Validação de espécies de Centropomus (Centropomidae, Perciformes) do litoral do Rio Grande do Norte através de caracterização citogenética e molecular

The Centropomidae family consists of three genera, Centropomus, Lates and Psammoperca. Centropomus is the most diverse group, with six Centropomus species occur in the Western Atlantic Ocean C. poeyi Chávez, 1961, C. parallelus Poey, 1860, C. mexicanus Bocourt, 1868, C. pectinatus Poey, 1860 and C. ensiferus Poey, 1860. Some of these species are considered cryptic, because of its morphological traits showed low resolution for identification purposes. Despite showing great interest as a natural resource and fish culture, aspects of their diversity and karyotypic patterns are poorly understood. In this work morphological identification and comparison of mitochondrial 16S gene sequence were used to identify the species of the genus Centropomus occurring in Rio Grande do Norte, northeastern Brazil. Two sepecies were identified, C. undecimalis and C. mexicanus, which had the chromosomal aspects analyzed, through Classical cytogenetic method analyzes (conventional staining, C-banding, Ag-NORs), fluorochrome staining AT- and GC-specific, replication bands by incorporating of the base analog 5-Bromo-2’-deoxyuridine (5-BrdU), in situ chromosomal mapping of (TTAGGG)n sequences and in situ chromosome mapping 18S and 5S rRNA genes. Both species show 2n=48 acrocentric chromosomes, with ribosomal sites (Ag-NOR/18S rDNA/ Mitramycin+) in second chromosomal pair, in telomeric position on the long arm in C. mexicanus and interstitial in C. undecimalis. The nuclear organization pair (pair 2) shown a resolutive cytotaxonomic marker for these two species. The generated data reveal a lower species diversity than previously believed, suggesting that greater attention should be paid in taxonomic identification of the species, in view of optimize commercial actions exploitation, biological conservation and cultivation.

Étude des interactions fonctionnelles de la tétraboucle 900 de l'ARN ribosomique 16S dans le ribosome bactérien

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Étude des interactions fonctionnelles de la tétraboucle 900 de l'ARN ribosomique 16S dans le ribosome bactérien

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

(Table S2) Relative abundance of archaeal 16S rRNA genes in sediment cores

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells (Parkes et al., 1994, doi:10.1038/371410a0; D'Hondt et al., 2004, doi:10.1126/science.1101155). Extrapolation of direct counts of stained microbial cells to the total volume of habitable marine subsurface sediments suggests that between 56 Pg (Parkes et al., 1994, doi:10.1038/371410a0) and 303 Pg (Whitman et al., 1998) of cellular carbon could be stored in this largely unexplored habitat. From recent studies using various culture-independent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem (Schippers et al., doi:10.1038/nature03302; Inagaki et al., doi:10.1073/pnas.0511033103 ; Mauclaire et al., doi:10.1111/j.1472-4677.2004.00035.x; Biddle et al., doi:10.1073/pnas.0600035103). Here we show that in subsurface sediments buried deeper than 1 m in a wide range of oceanographic settings at least 87% of intact polar membrane lipids, biomarkers for the presence of live cells (Biddle et al., doi:10.1073/pnas.0600035103; Sturt et al., 2004, doi:10.1002/rcm.1378), are attributable to archaeal membranes, suggesting that Archaea constitute a major fraction of the biomass. Results obtained from modified quantitative polymerase chain reaction and slot-blot hybridization protocols support the lipid-based evidence and indicate that these techniques have previously underestimated archaeal biomass. The lipid concentrations are proportional to those of total organic carbon. On the basis of this relationship, we derived an independent estimate of amounts of cellular carbon in the global marine subsurface biosphere. Our estimate of 90 Pg of cellular carbon is consistent, within an order of magnitude, with previous estimates, and underscores the importance of marine subsurface habitats for global biomass budgets.

Morphological and Phylogenetic Characterization of New Gephyrocapsa Isolates Suggests Introgressive Hybridization in the Emiliania/Gephyrocapsa Complex (Haptophyta)

The coccolithophore genus Gephyrocapsa contains a cosmopolitan assemblage of pelagic species, including the bloom-forming Gephyrocapsa oceanica, and is closely related to the emblematic coccolithophore Emiliania huxleyi within the Noëlaerhabdaceae. These two species have been extensively studied and are well represented in culture collections, whereas cultures of other species of this family are lacking. We report on three new strains of Gephyrocapsa isolated into culture from samples from the Chilean coastal upwelling zone using a novel flow cytometric single-cell sorting technique. The strains were characterized by morphological analysis using scanning electron microscopy and phylogenetic analysis of 6 genes (nuclear 18S and 28S rDNA, plastidial 16S and tufA, and mitochondrial cox1 and cox3 genes). Morphometric features of the coccoliths indicate that these isolates are distinct from G. oceanica and best correspond to G. muellerae. Surprisingly, both plastidial and mitochondrial gene phylogenies placed these strains within the E. huxleyi clade and well separated from G. oceanica isolates, making Emiliania appear polyphyletic. The only nuclear sequence difference, 1 bp in the 28S rDNA region, also grouped E. huxleyi with the new Gephyrocapsa isolates and apart from G. oceanica. Specifically, the G. muellerae morphotype strains clustered with the mitochondrial β clade of E. huxleyi, which, like G. muellerae, has been associated with cold (temperate and sub-polar) waters. Among putative evolutionary scenarios that could explain these results we discuss the possibility that E. huxleyi is not a valid taxonomic unit, or, alternatively the possibility of past hybridization and introgression between each E. huxleyi clade and older Gephyrocapsa clades. In either case, the results support the transfer of Emiliania to Gephyrocapsa. These results have important implications for relating morphological species concepts to ecological and evolutionary units of diversity.

Morphological and Phylogenetic Characterization of New Gephyrocapsa Isolates Suggests Introgressive Hybridization in the Emiliania/Gephyrocapsa Complex (Haptophyta)

The coccolithophore genus Gephyrocapsa contains a cosmopolitan assemblage of pelagic species, including the bloom-forming Gephyrocapsa oceanica, and is closely related to the emblematic coccolithophore Emiliania huxleyi within the Noëlaerhabdaceae. These two species have been extensively studied and are well represented in culture collections, whereas cultures of other species of this family are lacking. We report on three new strains of Gephyrocapsa isolated into culture from samples from the Chilean coastal upwelling zone using a novel flow cytometric single-cell sorting technique. The strains were characterized by morphological analysis using scanning electron microscopy and phylogenetic analysis of 6 genes (nuclear 18S and 28S rDNA, plastidial 16S and tufA, and mitochondrial cox1 and cox3 genes). Morphometric features of the coccoliths indicate that these isolates are distinct from G. oceanica and best correspond to G. muellerae. Surprisingly, both plastidial and mitochondrial gene phylogenies placed these strains within the E. huxleyi clade and well separated from G. oceanica isolates, making Emiliania appear polyphyletic. The only nuclear sequence difference, 1 bp in the 28S rDNA region, also grouped E. huxleyi with the new Gephyrocapsa isolates and apart from G. oceanica. Specifically, the G. muellerae morphotype strains clustered with the mitochondrial β clade of E. huxleyi, which, like G. muellerae, has been associated with cold (temperate and sub-polar) waters. Among putative evolutionary scenarios that could explain these results we discuss the possibility that E. huxleyi is not a valid taxonomic unit, or, alternatively the possibility of past hybridization and introgression between each E. huxleyi clade and older Gephyrocapsa clades. In either case, the results support the transfer of Emiliania to Gephyrocapsa. These results have important implications for relating morphological species concepts to ecological and evolutionary units of diversity.

Unexpected prevalence of parasite 18S rDNA sequences in winter among Antarctic marine protists

Parasites are not typically considered to be important components of polar marine ecosystems. It was therefore surprising when 18S rDNA surveys of protists in the West Antarctic Peninsula in winter revealed high abundances of parasite sequences. Parasite sequences made up, on average, over half (52%) of sequence reads in samples from deep water in winter. Winter surface water and sediment samples contained relatively fewer, but still strikingly high, parasite sequence reads (13 and 9%, respectively), while surface water samples in summer contained fewer parasite sequences (1.8%). A total of 1028 distinct parasite Operational Taxonomic Units were observed in winter, with the largest abundances and diversities within Syndiniales groups I and II, including Amoebophrya. Less abundant parasite sequence groups included Apicomplexa, Blastodinium, Chytriodinium, Cryptocaryon, Paradinium, Perkinsidae, Pirsonia and Ichthyophonae. Parasite sequence distributions suggested interactions with known hosts, such as diatom parasites which were mainly in the sediments, where resting spores of Chaetoceros spp. diatoms were abundant. Syndiniales sequences were correlated with radiolarian sequences, suggesting parasite–host interactions. The abundant proportions of parasite sequences indicate a potentially important role for parasites in the Antarctic marine ecosystem, with implications for plankton population dynamics, the role of the microbial loop, carbon flows and ecosystem responses to ongoing anthropogenic climate change.

Unexpected prevalence of parasite 18S rDNA sequences in winter among Antarctic marine protists

Parasites are not typically considered to be important components of polar marine ecosystems. It was therefore surprising when 18S rDNA surveys of protists in the West Antarctic Peninsula in winter revealed high abundances of parasite sequences. Parasite sequences made up, on average, over half (52%) of sequence reads in samples from deep water in winter. Winter surface water and sediment samples contained relatively fewer, but still strikingly high, parasite sequence reads (13 and 9%, respectively), while surface water samples in summer contained fewer parasite sequences (1.8%). A total of 1028 distinct parasite Operational Taxonomic Units were observed in winter, with the largest abundances and diversities within Syndiniales groups I and II, including Amoebophrya. Less abundant parasite sequence groups included Apicomplexa, Blastodinium, Chytriodinium, Cryptocaryon, Paradinium, Perkinsidae, Pirsonia and Ichthyophonae. Parasite sequence distributions suggested interactions with known hosts, such as diatom parasites which were mainly in the sediments, where resting spores of Chaetoceros spp. diatoms were abundant. Syndiniales sequences were correlated with radiolarian sequences, suggesting parasite–host interactions. The abundant proportions of parasite sequences indicate a potentially important role for parasites in the Antarctic marine ecosystem, with implications for plankton population dynamics, the role of the microbial loop, carbon flows and ecosystem responses to ongoing anthropogenic climate change.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis : C. parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis . In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Association of extended-spectrum β-lactamase VEB-5 and 16S rRNA methyltransferase armA in Salmonella enterica from the United Kingdom

Aminoglycosides and beta-lactams are used for the treatment of a wide range of infections due to both Gram-negative and Gram-positive. An emerging aminoglycoside resistance mechanism, methylation of the aminoacyl site of the 16S rRNA, confers high-level resistance to clinically important aminoglycosides such as amikacin, tobramycin and gentamicin. Eight 16S rRNA methyltransferase genes, armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF and npmA, have been identified in several species of enterobacteria worldwide (2, 6, 7, 9, 11, 13, 14). Resistance to extended spectrum β-lactams remains additionally an important clinical problem. Apart from the large TEM, SHV, and CTX-M families, several other extended-spectrum β-lactamases (ESBLs) have been identified, including VEB enzymes, which confer high-level resistance to cephalosporins and monobactams. Although 16S rRNA methyltransferases have been frequently identified associated with different ESBLs, there has been no report of association of a 16S rRNA methyltransferase with a VEB enzyme, except for the identification of rmtC with blaVEB-6 (14)

Phylogenetic placement of the sugarcane orange rust pathogen Puccinia kuehnii in a historical and regional context

Sugarcane orange rust, caused by Puccinia kuehnii, was once considered a minor disease in the Australian sugar industry. However, in 2000 a new race of the pathogen devastated the high-performing sugarcane cultivar Q124, and caused the industry Aus$150–210 million in yield losses. At the time of the epidemic, very little was known about the genetic and pathogenic diversity of the fungus in Australia and neighbouring sugar industries. DNA sequence data from three rDNA regions were used to determine the genetic relationships between isolates within two P. kuehnii collections. The first collection comprised only recent Australian field isolates and limited sequence variation was detected within this population. In the second study, Australian isolates were compared with isolates from Papua New Guinea, Indonesia, China and historical herbarium collections. Greater sequence variation was detected in this collection and phylogenetic analyses grouped the isolates into three clades. All isolates from commercial cane fields clustered together including the recent Australianfield isolates and the Australian historical isolate from 1898.The other two clades included rust isolates from wild and garden canes in Indonesia and PNG. These rusts appeared morphologically similar to P. kuehnii and could potentially pose a quarantine threat to the Australian sugar industry. The results have revealed greater diversity in sugarcane rusts than previously thought.

Deeply divergent mitochondrial lineages reveal patterns of local endemism in chironomids of the Australian Wet Tropics

The Wet Tropics bioregion of north-eastern Australia has been subject to extensive fluctuations in climate throughout the late Pliocene and Pleistocene. Cycles of rainforest contraction and expansion of dry sclerophyll forest associated with such climatic fluctuations are postulated to have played a major role in driving geographical endemism in terrestrial rainforest taxa. Consequences for the distributions of aquatic organisms, however, are poorly understood.The Australian non-biting midge species Echinocladius martini Cranston (Diptera: Chironomidae), although restricted to cool, well-forested freshwater streams, has been considered to be able to disperse among populations located in isolated rainforest pockets during periods of sclerophyllous forest expansion, potentially limiting the effect of climatic fluctuations on patterns of endemism. In this study, mitochondrial COI and 16S data were analysed for E. martini collected from eight sites spanning theWet Tropics bioregion to assess the scale and extent of phylogeographic structure. Analyses of genetic structure showed several highly divergent cryptic lineages with restricted geographical distributions. Within one of the identified lineages, strong genetic structure implied that dispersal among proximate (<1 km apart) streams was extremely restricted. The results suggest that vicariant processes, most likely due to the systemic drying of the Australian continent during the Plio-Pleistocene, might have fragmented historical E. martini populations and, hence, promoted divergence in allopatry.

Culture independent analysis of greyback canegrub-associated microflora and microbial community comparison using subtractive hybridisation

Greyback canegrubs cost the Australian sugarcane industry around $13 million per annum in damage and control. A novel and cost effective biocontrol bacterium could play an important role in the integrated pest management program currently in place to reduce damage and control associated costs. During the course of this project, terminal restriction fragment length polymorphism (TRFLP), 16-S rDNA cloning, suppressive subtractive hybridisation (SSH) and entomopathogen-specific PCR screening were used to investigate the little studied canegrub-associated microflora in an attempt to discover novel pathogens from putatively-diseased specimens. Microflora associated with these soil-dwelling insects was found to be both highly diverse and divergent between individual specimens. Dominant members detected in live specimens were predominantly from taxa of known insect symbionts while dominant sequences amplified from dead grubs were homologous to putativelysaprophytic bacteria and bacteria able to grow during refrigeration. A number of entomopathogenic bacteria were identified such as Photorhabdus luminescens and Pseudomonas fluorescens. Dead canegrubs prior to decomposition need to be analysed if these bacteria are to be isolated. Novel strategies to enrich putative pathogen-associated sequences (SSH and PCR screening) were shown to be promising approaches for pathogen discovery and the investigation of canegrubsassociated microflora. However, due to inter- and intra-grub-associated community diversity, dead grub decomposition and PCR-specific methodological limitations (PCR bias, primer specificity, BLAST database restrictions, 16-S gene copy number and heterogeneity), recommendations have been made to improve the efficiency of such techniques. Improved specimen collection procedures and utilisation of emerging high-throughput sequencing technologies may be required to examine these complex communities in more detail. This is the first study to perform a whole-grub analysis and comparison of greyback canegrub-associated microbial communities. This work also describes the development of a novel V3-PCR based SSH technique. This was the first SSH technique to use V3-PCR products as a starting material and specifically compare bacterial species present in a complex community.

Agglutinins from marine macroalgae of the southeastern United States

Protein extracts from 22 species of marine macroalgae from Florida and North Carolina were compared for their abilities to agglutinate sheep and rabbit erythrocytes. Protein extracts from 21 algal species agglutinated rabbit erythrocytes compared to 19 for sheep erythrocytes. However, agglutination by brown algal extracts was variable. The agglutination produced by protein extracts from Dictyota dichotoma could be blocked by addition of polyvinylpyrrolidone. Protein extracts from North Carolina macroalgae were also tested against five bacterial species. Three of these agglutinated bacterial cells. Ulva curvata and Bryopsis plumosa agglutinated all five species. Protein extracts from five species of Florida algae were tested for their effects on mitogenesis in mouse splenocytes and human lymphocytes. Gracilaria tikvahiae HBOI Strain G-5, Ulva rigida and Gracilaria verrucosa HBOI Strain G-16S stimulated mitogenesis in mouse splenocytes, while Gracilaria tikvahiae HBOI Strain G-16stimulated mitogenesis in human lymphocytes.