Microbiome diversity and composition in the phylogenetically related marine sponges S. spinosulus and I. variabilis

In this Thesis the theory of a uniform prokaryotic community associated with marine sponges was challenged. To this end, an in-depth inspection of the abundance, diversity, and composition of prokaryotic communities in the phylogenetically related marine sponges S. spinosulus and I. variabilis was undertaken. The within-habitat, between-habitat and temporal dynamics of these communities were disclosed. Further, an innovative approach to measure bacterial cultivation bias in the characterization of these communities was employed. Using state-of-the-art imaging technologies, both sponge species were classified as high microbe abundance sponges and bacterial cells were shown to be mainly associated with sponge cells and to neglect the sponge skeleton. PCR-DGGE fingerprinting was initially used and revealed that, within the same habitat, distinct bacterial communities were associated with S. spinosulus and I. variabilis. This was latter confirmed by 454 pyrosequencing of the same communities. Further, when two different cultivation-independent methods were applied to profile the bacterial communities associated with these hosts, a similar structure was obtained for S. spinosulus specimens, whereas the same was not true for I. variabilis. Using a common cultivation-dependent method, an alike bacterial community was detected in both sponge species, as opposed to the species-specific profiles obtained via cultivation-independent methods. Unexpectedly, around half of the OTUs recovered with the cultivation-dependent method was exclusive to this procedure. When between-habitat comparisons were made, replicates from the same sponge species were more similar to one another than replicates of different species independently of the sampling sites. Furthermore, the bacterial community associated with S. spinosulus displayed a state of dynamic stability over three consecutive years, whereby about half of the observed S. spinosulus could be detected in all sampling years. Remarkably, the archaeal community associated with S. spinosulus was dominated by one single OTU affiliated with Nitrosopumilus sp., a known ammonia-oxidizer. Overall, the prokaryotic community associated with S. spinosulus and I. variabilis was species-specific, and these communities were also maintained across biogeographical and temporal gradients, however the environmental also played a role.Esponjas marinhas são conhecidas por abrigar uma comunidade microbiana diversa e complexa. Inúmeros compostos bioativos têm sido isolados destes animais e acredita-se que os simbiontes sejam os produtores de pelo mesmo parte destes metabolitos, revelando assim um grande potencial biotecnológico. Uma das teorias desenvolvidas na área de microbiologia de esponjas marinhas estabelece que a composição da comunidade procariótica associada a estes animais é uniforme. Com o objectivo de testar esta teoria com acurácia, a abundância, diversidade e composição da comunidade procariótica em duas espécies de esponja pertencentes à família Irciniidae (classe Demospongiae, ordem Dictyoceratida), nomeadamente Sarcotragus spinosulus e Ircinia variabilis, foram investigadas em profundidade na presente Tese. Estas comunidades simbióticas foram caracterizadas em espécimes de ambas as espécies recolhidas em um mesmo habitat (costa do Algarve), em habitats diferentes (na costa do Algarve assim como nos arquipélagos da Madeira e dos Açores) e ao longo de três anos consecutivos (costa do Algarve). No último caso, foram coletadas apenas espécimes de S. spinosulus. Para além das espécimes de esponja, foram também recolhidas, em réplicas, amostras da água do mar circundante às esponjas e de sedimentos. Uma abordagem inovadora foi implementada para averiguar a eficiência de um método tradicional de cultivo na caracterização das comunidades procarióticas. Inúmeras técnicas foram empregadas à avaliação destas comunidades, especialmente de biologia molecular, tais como PCR-DGGE (reação em cadeia da polimerase – eletroforese em gel de gradiente denaturante) e pirosequenciação em massa com o uso da tecnologia 454. A abundância procariótica analisada com microscopia de epifluorescência em espécimes coletadas em um mesmo habitat revelou que: i) S. spinosulus abriga uma abundância procariótica significativamente maior quando comparada com I. variabilis e ii) a abundância detectada nas duas espécies de esponjas é significativamente superior, em 4 a 5 ordens de magnitude, à abundância procariótica encontrada na água do mar. Com base nesses resultados, S. spinosulus e I. variabilis foram classificadas como esponjas de alta abundância microbiana (“high microbial abundance sponges”). A comunidade bacteriana foi inicialmente investigada via PCR-DGGE, revelando em S. spinosulus uma menor variabilidade entre as réplicas e foi diferente da observada em I. variabilis, que mostrou maior variabilidade entre os replicados. Além disso, as comunidades bacterianas associadas às duas espécies de esponja foram distintas da observada na água do mar coletada nas proximidades das esponjas. Consequentemente, a comunidade bacteriana associada com S. spinosulus e I. variabilis é específica de cada espécie de esponja e distinta do ambiente. Os efeitos dos métodos independentes de cultivo e dependente de cultivo para a obtenção do DNA (ácido desoxirribonucleico) microbiano associado às esponjas foram determinados via PCR-DGGE e pirosequenciação em massa. A estrutura das comunidades bacterianas associadas à S. spinosulus acedidas pelos métodos independentes de cultivo foi semelhante, enquanto o oposto foi observado para I. variabilis. Portanto, os resultados obtidos com a pirosequenciação em massa confirmaram que a comunidade bacteriana é especificamente associada a cada espécie de esponja. Porém quando o método dependente de cultivo foi usado, a comunidade bacteriana nas duas espécies de esponja foi similar, em oposição aos perfis específicos das espécies obtidas por métodos independentes de cultivo. Surpreendentemente, por volta da metade das OTUs (Unidades Taxonómicas Operacionais definidas a uma similaridade de 97% entre sequências do gene 16S do RNA ribossômico) obtidas com este método foi exclusiva deste procedimento. Este resultado demonstra o potencial deste procedimento para a seleção e detecção de filotipos bacterianos menos abundantes que são enriquecidos pelo meio e condições de cultivo, como temperatura e tempo de incubação. Para além disso, a localização e distribuição de células bacterianas associadas às esponjas S. spinosulus e I. variabilis foram determinadas via hibridação in situ por fluorescência juntamente com microscopia eletrônica confocal de varredura (FISH-CLSM). Esta análise revelou que a grande maioria das células procarióticas foram encontradas associadas às células do mesoílo das esponjas e raramente associadas às fibras e filamentos (estruturas de suporte das esponjas), indicando existir uma troca de metabolitos entre a esponja e os simbiontes. De maneira geral a maioria das células bacterianas tinham a forma cocoíde e estavam entre as células das esponjas, de onde colônias bacterianas com alta abundância foram observadas. Quando as comparações entre habitat foram feitas, incluindo as espécies de esponjas, água do mar circundante às esponjas e de sedimentos, observou-se a formação de cinco grupos distintos. Independentemente dos locais onde as amostras foram recolhidas, todas as réplicas de S. spinosulus, Ircinia spp. e Spongia sp. agruparam entre si. O mesmo foi observado com as réplicas de sedimentos. Entretanto, as amostras de água do mar formaram três grupos distintos de acordo com o local de recolha. Para finalizar, a comunidade bacteriana associada à S. spinosulus exibiu um estado de estabilidade dinâmica ao longo de três anos consecutivos, sendo que cerca de metade dos simbiontes observados em S. spinosulus pode ser detectado em todos os anos de amostragem. Notavelmente, a comunidade de Archaea associada à S. spinosulus foi dominada por uma única OTU afiliada com Nitrosopumilus sp., conhecida por sua capacidade em oxidar amônia em condições aeróbicas. Todos os resultados apresentados nessa Tese sugerem uma importância fundamental dos simbiontes para a funcionalidade das esponjas marinhas e, devido à proximidade das células bacterianas com as células ativas da esponja é bastante provável que as mesmas executem funções vitais para manutenção da saúde e desenvolvimento das esponjas. Esta Tese suporta a teoria que a comunidade procariótica é na verdade específica de cada espécie de esponja, então refutando a visão de uniformidade. Está comunidade também foi mantida em diferentes gradientes biogeográficos e temporal. Finalmente, fatores ambientais também desempenham uma importante função como reservatório de bactérias simbiontes para as esponjas marinhas, com destaque para a comunidade encontrada nos sedimentos.Universidade do Algarve, Faculdade de Ciências e Tecnologi

Predicting the HMA-LMA status in marine sponges by machine learning

The dichotomy between high microbial abundance (HMA) and low microbial abundance (LMA) sponges has been observed in sponge-microbe symbiosis, although the extent of this pattern remains poorly unknown. We characterized the differences between the microbiomes of HMA (n=19) and LMA (n=17) sponges (575 specimens) present in the Sponge Microbiome Project. HMA sponges were associated with richer and more diverse microbiomes than LMA sponges, as indicated by the comparison of alpha diversity metrics. Microbial community structures differed between HMA and LMA sponges considering Operational Taxonomic Units (OTU) abundances and across microbial taxonomic levels, from phylum to species. The largest proportion of microbiome variation was explained by the host identity. Several phyla, classes, and OTUs were found differentially abundant in either group, which were considered “HMA indicators” and “LMA indicators”. Machine learning algorithms (classifiers) were trained to predict the HMA-LMA status of sponges. Among nine different classifiers, higher performances were achieved by Random Forest trained with phylum and class abundances. Random Forest with optimized parameters predicted the HMA-LMA status of additional 135 sponge species (1,232 specimens) without a priori knowledge. These sponges were grouped in four clusters, from which the largest two were composed of species consistently predicted as HMA (n=44) and LMA (n=74). In summary, our analyses shown distinct features of the microbial communities associated with HMA and LMA sponges. The prediction of the HMA-LMA status based on the microbiome profiles of sponges demonstrates the application of machine learning to explore patterns of host-associated microbial communities

Microbial communities and bioactive compounds in marine sponges of the family Irciniidae-a review

Marine sponges harbour complex microbial communities of ecological and biotechnological importance. Here, we propose the application of the widespread sponge family Irciniidae as an appropriate model in microbiology and biochemistry research. Half a gram of one Irciniidae specimen hosts hundreds of bacterial species-the vast majority of which are difficult to cultivate-and dozens of fungal and archaeal species. The structure of these symbiont assemblages is shaped by the sponge host and is highly stable over space and time. Two types of quorum-sensing molecules have been detected in these animals, hinting at microbe-microbe and host-microbe signalling being important processes governing the dynamics of the Irciniidae holobiont. Irciniids are vulnerable to disease outbreaks, and concerns have emerged about their conservation in a changing climate. They are nevertheless amenable to mariculture and laboratory maintenance, being attractive targets for metabolite harvesting and experimental biology endeavours. Several bioactive terpenoids and polyketides have been retrieved from Irciniidae sponges, but the actual producer (host or symbiont) of these compounds has rarely been clarified. To tackle this, and further pertinent questions concerning the functioning, resilience and physiology of these organisms, truly multi-layered approaches integrating cutting-edge microbiology, biochemistry, genetics and zoology research are needed.Portuguese Foundation [PTDC/MAR/101431/2008, PTDC/BIA-MIC/3865/2012]; European Regional Development Fund (ERDF) through the Operational Competitiveness Programme (COMPETE); national funds through FCT (Foundation for Science and Technology) [PEst-C/MAR/LA0015/2011]; FCT [SFRH/BD/60873/2009]info:eu-repo/semantics/publishedVersio

The gut microbiome and metabolome of two riparian communities in the Amazon

During the last decades it has become increasingly clear that the microbes that live on and in humans are critical for health. The communities they form, termed microbiomes, are involved in fundamental processes such as the maturation and constant regulation of the immune system. Additionally, they constitute a strong defense barrier to invading pathogens, and are also intricately linked to nutrition. The parameters that affect the establishment and maintenance of these microbial communities are diverse, and include the genetic background, mode of birth, nutrition, hygiene, and host lifestyle in general. Here, we describe the characterization of the gut microbiome of individuals living in the Amazon, and the comparison of these microbial communities to those found in individuals from an urban, industrialized setting. Our results showed striking differences in microbial communities from these two types of populations. Additionally, we used high-throughput metabolomics to study the chemical ecology of the gut environment and found significant metabolic changes between the two populations. Although we cannot point out a single cause for the microbial and metabolic changes observed between Amazonian and urban individuals, they are likely to include dietary differences as well as diverse patterns of environmental exposure. To our knowledge, this is the first description of gut microbial and metabolic profiles in Amazonian populations, and it provides a starting point for thorough characterizations of the impact of individual environmental conditions on the human microbiome and metabolome

Lifelong exposure to a low-dose of the glyphosate-based herbicide RoundUp® causes intestinal damage, gut dysbiosis, and behavioral changes in mice

RoundUp® (RUp) is a comercial formulation containing glyphosate (N-(phosphono-methyl) glycine), and is the world’s leading wide-spectrum herbicide used in agriculture. Supporters of the broad use of glyphosate-based herbicides (GBH) claim they are innocuous to humans, since the active compound acts on the inhibition of enzymes which are absent in human cells. However, the neurotoxic effects of GBH have already been shown in many animal models. Further, these formulations were shown to disrupt the microbiome of different species. Here, we investigated the effects of a lifelong exposure to low doses of the GBH-RUp on the gut environment, including morphological and microbiome changes. We also aimed to determine whether exposure to GBH-RUp could harm the developing brain and lead to behavioral changes in adult mice. To this end, animals were exposed to GBH-RUp in drinking water from pregnancy to adulthood. GBH-RUp-exposed mice had no changes in cognitive function, but developed impaired social behavior and increased repetitive behavior. GBH-Rup-exposed mice also showed an activation of phagocytic cells (Iba-1–positive) in the cortical brain tissue. GBH-RUp exposure caused increased mucus production and the infiltration of plama cells (CD138-positive), with a reduction in phagocytic cells. Long-term exposure to GBH-RUp also induced changes in intestinal integrity, as demonstrated by the altered expression of tight junction effector proteins (ZO-1 and ZO-2) and a change in the distribution of syndecan-1 proteoglycan. The herbicide also led to changes in the gut microbiome composition, which is also crucial for the establishment of the intestinal barrier. Altogether, our findings suggest that long-term GBH-RUp exposure leads to morphological and functional changes in the gut, which correlate with behavioral changes that are similar to those observed in patients with neurodevelopmental disorders

Dynamics of Seed-Borne Rice Endophytes on Early Plant Growth Stages

Bacterial endophytes are ubiquitous to virtually all terrestrial plants. With the increasing appreciation of studies that unravel the mutualistic interactions between plant and microbes, we increasingly value the beneficial functions of endophytes that improve plant growth and development. However, still little is known on the source of established endophytes as well as on how plants select specific microbial communities to establish associations. Here, we used cultivation-dependent and -independent approaches to assess the endophytic bacterrial community of surface-sterilized rice seeds, encompassing two consecutive rice generations. We isolated members of nine bacterial genera. In particular, organisms affiliated with Stenotrophomonas maltophilia and Ochrobactrum spp. were isolated from both seed generations. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) of seed-extracted DNA revealed that approximately 45% of the bacterial community from the first seed generation was found in the second generation as well. In addition, we set up a greenhouse experiment to investigate abiotic and biotic factors influencing the endophytic bacterial community structure. PCR-DGGE profiles performed with DNA extracted from different plant parts showed that soil type is a major effector of the bacterial endophytes. Rice plants cultivated in neutral-pH soil favoured the growth of seed-borne Pseudomonas oryzihabitans and Rhizobium radiobacter, whereas Enterobacter-like and Dyella ginsengisoli were dominant in plants cultivated in low-pH soil. The seed-borne Stenotrophomonas maltophilia was the only conspicuous bacterial endophyte found in plants cultivated in both soils. Several members of the endophytic community originating from seeds were observed in the rhizosphere and surrounding soils. Their impact on the soil community is further discussed

Molecular richness and biotechnological potential of bacteria cultured from Irciniidae sponges in the north-east Atlantic

Several bioactive compounds originally isolated from marine sponges have been later ascribed or suggested to be synthesized by their symbionts. The cultivation of sponge-associated bacteria provides one possible route to the discovery of these metabolites. Here, we determine the bacterial richness cultured from two irciniid sponge species, Sarcotragus spinosulus and Ircinia variabilis, and ascertain their biotechnological potential. A total of 279 isolates were identified from 13 sponge specimens. These were classified into 17 genera - with Pseudovibrio, Ruegeria and Vibrio as the most dominant - and 3 to 10 putatively new bacterial species. While 16S rRNA gene sequencing identified 29 bacterial phylotypes at the 'species' level (97% sequence similarity), whole-genome BOX-PCR fingerprinting uncovered 155 genotypes, unveiling patterns of specimen-dependent occurrence of prevailing bacterial genomes across sponge individuals. Among the BOX-PCR genotypes recovered, 34% were active against clinically relevant strains, with Vibrio isolates producing the most active antagonistic effect. Several Pseudovibrio genotypes showed the presence of polyketide synthase (PKS) genes, and these were for the first time detected in isolates of the genus Aquimarina (Bacteroidetes). Our results highlight great biotechnological potential and interest for the Irciniidae sponge family and their diversified bacterial genomes.Portuguese Foundation for Science and Technology (FCT) [PTDC/MAR/101431/2008]; FCT [SFRH/BD/60873/2009, SFRH/BPD/62946/2009

Phylogenetically and spatially close marine sponges harbour divergent bacterial communities

Recent studies have unravelled the diversity of sponge-associated bacteria that may play essential roles in sponge health and metabolism. Nevertheless, our understanding of this microbiota remains limited to a few host species found in restricted geographical localities, and the extent to which the sponge host determines the composition of its own microbiome remains a matter of debate. We address bacterial abundance and diversity of two temperate marine sponges belonging to the Irciniidae family - Sarcotragus spinosulus and Ircinia variabilis – in the Northeast Atlantic. Epifluorescence microscopy revealed that S. spinosulus hosted significantly more prokaryotic cells than I. variabilis and that prokaryotic abundance in both species was about 4 orders of magnitude higher than in seawater. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) profiles of S. spinosulus and I. variabilis differed markedly from each other – with higher number of ribotypes observed in S. spinosulus – and from those of seawater. Four PCR-DGGE bands, two specific to S. spinosulus, one specific to I. variabilis, and one present in both sponge species, affiliated with an uncultured sponge-specific phylogenetic cluster in the order Acidimicrobiales (Actinobacteria). Two PCR-DGGE bands present exclusively in S. spinosulus fingerprints affiliated with one sponge-specific phylogenetic cluster in the phylum Chloroflexi and with sponge-derived sequences in the order Chromatiales (Gammaproteobacteria), respectively. One Alphaproteobacteria band specific to S. spinosulus was placed in an uncultured sponge-specific phylogenetic cluster with a close relationship to the genus Rhodovulum. Our results confirm the hypothesized host-specific composition of bacterial communities between phylogenetically and spatially close sponge species in the Irciniidae family, with S. spinosulus displaying higher bacterial community diversity and distinctiveness than I. variabilis. These findings suggest a pivotal host-driven effect on the shape of the marine sponge microbiome, bearing implications to our current understanding of the distribution of microbial genetic resources in the marine realm.This work was financed by the Portuguese Foundation for Science and Technology (FCT - http://www.fct.pt) through the research project PTDC/MAR/101431/2008. CCPH has a PhD fellowship granted by FCT (Grant No. SFRH/BD/60873/2009). JRX’s research is funded by a FCT postdoctoral fellowship (grant no. SFRH/BPD/62946/2009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

IDENTIFICAÇÃO, SELEÇÃO E CARACTERIZAÇÃO DAS ESPÉCIES VEGETAIS DESTINADAS AO JARDIM SENSORIAL TUMUCUMAQUE, MUNICÍPIO DE SERRA DO NAVIO, AP/ BRASIL.

No jardim sensorial, as espécies utilizadas para paisagismo devem ter características específicas que despertem os sentidos dos seres humanos tais como olfato, tato e visão. Atualmente, o Jardim Sensorial (JS) tem sido objeto de estudos relacionados ao Ensino de Ciências como espaço destinado à aulas práticas nas escolas de Ensino Fundamental, além de constituir-se uma ferramenta de inclusão para alunos com necessidades educacionais especiais. A escolha de espécies que atendam tais características inerentes ao Jardim Sensorial é o objetivo deste trabalho. Desta forma, foi realizada a visita em cinco locais com JS para verificação das plantas ornamentais ali existentes, sendo posteriormente feita a análise e classificação destas para sua utilização no JS. As plantas selecionadas foram classificadas segundo seu hábito, habitat, grupo evolutivo e características sensoriais e os resultados agrupados em tabelas e quadros demonstrativos. Foram previamente selecionadas as seguintes espécies: Acalypha reptans Sw., Aloe vera (L.) Burm, Alternanthera dentata (Moench) Stuchlik ex RE F.., Anthurium andraeanum Linden, Arachis repens Handro, Catharanthus roseus (L.) G. Don., Episcia cupreata (Hook) Hanst., Ixora coccinea L., Melissa officinalis L., Sanseviera trifascinata Prain., Zoynia tenuifolia Thiele. Os resultados das informações obitidas demonstraram a predominância de espécies exóticas e introduzidas em relação as nativas, isto se deve a escassez de pesquisas direcionadas para a identificação de espécies nativas com potencial de uso paisagístico, e em especial para Jardim Sensorial.   Palavras-chave: Ensino de Ciências, Amazônia, etnobotânica.   DOI: http://dx.doi.org/10.18561/2179-5746/biotaamazonia.v3n1p22-3

Identification of isolated seed-borne strains.

a<p>Rice strains isolated from first (R1-R4) and second (R5-R16) generation of seeds.</p><p>*The 16S rRNA gene sequences of strains R6 and R8 were identical to PCR-DGGE products of the bands 12 and 9, respectively.</p>b<p>Source of the closest rice associated bacteria, LE – Leaf Endophyte <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano3" target="_blank">[21]</a>; LS – Leaf surface <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano3" target="_blank">[21]</a>; PF – Paddy Field (Islam et al., unpublished); PS – Paddy Soil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Shrestha1" target="_blank">[28]</a>; R - Rhizosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Steindler1" target="_blank">[25]</a>; RE1 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Hardoim2" target="_blank">[20]</a>; RE2 - Root Endosphere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano3" target="_blank">[21]</a> and SE – Seed endophyte <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030438#pone.0030438-Mano1" target="_blank">[5]</a>.</p