Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Ornithodoros peropteryx (Acari: Argasidae) in Bolivia: an argasid tick with a single nymphal stage

By the end of the 1960s, the argasid tick Ornithodoros peropteryx was described from larval specimens collected from the bat Peropteryx macrotis in Colombia. Since its original description, no additional record of O. peropteryx has been reported, and its post-larval stages have remained unknown. During July 2010, 18 larvae were collected from 9 bats (Centronycteris maximiliani), resulting in a mean infestation of 2.0 ± 2.2 ticks per bat (range 1–8). These bats were captured in a farm in northeastern Bolivia close to Guaporé River in the border with Brazil. Morphological examinations of the larvae revealed them to represent the species O. peropteryx. One engorged larva that was kept alive in the laboratory moulted to a nymph after 9 days. Fourteen days after the larval moulting, the nymph moulted to an adult female without taking any blood meal during the nymphal period. This adult female was used for a morphological description of the female stage of O. peropteryx. In addition, the larvae were used for a morphological redescription of this stage. One larva and two legs extirpated from the adult female were submitted to DNA extraction and PCR targeting a fragment of the mitochondrial 16S rDNA gene, which yielded DNA sequences at least 11 % divergent from any available argasid sequence in Genbank. We show that O. peropteryx ontogeny is characterized by a single, non-feeding, nymphal stage. This condition has never been reported for ticks.

The phylogeography of trypanosomes from South American alligatorids and African crocodilids is consistent with the geological history of South American river basins and the transoceanic dispersal of Crocodylus at the Miocene

Abstract Background Little is known about the diversity, phylogenetic relationships, and biogeography of trypanosomes infecting non-mammalian hosts. In this study, we investigated the influence of host species and biogeography on shaping the genetic diversity, phylogenetic relationship, and distribution of trypanosomes from South American alligatorids and African crocodilids. Methods Small Subunit rRNA (SSU rRNA) and glycosomal Glyceraldehyde Phosphate Dehydrogenase (gGAPDH) genes were employed for phylogenetic inferences. Trypanosomes from crocodilians were obtained by haemoculturing. Growth behaviour, morphology, and ultrastructural features complement the molecular description of two new species strongly supported by phylogenetic analyses. Results The inferred phylogenies disclosed a strongly supported crocodilian-restricted clade comprising three subclades. The subclade T. grayi comprised the African Trypanosoma grayi from Crocodylus niloticus and tsetse flies. The subclade T. ralphi comprised alligatorid trypanosomes represented by Trypanosoma ralphi n. sp. from Melanosuchus niger, Caiman crocodilus and Caiman yacare from Brazilian river basins. T. grayi and T. ralphi were sister subclades. The basal subclade T. terena comprised alligatorid trypanosomes represented by Trypanosoma terena n. sp. from Ca. yacare sharing hosts and basins with the distantly genetic related T. ralphi. This subclade also included the trypanosome from Ca. crocodilus from the Orinoco basin in Venezuela and, unexpectedly, a trypanosome from the African crocodilian Osteolaemus tetraspis. Conclusion The close relationship between South American and African trypanosomes is consistent with paleontological evidence of recent transoceanic dispersal of Crocodylus at the Miocene/Pliocene boundaries (4–5 mya), and host-switching of trypanosomes throughout the geological configuration of South American hydrographical basins shaping the evolutionary histories of the crocodilians and their trypanosomes.

Systematics of the Oswaldoi Complex (Anopheles, Nyssorhynchus) in South America

Abstract Background Effective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex. Methods DNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies. Results Separate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate. Conclusions: Through novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.

Endophytic fungi from the Amazonian plant Paullinia cupana and from Olea europaea isolated using cassava as an alternative starch media source

Endophytic fungi live inside plants, apparently do not cause any harm to their hosts and may play important roles in defense and growth promotion. Fungal growth is a routine practice at microbiological laboratories, and the Potato Dextrose Agar (PDA) is the most frequently used medium because it is a rich source of starch. However, the production of potatoes in some regions of the world can be costly. Aiming the development of a new medium source to tropical countries, in the present study, we used leaves from the guarana (a tropical plant from the Amazon region) and the olive (which grows in subtropical and temperate regions) to isolate endophytic fungi using PDA and Manihot Dextrose Agar (MDA). Cassava (Manihot esculenta) was evaluated as a substitute starch source. For guarana, the endophytic incidence (EI) was 90% and 98% on PDA and MDA media, respectively, and 65% and 70% for olive, respectively. The fungal isolates were sequenced using the ITS- rDNA region. The fungal identification demonstrated that the isolates varied according to the host plant and media source. In the guarana plant, 13 fungal genera were found using MDA and six were found using PDA. In the olive plant, six genera were obtained using PDA and 4 were obtained using MDA. The multivariate analysis results demonstrated the highest fungal diversity from guarana when using MDA medium. Interestingly, some genera were isolated from one specific host or in one specific media, suggesting the importance of these two factors in fungal isolation specificity. Thus, this study indicated that cassava is a feasible starch source that could serve as a potential alternative medium to potato medium.

The phylogeography of trypanosomes from South American alligatorids and African crocodilids is consistent with the geological history of South American river basins and the transoceanic dispersal of Crocodylus at the Miocene

Background: Little is known about the diversity, phylogenetic relationships, and biogeography of trypanosomes infecting non-mammalian hosts. In this study, we investigated the influence of host species and biogeography on shaping the genetic diversity, phylogenetic relationship, and distribution of trypanosomes from South American alligatorids and African crocodilids. Methods: Small Subunit rRNA (SSU rRNA) and glycosomal Glyceraldehyde Phosphate Dehydrogenase (gGAPDH) genes were employed for phylogenetic inferences. Trypanosomes from crocodilians were obtained by haemoculturing. Growth behaviour, morphology, and ultrastructural features complement the molecular description of two new species strongly supported by phylogenetic analyses. Results: The inferred phylogenies disclosed a strongly supported crocodilian-restricted clade comprising three subclades. The subclade T. grayi comprised the African Trypanosoma grayi from Crocodylus niloticus and tsetse flies. The subclade T. ralphi comprised alligatorid trypanosomes represented by Trypanosoma ralphi n. sp. From Melanosuchus niger, Caiman crocodilus and Caiman yacare from Brazilian river basins. T. grayi and T. ralphi were sister subclades. The basal subclade T. terena comprised alligatorid trypanosomes represented by Trypanosoma terena n. sp. from Ca. yacare sharing hosts and basins with the distantly genetic related T. ralphi. This subclade also included the trypanosome from Ca. crocodilus from the Orinoco basin in Venezuela and, unexpectedly, a trypanosome from the African crocodilian Osteolaemus tetraspis. Conclusion: The close relationship between South American and African trypanosomes is consistent with paleontological evidence of recent transoceanic dispersal of Crocodylus at the Miocene/Pliocene boundaries (4–5 mya), and host-switching of trypanosomes throughout the geological configuration of South American hydrographical basins shaping the evolutionary histories of the crocodilians and their trypanosomes.

Deltocyathiidae, an early-diverging family of Robust corals (Anthozoa, Scleractinia)

Over the last decade, molecular phylogenetics has called into question some fundamental aspects of coral systematics. Within the Scleractinia, most families composed exclusively by zooxanthellate species are polyphyletic on the basis of molecular data, and the second most speciose coral family, the Caryophylliidae (most members of which are azooxanthellate), is an unnatural grouping. As part of the process of resolving taxonomic affinities of caryophylliids', here a new Robust' scleractinian family (Deltocyathiidae fam. n.) is proposed on the basis of combined molecular (CO1 and 28S rDNA) and morphological data, accommodating the early-diverging clade of traditional caryophylliids (represented today by the genus Deltocyathus). Whereas this family captures the full morphological diversity of the genus Deltocyathus, one species, Deltocyathus magnificus, is an outlier in terms of molecular data, and groups with the Complex coral family Turbinoliidae. Ultrastructural data, however, place D.magnificus within Deltocyathiidae fam. nov. Unfortunately, limited ultrastructural data are as yet available for turbinoliids, but D.magnificus may represent the first documented case of morphological convergence at the microstructural level among scleractinian corals. Marcelo V.Kitahara, Centro de Biologia Marinha, Universidade de SAo Paulo, SAo SebastiAo, S.P. 11600-000, Brazil. E-mail:kitahara@usp.br

Trypanosoma livingstonei: a new species from African bats supports the bat seeding hypothesis for the Trypanosoma cruzi clade

BACKGROUND: Bat trypanosomes have been implicated in the evolutionary history of the T. cruzi clade, which comprises species from a wide geographic and host range in South America, Africa and Europe, including bat-restricted species and the generalist agents of human American trypanosomosis T. cruzi and T. rangeli. METHODS: Trypanosomes from bats (Rhinolophus landeri and Hipposideros caffer) captured in Mozambique, southeast Africa, were isolated by hemoculture. Barcoding was carried out through the V7V8 region of Small Subunit (SSU) rRNA and Fluorescent Fragment Length barcoding (FFLB). Phylogenetic inferences were based on SSU rRNA, glyceraldehyde phosphate dehydrogenase (gGAPDH) and Spliced Leader (SL) genes. Morphological characterization included light, scanning and transmission electron microscopy. RESULTS: New trypanosomes from bats clustered together forming a clade basal to a larger assemblage called the T. cruzi clade. Barcoding, phylogenetic analyses and genetic distances based on SSU rRNA and gGAPDH supported these trypanosomes as a new species, which we named Trypanosoma livingstonei n. sp. The large and highly polymorphic SL gene repeats of this species showed a copy of the 5S ribosomal RNA into the intergenic region. Unique morphological (large and broad blood trypomastigotes compatible to species of the subgenus Megatrypanum and cultures showing highly pleomorphic epimastigotes and long and slender trypomastigotes) and ultrastructural (cytostome and reservosomes) features and growth behaviour (when co-cultivated with HeLa cells at 37°C differentiated into trypomastigotes resembling the blood forms and do not invaded the cells) complemented the description of this species. CONCLUSION: Phylogenetic inferences supported the hypothesis that Trypanosoma livingstonei n. sp. diverged from a common ancestral bat trypanosome that evolved exclusively in Chiroptera or switched at independent opportunities to mammals of several orders forming the clade T. cruzi, hence, providing further support for the bat seeding hypothesis to explain the origin of T. cruzi and T. rangeli.

Ornithodoros guaporensis (Acari, Ixodida: Argasidae), a new tick species from the Guaporé River Basin in the Bolivian Amazon

The soft tick Ornithodoros guaporensis n. sp. (Acari: Ixodida: Argasidae) is described from larvae and adults. Morphological analysis and 16S rDNA sequences are provided. Adults were collected from a rocky fissure inhabited by bats located in the Amazonian forest in north-eastern Bolivia (Beni Department) close to the Guaporé River. Larvae were obtained from eggs laid by females collected in the field, and which were fed on rabbits in the laboratory. Larvae of O. guaporensis are morphologically closely related to Ornithodoros rioplatensis, Ornithodoros puertoricensis and Orni-thodoros talaje. Larvae of O. guaporensis and O. rioplatensis can be separated from O. puertoricensis and O. talaje by the number of pairs of dorsal setae (20 in O. guaporensis and O. rioplatensis, 18 in O. puertoricensis and 17 in O. talaje). Larvae of O. guaporensis and O. rioplatensis can be differentiated by the medial dental formula (2/2 in O. guaporensis and 3/3 in O. rioplatensis) and the apex of the hypostome, which is more pointed in O. rioplatensis than in O. guaporensis. The Principal Component Analysis performed with morphometric characters of larvae showed a clear separation among O. guaporensis, O. rioplatensis, O. puertoricensis and O. talaje. Significant morphological differences among adults of these four species were not found. The analysis of the 16S rDNA sequences allowed for the differentiation between O. guaporensis and the remaining Neotropical species of the family Argasidae.

Ornithodoros peropteryx (Acari Argasidae) in Bolivia an argasid tick with a single nymphal stage

By the end of the 1960s, the argasid tick Ornithodoros peropteryx was described from larval specimens collected from the bat Peropteryx macrotis in Colombia. Since its original description, no additional record of O. peropteryx has been reported, and its post-larval stages have remained unknown. During July 2010, 18 larvae were collected from 9 bats (Centronycteris maximiliani), resulting in a mean infestation of 2.0 ± 2.2 ticks per bat (range 1–8). These bats were captured in a farm in northeastern Bolivia close to Guapore´ River in the border with Brazil. Morphological examinations of the larvae revealed them to represent the species O. peropteryx. One engorged larva that was kept alive in the laboratory moulted to a nymph after 9 days. Fourteen days after the larval moulting, the nymph moulted to an adult female without taking any blood meal during the nymphal period. This adult female was used for a morphological description of the female stage of O. peropteryx. In addition, the larvae were used for a morphological redescription of this stage. One larva and two legs extirpated from the adult female were submitted to DNA extraction and PCR targeting a fragment of the mitochondrial 16S rDNA gene, which yielded DNA sequences at least 11 % divergent from any available argasid sequence in Genbank. We show that O. peropteryx ontogeny is characterized by a single, non-feeding, nymphal stage. This condition has never been reported for ticks.

Metodi molecolari nello studio della variabilità genetica di Tuber borchii

Tuber borchii (Ascomycota, order Pezizales) is highly valued truffle sold in local markets in Italy. Despite its economic importance, knowledge on its distribution and population variation is scarce. The objective of this work was to investigate the evolutionary forces shaping the genetic structure of this fungus using coalescent and phylogenetic methods to reconstruct the evolutionary history of populations in Italy. To assess population structure, 61 specimens were collected from 11 different Provinces of Italy. Sampling was stratified across hosts and habitats to maximize coverage in native oak and pine stands and both mychorrizae and fruiting bodies were collected. Samples were identified considering anatomo-morphological characters. DNA was extracted and both multilocus (AFLP) and single-locus (18 loci from rDNA, nDNA, and mtDNA) approaches were used to look for polymorphisms. Screening AFLP profiles, both Jaccard and Dice coefficients of similarity were utilized to transform binary matrix into a distance matrix and then to desume Neighbour-Joining trees. Though these are only preliminary examinations, phylogenetic trees were totally concordant with those deriving from single locus analyses. Phylogenetic analyses of the nuclear loci were performed using maximum likelihood with PAUP and a combined phylogenetic inference, using Bayesian estimation with all nuclear gene regions, was carried out. To reconstruct the evolutionary history, we estimated recurrent migration, migration across the history of the sample, and estimated the mutation and approximate age of mutations in each tree using SNAP Workbench. The combined phylogenetic tree using Bayesian estimation suggests that there are two main haplotypes that are difficult to be differentiated on the basis of morphology, of ecological parameters and symbiontic tree. Between these two lineages, that occur in sympatry within T. borchii populations, there is no evidence of recurrent migration. However, migration over the history of the sample was asymmetrical suggesting that isolation was a result of interrupted gene flow followed by range expansion. Low levels of divergence between the haplotypes indicate that there are likely to be two cryptic species within the T. borchii population sampled. Our results suggest that isolation between populations of T. borchii could have led to reproductive isolation between two lineages. This isolation is likely due to sympatric speciation caused by a multiple colonization from different refugia or a recent isolation. In attempting to determinate whether these haplotypes represent separate species or a partition of the same species we applied Biological and Mechanistic species Concepts. Notwithstanding, further analyses are necessary to evaluate if selection favoured premating or post-mating isolation.

Patologia ed epidemiologia molecolare delle infezioni associate all'impianto

Staphylococcus aureus and Staphylococcus epidermidis are leading pathogens of implant-related infections. This study aimed at investigating the diverse distribution of different bacterial pathogen factors in most prevalent S. aureus and S. epidermidis strain types causing orthopaedic implant infections. In this study the presence both of the ica genes, encoding for biofilm exopolysaccharide production, and the insertion sequence IS256, a mobile element frequently associated to transposons, was investigated in relationship with the prevalence of antibiotic resistance among Staphylococcus epidermidis strains. The investigation was conducted on 70 clinical isolates derived from orthopaedic implant infections. Among the clinical isolates investigated a dramatic high level of association was found between the presence of ica genes as well as of IS256 and multiple resistance to all the antibiotics tested. Noteworthy, a striking full association between the presence of IS256 and resistance to gentamicin was found, being none of the IS256-negative strain resistant to this antibiotic. This association is probably because of the link of the corresponding aminoglycoside-resistance genes, and IS256, often co-existing within the same staphylococcal transposon. Moreover we investigated the prevalence of aac(6’)-Ie-aph(2’’), aph (3’) IIIa, and ant(4’) genes, encoding for the three forms of aminoglycoside-modifying enzymes (AME), responsible for resistance to aminoglycoside antibiotics. All isolates were characterized by automated ribotyping, so that the presence of antibiotic resistance determinants was investigated in strains exhibiting different ribopatterns. Interestingly, combinations of coexisting AME genes appeared to be typical of specific ribopatterns. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes, accessory gene regulatory (agr) polymorphisms and toxins. For many ribogroups, characteristic tandem genes arrangements could be identified. Surprisingly, the isolates of the most prevalent cluster, enlisting 27 isolates, were susceptible to almost all antibiotics and never possessed the lukD/lukE gene, thus suggesting the role of factors other than antibiotic resistance and the here investigated toxins in driving the major epidemic clone to the larger success. Afterwards, .in the predominant S. aureus cluster, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. Moreover a PCR screening for the ebpS gene, conducted on over two hundred S. aureus clinical isolates from implant related infections revealed the detection of six strains exhibiting an altered amplicon size, shorter than expected. In order to elucidate the sequence changes present in these gene variants, the trait comprised between the primers was analyzed in all six isolates bearing the modification and in four isolates exhibiting the regular amplicon size. From nucleotide translation, the corresponding encoded protein was found to lack an entire peptide segment of 60 amino acids. These variants, missing an entire hydrophobic region, could actually facilitate current structural studies, helping to assess whether the absent domain is strictly necessary for a functional adhesin conformation and its contribution to the topology of the protein. This study suggests that epidemic clones appear to pursue different survival strategies, where adhesins, when present, exhibit diverse importance as virulence factors. A practical message arising from the present study is that strategies for the prevention and treatment of implant orthopaedic infections should target adhesins conjointly present in epidemic clones. Furthermore, the choice of reference strains for testing the anti-infective properties of biomaterials should focus on a selection of the most prevalent clones as they exhibit distinct profiles of adhesins.

Phylogenetische Charakterisierung von Mikroorganismen aus dem Intestinaltrakt von Insekten

Zusammenfassung Die komplexe Lebensgemeinschaft des Termitendarms fasziniert die Biologen schon seit langem. Es ist bekannt, dass Termiten ihre Nahrung mit Hilfe von symbiontischen Bakterien und Protozoen verdauen können. Ohne ihre Symbionten würden sie verhungern. Das Zusammenspiel von Termiten und darmbewohnenden Mikroorganismen, zu denen Flagellaten, Bakterien, Archaebakterien und Hefen gehören, ist trotz moderner Untersuchungstechniken keineswegs vollständig aufgeklärt. In der vorliegenden Arbeit wurden:1) Einige kultivierte und nicht-kultivierte Bakterien charakterisiert, die an der Darmwand von Mastotermes darwiniensis lokalisiert sind. Die Darmwandbakterien wurden entweder nach Kultivierung oder direkt von der Darmwand für die Analyse der 16S rDNA verwendet. Die Sequenzierung erfolgte entweder nach DGGE oder nach Klonierung der PCR-Produkte. Die identifizierten Bakterien kann man in 7 Gruppen teilen:1: Gram-positive Bakterien mit hohem GC-Gehalt 2: Gram-positive Bakterien mit niedrigem GC-Gehalt 3: Fusobakterien-ähnliche Bakterien 4: ß-Proteobakterien5: Verrucomicrobien6: Bacteroides-ähnliche Bakterien7: Methanogene Bakterien 2) Aufgrund des Vorhandenseins des Coenzyms Deazaflavin-Derivats F420, kann man Methanbakterien mikroskopisch identifizieren und von anderen Bakterien unterscheiden, weil Methanbakterien im kurzwelligen Blaulicht blaugrün aufleuchten. Untersuchungen haben gezeigt, dass mindestens zwei Morphotypen von Methanbakterien an der Darmwand von M. darwiniensis vorkommen. Sie wurden auch über 16S rDNA Sequenzanalyse identifiziert. Ihre Lokalisierung an der Darmwand wurde durch Fluoreszenz-in-situ-Hybridsierung mit spezifischen Oligonukleotiden nachgewiesen. Schließlich konnte gezeigt werden, dass pro Gramm Termite 2,6 µg Methan pro Stunde produziert werden. 3) Bis jetzt wurden aus verschiedenen Termiten sulfatreduzierende Bakterien (SRB) isoliert. Deshalb wurde in dieser Arbeit die Verbreitung der SRB in verschiedenen Insekten untersucht. Insgesamt wurden zwei Sequenzen aus Libellenlarven (FSBO4 und FSBRO2), drei Sequenzen aus Zuckmückenlarven (FSCI, FSCII und FSC4), eine Sequenz aus Rosenkäfern (FSPa4-5) und ebenfalls eine Sequenz aus Eintagsfliegenlarven (FSB6) identifiziert. Alle identifizierten Bakterien ausser Klon FSB6, gehören zur Gattung Desulfovibrio. Klon FSB6 gehört zu der Gram-positiven Gattung Desulfotomaculum.Außerdem wurde die Sulfatreduktionsrate der SRB im Darm von Rosenkäfern (Pachnoda marginata), Holz- bzw. Sulfat-gefütterten Termiten (Mastotermes darwiniensis) und einer Reinkultur von Desulfovibrio intestinalis gemessen. Dabei konnte gezeigt werden, dass die Aktivität pro Zelle in Holz-gefütterten Termite am höchsten ist (4,9 nmol/107 Bakterien x h).

Production, purification, properties and application of the cellulases from a wild type strain of a yeast isolate

Kurzzusammenfassung Produktion, Reinigung, Eigenschaften und Anwendung von Cellulasen eines Wildtyp-Hefeisolates. Die effiziente Verwendung von Cellulose wird in naher Zukonft ein wichtiges Instrument zur Vermeidung einer Nahrungsmittel- und Energieknappheit werden. Deshalb haben wir uns intensiv mit Cellulasen befaßt, die aus Hefestämmen isoliert wurden. Die Fähigkeit der Cellulase-produktion eines Hefe-Stammes der Feuerwanze Pyrrhocoris apterus wurde genauer untersucht. Die systematische Stellung des Hefe-Isolates PAG1 wurde durch Sequenzierung der 18S rDNA bestimmt. Es zeigte eine nahe Verwandtschaft zu einem bereits beschriebenen Stämme der Gattung Trichosporon. Außerdem wurden die Wachstums-bedingungen für eine optimale Cellulase–Produktion bestimmt. Anschließend konnte eine der produzierten Cellulasen mit FPLC aufgereinigt und deren biochemische Eigenschaften (z.B. Substratspezifität, Temperatur optimum, optimaler pH-Wert, Einfluß von Chemikalien) untersucht werden. Eine Analyse der Abbau-Produkte zeigte, daß kristalline Cellulose und CMC zu Cellobiose, Cellulotriose, Cellulotetraose und Cellulopentaose in einem molaren Verhältnis von 32:16:8:1 umgesetezt wurden. Bei Zusatz von ?-Glykosidase aus demselben Hefestamm entstand nur Glucose und Cellobiose in einem molaren Verhältnis von 1:10. Da bisher nur eine Publikation über Cellulase-produzierende Hefe-Stämme erschienen ist, zeigen auch unsere Untersuchungen, daß Wildtyp-Hefestämme Cellulasen mit interessanten Eigenschaften produzieren können.

Transposable elements: structure and dynamic of the autonomous retrotransposon R2 in Arthropoda with non-canonical reproduction

Eukaryotic ribosomal DNA constitutes a multi gene family organized in a cluster called nucleolar organizer region (NOR); this region is composed usually by hundreds to thousands of tandemly repeated units. Ribosomal genes, being repeated sequences, evolve following the typical pattern of concerted evolution. The autonomous retroelement R2 inserts in the ribosomal gene 28S, leading to defective 28S rDNA genes. R2 element, being a retrotransposon, performs its activity in the genome multiplying its copy number through a “copy and paste” mechanism called target primed reverse transcription. It consists in the retrotranscription of the element’s mRNA into DNA, then the DNA is integrated in the target site. Since the retrotranscription can be interrupted, but the integration will be carried out anyway, truncated copies of the element will also be present in the genome. The study of these truncated variants is a tool to examine the activity of the element. R2 phylogeny appears, in general, not consistent with that of its hosts, except some cases (e.g. Drosophila spp. and Reticulitermes spp.); moreover R2 is absent in some species (Fugu rubripes, human, mouse, etc.), while other species have more R2 lineages in their genome (the turtle Mauremys reevesii, the Japanese beetle Popilia japonica, etc). R2 elements here presented are isolated in 4 species of notostracan branchiopods and in two species of stick insects, whose reproductive strategies range from strict gonochorism to unisexuality. From sequencing data emerges that in Triops cancriformis (Spanish gonochoric population), in Lepidurus arcticus (two putatively unisexual populations from Iceland) and in Bacillus rossius (gonochoric population from Capalbio) the R2 elements are complete and encode functional proteins, reflecting the general features of this family of transposable elements. On the other hand, R2 from Italian and Austrian populations of T. cancriformis (respectively unisexual and hermaphroditic), Lepidurus lubbocki (two elements within the same Italian population, gonochoric but with unfunctional males) and Bacillus grandii grandii (gonochoric population from Ponte Manghisi) have sequences that encode incomplete or non-functional proteins in which it is possible to recognize only part of the characteristic domains. In Lepidurus couesii (Italian gonochoric populations) different elements were found as in L. lubbocki, and the sequencing is still in progress. Two hypothesis are given to explain the inconsistency of R2/host phylogeny: vertical inheritance of the element followed by extinction/diversification or horizontal transmission. My data support previous study that state the vertical transmission as the most likely explanation; nevertheless horizontal transfer events can’t be excluded. I also studied the element’s activity in Spanish populations of T. cancriformis, in L. lubbocki, in L. arcticus and in gonochoric and parthenogenetic populations of B. rossius. In gonochoric populations of T. cancriformis and B. rossius I found that each individual has its own private set of truncated variants. The situation is the opposite for the remaining hermaphroditic/parthenogenetic species and populations, all individuals sharing – in the so far analyzed samples - the majority of variants. This situation is very interesting, because it isn’t concordant with the Muller’s ratchet theory that hypothesizes the parthenogenetic populations being either devoided of transposable elements or TEs overloaded. My data suggest a possible epigenetic mechanism that can block the retrotransposon activity, and in this way deleterious mutations don’t accumulate.