MGPCs by activating a signaling network that includes Wnt/β‐catenin. In FGF2‐treated retinas, inhibition of Wnt‐signaling reduced numbers of proliferating MGPCs, whereas activation of Wnt‐signaling failed to influence the formation of proliferating MGPCs. Our findings indicate that Wnt‐signaling is part of a network initiated by FGF2/MAPK or retinal damage, and activation of canonical Wnt‐signaling is required for the formation of proliferating MGPCs. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 983–1002, 2016     

A Proteomics Approach to Identify Candidate Proteins Secreted by Müller Glia that Protect Ganglion Cells in the Retina

The retinal Müller glial cells, can enhance the survival and activity of neurons, especially of retinal ganglion cells (RGCs), which are the neurons affected in diseases such as glaucoma, diabetes, and retinal ischemia. It has been demonstrated that Müller glia release neurotrophic factors that support RGC survival, yet many of these factors remain to be elucidated. To define these neurotrophic factors, a quantitative proteomic approach was adopted aiming at identifying neuroprotective proteins. First, the conditioned medium from porcine Müller cells cultured in vitro under three different conditions were isolated and these conditioned media were tested for their capacity to promote survival of primary adult RGCs in culture. Mass spectrometry was used to identify and quantify proteins in the conditioned medium, and osteopontin (SPP1), clusterin (CLU), and basigin (BSG) were selected as candidate neuroprotective factors. SPP1 and BSG significantly enhance RGC survival in vitro, indicating that the survival‐promoting activity of the Müller cell secretome is multifactorial, and that SPP1 and BSG contribute to this activity. Thus, the quantitative proteomics strategy identify proteins secreted by Müller glia that are potentially novel neuroprotectants, and it may also serve to identify other bioactive proteins or molecular markers.     

Feeding habits of ocellate spot skate,Okamejei kenojei (Müller & Henle, 1841), in coastal waters of Taean,Korea

The feeding habits of Okamejei kenojei were studied using 592 specimens collected in the coastal waters of Taean, Korea from April 2008 to March 2009. O. kenojei is a bottom‐feeding carnivore that consumes mainly shrimp, fishes, and crabs. Its diet also includes small quantities of amphipods, mysids, cephalopods, euphausiids, copepods, isopods, and polychaetes. The total length (TL) of individuals in this study ranged from 8.2 to 49.0 cm. Cluster analysis based on %IRI (index of relative importance) identified three size classes. Group A (< 20 cm TL) ate primarily caridean shrimp and amphipods; group B (20–30 cm TL) ate exclusively shrimp; and group C (> 30 cm TL) ate penaeoidean shrimp, fishes, and crabs. O. kenojei showed ontogenetic changes in feeding habits. Although shrimps were the primary food consumed by all size groups, the proportion of shrimp in the total diet decreased and the consumption of fishes and crabs gradually increased with the body size of O. kenojei. Size of the prey organisms also increased. Smaller individuals fed mainly on small prey, such as amphipods, mysids, and small shrimp, whereas larger individuals preferred larger prey, such as larger shrimp, fishes, and crabs. The size‐related diet breadth and the percentage of empty stomachs were significant; the diet breadth gradually increased with body size, whereas the percentage of empty stomachs decreased. Seasonal changes in the O. kenojei diet were not significant, but shrimp constituted 97.3% of the summer diet by %IRI. Seasonal changes in diet breadth and the percentage of empty stomachs were not significant.     

Hypoosmotic and glutamate‐induced swelling of bipolar cells in the rat retina: comparison with swelling of Müller glial cells

Regulation of cellular volume is of great importance to avoid changes in neuronal excitability resulting from a decrease in the extracellular space volume. We compared the volume regulation of retinal glial (Müller) and neuronal (bipolar) cells under hypoosmotic and glutamate‐stimulated conditions. Freshly isolated slices of the rat retina were superfused with a hypoosmotic solution (60% osmolarity; 4 min) or with a glutamate (1 mM)‐containing isoosmotic solution (15 min), and the size changes of Müller and bipolar cell somata were recorded. Bipolar cell somata, but not Müller cell somata, swelled under hypoosmotic conditions and in the presence of glutamate. The hypoosmotic swelling of bipolar cell somata might be mediated by sodium flux into the cells, because it was not observed under extracellular sodium‐free conditions, and was induced by activation of metabotropic glutamate receptors and sodium‐dependent glutamate transporters. The glutamate‐induced swelling of bipolar cell somata was mediated by sodium chloride flux into the cells induced by activation of NMDA‐ and non‐NMDA glutamate receptors, glutamate transporters, and voltage‐gated sodium channels. The glutamate‐induced swelling of bipolar cell somata was abrogated by adenosine and γ‐aminobutyric acid, but not by vascular endothelial growth factor and ATP. The data may suggest that Müller cells, in contrast to bipolar cells, possess endogenous mechanisms which tightly regulate the cellular volume in response to hypoosmolarity and prolonged glutamate exposure. Inhibitory retinal transmission may regulate the volume of bipolar cells, likely by inhibition of the excitatory action of glutamate.     

Development,morphology and ultrastructure of the branchial crown of Fabricia stellaris (Müller, 1774) (Polychaeta: Sabellida: Fabriciinae)

Randel, N. and Bick, A. 2011. Development, morphology and ultrastructure of the branchial crown of Fabricia stellaris (Müller, 1774) (Polychaeta: Sabellida: Fabriciinae). —Acta Zoologica (Stockholm) 93 : 409–421. Sabellidae and Serpulidae are well‐known tube‐building polychaetes with a distinctive and often spectacularly colourful branchial crown. Morphological investigations suggest that these taxa form the monophyletic clade Sabellida, with the adelphotaxa Sabellidae and Serpulidae, but the relationship between these taxa remains ambiguous. Molecular investigations have indicated that the Fabriciinae, major taxon of Sabellidae, belongs to Serpulidae, thereby making Sabellidae paraphyletic; however, morphological characters are absent to support this result. We investigate the development, anatomy and ultrastructure of the branchial crown of Fabricia stellaris (Müller, 1774), describing morphological characteristics useful not only for constructing morphological phylogenies but also for understanding the evolution of the branchial crown. The morphology of the radioles and pinnules does not differ from each other. The supporting tissue of the branchial crown consists of myoepithelial cells and a solid extracellular matrix (ECM). Both ciliated and non‐ciliated cells form the epidermal layer; ciliated cells shape the food groove. Most important is the result that radioles and pinnules within Sabellida may not be homologous, because the morphology and the branching of radioles and pinnules are completely different between Sabellinae, Fabriciinae and Serpulidae. The terms ‘primary branch’ for radioles and ‘secondary branch’ for pinnules are proposed for Fabriciinae. The phylogeny of the Sabellida is discussed.     

Eudiaptomus anatolicus n.sp. (Copepoda: Calanoida) from Turkey

Eudiaptomus anatolicus n.sp. displays similarity to E. transylvanicus (Daday, 1890) by possessing an outer marginal spine located proximally on the second exopodite segment of the male right P₅, but it differs from this closest species in the presence of a chitinous projection on the second exopodite segment of the male right P₅, and in the typical shape of the female's thoracic wings. E. transylvanicus (Daday, 1890) has three chitinous processes located on the basipodite segment of the male right P₅, whereas Eudiaptomus anatolicus n.sp. has only one small process on this part. This new species is living in lake Poyraz, which is small and shallow. Some cladoceran species, namely, Pleuroxus truncatus (O.F. Müller, 1785), Pleuroxus laevis (Sars, 1862), Pleuroxus trigonellus (O.F. Müller, 1785), Simocephalus exspinosus (Koch, 1841), Eurycercus lamellatus (O.F. Müller, 1785), Alonella excisa (Fischer, 1854), Lathonura rectirostris (O.F. Müller, 1785), Pseudochydorus globosus (Baird, 1843) and two cyclopoid copepods, Megacyclops viridis (Jurine, 1820) and Eucyclops serrulatus (Fischer, 1851) share the same habitat. This revised version was published online in July 2006 with corrections to the Cover Date.     

Morphological and Molecular Redefinition of Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and Aspidisca lynceus (Müller,1773) Ehrenberg, 1859, with Reconsideration of a “Well‐known” Euplotes Ciliate,Euplotes harpa Stein, 1859 (Ciliophora,Euplotida)

We documented the morphology, infraciliature, silverline system, and molecular data of two euplotid species isolated from China, including two populations of the poorly known Euplotes platystoma Dragesco & Dragesco‐Kernéis, 1986 and the previously well described Aspidisca lynceus (Müller, 1773 ) Ehrenberg, 1830. Based on the information available, an improved diagnosis of Euplotes platystoma is given, including: a narrow adoral zone with 44–68 membranelles, 10 frontoventral, 5 transverse, 2 left marginal and 2 caudal cirri, 11–13 dorsal kineties with 17–25 dikinetids in the mid‐dorsal row, and dorsal silverline system of the double‐eurystomus type. The Chinese population of Aspidisca lynceus closely resembles previously described populations. Phylogenetic analyses inferred from SSU rDNA sequences show that E. platystoma is closely related with E. neapolitanus, and the internal position of A. lynceus within this genus is still not robust. A reconsideration of the “well‐known” Euplotes harpa and a comparison of all SSU rDNA sequences of E. harpa in GenBank are provided. We speculate that the sequences available from GenBank under the name of E. harpa are very likely from misidentified materials, that is, the identity of the species currently associated with the SSU rDNA of this “well‐known” form in molecular databases requires further confirmation.     

Distribution Patterns and Habitat Characterization of Elmidae and Hydraenidae (Insecta: Coleoptera) in the Weidlingbach Near Vienna,Austria

A total of 870 adult and 831 larval Elmidae and Hydraenidae were caught from September 1995 to September 1996 at the Weidlingbach, a fourth order tributary of the Danube near Vienna, Austria, using a Heß sampler at 14 sampling stations from source to mouth. Elmis maugetii (Latreille ) and Riolus subviolaceus (Müller ) accounted for 66.3% of the total, whereas Hydraena gracilis (Germar ) was the most abundant Hydraenidae species. Based on head width, instars 1–6 were collected in E. maugetii, instars 2–6 in R. subviolaceus and instars 3–6 in Limnius volckmari (Panzer ) and Riolus cupreus (Müller ); from the remaining species, only instars 4–6 were sampled. E. maugetii was most abundant on coarse, moss-covered substrates (median = 22.2 mm) exposed to high water velocity (median = 69.4 cm/s; range = 6.0–117.6 cm/s); the latter was also true for R. subviolaceus although it favoured smaller sediment grain sizes (median = 10.7 mm). Sites exposed to only moderate flow and with abundant filiform algae were preferred by Esolus parallelepipedus (Müller ) and L. volckmari, whereas the Hydraenidae species, Esolus angustatus (Müller ), R. cupreus and Oulimnius tuberculatus (Müller ) were collected mostly at sites with moderate current speed and abundant moss-covered pebbles of various size. Species richness and population density increased from source to mouth. At the spring sampling site Elmidae and Hydraenidae were completely lacking.