Bacterial and pathological study of caudal fin rot in brood stocks of Salmo trutta caspius from the propagation and breeding center of Shahid Bahonar of Kelardasht region

In order to study caudal fin rot with emphasis on Aeromonas hydrophila and Pseudomonas fluorescens in Salmo trutta caspius from the salmonids propagation and breeding center of Shahid Bahonar of kelardasht region, One hundred and eighty brood stocks having fin damage symptoms were chosen. Two bacterial samples from each fish were cultured on Aeromonas and Pseudomonas specific media. Biochemical tests, API2OE identification system and antibiogram test using six antibiotic disks were performed for diagnosing isolates bacteria and finding suitable antibiotic. Thirty samples from caudal fin of damaged fishes were fixed in 10% formalin and 51.tm microscopic sections were prepared using standard scatological methods and then stained by Haematoxylin-Eosin staining method to observe the pathological changes and also Maccallum-Goodpasture staining method to observe the bacterial colonies. In second stage of the study, bacterial samples were taken from thirty brood stocks using similar method at the first stage of sampling. For isolation and biochemical diagnosis of Aeromonas and Pseudormonas genus, the samples were analyzed by molecular research included PCR amplification (using 16S rDNA genes of the genus pseudomonas and 16S-23S rDNA intergenic spacer of the genus Aeromonas) and restriction analysis by four restriction enzymes for each genus. The results of biochemical tests showed that isolated bacteria were belonged to Aeromonas caviae and Aeromonas hydrophila (subspecies anaerogenes), Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas alcaligenes while the results of API2OE identification system showed that the isolated bacteria belonged to Aeromonas hydrophila, Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas aeruginosa. Restriction analysis of Aeromonas samples with Hin6l, Csp6I, Taql, and Tasl revealed three samples were different from others while restriction analysis of Pseudomonas samples with Alul, Hinfl, Rsal, and Trull showed at least five species or biovars. The results of antibiogram test showed all Aeromonas samples were sensitive to Trimethoprim, Chloramphenicol and Nitrofurazone, mostly to Nalidixic acid and Chloramphenicol, while most of samples were resistant to Erythromycin and Oxytetracycline. Pseudomonas samples were only sensitive to Nitrofurazone and mostly resistant to Oxytetracycline, Nalidixic acid, Erythromycin, Trimethoprim and Chloramphenicol. The results of light microscope study showed hyperplasia and spongiosis of the malpigian cells of epidermis, increasing of melanin pigments underlying epidermis; sever necrosis in both epidermis and dermis and also sloughing the epidermis in some cases. Occurrence of clefts through the epithelium, neovascularization, hyperemia and mild inflammatory response in dermis and separation of the fin rays also were observed. No bacterial colonies were found in the sections.

What influences fungal communities in cotton soils

Soilborne diseases such as Fusarium wilt, Black root rot and Verticillium wilt have significant impact on cotton production. Fungi are an important component of soil biota with capacity to affect pathogen inoculum levels and their disease causing potential. Very little is known about the soil fungal community structure and management effects in Australian cotton soils. We analysed surface soils from ongoing field experiments monitoring cotton performance and disease incidence in three cotton growing regions, collected prior to 2013 planting, for the genetic diversity and abundance as influenced by soil type, environment and management practices and link it with disease incidence and suppression. Results from the 28S LSU rRNA sequencing based analysis indicated a total of 370 fungal genera in all the cotton soils and the top 25 genera in abundance accounted for the major portion of total fungal community. There were significant differences in the composition and genetic diversity of soil fungi between the different field sites from the three cotton growing regions. Results for diversity indices showed significantly greater diversity in the long-term crop rotation experiment at Narrabri (F6E) and experiments at Cowan and Goondiwindi compared to the Biofumigation and D1 field experiments at ACRI, Narrabri. Diversity was lowest in the soils under brassica crop rotation in Biofumigation experiment. Overall, the diversity and abundance of soil fungal community varied significantly in the three cotton growing regions indicating soil type and environmental effects. These results suggest that changes in soil fungal community may play a notable role in soilborne disease incidence in cotton.

Analiza proteomiczna kompleksów tworzonych przez białko hDPY30 w komórkach ludzkich

Wydział Biologii

Parâmetros citogenéticos de espécies comerciais de carangidae (perciformes), com vistas a sua empregabilidade na conservação biológica e piscicultura marinha

Worldwide, families Carangidae and Rachycentridae represent one of the groups most important commercial fish, used for food, and great potential for marine aquaculture. However, the genetic bases that can underpin the future cultivation of these species, cytogenetic between these aspects are very weak. The chromosomal patterns have provided basic data for the exploration of biotechnological processes aimed at handling chromosomal genetic improvement, such as induction of polyploidy, androgenesis and ginogenesis, as well as obtaining monosex stocks and interspecific hybridizations. This paper presents a comprehensive cytogenetic survey in 10 species, seven of the family Carangidae and the monotypic family Rachycentridae. Classical cytogenetic analysis and in situ mapping of multigene sequences were employed, and additionally for the genus Selene and morphotypes of Caranx lugubris, comparisons were made using geometric morphometrics. In general, conservative species exhibit a marked chromosome number (2n=48). Although present in large part, different karyotypic form, retain many characteristics typical of chromosomal Order Perciformes, the high number of elements monobrachyal, Ag-NORs/18S rDNA sites and heterochromatin simply reduced, preferably centromeric. The main mechanisms involved in karyotypic diversification are the pericentric inversions, with secondary action of centric fusions. In addition to physical mapping and chromosome detail for the species are presented and discussed patterns of intra-and interspecific diversity, cytotaxonomic markers. This data set provides a better understanding of these patterns caryoevolutyonary groups and conditions for the development of protocols based on Biotechnology for chromosomal manipulation Atlantic these species

Mapeamento cromossômico de genes ribossomais em espécies estuarinas da família Gerreidae Identificação de uniformidade cariotípica e sua empregabilidade com fins biotecnológicos - REPROGEN

Perciformes are dominant in the marine environment, characterized as the largest and most diverse fish group. Some families, as Gerreidae, popularly known as silver jennies, carapebas, or mojarras have a high economic potential to marine fish farming, natural explotation and game fishing. Genetic information of these species are of fundamental importance for their management and production. Despite exist over 13,000 marine fish species described, only 2% were cytogenetically analyzed and less than 1% have some reproductive characteristics known. Induced breeding, cytogenetic characterization and cryopreservation of gametes, represent important areas in applied fish studies. In this project cytogenetic analyzes were performed to acess genetic aspects of Gerreidae species, distributed in coastal and estuarine regions of Northeast Brazil. Different methods for identifying chromosomal regions were employed using conventional techniques (Ag-NORs, C-banding), staining with base-specific fluorochromes (DAPI-CMA3), and physical mapping of ribosomal genes 18S and 5S rDNA, through hybridization in situ with fluorescent probes (FISH). The six species analyzed showed remarkable chromosome conservatism. The 18S and 5S ribosomal genes when analyzed in phylogenetic perspective demonstrate varied evolutionary dynamics, suggesting ocurrence of stasis process in some groups and greater dynamism in others. Double FISH with 18S and 5S probes showed both how efficient cytotaxonomic markers in the homogeneous karyotypes of this group of species. The karyotypic pattern identified in addition to the evolutionary aspects of karyotype, are suggestive of existence of low potential of post-zygotic barrier, prompting further research to prospect for artificial interspecific hybridization of these species of commercial importance

Detecção parasitológica e molecular de tripanossomatídeos em triatomíneos sinantrópicos e primatas neotropicais no Brasil central

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Medicina Tropical, 2016.

Asexual origin of brooding in the sea anemones Actinia equina and A. schmidti: Molecular evidence from the portuguese shore

Reproduction by sexual or asexual viviparity is a common phenomenon in some anemone species. In this short communication, the origin of the brooded young of Actinia equina and A. schmidti from the Portuguese shore was investigated. DNA was extracted from 56 brooding adult Actinia sp. and the nuclear gene that codes for the 28S ribosomal subunit was sequenced. Species identity was then assessed using GenBank. In total, 50 individuals were A. schmidti, five were A. equina and one had a hybrid origin. Three adult anemones (the hybrid, one A. equina and one A. schmidti) possessed two different 28S sequences and so their offspring was selected for further analysis using the same molecular procedure. Each brooded polyp was found to possess the exact same sequence as its parent, strongly suggesting the asexual origin of broods in A. equina and A. schmidti.

Diversidade de bactérias de solo sob vegetação natural e cultivo de hortaliças.

Os microrganismos do solo são componentes essenciais na manutenção do equilíbrio físico-químico e biológico do mesmo e exercem importante função que inclui a degradação de resíduos de plantas e animais e a liberação de nutrientes na cadeia alimentar. Este trabalho teve como objetivo comparar a microbiota de um solo com cobertura de mata (SMS) e outro cultivado com hortaliças (SHC), supressivos ou não a Rhizoctonia solani. Foram feitas extrações do DNA total dos solos e a partir dos mesmos, amplificação por PCR dos genes 16S rDNA, clonagem dos fragmentos e seqüenciamento dos genes do RNA ribossomal. A análise dos resultados demonstrou que essa metodologia foi eficiente para avaliação de bactérias. No solo supressivo de mata os filos mais encontrados pertencem aos das Acidobactérias, Verrucomicrobia e Actinobactérias e no solo conducente cultivado com hortaliças a maioria pertence aos filos das Proteobactérias, Firmicutes e Bacteroidetes.

Potencial biotecnológico de bactérias endofíticas de milho.

Bactérias endofíticas são microrganismos que habitam o interior dos tecidos vegetais e são conhecidas promotoras do crescimento de plantas, favorecendo sua nutrição, pela solubilização de fosfatos, além de produzirem fito-hormônio, enzimas e sideróforos, apresentando potencial para uso como bioinoculantes. O objetivo deste trabalho foi isolar e caracterizar bactérias endofíticas de milho eficientes na solubilização de fosfatos de cálcio e de ferro e na produção de sideróforos, substâncias que atuam na captação de ferro.Para o isolamento das bactérias, foram coletadas plantas de milho no florescimento em área de cerrado, sendo estas posteriormente desinfestadas e maceradas para o isolamento dos endófitos. Foram isoladas 113 bactérias provenientes de raízes (54,9%), folhas (20,4%) e seiva de milho (24,8%). Dessas bactérias, 49 foram caracterizadas geneticamente com base na sequência do 16S rDNA e avaliadas quanto à capacidade de solubilização de fosfatos em meio de cultura líquido contendo fosfato de cálcio e de ferro e produção de sideróforos. Os isolados mais eficientes na solubilização de fosfato de cálcio e fosfato de ferro produziram entre 179,4 a 193,7 mgP. L-1 e 62,7 a 68,7 mg P. L-1, respectivamente. Foi observada uma correlação negativa de -0,58 e -0,48 entre a solubilização de fósforo nos dois fosfatos testados, P-Ca e P-Fe, e os valores de pH, respectivamente, indicando a produção de ácidos orgânicos como o principal mecanismo de solubilização de P. O sideróforo produzido por 69% dos microrganismos avaliados foi do tipo carboxilato. As bactérias endofíticas eficientes na solubilização de P foram identificadas principalmente como pertencentes ao gênero Bacillus e Pantoea. Foi possível concluir que as estirpes bacterianas provenientes do microbioma interno de milho cultivado em solo de cerrado possuem características promissoras de biossolubilização de fosfatos e promoção de crescimento de plantas, sendo possível neste estudo a seleção de seis isolados do gênero Bacillus para futuros testes de inoculação em plantas.

Leishmaniose visceral canina em cães atendidos em hospital veterinário particular do Distrito Federal, Brasil

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Medicina Tropical, 2016.

Ecogenômica de archaea e monitoramento de comunidades de procariotos redutores de sulfato: aplicações na indústria de petróleo e gás

The human activities responsible for the ambient degradation in the modern world are diverse. The industrial activities are preponderant in the question of the impact consequences for brazilian ecosystems. Amongst the human activities, the petroliferous industry in operation in Potiguar Petroliferous Basin (PPB) displays the constant risk of ambient impacts in the integrant cities, not only for the human populations and the environment, but also it reaches the native microorganisms of Caatinga ground and in the mangrove sediment. Not hindering, the elaboration of strategies of bioremediation for impacted areas pass through the knowledge of microbiota and its relations with the environment. Moreover, in the microorganism groups associated to oil, are emphasized the sulfate-reducing prokaryotes (SRP) that, in its anaerobic metabolism, these organisms participate of the sulfate reduction, discharging H2S, causing ambient risks and causing the corrosion of surfaces, as pipelines and tanks, resulting in damages for the industry. Some ancestries of PRS integrate the Archaea domain, group of microorganisms whose sequenced genomes present predominance of extremophilic adaptations, including surrounding with oil presence. This work has two correlated objectives: i) the detection and monitoring of the gene dsrB, gift in sulfate-reducing prokaryotes, through DGGE analysis in samples of mDNA of a mangrove sediment and semiarid soil, both in the BPP; ii) to relate genomic characteristics to the ecological aspects of Archaea through in silico studies, standing out the importance to the oil and gas industry. The results of the first work suggest that the petrodegraders communities of SRP persist after the contamination with oil in mangrove sediment and in semiarid soil. Comparing the populations of both sites, it reveals that there are variations in the size and composition during one year of experiments. In the second work, functional and structural factors are the probable cause to the pressure in maintenance of the conservation of the sequences in the multiple copies of the 16S rDNA gene. Is verified also the discrepancy established between total content GC and content GC of the same gene. Such results relating ribosomal genes and the ambient factors are important for metagenomic evaluations using PCR-DGGE. The knowledge of microbiota associated to the oil can contribute for a better destination of resources by the petroliferous industry and the development of bioremediation strategies. Likewise, search to lead to the best agreement of the performance of native microbiota in biogeochemical cycles in Potiguar Petroliferous Basin ecosystem

Climate variability of the last 1000 years in the NW Pacific: high resolution, multi-biomarker records from Lake Toyoni

The East Asian Monsoon (EAM) is an active component of the global climate system and has a profound social and economic impact in East Asia and its surrounding countries. Its impact on regional hydrological processes may influence society through industrial water supplies, food productivity and energy use. In order to predict future rates of climate change, reliable and accurate reconstructions of regional temperature and rainfall are required from all over the world to test climate models and better predict future climate variability. Hokkaido is a region which has limited palaeo-climate data and is sensitive to climate change. Instrumental data show that the climate in Hokkaido is influenced by the East Asian Monsoon (EAM), however, instrumental data is limited to the past ~150 years. Therefore down-core climate reconstructions, prior to instrumental records, are required to provide a better understanding of the long-term behaviour of the climate drivers (e.g. the EAM, Westerlies, and teleconnections) in this region. The present study develops multi-proxy reconstructions to determine past climatic and hydrologic variability in Japan over the past 1000 years and aid in understanding the effects of the EAM and the Westerlies independently and interactively. A 250-cm long sediment core from Lake Toyoni, Hokkaido was retrieved to investigate terrestrial and aquatic input, lake temperature and hydrological changes over the past 1000-years within Lake Toyoni and its catchment using X-Ray Fluorescence (XRF) data, alkenone palaeothermometry, the molecular and hydrogen isotopic composition of higher plant waxes (δD(HPW)). Here, we conducted the first survey for alkenone biomarkers in eight lakes in the Hokkaido, Japan. We detected the occurrence of alkenones within the sediments of Lake Toyoni. We present the first lacustrine alkenone record from Japan, including genetic analysis of the alkenone producer. C37 alkenone concentrations in surface sediments are 18µg C37 g−1 of dry sediment and the dominant alkenone is C37:4. 18S rDNA analysis revealed the presence of a single alkenone producer in Lake Toyoni and thus a single calibration is used for reconstructing lake temperature based on alkenone unsaturation patterns. Temperature reconstructions over the past 1000 years suggest that lake water temperatures varies between 8 and 19°C which is in line with water temperature changes observed in the modern Lake Toyoni. The alkenone-based temperature reconstruction provides evidence for the variability of the EAM over the past 1000 years. The δD(HPW) suggest that the large fluctuations (∼40‰) represent changes in temperature and source precipitation in this region, which is ultimately controlled by the EAM system and therefore a proxy for the EAM system. In order to complement the biomarker reconstructions, the XRF data strengthen the lake temperature and hydrological reconstructions by providing information on past productivity, which is controlled by the East Asian Summer monsoon (EASM) and wind input into Lake Toyoni, which is controlled by the East Asian Winter Monsoon (EAWM) and the Westerlies. By combining the data generated from XRF, alkenone palaeothermometry and the δD(HPW) reconstructions, we provide valuable information on the EAM and the Westerlies, including; the timing of intensification and weakening, the teleconnections influencing them and the relationship between them. During the Medieval Warm Period (MWP), we find that the EASM dominated and the EAWM was suppressed, whereas, during the Little Ice Age (LIA), the influence of the EAWM dominated with time periods of increased EASM and Westerlies intensification. The El Niño Southern Oscillation (ENSO) significantly influenced the EAM; a strong EASM occurred during El Niño conditions and a strong EAWM occurred during La Niña. The North Atlantic Oscillation, on the other hand, was a key driver of the Westerlies intensification; strengthening of the Westerlies during a positive NAO phase and weakening of the Westerlies during a negative NAO phase. A key finding from this study is that our data support an anti-phase relationship between the EASM and the EAWM (e.g. the intensification of the EASM and weakening of the EAWM and vice versa) and that the EAWM and the Westerlies vary independently from each other, rather than coincide as previously suggested in other studies.

Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

IDENTIFICAÇÃO POLIFÁSICA DE GONGRONELLA SP. E DE RHIZOPUS SP. DEGRADADORES DO FUNGICIDA SISTÉMICO METALAXIL

A capacidade destes fungos degradarem compostos xenobióticos e recalcitrantes é uma característica biotecnologicamente importante tornando-os potencialmente úteis para processos de biorremediação. Gongronella sp. e Rhizopus sp. foram isolados do solo de vinhas da região do Alentejo, Portugal. Estes isolados mostraram elevada capacidade de degradarem o fungicida acilalanina metalaxil. No presente estudo, para a identificação polifásica de Gongronella sp. e Rhizopus sp., presuntivamente identificado como R. stolonifer, várias linhagens de referência da ordem Mucorales (Absidia, Circinella, Gongronella e Rhizopus) foram incluídas. A abordagem polifásica combinou a análise das macro- e micromorfologias, a análise da sequência inteira da região ITS ribossomal (i.e., ITS1/5.8S rDNA/ITS2) e ainda o uso da espectrometria de massas por Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF).