A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome

Oocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological. criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

Production of Bovine Hand-Made Cloned Embryos by Zygote-Oocyte Cytoplasmic Hemi-complementation

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; handmade cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

Single embryo and oocyte lipid fingerprinting by mass spectrometry

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid ( represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species.-Ferreira, C. R., S. A. Saraiva, R. R. Catharino, J. S. Garcia, F. C. Gozzo, G. B. Sanvido, L. F. A. Santos, E. G. Lo Turco, J. H. F. Pontes, A. C. Basso, R. P. Bertolla, R. Sartori, M. M. Guardieiro, F. Perecin, F. V. Meirelles, J. R. Sangalli, and M. N. Eberlin. Single embryo and oocyte lipid fingerprinting by mass spectrometry. J. Lipid Res. 2010. 51: 1218-1227.

Myxobolus cordeiroi n. sp., a parasite of Zungaro jahu (Siluriformes: Pimelodiade) from Brazilian Pantanal: Morphology, phylogeny and histopathology

This work is part of an ongoing investigation into the characteristics of Myxozoan parasites of freshwater fish in Brazil and was carried out using morphology, histopathology and molecular analysis. A new Myxosporea species (Myxobolus cordeiroi) is described infecting the jau catfish (Zungaro jahu). Fifty jau specimens were examined and 78% exhibited plasmodia of the parasite. The plasmodia were white and round, measuring 0.3-2.0 mm in diameter and the development occurred in the gill arch, skin, serosa of the body cavity, urinary bladder and eye. The spores had an oval body and the spore wall was smooth. Partial sequencing of the 18S rDNA gene resulted in a total of 505 bp and the alignment of the sequences obtained from samples in different organs revealed 100% identity. In the phylogenetic analysis, the Myxobolus species clustered into two clades-one primarily parasites of freshwater fish and the other primarily parasites of marine fish. M. cordeiroi n. sp. was clustered in a basal position in the freshwater fish species clade. The histological analysis revealed the parasite in the connective tissue of the different infected sites, thereby exhibiting affinity to this tissue. The plasmodium was surrounded by an outer collagen capsule of fibers with distinct orientation from the adjacent connective tissue and an inner layer composed of delicate collagen fibrils-more precisely reticular fibers. The development of the parasite in the cornea and urinary bladder caused considerable stretching of the epithelium. (C) 2009 Elsevier B.V. All rights reserved.

Influence of nitric oxide during maturation on bovine oocyte meiosis and embryo development in vitro

The effect of s-nitroso-N-acetyl-1,1-penicillamine (SNAP, a nitric oxide donor) during in vitro maturation (IVM) on nuclear maturation and embryo development was investigated. The effect of increasing nitric oxide (NO) during prematuration or maturation, or both, on embryo development was also assessed. 10(-3) M SNAP nearly blocked oocytes reaching metaphase II (MII) (7%, P < 0.05) while 10(-5) M SNAP showed intermediate proportions (55%). For 10(-7) M SNAP and controls (without SNAP), MII percentages were similar (72% for both, P > 0.05), but superior to the other treatment groups (P < 0.05). Blastocyst development, however, was not affected (38% for all treatments, P < 0.05). TUNEL-positive cells in hatched blastocysts (Day 9) increased when IVM included 10(-5) M SNAP (8 v. 3 to 4 cells in the other treatments, P > 0.05), without affecting total cell numbers (240 to 291 cells, P > 0.05). When oocytes were prematured followed by IVM with or without 10(-7) M SNAP, during either culture period or both, blastocyst development was similar (26 to 40%, P > 0.05). When SNAP was included during both prematuration and IVM, the proportion of Day 9 hatched embryos increased (28% v. 14 to 19% in the other treatments, P < 0.05). Apoptotic cells, however, increased when SNAP was included (6 to 10 cells) in comparison to prematuration and maturation without SNAP (3 cells, P < 0.05). NO may be involved in meiotic progression and apoptosis during embryo development.

Host-parasite-environment relationship, morphology and molecular analyses of Henneguya eirasi n. sp parasite of two wild Pseudoplatystoma spp. in Pantanal Wetland, Brazil

A new myxosporean species, Henneguya eirasi n. sp., is described parasitizing the gill filaments of Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum (Siluriformes: Pimelodidae) caught in the Patanal Wetland of the state of Mato Grosso, Brazil. The parasite formed white, elongated plasmodia measuring up to 3 mm. Mature spores were ellipsoidal in the frontal view, measuring 37.1 +/- 1.8 mu m in total length, 12.9 +/- 0.8 mu m in body length, 3.4 +/- 0.3 mu m in width, 3.1 +/- 0.1 mu m in thickness and 24.6 +/- 2.2 mu m in the caudal process. Polar capsules were elongated and equal in size, measuring 5.4 +/- 0.5 mu m in length and 0.7 +/- 0.1 mu m in width. Polar filaments had 12-13 coils. Histopathological analysis revealed that the parasite developed in the sub-epithelial connective tissue of the gill filaments and the plasmodia were surrounded by a capsule of host connective tissue. The plasmodia caused slight compression of the adjacent tissues, but no inflammatory response was observed in the infection site. Ultrastructure analysis revealed a single plasmodial wall connected to the ectoplasmic zone through numerous pinocytotic canals. The plasmodial wall exhibited numerous projections and slightly electron-dense material was found in the ectoplasm next to the plasmodial wall, forming a line just below the wall. Partial sequencing of the 18S rDNA gene of H. eirasi n. sp. obtained from P. fasciatum resulted in a total of 1066 bp and this sequence did not match any of the Myxozoa available in the GenBank. Phylogenetic analysis revealed the Henneguya species clustering into clades following the order and family of the host fishes. H. eirasi n. sp. clustered alone in one clade, which was the basal unit for the clade composed of Henneguya species parasites of siluriform ictalurids. The prevalence of the parasite was 17.1% in both fish species examined. Parasite prevalence was not influenced by season, host sex or host size. (C) 2011 Elsevier B.V. All rights reserved.

Phylogenetic analysis of the Brazilian microendemic Paepalanthus subgenus Xeractis (Eriocaulaceae) inferred from morphology

Paepalanthus subgenus Xeractis (Eriocaulaceae) comprises 28 recognized species endemic to the Espinhaco Range, in Minas Gerais state, Brazil. Most species of the subgenus are restricted to small localities and critically endangered, but still in need of systematic study. The monophyly of the subgenus has already been tested, but only with a few species. Our study presents the first phylogenetic hypothesis within the group, based on morphology. A maximum parsimony analysis was conducted on a matrix of 30 characters for 30 terminal taxa, including all species of the subgenus and two outgroups. The biogeographical hypotheses for the subgenus were inferred based on dispersal-vicariance analysis (DIVA). The analysis provided one most-parsimonious hypothesis that supports most of the latest published subdivisions (sections and series). However, some conflicts remain concerning the position of a few species and the relationships between sections. The distribution and origin(s) of microendemism are also discussed, providing the ground for conservation strategies to be developed in the region. (C) 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167, 137-152.

Comparative leaf anatomy and morphology of some neotropical Rutaceae: Pilocarpus Vahl and related genera

Previous anatomical studies have been restricted to the foliar aspects of Pilocarpus. However, no anatomical studies analyzing the foliar aspects of Pilocarpus in relation to related genera have been carried out. Therefore, the aim of this study was to identify characters for future taxonomic and phylogenetic studies in Rutaceae, particularly in Pilocarpus, and to discuss the characteristics associated with the simple or compound leaf condition for the group. The petiole and the leaf blade of 14 neotropical Rutaceae species were analyzed, and the following characteristics were observed in all leaves studied: stomata on both surfaces; secretory cavities, including mesophyll type; camptodromous-brochidodromous venation pattern; and free vascular cylinder in the basal region of the petiole. Additional promising characters were identified for future taxonomic and phylogenetic studies in the Rutaceae family, especially for the Pilocarpus genera.

Placement of Kuhlmanniodendron Fiaschi & Groppo in Lindackerieae (Achariaceae, Malpighiales) confirmed by analyses of rbcL sequences, with notes on pollen morphology and wood anatomy

The phylogenetic placement of Kuhlmanniodendron Fiaschi & Groppo (Achariaceae) within Malpighiales was investigated with rbcL sequence data. This genus was recently created to accommodate Carpotroche apterocarpa Kuhlm., a poorly known species from the rainforests of Espirito Santo, Brazil. One rbcL sequence was obtained from Kuhlmanniodendron and analyzed with 73 additional sequences from Malpighiales, and 8 from two closer orders, Oxalidales and Celastrales, all of which were available at Genbank. Phylogenetic analyses were carried out with maximum parsimony and Bayesian inference; bootstrap analyses were used in maximum parsimony to evaluate branch support. The results confirmed the placement of Kuhlmanniodendron together with Camptostylus, Lindackeria, Xylotheca, and Caloncoba in a strongly supported clade (posterior probability = 0.99) that corresponds with the tribe Lindackerieae of Achariaceae (Malpighiales). Kuhlmanniodendron also does not appear to be closely related to Oncoba (Salicaceae), an African genus with similar floral and fruit morphology that has been traditionally placed among cyanogenic Flacourtiaceae (now Achariaceae). A picrosodic paper test was performed in herbarium dry leaves, and the presence of cyanogenic glycosides, a class of compounds usually found in Achariaceae, was detected. Pollen morphology and wood anatomy of Kuhlmanniodendron were also investigated, but both pollen (3-colporate and microreticulate) and wood, with solitary to multiple vessels, scalariform perforation plates and other features, do not seem to be useful to distinguish this genus from other members of the Achariaceae and are rather common among the eudicotyledons as a whole. However, perforated ray cells with scalariform plates, an uncommon wood character, present in Kuhlmanniodendron are similar to those found in Kiggelaria africana (Pangieae, Achariaceae), but the occurrence of such cells is not mapped among the angiosperms, and it is not clear how homoplastic this character could be.

Morphology and anatomy of inflorescence and inflorescence axis in Paepalanthus sect. Diphyomene Ruhland (Eriocaulaceae, Poales) and its taxonomic implications

Paepalanthus sect. Diphyomene has inflorescences arranged in umbels. The underlying bauplan seems however to be more complex and composed of several distinct subunits. Despite appearing superficially very similar, the morphology and anatomy of the inflorescences can supply useful information for the understanding of the phylogeny and taxonomy of the group. Inflorescences of Paepalanthus erectifolius, Paepalanthus flaccidus, Paepalanthus giganteus, and Paepalanthus polycladus were analyzed in regard to branching pattern and anatomy. In P. erectifolius, P. giganteus and P. polycladus the structure is a tribotryum, with terminal dibotryum, and with pherophylls bearing lateral dibotrya. In P. flaccidus, the inflorescence is a pleiobotryum, with terminal subunit, and without pherophylls. Secondary inflorescences may occur in all species without regular pattern. Especially when grown in sites without a pronounced seasonality, the distinction between enrichment zone (part of the same inflorescence) and new inflorescences may be obscured. The main anatomical features supplying diagnostic and phylogenetic information are as follows: (a) in the elongated axis, the thickness of the epidermal cell walls and the cortex size; (b) in the bracts, the quantity of parenchyma cells (c) in the scapes, the shape and the presence of a pith tissue. Therefore, P. sect. Diphyomene can be divided in two groups; group A is represented by P. erectifolius, P. giganteus and P. polycladus, and group B is represented by P. flaccidus. The differentiation is based in both, inflorescence structure and anatomy. Group A presents a life cycle and anatomical features similar to species of Actinocephalus. Molecular trees also point that these two groups are closely related. However, inflorescence morphology and blooming sequence are different. Species of group B present an inflorescence structure and anatomical features shared with many genera and species in Eriocaulaceae. The available molecular and morphology based phylogenies still do not allow a precise allocation of the group in the bulk of basal species of Paepalanthus collocated in P. sect. Variabiles. The characters described and used here supply however important information towards this goal. (C) 2009 Elsevier GmbH. All rights reserved.

Paleoamerican Morphology in the Context of European and East Asian Late Pleistocene Variation: Implications for Human Dispersion Into the New World

Early American crania show a different morphological pattern from the one shared by late Native Americans. Although the origin of the diachronic morphological diversity seen on the continents is still debated, the distinct morphology of early Americans is well documented and widely dispersed. This morphology has been described extensively for South America, where larger samples are available. Here we test the hypotheses that the morphology of Early Americans results from retention of the morphological pattern of Late Pleistocene modern humans and that the occupation of the New World precedes the morphological differentiation that gave rise to recent Eurasian and American morphology. We compare Early American samples with European Upper Paleolithic skulls, the East Asian Zhoukoudian Upper Cave specimens and a series of 20 modern human reference crania. Canonical Analysis and Minimum Spanning Tree were used to assess the morphological affinities among the series, while Mantel and Dow-Cheverud tests based on Mahalanobis Squared Distances were used to test different evolutionary scenarios. Our results show strong morphological affinities among the early series irrespective of geographical origin, which together with the matrix analyses results favor the scenario of a late morphological differentiation of modern humans. We conclude that the geographic differentiation of modern human morphology is a late phenomenon that occurred after the initial settlement of the Americas. Am J Phys Anthropol 144:442-453, 2011. (c) 2010 Wiley-Liss, Inc.

Morphology, molecular phylogeny, and taxonomic inconsistencies in the study of Bradypus sloths (Pilosa: Bradypodidae)

This study focuses on morphological and molecular data analyses, misidentifications, and phylogenetic inconsistencies regarding Bradypus variegatus (the brown-throated sloth) and B. tridactylus (the pale-throated sloth). Misidentifications were recorded on 75 of 313 museum specimens of Bradypus. Almost 90% of the misidentified specimens were B. variegatus from north-central Brazil, erroneously attributed to B. tridactylus. These misidentified specimens are reported in taxonomic reviews as the southernmost records of B. tridactylus. A history of confusing nomenclature regarding sloth species exists, and these particular misidentifications could be attributable to the similarity in face and throat color between B. variegatus from north-central Brazil and B. tridactylus. The molecular phylogeny of morphologically confirmed sloth specimens exhibits 2 monophyletic lineages representing B. variegatus and B. tridactylus. The split time between these 2 lineages was estimated at 6 million years ago (mya), contradicting previous studies that estimated this divergence to be 0.4 mya. Taxonomic inconsistencies were detected when comparing the molecular phylogeny to previously published DNA sequences ascribed to B. tridactylus. Misidentification or introgression could underlie such phylogenetic incongruities. Regardless of their causes, these discrepancies lead to misstatements regarding geographic distribution, phylogeny, and taxonomy of B. variegatus and B. tridactylus.

Phylogeny of the Neotropical genus Acestrorhynchus (Ostariophysi: Characiformes) based on nuclear and mitochondrial gene sequences and morphology: A total evidence approach

Acestrorhynchus is the sole genus of the family Acestrorhynchidae which includes 14 species currently recognized as valid. Species of Acestrorhynchus comprise small-to-medium sized piscivorous fishes and have been traditionally grouped on the basis of well-defined color patterns. A recent phylogeny, based on morphological characters, could not resolve the phylogenetic affinities of A. heterolepis and the relationships among the species of the clade formed by A. abbreviatus, A. altus, A. falcatus, A. lacustris, and A. pantaneiro. The simultaneous analysis of two mitochondrial genes (16S and ATP synthase subunits 6 and 8) and one nuclear intron (S7) was able to resolve the latter clade, but the position of A. heterolepis remained unresolved. The combination of the molecular and morphological data sets in a total evidence analysis resulted in a well-resolved hypothesis regarding the phylogenetic relationships of Acestrorhynchus species. (C) 2009 Elsevier Inc. All rights reserved.

Morphology and phylogenetic relationships of a remarkable new genus and two new species of Neotropical freshwater stingrays from the Amazon basin (Chondrichthyes: Potamotrygonidae)

The morphology and phylogenetic relationships of a new genus and two new species of Neotropical freshwater stingrays, family Potamotrygonidae, are investigated and described in detail. The new genus, Heliotrygon, n. gen., and its two new species, Heliotrygon gomesi, n. sp. (type-species) and Heliotrygon rosai, n. sp., are compared to all genera and species of potamotrygonids, based on revisions in progress. Some of the derived features of Heliotrygon include its unique disc proportions (disc highly circular, convex anteriorly at snout region, its width and length very similar), extreme subdivision of suborbital canal (forming a complex honeycomb-like pattern anterolaterally on disc), stout and triangular pelvic girdle, extremely reduced caudal sting, basibranchial copula with very slender and acute anterior extension, and precerebral and frontoparietal fontanellae of about equal width, tapering very little posteriorly. Both new species can be distinguished by their unique color patterns: Heliotrygon gomesi is uniform gray to light tan or brownish dorsally, without distinct patterns, whereas Heliotrygon rosai is characterized by numerous white to creamy-white vermiculate markings over a light brown, tan or gray background color. Additional proportional characters that may further distinguish both species are also discussed. Morphological descriptions are provided for dermal denticles, ventral lateral-line canals, skeleton, and cranial, hyoid and mandibular muscles of Heliotrygon, which clearly corroborate it as the sister group of Paratrygon. Both genera share numerous derived features of the ventral lateral-line canals, neurocranium, scapulocoracoid, pectoral basals, clasper morphology, and specific patterns of the adductor mandibulae and spiracularis medialis muscles. Potamotrygon and Plesiotrygon are demonstrated to share derived characters of their ventral lateral-line canals, in addition to the presence of angular cartilages. Our morphological phylogeny is further corroborated by a molecular phylogenetic analysis of cytochrome b based on four sequences (637 base pairs in length), representing two distinct haplotypes for Heliotrygon gomesi. Parsimony analysis produced a single most parsimonious tree revealing Heliotrygon and Paratrygon as sister taxa (boot-strap proportion of 70%), which together are the sister group to a clade including Plesiotrygon and species of Potamotrygon. These unusual stingrays highlight that potamotrygonid diversity, both in terms of species composition and undetected morphological and molecular patterns, is still poorly known.

Ontogenetic Changes in the Bell Morphology and Kinematics and Swimming Behavior of Rowing Medusae: the Special Case of the Limnomedusa Liriope tetraphylla

Swimming animals may experience significant changes in the Reynolds number (Re) of their surrounding fluid flows throughout ontogeny. Many medusae experience Re environments with significant viscous forces as small juveniles but inertially dominated Re environments as adults. These different environments may affect their propulsive strategies. In particular, rowing, a propulsive strategy with ecological advantages for large adults, may be constrained by viscosity for small juvenile medusae. We examined changes in the bell morphology and swimming kinematics of the limnomedusa Liriope tetraphylla at different stages of development. L. tetraphylla maintained an oblate bell (fineness ratio approximate to 0.5-0.6), large velar aperture ratio (R(v) approximate to 0.5-0.8), and rapid bell kinematics throughout development. These traits enabled it to use rowing propulsion at all stages except the very smallest sizes observed (diameter = 0.14 cm). During the juvenile stage, very rapid bell kinematics served to increase Re sufficiently for rowing propulsion. Other taxa that use rowing propulsion as adults, such as leptomedusae and scyphomedusae, typically utilize different propulsive strategies as small juveniles to function in low Re environments. We compared the performance values of the different propulsive modes observed among juvenile medusae.