Systemic alterations in neutrophils and their precursors in early-stage chronic obstructive pulmonary disease.

Systemic inflammation is established as part of late-stage severe lung disease, but molecular, functional, and phenotypic changes in peripheral immune cells in early disease stages remain ill defined. Chronic obstructive pulmonary disease (COPD) is a major respiratory disease characterized by small-airway inflammation, emphysema, and severe breathing difficulties. Using single-cell analyses we demonstrate that blood neutrophils are already increased in early-stage COPD, and changes in molecular and functional neutrophil states correlate with lung function decline. Assessing neutrophils and their bone marrow precursors in a murine cigarette smoke exposure model identified similar molecular changes in blood neutrophils and precursor populations that also occur in the blood and lung. Our study shows that systemic molecular alterations in neutrophils and their precursors are part of early-stage COPD, a finding to be further explored for potential therapeutic targets and biomarkers for early diagnosis and patient stratification.

hCoCena: horizontal integration and analysis of transcriptomics datasets.

MOTIVATION: Transcriptome-based gene co-expression analysis has become a standard procedure for structured and contextualized understanding and comparison of different conditions and phenotypes. Since large study designs with a broad variety of conditions are costly and laborious, extensive comparisons are hindered when utilizing only a single dataset. Thus, there is an increased need for tools that allow the integration of multiple transcriptomic datasets with subsequent joint analysis, which can provide a more systematic understanding of gene co-expression and co-functionality within and across conditions. To make such an integrative analysis accessible to a wide spectrum of users with differing levels of programming expertise it is essential to provide user-friendliness and customizability as well as thorough documentation. RESULTS: This article introduces horizontal CoCena (hCoCena: horizontal construction of co-expression networks and analysis), an R-package for network-based co-expression analysis that allows the analysis of a single transcriptomic dataset as well as the joint analysis of multiple datasets. With hCoCena, we provide a freely available, user-friendly and adaptable tool for integrative multi-study or single-study transcriptomics analyses alongside extensive comparisons to other existing tools. AVAILABILITY AND IMPLEMENTATION: The hCoCena R-package is provided together with R Markdowns that implement an exemplary analysis workflow including extensive documentation and detailed descriptions of data structures and objects. Such efforts not only make the tool easy to use but also enable the seamless integration of user-written scripts and functions into the workflow, creating a tool that provides a clear design while remaining flexible and highly customizable. The package and additional information including an extensive Wiki are freely available on GitHub: https://github.com/MarieOestreich/hCoCena. The version at the time of writing has been added to Zenodo under the following link: https://doi.org/10.5281/zenodo.6911782. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Disease severity-specific neutrophil signatures in blood transcriptomes stratify COVID-19 patients.

BACKGROUND: The SARS-CoV-2 pandemic is currently leading to increasing numbers of COVID-19 patients all over the world. Clinical presentations range from asymptomatic, mild respiratory tract infection, to severe cases with acute respiratory distress syndrome, respiratory failure, and death. Reports on a dysregulated immune system in the severe cases call for a better characterization and understanding of the changes in the immune system. METHODS: In order to dissect COVID-19-driven immune host responses, we performed RNA-seq of whole blood cell transcriptomes and granulocyte preparations from mild and severe COVID-19 patients and analyzed the data using a combination of conventional and data-driven co-expression analysis. Additionally, publicly available data was used to show the distinction from COVID-19 to other diseases. Reverse drug target prediction was used to identify known or novel drug candidates based on finding from data-driven findings. RESULTS: Here, we profiled whole blood transcriptomes of 39 COVID-19 patients and 10 control donors enabling a data-driven stratification based on molecular phenotype. Neutrophil activation-associated signatures were prominently enriched in severe patient groups, which was corroborated in whole blood transcriptomes from an independent second cohort of 30 as well as in granulocyte samples from a third cohort of 16 COVID-19 patients (44 samples). Comparison of COVID-19 blood transcriptomes with those of a collection of over 3100 samples derived from 12 different viral infections, inflammatory diseases, and independent control samples revealed highly specific transcriptome signatures for COVID-19. Further, stratified transcriptomes predicted patient subgroup-specific drug candidates targeting the dysregulated systemic immune response of the host. CONCLUSIONS: Our study provides novel insights in the distinct molecular subgroups or phenotypes that are not simply explained by clinical parameters. We show that whole blood transcriptomes are extremely informative for COVID-19 since they capture granulocytes which are major drivers of disease severity.

Association of coral algal symbionts with a diverse viral community responsive to heat shock.

BACKGROUND: Stony corals provide the structural foundation of coral reef ecosystems and are termed holobionts given they engage in symbioses, in particular with photosynthetic dinoflagellates of the genus Symbiodinium. Besides Symbiodinium, corals also engage with bacteria affecting metabolism, immunity, and resilience of the coral holobiont, but the role of associated viruses is largely unknown. In this regard, the increase of studies using RNA sequencing (RNA-Seq) to assess gene expression provides an opportunity to elucidate viral signatures encompassed within the data via careful delineation of sequence reads and their source of origin. RESULTS: Here, we re-analyzed an RNA-Seq dataset from a cultured coral symbiont (Symbiodinium microadriaticum, Clade A1) across four experimental treatments (control, cold shock, heat shock, dark shock) to characterize associated viral diversity, abundance, and gene expression. Our approach comprised the filtering and removal of host sequence reads, subsequent phylogenetic assignment of sequence reads of putative viral origin, and the assembly and analysis of differentially expressed viral genes. About 15.46% (123 million) of all sequence reads were non-host-related, of which <1% could be classified as archaea, bacteria, or virus. Of these, 18.78% were annotated as virus and comprised a diverse community consistent across experimental treatments. Further, non-host related sequence reads assembled into 56,064 contigs, including 4856 contigs of putative viral origin that featured 43 differentially expressed genes during heat shock. The differentially expressed genes included viral kinases, ubiquitin, and ankyrin repeat proteins (amongst others), which are suggested to help the virus proliferate and inhibit the algal host's antiviral response. CONCLUSION: Our results suggest that a diverse viral community is associated with coral algal endosymbionts of the genus Symbiodinium, which prompts further research on their ecological role in coral health and resilience.

Engineering Strategies to Decode and Enhance the Genomes of Coral Symbionts.

Elevated sea surface temperatures from a severe and prolonged El Niño event (2014-2016) fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts, Symbiodinium spp., from coral tissues) and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium, and in turn, coral reefs.

Discrete phenotypes are not underpinned by genome-wide genetic differentiation in the squat lobster Munida gregaria (Crustacea: Decapoda: Munididae): a multi-marker study covering the Patagonian shelf.

BACKGROUND: DNA barcoding has demonstrated that many discrete phenotypes are in fact genetically distinct (pseudo)cryptic species. Genetically identical, isogenic individuals, however, can also express similarly different phenotypes in response to a trigger condition, e.g. in the environment. This alternative explanation to cryptic speciation often remains untested because it requires considerable effort to reject the hypothesis that the observed underlying genetic homogeneity of the different phenotypes may be trivially caused by too slowly evolving molecular markers. The widespread squat lobster Munida gregaria comprises two discrete ecotypes, gregaria s. str. and subrugosa, which were long regarded as different species due to marked differences in morphological, ecological and behavioral traits. We studied the morphometry and genetics of M. gregaria s. l. and tested (1) whether the phenotypic differences remain stable after continental-scale sampling and inclusion of different life stages, (2) and whether each phenotype is underpinned by a specific genotype. RESULTS: A total number of 219 gregaria s. str. and subrugosa individuals from 25 stations encompassing almost entire range in South America were included in morphological and genetic analyses using nine unlinked hypervariable microsatellites and new COI sequences. Results from the PCA and using discriminant functions demonstrated that the morphology of the two forms remains discrete. The mitochondrial data showed a shallow, star-like haplotype network and complete overlap of genetic distances within and among ecotypes. Coalescent-based species delimitation methods, PTP and GMYC, coherently suggested that haplotypes of both ecotypes forms a single species. Although all microsatellite markers possess sufficient genetic variation, AMOVA, PCoA and Bayesian clustering approaches revealed no genetic clusters corresponding to ecotypes or geographic units across the entire South-American distribution. No evidence of isolation-by-distance could be detected for this species in South America. CONCLUSIONS: Despite their pronounced bimodal morphologies and different lifestyles, the gregaria s. str. and subrugosa ecotypes form a single, dimorphic species M. gregaria s. l.. Based on adequate geographic coverage and multiple independent polymorphic loci, there is no indication that each phenotype may have a unique genetic basis, leaving phenotypic plasticity or localized genomic islands of speciation as possible explanations.

Phylogeography of social polymorphism in a boreo-montane ant.

BACKGROUND: The disjunct distribution of several Palearctic species has been widely shaped by the changes in climatic conditions during the Quaternary. The observed genetic differentiation or reproductive isolation between extant populations may be the outcome of their contemporary geographic separation or reproductive incompatibility due to differences in phenotypic traits which have evolved in isolated refugia. In the boreal ant Leptothorax acervorum, colonies from central and peripheral populations differ in social structure: colonies from Central and Northern Europe may contain several equally reproductive queens (facultative polygyny), while in colonies from peripheral populations in Spain only one the most dominant of several queens lays eggs (functional monogyny). By reconstructing the specie's evolutionary and demographic history in Southwestern Europe we examine whether variation in social organization is associated with restricted gene flow between the two social forms. RESULTS: We show that multi-queen colonies from all so far known inner Iberian populations of L. acervorum are functionally monogynous, whereas multi-queen colonies from all Pyrenean populations are polygynous, like those from other previously studied areas in Europe. Our analyses revealed complex spatial-genetic structure, but no association between spatial-genetic structure and social organization in SW-Europe. The population in the western Pyrenees diverged most strongly from other Iberian populations. Moreover, microsatellite data suggest the occurrence of recent bottlenecks in Pyrenean and inner Iberian populations. CONCLUSIONS: Our study shows a lack of reproductive isolation between the two social forms in SW-Europe. This in turn suggests that demographic and spatial patterns in genetic variation as well as the distribution of social phenotypes are better explained by co-variation with climatic, ecological, and historical factors. Moreover, we for the first time show the existence of substantial spatial-genetic structure in L. acervorum, suggesting the existence of multiple refugia in SW-Europe, including two extra-Mediterranean refugia in France. While gene flow among inner Iberian refugia may have been larger during the late glacial, extra-Mediterranean refugia in southern France may have contributed to the post-glacial recolonization of W-Europe.

Comparative genomics explains the evolutionary success of reef-forming corals.

Transcriptome and genome data from twenty stony coral species and a selection of reference bilaterians were studied to elucidate coral evolutionary history. We identified genes that encode the proteins responsible for the precipitation and aggregation of the aragonite skeleton on which the organisms live, and revealed a network of environmental sensors that coordinate responses of the host animals to temperature, light, and pH. Furthermore, we describe a variety of stress-related pathways, including apoptotic pathways that allow the host animals to detoxify reactive oxygen and nitrogen species that are generated by their intracellular photosynthetic symbionts, and determine the fate of corals under environmental stress. Some of these genes arose through horizontal gene transfer and comprise at least 0.2% of the animal gene inventory. Our analysis elucidates the evolutionary strategies that have allowed symbiotic corals to adapt and thrive for hundreds of millions of years.

Genetic and morphological divergences in the cosmopolitan deep-sea amphipod Eurythenes gryllus reveal a diverse abyss and a bipolar species.

Eurythenes gryllus is one of the most widespread amphipod species, occurring in every ocean with a depth range covering the bathyal, abyssal and hadal zones. Previous studies, however, indicated the existence of several genetically and morphologically divergent lineages, questioning the assumption of its cosmopolitan and eurybathic distribution. For the first time, its genetic diversity was explored at the global scale (Arctic, Atlantic, Pacific and Southern oceans) by analyzing nuclear (28S rDNA) and mitochondrial (COI, 16S rDNA) sequence data using various species delimitation methods in a phylogeographic context. Nine putative species-level clades were identified within E. gryllus. A clear distinction was observed between samples collected at bathyal versus abyssal depths, with a genetic break occurring around 3,000 m. Two bathyal and two abyssal lineages showed a widespread distribution, while five other abyssal lineages each seemed to be restricted to a single ocean basin. The observed higher diversity in the abyss compared to the bathyal zone stands in contrast to the depth-differentiation hypothesis. Our results indicate that, despite the more uniform environment of the abyss and its presumed lack of obvious isolating barriers, abyssal populations might be more likely to show population differentiation and undergo speciation events than previously assumed. Potential factors influencing species' origins and distributions, such as hydrostatic pressure, are discussed. In addition, morphological findings coincided with the molecular clades. Of all specimens available for examination, those of the bipolar bathyal clade seemed the most similar to the 'true' E. gryllus. We present the first molecular evidence for a bipolar distribution in a macro-benthic deep-sea organism.

Exploring Pandora's box: potential and pitfalls of low coverage genome surveys for evolutionary biology.

High throughput sequencing technologies are revolutionizing genetic research. With this "rise of the machines", genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02-25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers.

Long-distance island hopping without dispersal stages: transportation across major zoogeographic barriers in a Southern Ocean isopod.

Species integrity is maintained only if recurrent allelic exchange between subpopulations occurs by means of migrating specimens. Predictions of this gene flow on the basis of observed or assumed mobility of marine species have proven to be error-prone. Using one mitochondrial gene and seven microsatellite markers, we studied the genetic structure and gene flow in Septemserolis septemcarinata, a strictly benthic species lacking pelagic larvae and the ability to swim. Suitable shallow-water habitats around three remote islands (South Georgia, Bouvet, and Marion Island) are geographically disjunct, isolated by more than 2,000 km of uninhabitable deep sea (east-west) and also separated by the Polar Front (north-south), which serves as a strong demarcation line in many marine taxa. Although we did find genetic differentiation among the three island populations, our results also revealed that a scenario with recent gene flow explains our data best. A model assuming no gene flow after initial colonization of the islands performs significantly worse. The tests also favor an asymmetric gene flow pattern (west to east >> east to west) thus mirroring the directionality of major oceanographic currents in the area. We conclude that rare long-distance dispersal rather than vicariance or human-mediated transport must be responsible for the observed patterns. As a mechanism, we propose passive rafting on floating substrata in the Antarctic Circumpolar Current. The results demonstrate that the effectiveness of a physical barrier is not solely a function of its physical parameters but strongly depends on how organisms interact with their environment.

Isolation and characterization of microsatellite markers from the marine isopods Serolis paradoxa and Septemserolis septemcarinata (Crustacea: Peracarida).

This study reports the successful isolation of highly informative microsatellite marker sets for two marine serolid isopod species. For Serolis paradoxa (Fabricius, 1775), 13, and for Septemserolis septemcarinata (Miers, 1875), eight polymorphic microsatellite markers were isolated using the reporter genome enrichment protocol. The number of alleles per locus (N(A) ) and the observed heterozygosity (H(O) ) encompass a wide range of variation within S. paradoxa (N(A) 3-31, H(O) 6-89%) and S. septemcarinata (N(A) 2-18, H(O) 9-94%). The suitability of the newly isolated markers for population genetic studies is evaluated.