Gut microbiota of homing pigeons shows summer-winter variation under constant diet indicating a substantial effect of temperature.

BACKGROUND: Gut microbiotas play a pivotal role in host physiology and behaviour, and may affect host life-history traits such as seasonal variation in host phenotypic state. Generally, seasonal gut microbiota variation is attributed to seasonal diet variation. However, seasonal temperature and day length variation may also drive gut microbiota variation. We investigated summer-winter differences in the gut bacterial community (GBC) in 14 homing pigeons living outdoors under a constant diet by collecting cloacal swabs in both seasons during two years. Because temperature effects may be mediated by host metabolism, we determined basal metabolic rate (BMR) and body mass. Immune competence is influenced by day length and has a close relationship with the GBC, and it may thus be a link between day length and gut microbiota. Therefore, we measured seven innate immune indices. We expected the GBC to show summer-winter differences and to correlate with metabolism and immune indices. RESULTS: BMR, body mass, and two immune indices varied seasonally, other host factors did not. The GBC showed differences between seasons and sexes, and correlated with metabolism and immune indices. The most abundant genus (Lachnoclostridium 12, 12%) and associated higher taxa, were more abundant in winter, though not significantly at the phylum level, Firmicutes. Bacteroidetes were more abundant in summer. The Firmicutes:Bacteroidetes ratio tended to be higher in winter. The KEGG ortholog functions for fatty acid biosynthesis and linoleic acid metabolism (PICRUSt2) had increased abundances in winter. CONCLUSIONS: The GBC of homing pigeons varied seasonally, even under a constant diet. The correlations between immune indices and the GBC did not involve consistently specific immune indices and included only one of the two immune indices that showed seasonal differences, suggesting that immune competence may be an unlikely link between day length and the GBC. The correlations between the GBC and metabolism indices, the higher Firmicutes:Bacteroidetes ratio in winter, and the resemblance of the summer-winter differences in the GBC with the general temperature effects on the GBC in the literature, suggest that temperature partly drove the summer-winter differences in the GBC in homing pigeons.

Microbiome Heritability and Its Role in Adaptation of Hosts to Novel Resources.

Microbiomes are involved in most vital processes, such as immune response, detoxification, and digestion and are thereby elementary to organismal functioning and ultimately the host's fitness. In turn, the microbiome may be influenced by the host and by the host's environment. To understand microbiome dynamics during the process of adaptation to new resources, we performed an evolutionary experiment with the two-spotted spider mite, Tetranychus urticae. We generated genetically depleted strains of the two-spotted spider mite and reared them on their ancestral host plant and two novel host plants for approximately 12 generations. The use of genetically depleted strains reduced the magnitude of genetic adaptation of the spider mite host to the new resource and, hence, allowed for better detection of signals of adaptation via the microbiome. During the course of adaptation, we tested spider mite performance (number of eggs laid and longevity) and characterized the bacterial component of its microbiome (16S rRNA gene sequencing) to determine: (1) whether the bacterial communities were shaped by mite ancestry or plant environment and (2) whether the spider mites' performance and microbiome composition were related. We found that spider mite performance on the novel host plants was clearly correlated with microbiome composition. Because our results show that only little of the total variation in the microbiome can be explained by the properties of the host (spider mite) and the environment (plant species) we studied, we argue that the bacterial community within hosts could be valuable for understanding a species' performance on multiple resources.

Prenatal Transfer of Gut Bacteria in Rock Pigeon.

Vertebrates evolved in concert with bacteria and have developed essential mutualistic relationships. Gut bacteria are vital for the postnatal development of most organs and the immune and metabolic systems and may likewise play a role during prenatal development. Prenatal transfer of gut bacteria is shown in four mammalian species, including humans. For the 92% of the vertebrates that are oviparous, prenatal transfer is debated, but it has been demonstrated in domestic chicken. We hypothesize that also non-domestic birds can prenatally transmit gut bacteria. We investigated this in medium-sized Rock pigeon (Columba livia), ensuring neonates producing fair-sized first faeces. The first faeces of 21 neonate rock pigeons hatched in an incubator, contained a microbiome (bacterial community) the composition of which resembled the cloacal microbiome of females sampled from the same population (N = 5) as indicated by multiple shared phyla, orders, families, and genera. Neonates and females shared 16.1% of the total number of OTUs present (2881), and neonates shared 45.5% of their core microbiome with females. In contrast, the five females shared only 0.3% of the 1030 female OTUs present. These findings suggest that prenatal gut bacterial transfer may occur in birds. Our results support the hypothesis that gut bacteria may be important for prenatal development and present a heritability pathway of gut bacteria in vertebrates.

Coral Bacterial-Core Abundance and Network Complexity as Proxies for Anthropogenic Pollution.

Acclimatization via changes in the stable (core) or the variable microbial diversity and/or abundance is an important element in the adaptation of coral species to environmental changes. Here, we explored the spatial-temporal dynamics, diversity and interactions of variable and core bacterial populations associated with the coral Mussismilia hispida and the surrounding water. This survey was performed on five reefs along a transect from the coast (Reef 1) to offshore (Reef 5), representing a gradient of influence of the river mouth, for almost 12 months (4 sampling times), in the dry and rainy seasons. A clear increasing gradient of organic-pollution proxies (nitrogen content and fecal coliforms) was observed from Reef 1 to Reef 5, during both seasons, and was highest at the Buranhém River mouth (Reef 1). Conversely, a clear inverse gradient of the network analysis of the whole bacterial communities also revealed more-complex network relationships at Reef 5. Our data also indicated a higher relative abundance of members of the bacterial core, dominated by Acinetobacter sp., at Reef 5, and higher diversity of site-stable bacterial populations, likely related to the higher abundance of total coliforms and N content (proxies of sewage or organic pollution) at Reef 1, during the rainy season. Thus, the less "polluted" areas may show a more-complex network and a high relative abundance of members of the bacterial core (almost 97% in some cases), resulting in a more-homogeneous and well-established bacteriome among sites/samples, when the influence of the river is stronger (rainy seasons).

Specific plasmid patterns and high rates of bacterial co-occurrence within the coral holobiont.

Despite the importance of coral microbiomes for holobiont persistence, the interactions among these are not well understood. In particular, knowledge of the co-occurrence and taxonomic importance of specific members of the microbial core, as well as patterns of specific mobile genetic elements (MGEs), is lacking. We used seawater and mucus samples collected from Mussismilia hispida colonies on two reefs located in Bahia, Brazil, to disentangle their associated bacterial communities, intertaxa correlations, and plasmid patterns. Proxies for two broad-host-range (BHR) plasmid groups, IncP-1ß and PromA, were screened. Both groups were significantly (up to 252 and 100%, respectively) more abundant in coral mucus than in seawater. Notably, the PromA plasmid group was detected only in coral mucus samples. The core bacteriome of M. hispida mucus was composed primarily of members of the Proteobacteria, followed by those of Firmicutes. Significant host specificity and co-occurrences among different groups of the dominant phyla (e.g., Bacillaceae and Pseudoalteromonadaceae and the genera Pseudomonas, Bacillus, and Vibrio) were detected. These relationships were observed for both the most abundant phyla and the bacteriome core, in which most of the operational taxonomic units showed intertaxa correlations. The observed evidence of host-specific bacteriome and co-occurrence (and potential symbioses or niche space co-dominance) among the most dominant members indicates a taxonomic selection of members of the stable bacterial community. In parallel, host-specific plasmid patterns could also be, independently, related to the assembly of members of the coral microbiome.

Functionality of Root-Associated Bacteria along a Salt Marsh Primary Succession.

Plant-associated bacteria are known for their high functional trait diversity, from which many are likely to play a role in primary and secondary succession, facilitating plant establishment in suboptimal soils conditions. Here we used an undisturbed salt marsh chronosequence that represents over 100 years of soil development to assess how the functional traits of plant associated bacteria respond to soil type, plant species and plant compartment. We isolated and characterized 808 bacterial colonies from the rhizosphere soil and root endosphere of two salt marsh plants, Limonium vulgare and Artemisia maritima, along the chronosequence. From these, a set of 59 strains (with unique BOX-PCR patterns, 16S rRNA sequence and unique to one of the treatments) were further screened for their plant growth promoting traits (siderophore production, IAA production, exoprotease production and biofilm formation), traits associated with bacterial fitness (antibiotic and abiotic stress resistance - pH, osmotic and oxidative stress, and salinity) and metabolic potential. An overall view of functional diversity (multivariate analysis) indicated that the distributional pattern of bacterial functional traits was driven by soil type. Samples from the late succession (Stage 105 year) showed the most restricted distribution, harboring strains with relatively low functionalities, whereas the isolates from intermediate stage (35 year) showed a broad functional profiles. However, strains with high trait performance were largely from stage 65 year. Grouping the traits according to category revealed that the functionality of plant endophytes did not vary along the succession, thus being driven by plant rather than soil type. In opposition, the functionality of rhizosphere isolates responded strongly to variations in soil type as observed for antibiotic resistance (P = 0.014). Specifically, certain Pseudomonas sp. and Serratia sp. strains revealed high resistance against abiotic stress and antibiotics and produce more siderophores, confirming the high plant-growth promoting activity of these two genera. Overall, this study contributes to a better understanding of the functional diversity and adaptation of the microbiome at typical salt marsh plant species across soil types. Specifically, soil type was influential only in the rhizosphere but not on the endosphere, indicating a strong plant-driven effect on the functionality of endophytes.

Legacy Effects on the Recovery of Soil Bacterial Communities from Extreme Temperature Perturbation.

The type and frequency of disturbances experienced by soil microbiomes is expected to increase given predicted global climate change scenarios and intensified anthropogenic pressures on ecosystems. While the direct effect of multiple disturbances to soil microbes has been explored in terms of function, their effect on the recovery of microbial community composition remains unclear. Here, we used soil microcosm experiments and multiple model disturbances to explore their short-term effect on the recovery of soil microbiota after identical or novel stresses. Soil microcosms were exposed to a heat shock to create an initial effect. Upon initial community recovery (25 days after stress), they were subjected to a second stress, either a heat or a cold shock, and they were monitored for additional 25 days. To carefully verify the bacterial response to the disturbances, we monitored changes in community composition throughout the experiment using 16S rRNA gene transcript amplicon sequencing. The application of a heat shock to soils with or without the initial heat shock resulted in similar successional dynamics, but these dynamics were faster in soils with a prior heat shock. The application of a cold shock had negligible effects on previously undisturbed soils but, in combination with an initial heat shock, caused the largest shift in the community composition. Our findings show that compounded perturbation affects bacterial community recovery by altering community structure and thus, the community's response during succession. By altering dominance patterns, disturbance legacy affects the microbiome's ability to recover from further perturbation within the 25 days studied. Our results highlight the need to consider the soil's disturbance history in the development of soil management practices in order to maintain the system's resilience.

Bacterial Synergism in Lignocellulose Biomass Degradation - Complementary Roles of Degraders As Influenced by Complexity of the Carbon Source.

Lignocellulosic biomass (LCB) is an attractive source of carbon for the production of sugars and other chemicals. Due to its inherent complexity and heterogeneity, efficient biodegradation requires the actions of different types of hydrolytic enzymes. In nature, complex microbial communities that work efficiently and often synergistically accomplish degradation. Studying such synergisms in LCB degradation is fundamental for the establishment of an optimal biological degradation process. Here, we examine the wheat straw degradation potential of synthetic microbial consortia composed of bacteria and fungi. Growth of, and enzyme secretion by, monocultures of degrader strains were studied in aerobic cultures using wheat straw as the sole carbon and energy source. To investigate synergism, co-cultures were constructed from selected strains and their performance was tested in comparison with the respective monocultures. In monoculture, each organism - with a typical enzymatic profile - was found to mainly consume the cellulose part of the substrate. One strain, Flavobacterium ginsengisoli so9, displayed an extremely high degradation capacity, as measured by its secreted enzymes. Among 13 different co-cultures, five presented synergisms. These included four bacterial bicultures and one bacterial-fungal triculture. The highest level of synergism was found in a Citrobacter freundii/Sphingobacterium multivorum biculture, which revealed an 18.2-fold increase of the produced biomass. As compared to both monocultures, this bacterial pair showed significantly increased enzymatic activities, in particular of cellobiohydrolases, mannosidases, and xylosidases. Moreover, the synergism was unique to growth on wheat straw, as it was completely absent in glucose-grown bicultures. Spent supernatants of either of the two partners were found to stimulate the growth on wheat straw of the counterpart organism, in a directional manner. Thus, the basis of the LCB-specific synergism might lie in the specific release of compounds or agents by S. multivorum w15 that promote the activity of C. freundii so4 and vice versa.

Serotonin Transporter Genotype Modulates the Gut Microbiota Composition in Young Rats, an Effect Augmented by Early Life Stress.

The neurotransmitter serotonin (5-HT) plays a vital regulatory role in both the brain and gut. 5-HT is crucial for regulating mood in the brain as well as gastrointestinal motility and secretion peripherally. Alterations in 5-HT transmission have been linked to pathological symptoms in both intestinal and psychiatric disorders and selective 5-HT transporter (5-HTT) inhibitors, affecting the 5-HT system by blocking the 5-HT transporter (5-HTT) have been successfully used to treat CNS- and intestinal disorders. Humans that carry the short allele of the 5-HTT-linked polymorphic region (5-HTTLPR) are more vulnerable to adverse environmental stressors, in particular early life stress. Although, early life stress has been shown to alter the composition of the gut microbiota, it is not known whether a lower 5-HTT expression is also associated with an altered microbiome composition. To investigate this, male and female wild type (5-HTT+/+), heterozygous (5-HTT+/-), and knockout (5-HTT-/-) 5-HT transporter rats were maternally separated for 6 h a day from postnatal day 2 till 15. On postnatal day 21, fecal samples were collected and the impact of 5-HTT genotype and maternal separation (MS) on the microbiome was analyzed using high-throughput sequencing of the bacterial 16S rRNA gene. MS showed a shift in the ratio between the two main bacterial phyla characterized by a decrease in Bacteroidetes and an increase in Firmicutes. Interestingly, the 5-HTT genotype caused a greater microbal dysbiosis (microbial imbalance) compared with MS. A significant difference in microbiota composition was found segregating 5-HTT-/- apart from 5-HTT+/- and 5-HTT+/+ rats. Moreover, exposure of rats with 5-HTT diminished expression to MS swayed the balance of their microbiota away from homeostasis to 'inflammatory' type microbiota characterized by higher abundance of members of the gut microbiome including Desulfovibrio, Mucispirillum, and Fusobacterium, all of which are previously reported to be associated with a state of intestinal inflammation, including inflammation associated with MS and brain disorders like multiple depressive disorders. Overall, our data show for the first time that altered expression of 5-HTT induces disruptions in male and female rat gut microbes and these 5-HTT genotype-related disruptions are augmented when combined with early life stress.

Where less may be more: how the rare biosphere pulls ecosystems strings.

Rare species are increasingly recognized as crucial, yet vulnerable components of Earth's ecosystems. This is also true for microbial communities, which are typically composed of a high number of relatively rare species. Recent studies have demonstrated that rare species can have an over-proportional role in biogeochemical cycles and may be a hidden driver of microbiome function. In this review, we provide an ecological overview of the rare microbial biosphere, including causes of rarity and the impacts of rare species on ecosystem functioning. We discuss how rare species can have a preponderant role for local biodiversity and species turnover with rarity potentially bound to phylogenetically conserved features. Rare microbes may therefore be overlooked keystone species regulating the functioning of host-associated, terrestrial and aquatic environments. We conclude this review with recommendations to guide scientists interested in investigating this rapidly emerging research area.

Reconstructing the Genetic Potential of the Microbially-Mediated Nitrogen Cycle in a Salt Marsh Ecosystem.

Coastal ecosystems are considered buffer zones for the discharge of land-derived nutrients without accounting for potential negative side effects. Hence, there is an urgent need to better understand the ecological assembly and dynamics of the microorganisms that are involved in nitrogen (N) cycling in such systems. Here, we employed two complementary methodological approaches (i.e., shotgun metagenomics and quantitative PCR) to examine the distribution and abundance of selected microbial genes involved in N transformations. We used soil samples collected along a well-established pristine salt marsh soil chronosequence that spans over a century of ecosystem development at the island of Schiermonnikoog, The Netherlands. Across the examined soil successional stages, the structure of the populations of genes involved in N cycling processes was strongly related to (shifts in the) soil nitrogen levels (i.e., [Formula: see text], [Formula: see text]), salinity and pH (explaining 73.8% of the total variation, R (2) = 0.71). Quantification of the genes used as proxies for N fixation, nitrification and denitrification revealed clear successional signatures that corroborated the taxonomic assignments obtained by metagenomics. Notably, we found strong evidence for niche partitioning, as revealed by the abundance and distribution of marker genes for nitrification (ammonia-oxidizing bacteria and archaea) and denitrification (nitrite reductase nirK, nirS and nitrous oxide reductase nosZ clades I and II). This was supported by a distinct correlation between these genes and soil physico-chemical properties, such as soil physical structure, pH, salinity, organic matter, total N, [Formula: see text], [Formula: see text] and [Formula: see text], across four seasonal samplings. Overall, this study sheds light on the successional trajectories of microbial N cycle genes along a naturally developing salt marsh ecosystem. The data obtained serve as a foundation to guide the formulation of ecological models that aim to effectively monitor and manage pristine and impacted salt marsh areas. Such models should account for the ecology as well as the historical contingency of N cycling communities.

Shifts in bacterial communities of eggshells and antimicrobial activities in eggs during incubation in a ground-nesting passerine.

Microbial invasion of egg contents is a cause of embryonic death. To counter infection risks, the embryo is protected physically by the eggshell and chemically by antimicrobial proteins. If microbial pressure drives embryo mortality, then females may have evolved, through natural selection, to adapt their immune investment into eggs. Although frequently hypothesized, this match between immune allocation and microorganisms has not been explored yet. To examine if correlations between microbes on eggs and immunity in eggs exist, we collected eggs from red-capped larks (Calandrella cinerea) and simultaneously examined their bacterial communities and antimicrobial components--pH, lysozyme and ovotransferrin--during natural incubation. Using molecular techniques, we find that bacterial communities are highly dynamic: bacterial abundance increases from the onset to late incubation, Shannon's α-diversity index increases during early incubation stages, and ß-diversity analysis shows that communities from 1 day-old clutches are phylogenetically more similar to each other than the older ones. Regarding the antimicrobials, we notice a decrease of pH and lysozyme concentration, while ovotransferrin concentration increases during incubation. Interestingly, we show that two eggs of the same clutch share equivalent immune protection, independent of clutch age. Lastly, our results provide limited evidence of significant correlation between antimicrobial compounds and bacterial communities. Our study examined simultaneously, for the first time in a wild bird, the dynamics of bacterial communities present on eggshells and of albumen-associated antimicrobial components during incubation and investigated their relationship. However, the link between microorganisms and immunity of eggs remains to be elucidated further. Identifying invading microbes and their roles in embryo mortality, as well as understanding the role of the eggshell microbiome, might be key to better understand avian strategies of immune maternal investment.

Dynamics of bacterial and fungal communities associated with eggshells during incubation.

Microorganisms are closely associated with eggs and may play a determinant role in embryo survival. Yet, the majority of studies focusing on this association relied on culture-based methodology, eventually leading to a skewed assessment of microbial communities. By targeting the 16S rRNA gene and internal transcribed spacer (ITS) region, we, respectively, described bacterial and fungal communities on eggshells of the homing pigeon Columba livia. We explored their structure, abundance, and composition. Firstly, we showed that sampling technique affected the outcome of the results. While broadly used, the egg swabbing procedure led to a lower DNA extraction efficiency and provided different profiles of bacterial communities than those based on crushed eggshell pieces. Secondly, we observed shifts in bacterial and fungal communities during incubation. At late incubation, bacterial communities showed a reduction in diversity, while their abundance increased, possibly due to the competitive advantage of some species. When compared to their bacterial counterparts, fungal communities also decreased in diversity at late incubation. In that case, however, the decline was associated with a diminution of their overall abundance. Conclusively, our results showed that although incubation might inhibit microbial growth when compared to unincubated eggs, we observed the selective growth of specific bacterial species during incubation. Moreover, we showed that fungi are a substantial component of the microbial communities associated with eggshells and require further investigations in avian ecology. Identifying the functional roles of these microorganisms is likely to provide news insights into the evolutionary strategies that control embryo survival. We aimed to describe the dynamics of bacterial and fungal communities on homing pigeon eggshell surfaces. We investigated these communities at early and late incubation stages.

Effect of soil sample preservation, compared to the effect of other environmental variables, on bacterial and eukaryotic diversity.

Archived soil samples are a valuable source for retrospective ecological studies, and their recent analysis using molecular ecological approaches has drawn significant attention within the scientific community. However, the possibility of addressing ecological questions regarding detectable microbiota in dried and extensively stored soils has not yet been fully evaluated. To achieve this, soil samples collected from two long-term grassland experiments in the United Kingdom and The Netherlands were subjected to air-drying at 40-42 degrees C and stored at room temperature. Total bacterial, Bacillus benzoevorans-related and eukaryotic communities associated with these samples were analyzed by DGGE-fingerprinting of PCR-amplified ribosomal RNA gene fragments. Changes in microbial community structure due to drying and storage were evaluated by multivariate analysis in relation to changes caused by other environmental conditions, such as soil pH, type of fertilizer and vegetation. Soil drying and storage affected the detectable community structure, but did not materially impair our capacity to identify the effect of soil parameters studied in long-term grassland experiments. Although, in some cases, the amplitude of the influence of a given parameter changed due to sample preservation, analyses revealed that pH, fertilization and soil type significantly influenced microbial community structure in the analyzed samples.

Community niche predicts the functioning of denitrifying bacterial assemblages.

Predicting biodiversity effects on ecosystem functioning requires adequate evaluation of the mechanisms explaining why more diverse systems could perform better than less diverse ones. In this context, tackling functional diversity has become an important issue. Even though the aggregation of species into functional groups supposes niche differences among groups, the concept of niche has not been fully exploited in the context of the biodiversity-ecosystem functioning research. Here we report the results of microcosm experiments where we used bacteria as a model to explore whether niche differences among species provide a good estimation of community functioning. For that we used experimental communities of denitrifying bacterial species and investigated the effects of bacterial diversity on two community processes, denitrification and anaerobic CO2-production. We first measured the activities of 16 bacterial species grown individually on six different carbon sources. We then used the same set of species to assemble communities varying in both species richness and composition in microcosms containing a mixture of all six carbon sources. The performances of individual species on individual carbon sources were used to calculate, for each process measured, an a priori index called "community niche" that accounted for the performances of the species present in a given community across the entire range of the six resources. We found that species richness had a positive but small effect on both community processes whereas community niche explained a much larger proportion of the variation. According to the results of a path analysis, community niche was the main driver for the corresponding community process, but species richness affected community niche and thus had an indirect effect on denitrification and CO2 production. In addition to community niche, the presence of particular bacterial species also influenced community functioning, indicating that other effects than the capacity to use carbon sources played a, albeit minor, role in our experiment. Our study provides evidence for the importance of resource niches in shaping biodiversity-ecosystem functioning relationships of bacterial communities.

Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays.

BACKGROUND: Diagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex, quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput, ligation-based assay for simultaneous quantitative detection of multiple plant pathogens. The ligation probes, designated Plant Research International-lock probes (PRI-lock probes), are long oligonucleotides with target complementary regions at their 5' and 3' ends. Upon perfect target hybridization, the PRI-lock probes are circularized via enzymatic ligation, subsequently serving as template for individual, standardized amplification via unique probe-specific primers. Adaptation to OpenArrays, which can accommodate up to 3072 33 nl PCR amplifications, allowed high-throughput real-time quantification. The assay combines the multiplex capabilities and specificity of ligation reactions with high-throughput real-time PCR in the OpenArray, resulting in a flexible, quantitative multiplex diagnostic system. RESULTS: The performance of the PRI-lock detection system was demonstrated using 13 probes targeting several significant plant pathogens at different taxonomic levels. All probes specifically detected their corresponding targets and provided perfect discrimination against non-target organisms with very similar ligation target sites. The nucleic acid targets could be reliably quantified over 5 orders of magnitude with a dynamic detection range of more than 104. Pathogen quantification was equally robust in single target versus mixed target assays. CONCLUSION: This novel assay enables very specific, high-throughput, quantitative detection of multiple pathogens over a wide range of target concentrations and should be easily adaptable for versatile diagnostic purposes.