A comparison of short-read, HiFi long-read, and hybrid strategies for genome-resolved metagenomics.

Shotgun metagenomics enables the reconstruction of complex microbial communities at a high level of detail. Such an approach can be conducted using both short-read and long-read sequencing data, as well as a combination of both. To assess the pros and cons of these different approaches, we used 22 fecal DNA extracts collected weekly for 11 weeks from two respective lab mice to study seven performance metrics over four combinations of sequencing depth and technology: (i) 20 Gbp of Illumina short-read data, (ii) 40 Gbp of short-read data, (iii) 20 Gbp of PacBio HiFi long-read data, and (iv) 40 Gbp of hybrid (20 Gbp of short-read +20 Gbp of long-read) data. No strategy was best for all metrics; instead, each one excelled across different metrics. The long-read approach yielded the best assembly statistics, with the highest N50 and lowest number of contigs. The 40 Gbp short-read approach yielded the highest number of refined bins. Finally, the hybrid approach yielded the longest assemblies and the highest mapping rate to the bacterial genomes. Our results suggest that while long-read sequencing significantly improves the quality of reconstructed bacterial genomes, it is more expensive and requires deeper sequencing than short-read approaches to recover a comparable amount of reconstructed genomes. The most optimal strategy is study-specific and depends on how researchers assess the trade-off between the quantity and quality of recovered genomes.IMPORTANCEMice are an important model organism for understanding the gut microbiome. When studying these gut microbiomes using DNA techniques, researchers can choose from technologies that use short or long DNA reads. In this study, we perform an extensive benchmark between short- and long-read DNA sequencing for studying mice gut microbiomes. We find that no one approach was best for all metrics and provide information that can help guide researchers in planning their experiments.

Field and laboratory guidelines for reliable bioinformatic and statistical analysis of bacterial shotgun metagenomic data.

Shotgun metagenomics is an increasingly cost-effective approach for profiling environmental and host-associated microbial communities. However, due to the complexity of both microbiomes and the molecular techniques required to analyze them, the reliability and representativeness of the results are contingent upon the field, laboratory, and bioinformatic procedures employed. Here, we consider 15 field and laboratory issues that critically impact downstream bioinformatic and statistical data processing, as well as result interpretation, in bacterial shotgun metagenomic studies. The issues we consider encompass intrinsic properties of samples, study design, and laboratory-processing strategies. We identify the links of field and laboratory steps with downstream analytical procedures, explain the means for detecting potential pitfalls, and propose mitigation measures to overcome or minimize their impact in metagenomic studies. We anticipate that our guidelines will assist data scientists in appropriately processing and interpreting their data, while aiding field and laboratory researchers to implement strategies for improving the quality of the generated results.

Mammals show distinct functional gut microbiome dynamics to identical series of environmental stressors.

IMPORTANCE: In our manuscript, we report the first interspecific comparative study about the plasticity of the gut microbiota. We conducted a captivity experiment that exposed wild-captured mammals to a series of environmental challenges over 45 days. We characterized their gut microbial communities using genome-resolved metagenomics and modeled how the taxonomic, phylogenetic, and functional microbial dynamics varied across a series of disturbances in both species. Our results indicate that the intrinsic properties (e.g., diversity and functional redundancy) of microbial communities coupled with physiological attributes (e.g., thermal plasticity) of hosts shape the taxonomic, phylogenetic, and functional response of gut microbiomes to environmental stressors, which might influence their contribution to the acclimation and adaptation capacity of animal hosts.

Unravelling animal-microbiota evolution on a chip.

Whether and how microorganisms have shaped the evolution of their animal hosts is a major question in biology. Although many animal evolutionary processes appear to correlate with changes in their associated microbial communities, the mechanistic processes leading to these patterns and their causal relationships are still far from being resolved. Gut-on-a-chip models provide an innovative approach that expands beyond the potential of conventional microbiome profiling to study how different animals sense and react to microbes by comparing responses of animal intestinal tissue models to different microbial stimuli. This complementary knowledge can contribute to our understanding of how host genetic features facilitate or prevent different microbiomes from being assembled, and in doing so elucidate the role of host-microbiota interactions in animal evolution.

A simplified protocol for DNA extraction from FTA cards for faecal microbiome studies.

As metagenomic studies continue to increase in size and complexity, they are often required to incorporate data from geographically isolated locations or longitudinal time samples. This represents a technical challenge, given that many of the commonly used methods used for sample collection, storage, and DNA extraction are sensitive to differences related to the time, storage and chemistry involved. FTA cards have been previously proposed as a simple, reliable and cost-efficient method for the preservation of animal faecal microbiomes. In this study, we report a simplified extraction methodology for recovering microbiome DNA from faeces stored on FTA cards and compare its performance to a common alternative means of characterising such microbiomes; namely, immediate freezing of the faeces followed by DNA extraction using the Qiagen PowerSoil DNA isolation kit. Our results show that overall the application of our simplified DNA extraction methodology yields microbial community results that have higher diversity and an expanded core microbiome than that found using the PowerSoil methodology. This suggests that the FTA card extraction method presented here is a viable alternative for metagenomic studies using faecal material when traditional freeze-based storage methods are not feasible.

Enriching captivity conditions with natural elements does not prevent the loss of wild-like gut microbiota but shapes its compositional variation in two small mammals.

As continued growth in gut microbiota studies in captive and model animals elucidates the importance of their role in host biology, further pursuit of how to retain a wild-like microbial community is becoming increasingly important to obtain representative results from captive animals. In this study, we assessed how the gut microbiota of two wild-caught small mammals, namely Crocidura russula (Eulipotyphla, insectivore) and Apodemus sylvaticus (Rodentia, omnivore), changed when bringing them into captivity. We analyzed fecal samples of 15 A. sylvaticus and 21 C. russula, immediately after bringing them into captivity and 5 weeks later, spread over two housing treatments: a "natural" setup enriched with elements freshly collected from nature and a "laboratory" setup with sterile artificial elements. Through sequencing of the V3-V4 region of the 16S recombinant RNA gene, we found that the initial microbial diversity dropped during captivity in both species, regardless of treatment. Community composition underwent a change of similar magnitude in both species and under both treatments. However, we did observe that the temporal development of the gut microbiome took different trajectories (i.e., changed in different directions) under different treatments, particularly in C. russula, suggesting that C. russula may be more susceptible to environmental change. The results of this experiment do not support the use of microbially enriched environments to retain wild-like microbial diversities and compositions, yet show that specific housing conditions can significantly affect the drift of microbial communities under captivity.

Gut microbiota differences between paired intestinal wall and digesta samples in three small species of fish.

The microbial gut communities of fish are receiving increased attention for their relevance, among others, in a growing aquaculture industry. The members of these communities are often split into resident (long-term colonisers specialised to grow in and adhere to the mucus lining of the gut) and transient (short-term colonisers originated from food items and the surrounding water) microorganisms. Separating these two communities in small fish are impeded by the small size and fragility of the gastrointestinal tract. With the aim of testing whether it is possible to recover two distinct communities in small species of fish using a simple sampling technique, we used 16S amplicon sequencing of paired intestinal wall and digesta samples from three small Cyprinodontiformes fish. We examined the diversity and compositional variation of the two recovered communities, and we used joint species distribution modelling to identify microbes that are most likely to be a part of the resident community. For all three species we found that the diversity of intestinal wall samples was significantly lower compared to digesta samples and that the community composition between sample types was significantly different. Across the three species we found seven unique families of bacteria to be significantly enriched in samples from the intestinal wall, encompassing most of the 89 ASVs enriched in intestinal wall samples. We conclude that it is possible to characterise two different microbial communities and identify potentially resident microbes through separately analysing samples from the intestinal wall and digesta from small species of fish. We encourage researchers to be aware that different sampling procedures for gut microbiome characterization will capture different parts of the microbiome and that this should be taken into consideration when reporting results from such studies on small species of fish.

Diversity and compositional changes in the gut microbiota of wild and captive vertebrates: a meta-analysis.

The gut microbiota is recognised as an essential asset for the normal functioning of animal biology. When wild animals are moved into captivity, the modified environmental pressures are expected to rewire the gut microbiota, yet whether this transition follows similar patterns across vertebrates is still unresolved due to the absence of systematic multi-species analyses. We performed a meta-analysis of gut microbiota profiles of 322 captive and 322 wild specimens from 24 vertebrate species. Our analyses yielded no overall pattern of diversity and compositional variation between wild and captive vertebrates, but a heterogeneous landscape of responses, which differed depending on the components of diversity considered. Captive populations showed enrichment patterns of human-associated microorganisms, and the minimal host phylogenetic signal suggests that changes between wild and captive populations are mainly driven by case-specific captivity conditions. Finally, we show that microbiota differences between wild and captive populations can impact evolutionary and ecological inferences that rely on hierarchical clustering-based comparative analyses of gut microbial communities across species.

The role of the gut microbiota in the dietary niche expansion of fishing bats.

BACKGROUND: Due to its central role in animal nutrition, the gut microbiota is likely a relevant factor shaping dietary niche shifts. We analysed both the impact and contribution of the gut microbiota to the dietary niche expansion of the only four bat species that have incorporated fish into their primarily arthropodophage diet. RESULTS: We first compared the taxonomic and functional features of the gut microbiota of the four piscivorous bats to that of 11 strictly arthropodophagous species using 16S rRNA targeted amplicon sequencing. Second, we increased the resolution of our analyses for one of the piscivorous bat species, namely Myotis capaccinii, and analysed multiple populations combining targeted approaches with shotgun sequencing. To better understand the origin of gut microorganisms, we also analysed the gut microbiota of their fish prey (Gambusia holbrooki). Our analyses showed that piscivorous bats carry a characteristic gut microbiota that differs from that of their strict arthropodophagous counterparts, in which the most relevant bacteria have been directly acquired from their fish prey. This characteristic microbiota exhibits enrichment of genes involved in vitamin biosynthesis, as well as complex carbohydrate and lipid metabolism, likely providing their hosts with an enhanced capacity to metabolise the glycosphingolipids and long-chain fatty acids that are particularly abundant in fish. CONCLUSIONS: Our results depict the gut microbiota as a relevant element in facilitating the dietary transition from arthropodophagy to piscivory.

Gut Microbiota Linked with Reduced Fear of Humans in Red Junglefowl Has Implications for Early Domestication.

Domestication of animals can lead to profound phenotypic modifications within short evolutionary time periods, and for many species behavioral selection is likely at the forefront of this process. Animal studies have strongly implicated that the gut microbiome plays a major role in host behavior and cognition through the microbiome-gut-brain axis. Consequently, herein, it is hypothesized that host gut microbiota may be one of the earliest phenotypes to change as wild animals were domesticated. Here, the gut microbiome community in two selected lines of red junglefowl that are selected for either high or low fear of humans up to eight generations is examined. Microbiota profiles reveal taxonomic differences in gut bacteria known to produce neuroactive compounds between the two selection lines. Gut-brain module analysis by means of genome-resolved metagenomics identifies enrichment in the microbial synthesis and degradation potential of metabolites associated with fear extinction and reduces anxiety-like behaviors in low fear fowls. In contrast, high fear fowls are enriched in gut-brain modules from the butyrate and glutamate pathways, metabolites associated with fear conditioning. Overall, the results identify differences in the composition and functional potential of the gut microbiota across selection lines that may provide insights into the mechanistic explanations of the domestication process.

Holo-Omics: Integrated Host-Microbiota Multi-omics for Basic and Applied Biological Research.

From ontogenesis to homeostasis, the phenotypes of complex organisms are shaped by the bidirectional interactions between the host organisms and their associated microbiota. Current technology can reveal many such interactions by combining multi-omic data from both hosts and microbes. However, exploring the full extent of these interactions requires careful consideration of study design for the efficient generation and optimal integration of data derived from (meta)genomics, (meta)transcriptomics, (meta)proteomics, and (meta)metabolomics. In this perspective, we introduce the holo-omic approach that incorporates multi-omic data from both host and microbiota domains to untangle the interplay between the two. We revisit the recent literature on biomolecular host-microbe interactions and discuss the implementation and current limitations of the holo-omic approach. We anticipate that the application of this approach can contribute to opening new research avenues and discoveries in biomedicine, biotechnology, agricultural and aquacultural sciences, nature conservation, as well as basic ecological and evolutionary research.

DNA metabarcoding and spatial modelling link diet diversification with distribution homogeneity in European bats.

Inferences of the interactions between species' ecological niches and spatial distribution have been historically based on simple metrics such as low-resolution dietary breadth and range size, which might have impeded the identification of meaningful links between niche features and spatial patterns. We analysed the relationship between dietary niche breadth and spatial distribution features of European bats, by combining continent-wide DNA metabarcoding of faecal samples with species distribution modelling. Our results show that while range size is not correlated with dietary features of bats, the homogeneity of the spatial distribution of species exhibits a strong correlation with dietary breadth. We also found that dietary breadth is correlated with bats' hunting flexibility. However, these two patterns only stand when the phylogenetic relations between prey are accounted for when measuring dietary breadth. Our results suggest that the capacity to exploit different prey types enables species to thrive in more distinct environments and therefore exhibit more homogeneous distributions within their ranges.

Trait-based functional dietary analysis provides a better insight into the foraging ecology of bats.

The degree of trophic specialization determines the ability of predators to cope with changing foraging conditions, but in predators that prey on hundreds of species it is challenging to assess, especially when prey identity varies among predator individuals and across space and time. Here, we test the hypothesis that a bat species foraging on flying insects like moths will show ample flexibility in trophic niche, and this irrespective of phylogenetic relationships among moths, so as to cope with a high diversity of prey types that vary across seasons. We predict that individual bats will show functional dietary differences consistent with energetic requirements and hunting skills. We used DNA metabarcoding to determine the diet of 126 Mediterranean horseshoe bats (Rhinolophus euryale) from two different sites during three seasons. Simultaneously, we measured moth availability and characterized the traits of 290 moth taxa. Next, we explored the relationship between phylogeny and traits of all consumed and available moth taxa. Finally, we assessed the relationship between individual traits of bats and traits related to prey profitability, for which we used the RLQ and fourth-corner statistical techniques. Seasonality was the main factor explaining the functional dietary variation in adult bats, with moths consumed irrespective of their phylogenetic relationships. While adults consumed moths with a broad range in wing loading, body mass and echolocation detection ability, juveniles consumed slower, smaller and lighter moths, which suggests that young individuals may undergo some fitness gain and/or psychomotor learning process during which they would acquire more effective foraging skills. Our approach revealed a degree of functional flexibility in the trophic niche previously unknown for an insectivorous bat. Rhinolophus euryale consumed a wide variety of moth taxa differing in profitability throughout seasons and between ontogenetic stages. We showed the validity of trait-based approaches to gain new insights in the trophic specialization of predators consuming hundreds of species of prey.

Promises and pitfalls of using high-throughput sequencing for diet analysis.

The application of high-throughput sequencing-based approaches to DNA extracted from environmental samples such as gut contents and faeces has become a popular tool for studying dietary habits of animals. Due to the high resolution and prey detection capacity they provide, both metabarcoding and shotgun sequencing are increasingly used to address ecological questions grounded in dietary relationships. Despite their great promise in this context, recent research has unveiled how a wealth of biological (related to the study system) and technical (related to the methodology) factors can distort the signal of taxonomic composition and diversity. Here, we review these studies in the light of high-throughput sequencing-based assessment of trophic interactions. We address how the study design can account for distortion factors, and how acknowledging limitations and biases inherent to sequencing-based diet analyses are essential for obtaining reliable results, thus drawing appropriate conclusions. Furthermore, we suggest strategies to minimize the effect of distortion factors, measures to increase reproducibility, replicability and comparability of studies, and options to scale up DNA sequencing-based diet analyses. In doing so, we aim to aid end-users in designing reliable diet studies by informing them about the complexity and limitations of DNA sequencing-based diet analyses, and encourage researchers to create and improve tools that will eventually drive this field to its maturity.

Agriculture shapes the trophic niche of a bat preying on multiple pest arthropods across Europe: Evidence from DNA metabarcoding.

The interaction between agricultural production and wildlife can shape, and even condition, the functioning of both systems. In this study, we i) explored the degree to which a widespread European bat, namely the common bent-wing bat Miniopterus schreibersii, consumes crop-damaging insects at a continental scale, and ii) tested whether its dietary niche is shaped by the extension and type of agricultural fields. We employed a dual-primer DNA metabarcoding approach to characterize arthropod 16S and COI DNA sequences within bat faecal pellets collected across 16 Southern European localities, to first characterize the bat species' dietary niche, second measure the incidence of agricultural pests across their ranges and third assess whether geographical dietary variation responds to climatic, landscape diversity, agriculture type and vegetation productivity factors. We detected 12 arthropod orders, among which lepidopterans were predominant. We identified >200 species, 44 of which are known to cause agricultural damage. Pest species were detected at all but one sampling site and in 94% of the analysed samples. Furthermore, the dietary diversity of M. schreibersii exhibited a negative linear relation with the area of intensive agricultural fields, thus suggesting crops restrict the dietary niche of bats to prey taxa associated with agricultural production within their foraging range. Overall, our results imply that M. schreibersii might be a valuable asset for biological pest suppression in a variety of agricultural productions and highlight the dynamic interplay between wildlife and agricultural systems.

Fishing Technique of Long-Fingered Bats Was Developed from a Primary Reaction to Disappearing Target Stimuli.

Behavioral plasticity is a key feature allowing animals to broaden their dietary niche when novel food resources become available, and long-fingered bats provide an appropriate model system to study the underpinnings of behavioral plasticity, since although generally being an insectivorous species, some individuals have been reported to catch fish. Aiming to get insight into the origin of fishing behavior in long-fingered bats, we studied in the field the differences in sensorial and mechanical reactions to insect-like (stationary) and fish-like (temporary) prey stimuli between well-known piscivorous and strictly insectivorous individuals. Both piscivorous and insectivorous individuals exhibited a qualitatively similar reaction to temporary target stimuli (longer and deeper dips and terminal echolocation phase skewed towards buzz I compared to stationary stimuli). Nevertheless, the quantitative differences observed in the sensorial and mechanical features (the intensity of the shift was significantly greater in piscivorous than in insectivorous individuals) show that piscivorous individuals have honed their capture technique likely enhancing the fishing success. Thus, our results suggest that the fishing technique was developed from a primary reaction shared by all long-fingered bats. All individuals seem to be mechanically and sensorially adapted to detect and capture fish, although under appropriate environmental conditions, they would further improve their technique by experience and/or social learning.

Do Vertebrate Gut Metagenomes Confer Rapid Ecological Adaptation?

During times of rapid environmental change, survival of most vertebrate populations depends on their phenomic plasticity. Although differential gene-expression and post-transcriptional processes of the host genome receive focus as the main molecular mechanisms, growing evidence points to the gut microbiota as a key driver defining hosts' phenotypes. We propose that the plasticity of the gut microbiota might be an essential factor determining phenomic plasticity of vertebrates, and that it might play a pivotal role when vertebrates acclimate and adapt to fast environmental variation. We contemplate some key questions and suggest methodological approaches and experimental designs that can be used to evaluate whether gut microorganisms provide a boost of plasticity to vertebrates' phenomes, thereby increasing their acclimation and adaptation capacity.

Insight on how fishing bats discern prey and adjust their mechanic and sensorial features during the attack sequence.

Several insectivorous bats have included fish in their diet, yet little is known about the processes underlying this trophic shift. We performed three field experiments with wild fishing bats to address how they manage to discern fish from insects and adapt their hunting technique to capture fish. We show that bats react only to targets protruding above the water and discern fish from insects based on prey disappearance patterns. Stationary fish trigger short and shallow dips and a terminal echolocation pattern with an important component of the narrowband and low frequency calls. When the fish disappears during the attack process, bats regulate their attack increasing the number of broadband and high frequency calls in the last phase of the echolocation as well as by lengthening and deepening their dips. These adjustments may allow bats to obtain more valuable sensorial information and to perform dips adjusted to the level of uncertainty on the location of the submerged prey. The observed ultrafast regulation may be essential for enabling fishing to become cost-effective in bats, and demonstrates the ability of bats to rapidly modify and synchronise their sensorial and motor features as a response to last minute stimulus variations.

Unveiling the factors shaping the distribution of widely distributed alpine vertebrates, using multi-scale ecological niche modelling of the bat Plecotus macrobullaris.

Several alpine vertebrates share a distribution pattern that extends across the South-western Palearctic but is limited to the main mountain massifs. Although they are usually regarded as cold-adapted species, the range of many alpine vertebrates also includes relatively warm areas, suggesting that factors beyond climatic conditions may be driving their distribution. In this work we first recognize the species belonging to the mentioned biogeographic group and, based on the environmental niche analysis of Plecotus macrobullaris, we identify and characterize the environmental factors constraining their ranges. Distribution overlap analysis of 504 European vertebrates was done using the Sorensen Similarity Index, and we identified four birds and one mammal that share the distribution with P. macrobullaris. We generated 135 environmental niche models including different variable combinations and regularization values for P. macrobullaris at two different scales and resolutions. After selecting the best models, we observed that topographic variables outperformed climatic predictors, and the abruptness of the landscape showed better predictive ability than elevation. The best explanatory climatic variable was mean summer temperature, which showed that P. macrobullaris is able to cope with mean temperature ranges spanning up to 16°C. The models showed that the distribution of P. macrobullaris is mainly shaped by topographic factors that provide rock-abundant and open-space habitats rather than climatic determinants, and that the species is not a cold-adapted, but rather a cold-tolerant eurithermic organism. P. macrobullaris shares its distribution pattern as well as several ecological features with five other alpine vertebrates, suggesting that the conclusions obtained from this study might be extensible to them. We concluded that rock-dwelling and open-space foraging vertebrates with broad temperature tolerance are the best candidates to show wide alpine distribution in the Western Palearctic.

Fine-tuned echolocation and capture-flight of Myotis capaccinii when facing different-sized insect and fish prey.

Formerly thought to be a strictly insectivorous trawling bat, recent studies have shown that Myotis capaccinii also preys on fish. To determine whether differences exist in bat flight behaviour, prey handling and echolocation characteristics when catching fish and insects of different size, we conducted a field experiment focused on the last stage of prey capture. We used synchronized video and ultrasound recordings to measure several flight and dip features as well as echolocation characteristics, focusing on terminal buzz phase I, characterized by a call rate exceeding 100 Hz, and buzz phase II, characterized by a drop in the fundamental well below 20 kHz and a repetition rate exceeding 150 Hz. When capturing insects, bats used both parts of the terminal phase to the same extent, and performed short and superficial drags on the water surface. In contrast, when preying on fish, buzz I was longer and buzz II shorter, and the bats made longer and deeper dips. These variations suggest that lengthening buzz I and shortening buzz II when fishing is beneficial, probably because buzz I gives better discrimination ability and the broader sonar beam provided by buzz II is useless when no evasive flight of the prey is expected. Additionally, bats continued emitting calls beyond the theoretical signal-overlap zone, suggesting that they might obtain information even when they have surpassed that threshold, at least initially. This study shows that M. capaccinii can regulate the temporal components of its feeding buzzes and modify prey capture technique according to the target.