Elevated resource availability sufficient to turn opportunistic into virulent fish pathogens.

There is mounting evidence that organic or inorganic enrichment of aquatic environments increases the risk of infectious diseases, with disease agents ranging from helminth parasites to fungal, bacterial, and viral pathogens. The causal link between microbial resource availability and disease risk is thought to be complex and, in the case of so-called "opportunistic pathogens," to involve additional stressors that weaken host resistance (e.g., temperature shifts or oxygen deficiencies). In contrast to this perception, our experiment shows that the link between resource levels and infection of fish embryos can be very direct: increased resource availability can transform benign microbial communities into virulent ones. We find that embryos can be harmed before further stresses (e.g., oxygen depletion) weaken them, and treatment with antibiotics and fungicides cancels the detrimental effects. The changed characteristics of symbiotic microbial communities could simply reflect density-dependent relationships or be due to a transition in life-history strategy. Our findings demonstrate that simple microhabitat changes can be sufficient to turn "opportunistic" into virulent pathogens.

Coevolutionary networks: a novel approach to understanding the relationships of humans with the infectious agents

Human organism is interpenetrated by the world of microorganisms, from the conception until the death. This interpenetration involves different levels of interactions between the partners including trophic exchanges, bi-directional cell signaling and gene activation, besides genetic and epigenetic phenomena, and tends towards mutual adaptation and coevolution. Since these processes are critical for the survival of individuals and species, they rely on the existence of a complex organization of adaptive systems aiming at two apparently conflicting purposes: the maintenance of the internal coherence of each partner, and a mutually advantageous coexistence and progressive adaptation between them. Humans possess three adaptive systems: the nervous, the endocrine and the immune system, each internally organized into subsystems functionally connected by intraconnections, to maintain the internal coherence of the system. The three adaptive systems aim at the maintenance of the internal coherence of the organism and are functionally linked by interconnections, in such way that what happens to one is immediately sensed by the others. The different communities of infectious agents that live within the organism are also organized into functional networks. The members of each community are linked by intraconnections, represented by the mutual trophic, metabolic and other influences, while the different infectious communities affect each other through interconnections. Furthermore, by means of its adaptive systems, the organism influences and is influenced by the microbial communities through the existence of transconnections. It is proposed that these highly complex and dynamic networks, involving gene exchange and epigenetic phenomena, represent major coevolutionary forces for humans and microorganisms.

Microbiological activities in moonmilk monitored using isothermal microcalorimetry (cave of Vers Chez le Brandt, Neuchatel, Switzerland)

Studies of the influence of microbial communities on calcium carbonate deposits mostly rely on classical or molecular microbiology, isotopic analyses, and microscopy. Using these techniques, it is difficult to infer microbial activities in such deposits. In this context, we used isothermal microcalorimetry, a sensitive and nondestructive tool, to measure microbial activities associated with moonmilk ex-situ. Upon the addition of diluted LB medium and other carbon sources to fresh moonmilk samples, we estimated the number of colony forming units per gram of moonmilk to be 4.8 3 105 6 0.2 3 105. This number was close to the classical plate counts, but one cannot assume that all active cells producing metabolic heat were culturable. Using a similar approach, we estimated the overall growth rate and generation time of the microbial community associated with the moonmilk upon addition of various carbon sources. The range of apparent growth rates of the chemoheterotrophic microbial community observed was between 0.025 and 0.067 h21 and generation times were between 10 and 27 hours. The highest growth rates were observed for citrate and diluted LB medium, while the highest carbon-source consumption rates were observed for low molecular weight organic acids (oxalate and acetate) and glycerol. Considering the rapid degradation of organic acids, glucose, and other carbon sources observed in the moonmilk, it is obvious that upon addition of nutrients during snow melting or rainfall these communities can have high overall activities comparable to those observed in some soils. Such communities can influence the physico-chemical conditions and participate directly or indirectly to the formation of moonmilk.

Seasonal fluctuations of bacterial community diversity in agricultural soil and experimental validation by laboratory disturbance experiments.

Natural fluctuations in soil microbial communities are poorly documented because of the inherent difficulty to perform a simultaneous analysis of the relative abundances of multiple populations over a long time period. Yet, it is important to understand the magnitudes of community composition variability as a function of natural influences (e.g., temperature, plant growth, or rainfall) because this forms the reference or baseline against which external disturbances (e.g., anthropogenic emissions) can be judged. Second, definition of baseline fluctuations in complex microbial communities may help to understand at which point the systems become unbalanced and cannot return to their original composition. In this paper, we examined the seasonal fluctuations in the bacterial community of an agricultural soil used for regular plant crop production by using terminal restriction fragment length polymorphism profiling (T-RFLP) of the amplified 16S ribosomal ribonucleic acid (rRNA) gene diversity. Cluster and statistical analysis of T-RFLP data showed that soil bacterial communities fluctuated very little during the seasons (similarity indices between 0.835 and 0.997) with insignificant variations in 16S rRNA gene richness and diversity indices. Despite overall insignificant fluctuations, between 8 and 30% of all terminal restriction fragments changed their relative intensity in a significant manner among consecutive time samples. To determine the magnitude of community variations induced by external factors, soil samples were subjected to either inoculation with a pure bacterial culture, addition of the herbicide mecoprop, or addition of nutrients. All treatments resulted in statistically measurable changes of T-RFLP profiles of the communities. Addition of nutrients or bacteria plus mecoprop resulted in bacteria composition, which did not return to the original profile within 14 days. We propose that at less than 70% similarity in T-RFLP, the bacterial communities risk to drift apart to inherently different states.

Diversity of the bacterial community in the surface soil of a pear orchard based on 16S rRNA gene analysis

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes was used to characterize bacterial populations in the surface soil of a commercial pear orchard consisting of different pear cultivars during two consecutive growing seasons. Pyrus communis L. cvs Blanquilla, Conference, and Williams are among the most widely cultivated cultivars in Europe and account for the majority of pear production in Northeastern Spain. To assess the heterogeneity of the community structure in response to environmental variables and tree phenology, bacterial populations were examined using PCR-denaturing gradient gel electrophoresis (DGGE) followed by cluster analysis of the 16S ribosomal DNA profiles by means of the unweighted pair group method with arithmetic means. Similarity analysis of the band patterns failed to identify characteristic fingerprints associated with the pear cultivars. Both environmentally and biologically based principal-component analyses showed that the microbial communities changed significantly throughout the year depending on temperature and, to a lesser extent, on tree phenology and rainfall. Prominent DGGE bands were excised and sequenced to gain insight into the identities of the predominant bacterial populations. Most DGGE band sequences were related to bacterial phyla, such as Bacteroidetes, Cyanobacteria, Acidobacteria, Proteobacteria, Nitrospirae, and Gemmatimonadetes, previously associated with typical agronomic crop environments

Diversitat bacteriana a les esponges marines

L’objectiu principal del projecte era aprofundir en el coneixement de les malalties de les esponges, centrant-nos en la diversitat bacteriana que acullen. Als estius 2008 i 2009 va haver episodis d’elevades temperatures de l’aigua de mar que van suposar importants mortalitats massives d’esponges a la Mediterrània. Contra tot pronòstic, els estius del projecte, el 2010 i el 2011, no van resultar especialment calorosos i no hi va haver episodis de mortalitat. És un fet molt positiu per les comunitats bentòniques però algun dels objectius específics de la meva proposta no es va poder dur a terme com havien estat platejats inicialment. Disposàvem de diferents mostres d’esponges malaltes de l’espècie Ircinia fasciculata, sanes i aigua circumdant recollides l’estiu 2009 i fixades per DNA, per això s’han pogut identificar i caracteritzar els canvis en la comunitat microbiològica de les esponges malaltes respecte de les sanes. Hem constatat que més que una infecció per un o pocs patògens puntuals, com havien proposat alguns autors, hi ha un canvi dràstic en la comunitat microbiològica associada a les esponges. Les esponges malaltes, on dominen els bacteris heterotròfics, presenten una major diversitat bacteriana que les sanes, on dominen els autòtrofs. Tot i que no es va poder realitzar un nou mostreig d’individus malalts, i algun objectiu específic no es va poder desenvolupar, vaig aprofitar l’estada en un centre de referència en estudis de la diversitat microbiana, per ampliar el coneixement general de les comunitats bacterianes associades a esponges. Es van realitzar nous estudis en què vam testar si totes les esponges (bacteriosponges i no) presenten simbionts reals o si les no-bacteriosponges presenten un enriquiment de les comunitats de l’aigua de mar però no una flora específica. El resultat és que totes les esponges presenten associacions molt específiques malgrat hi ha fortes diferències entre les comunitats microbianes associades a bacteriosponges i a no-bacteriosponges.

Land-use systems affect Archaeal community structure and functional diversity in western Amazon soils

The study of the ecology of soil microbial communities at relevant spatial scales is primordial in the wide Amazon region due to the current land use changes. In this study, the diversity of the Archaea domain (community structure) and ammonia-oxidizing Archaea (richness and community composition) were investigated using molecular biology-based techniques in different land-use systems in western Amazonia, Brazil. Soil samples were collected in two periods with high precipitation (March 2008 and January 2009) from Inceptisols under primary tropical rainforest, secondary forest (5-20 year old), agricultural systems of indigenous people and cattle pasture. Denaturing gradient gel electrophoresis of polymerase chain reaction-amplified DNA (PCR-DGGE) using the 16S rRNA gene as a biomarker showed that archaeal community structures in crops and pasture soils are different from those in primary forest soil, which is more similar to the community structure in secondary forest soil. Sequence analysis of excised DGGE bands indicated the presence of crenarchaeal and euryarchaeal organisms. Based on clone library analysis of the gene coding the subunit of the enzyme ammonia monooxygenase (amoA) of Archaea (306 sequences), the Shannon-Wiener function and Simpson's index showed a greater ammonia-oxidizing archaeal diversity in primary forest soils (H' = 2.1486; D = 0.1366), followed by a lower diversity in soils under pasture (H' = 1.9629; D = 0.1715), crops (H' = 1.4613; D = 0.3309) and secondary forest (H' = 0.8633; D = 0.5405). All cloned inserts were similar to the Crenarchaeota amoA gene clones (identity > 95 %) previously found in soils and sediments and distributed primarily in three major phylogenetic clusters. The findings indicate that agricultural systems of indigenous people and cattle pasture affect the archaeal community structure and diversity of ammonia-oxidizing Archaea in western Amazon soils.

Factors promoting the exposure to bioaerosols among crop workers

Agricultural workers are among the professional groups most at risk of developing acute or chronic respiratory problems. Despite this fact, the etiology of these occupational diseases is poorly known, even in important sectors of agriculture such as the crops sector. A chronic exposure to multiple microorganisms, such as different bacterial and fungal species, has been proposed to be the cause of these multiple respiratory pathologies. Nevertheless, these microbial communities are still partially known. The aim of this study is to characterize all fungal species inhaled by the crops workers during different grain related activities and identify the abiotic and biotic factors that reduce the growth of the toxigenic, irritative or allergenic microbial species. Here, we are presenting the factors promoting the exposure to bioaerosols during different wheat related activities: harvesting, grain unload, baling straw, the cleaning of harvesters and silos. Total dust has been quantified following NIOSH 0500 method. Reactive endotoxin activity has been determined with Limulus Amebocyte Lysate Assay. All molds have been identified by the pyrosequencing of ITS2 amplicons generated from bioaerosol.

Community-wide plasmid gene mobilization and selection.

Plasmids have long been recognized as an important driver of DNA exchange and genetic innovation in prokaryotes. The success of plasmids has been attributed to their independent replication from the host's chromosome and their frequent self-transfer. It is thought that plasmids accumulate, rearrange and distribute nonessential genes, which may provide an advantage for host proliferation under selective conditions. In order to test this hypothesis independently of biases from culture selection, we study the plasmid metagenome from microbial communities in two activated sludge systems, one of which receives mostly household and the other chemical industry wastewater. We find that plasmids from activated sludge microbial communities carry among the largest proportion of unknown gene pools so far detected in metagenomic DNA, confirming their presumed role of DNA innovators. At a system level both plasmid metagenomes were dominated by functions associated with replication and transposition, and contained a wide variety of antibiotic and heavy metal resistances. Plasmid families were very different in the two metagenomes and grouped in deep-branching new families compared with known plasmid replicons. A number of abundant plasmid replicons could be completely assembled directly from the metagenome, providing insight in plasmid composition without culturing bias. Functionally, the two metagenomes strongly differed in several ways, including a greater abundance of genes for carbohydrate metabolism in the industrial and of general defense factors in the household activated sludge plasmid metagenome. This suggests that plasmids not only contribute to the adaptation of single individual prokaryotic species, but of the prokaryotic community as a whole under local selective conditions.

Phenotypic plasticity in salmonid embryos : characterizing pathogen-induced stress effects on heritable variation in traits and reaction norms

Les pressions écologiques peuvent varier tant en nature qu'en intensité dans le temps et l'espace. C'est pourquoi, un phénotype unique ne peut pas forcément conférer la meilleure valeur sélective. La plasticité phénotypique peut être un moyen de s'accommoder de cette situation, en augmentant globalement la tolérance aux changements environnementaux. Comme pour tout trait de caractère, une variation génétique doit persister pour qu'évoluent les traits plastiques dans une population donnée. Cependant, les pressions extérieures peuvent affecter l'héritabilité, et la direction de ces changements peut dépendre du caractère en question, de l'espèce mais aussi du type de stress. Dans la présente thèse, nous avons cherché à élucider les effets des pressions pathogéniques sur les phénotypes et la génétique quantitative de plusieurs traits plastiques chez les embryons de deux salmonidés, la palée (Coregonus palaea), et la truite de rivière (Salmo trutta). Les salmonidés se prêtent à de telles études du fait de leur extraordinaire variabilité morphologique, comportementale et des traits d'histoire de vie. Par ailleurs, avec le déclin des salmonidés dans le monde, il est important de savoir combien la variabilité génétique persiste dans les normes de réaction afin d'aider à prédire leur capacité à répondre aux changements de leur milieu. Nous avons observé qu'une augmentation de la croissance des communautés microbiennes symbiotiques entraînait une mortalité accrue et une éclosion précoce chez la palée, et dévoilait la variance génétique additive pour ces deux caractères (Chapitres 1-2). Bien qu'aucune variation génétique n'ait été trouvée pour les normes de réaction, nous avons observé une variabilité de la plasticité d'éclosion. Néanmoins, on a trouvé que les temps d'éclosion étaient corrélés entre les environnements, ce qui pourrait limiter l'évolution de la norme de réaction. Le temps d'éclosion des embryons est lié à la taille des géniteurs mâles, ce qui indique des effets pléiotropiques. Dans le Chapitre 3, nous avons montré qu'une interaction triple entre la souche bactérienne {Pseudomonas fluorescens}, l'état de dévelopement de l'hôte ainsi que ses gènes ont une influence sur la mortalité, le temps d'éclosion et la taille des alevins de la palée. Nous avons démontré qu'une variation génétique subsistait généralement dans les normes de réaction des temps d'éclosion, mais rarement pour la taille des alevins, et jamais pour la mortalité. Dans le même temps, nous avons exhibé que des corrélations entre environnements dépendaient des caractères phénotypiques, mais contrairement au Chapitre 2, nous n'avons pas trouvé de preuve de corrélations transgénérationnelles. Le Chapitre 4 complète le chapitre précédent, en se plaçant du point de vue moléculaire, et décrit comment le traitement d'embryons avec P. fluorescens s'est traduit par une régulation négative d'expression du CMH-I indépendemment de la souche bactérienne. Nous avons non seulement trouvé une variation génétique des caractères phénotypiques moyens, mais aussi de la plasticité. Les deux derniers chapitres traitent de l'investigation, chez la truite de rivière, des différences spécifiques entre populations pour des normes de réaction induites par les pathogènes. Dans le Chapitre 5, nous avons illustré que le métissage entre des populations génétiquement distinctes n'affectait en rien la hauteur ou la forme des normes de réaction d'un trait précoce d'histoire de vie suite au traitement pathogénique. De surcroît, en dépit de l'éclosion tardive et de la réduction de la taille des alevins, le traitement n'a pas modifié la variation héritable des traits de caractère. D'autre part, dans le Chapitre 6, nous avons démontré que le traitement d'embryons avec des stimuli contenus dans l'eau de conspécifiques infectés a entraîné des réponses propre à chaque population en terme de temps d'éclosion ; néanmoins, nous avons observé peu de variabilité génétique des normes de réaction pour ce temps d'éclosion au sein des populations. - Ecological stressors can vary in type and intensity over space and time, and as such, a single phenotype may not confer the highest fitness. Phenotypic plasticity can act as a means to accommodate this situation, increasing overall tolerance to environmental change. As with any trait, for plastic traits to evolve in a population, genetic variation must persist. However, environmental stress can alter trait heritability, and the direction of this shift can be trait, species, and stressor-dependent. In this thesis, we sought to understand the effects of pathogen stressors on the phenotypes and genetic architecture of several plastic traits in the embryos of two salmonids, the whitefish (Coregonus palaea), and the brown trout (Salmo trutta). Salmonids lend themselves to such studies because their extraordinary variability in morphological, behavioral, and life-history traits. Also, with declines in salmonids worldwide, knowing how much genetic variability persists in reaction norms may help predict their ability to respond to environmental change. We found that increasing growth of symbiotic microbial communities increased mortality and induced hatching in whitefish, and released additive genetic variance for both traits (Chapters 1-2). While no genetic variation was found for survival reaction norms, we did find variability in hatching plasticity. Nevertheless, hatching time was correlated across environments, which could constrain evolution of the reaction norm. Hatching time in the induced environment was also correlated to sire size, indicating pleiotropic effects. In Chapter 3 we report that a three-way interaction between bacterial strain (Pseudomonas fluorescens), host developmental stage, and host genetics impacted mortality, hatching time, and hatchling size in whitefish. We also showed that genetic variation generally persisted in hatching age reaction norms, but rarely for hatchling length, and never for mortality. At the same time, we demonstrated that cross-environmental correlations were trait-dependent, and unlike Chapter 2, we found no evidence of cross-generational correlations. Chapter 4 expands on the previous chapter, moving to the molecular level, and describes how treatment of embryos with P. fluorescens resulted in strain-independent downregulation of MHC class I. Genetic variation was evident not only in trait means, but also in plasticity. In the last two chapters, we investigated population level differences in pathogen- induced reaction norms in brown trout. In Chapter 5, we found that interbreeding between genetically distinct populations did not affect the elevation or shapes of the reaction norms of early life-history traits after pathogen challenge. Moreover, despite delaying hatching and reducing larval length, treatment produced no discernable shifts in heritable variation in traits. On the other hand, in Chapter 6, we found that treatment of embryos with water-borne cues from infected conspecifics elicited population-specific responses in terms of hatching time; however, we found little evidence of genetic variability in hatching reaction norms within populations. We have made considerable progress in understanding how pathogen stressors affect various early life-history traits in salmonid embryos. We have demonstrated that the effect of a particular stressor on heritable variation in these traits can vary according to the trait and species under consideration, in addition to the developmental stage of the host. Moreover, we found evidence of genetic variability in some, but not all reaction norms in whitefish and brown trout.

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA) and an eucalyptus arboretum (EAA). PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.

Hidden diversity in honey bee gut symbionts detected by single-cell genomics.

Microbial communities in animal guts are composed of diverse, specialized bacterial species, but little is known about how gut bacteria diversify to produce genetically and ecologically distinct entities. The gut microbiota of the honey bee, Apis mellifera, presents a useful model, because it consists of a small number of characteristic bacterial species, each showing signs of diversification. Here, we used single-cell genomics to study the variation within two species of the bee gut microbiota: Gilliamella apicola and Snodgrassella alvi. For both species, our analyses revealed extensive variation in intraspecific divergence of protein-coding genes but uniformly high levels of 16S rRNA similarity. In both species, the divergence of 16S rRNA loci appears to have been curtailed by frequent recombination within populations, while other genomic regions have continuously diverged. Furthermore, gene repertoires differ markedly among strains in both species, implying distinct metabolic capabilities. Our results show that, despite minimal divergence at 16S rRNA genes, in situ diversification occurs within gut communities and generates bacterial lineages with distinct ecological niches. Therefore, important dimensions of microbial diversity are not evident from analyses of 16S rRNA, and single cell genomics has potential to elucidate processes of bacterial diversification.

Declining diversity of egg-associated bacteria during development of naturally spawned whitefish embryos (Coregonus spp.)

Fish eggs are associated with microbes, whose roles range from mutualism to parasitism. Recent laboratory experiments have shown that the taxonomic composition of associated microbial communities on the egg influences embryonic development. Host genetics also plays an important role in determining the consequences for embryonic growth and survival in this interaction. Moreover, it has been found that the importance of host genetics increases during embryogenesis. These findings suggest that during embryogenesis, the host increasingly influences the composition of its associated microbial community. However, little is known about the composition of microbial communities associated with naturally spawned eggs in the wild. We sampled fertilized whitefish eggs (Coregonus spp.) of different developmental stages from six sub-Alpine lakes and used a universal primer pair and 454 pyrosequencing in order to describe the taxonomic composition of egg-associated bacterial communities. We found bacterial communities on early embryos to be very diverse and to resemble the bacterial composition of the surrounding water environment. The bacterial communities on late embryos were significantly less diverse than on early embryos and displayed a clear shift in taxonomic composition that corresponded poorly with the bacterial composition of the surrounding water environment. The main bacterial components on whitefish eggs in this study were Proteobacteria, Actinobacteria, and Firmicutes, while the five most common families were Leuconostocaceae, Streptococcaceae, Comamonadaceae, Oxalobacteraceae and Moraxellaceae. Their putative relationships with the host are discussed. We conclude that natural symbiotic bacterial communities become more specialized during embryogenesis because of specific interactions with their embryo host.

Has the airway microbiome been overlooked in respiratory disease?

The respiratory disease field is changing because of recent advances in our understanding of the airway microbiome. Central to this is dysbiosis, an imbalance of microbial communities that can lead to and flag inflammation in the airways. The increasing momentum of research in this area holds promise for novel treatment strategies.

Increased diversity of egg-associated bacteria on brown trout (Salmo trutta) at elevated temperatures.

The taxonomic composition of egg-associated microbial communities can play a crucial role in the development of fish embryos. In response, hosts increasingly influence the composition of their associated microbial communities during embryogenesis, as concluded from recent field studies and laboratory experiments. However, little is known about the taxonomic composition and the diversity of egg-associated microbial communities within ecosystems; e.g., river networks. We sampled late embryonic stages of naturally spawned brown trout at nine locations within two different river networks and applied 16S rRNA pyrosequencing to describe their bacterial communities. We found no evidence for a significant isolation-by-distance effect on the composition of bacterial communities, and no association between neutral genetic divergence of fish host (based on 11 microsatellites) and phylogenetic distances of the composition of their associated bacterial communities. We characterized core bacterial communities on brown trout eggs and compared them to corresponding water samples with regard to bacterial composition and its presumptive function. Bacterial diversity was positively correlated with water temperature at the spawning locations. We discuss this finding in the context of the increased water temperatures that have been recorded during the last 25 years in the study area.