Groundwater environmental DNA metabarcoding reveals hidden diversity and reflects land-use and geology.

Despite being the most important source of liquid freshwater on the planet, groundwater is severely threatened by climate change, agriculture, or industrial mining. It is thus extensively monitored for pollutants and declines in quantity. The organisms living in groundwater, however, are rarely the target of surveillance programmes and little is known about the fauna inhabiting underground habitats. The difficulties accessing groundwater, the lack of expertise, and the apparent scarcity of these organisms challenge sampling and prohibit adequate knowledge on groundwater fauna. Environmental DNA (eDNA) metabarcoding provides an approach to overcome these limitations but is largely unexplored. Here, we sampled water in 20 communal spring catchment boxes used for drinking water provisioning in Switzerland, with a high level of replication at both filtration and amplification steps. We sequenced a portion of the COI mitochondrial gene, which resulted in 4917 ASVs, yet only 3% of the reads could be assigned to a species, genus, or family with more than 90% identity. Careful evaluation of the unassigned reads corroborated that these sequences were true COI sequences belonging mostly to diverse eukaryotic groups, not present in the reference databases. Principal component analyses showed a strong correlation of the community composition with the surface land-use (agriculture vs. forest) and geology (fissured rock vs. unconsolidated sediment). While incomplete reference databases limit the assignment of taxa in groundwater eDNA metabarcoding, we showed that taxonomy-free approaches can reveal large hidden diversity and couple it with major land-use drivers, revealing their imprint on chemical and biological properties of groundwater.

CelloSelect - A synthetic cellobiose metabolic pathway for selection of stable transgenic CHO cell lines.

Current protocols for generating stable transgenic cell lines mostly rely on antibiotic selection or the use of specialized cell lines lacking an essential part of their metabolic machinery, but these approaches require working with either toxic chemicals or knockout cell lines, which can reduce productivity. Since most mammalian cells cannot utilize cellobiose, a disaccharide consisting of two ß-1,4-linked glucose molecules, we designed an antibiotic-free selection system, CelloSelect, which consists of a selection cassette encoding Neurospora crassa cellodextrin transporter CDT1 and ß-glucosidase GH1-1. When cultivated in glucose-free culture medium containing cellobiose, CelloSelect-transfected cells proliferate by metabolizing cellobiose as a primary energy source, and are protected from glucose starvation. We show that the combination of CelloSelect with a PiggyBac transposase-based integration strategy provides a platform for the swift and efficient generation of stable transgenic cell lines. Growth rate analysis of metabolically engineered cells in cellobiose medium confirmed the expansion of cells stably expressing high levels of a cargo fluorescent marker protein. We further validated this strategy by applying the CelloSelect system for stable integration of sequences encoding two biopharmaceutical proteins, erythropoietin and the monoclonal antibody rituximab, and confirmed that the proteins are efficiently produced in either cellobiose- or glucose-containing medium in suspension-adapted CHO cells cultured in chemically defined media. We believe coupling heterologous metabolic pathways additively to the endogenous metabolism of mammalian cells has the potential to complement or to replace current cell-line selection systems.

Meta-analysis shows both congruence and complementarity of DNA and eDNA metabarcoding to traditional methods for biological community assessment.

DNA metabarcoding is increasingly used for the assessment of aquatic communities, and numerous studies have investigated the consistency of this technique with traditional morpho-taxonomic approaches. These individual studies have used DNA metabarcoding to assess diversity and community structure of aquatic organisms both in marine and freshwater systems globally over the last decade. However, a systematic analysis of the comparability and effectiveness of DNA-based community assessment across all of these studies has hitherto been lacking. Here, we performed the first meta-analysis of available studies comparing traditional methods and DNA metabarcoding to measure and assess biological diversity of key aquatic groups, including plankton, microphytobentos, macroinvertebrates, and fish. Across 215 data sets, we found that DNA metabarcoding provides richness estimates that are globally consistent to those obtained using traditional methods, both at local and regional scale. DNA metabarcoding also generates species inventories that are highly congruent with traditional methods for fish. Contrastingly, species inventories of plankton, microphytobenthos and macroinvertebrates obtained by DNA metabarcoding showed pronounced differences to traditional methods, missing some taxa but at the same time detecting otherwise overseen diversity. The method is generally sufficiently advanced to study the composition of fish communities and replace more invasive traditional methods. For smaller organisms, like macroinvertebrates, plankton and microphytobenthos, DNA metabarcoding may continue to give complementary rather than identical estimates compared to traditional approaches. Systematic and comparable data collection will increase the understanding of different aspects of this complementarity, and increase the effectiveness of the method and adequate interpretation of the results.

Species Interactions Limit the Predictability of Community Responses to Environmental Change.

AbstractPredicting how ecological communities will respond to environmental change is challenging but highly relevant in this era of global change. Ecologists commonly use current spatial relationships between species and environmental conditions to make predictions about the future. This assumes that species will track conditions by shifting their distributions. However, theory and experimental evidence suggest that species interactions prevent communities from predictably tracking temporal changes in environmental conditions on the basis of current spatial relationships between species and environmental gradients. We tested this hypothesis by assessing the dynamics of protist species in replicated two-patch microcosm landscapes that experienced different regimes of spatial and temporal environmental heterogeneity (light vs. dark). Populations were kept in monocultures or polycultures to assess the effect of species interactions. In monocultures, abundances were predictable on the basis of current environmental conditions, regardless of whether the populations had experienced temporal environmental change. But in polycultures, abundances also depended on the history of the environmental conditions experienced. This suggests that because of species interactions, communities should respond differently to spatial versus temporal environmental changes. Thus, species interactions likely reduce the accuracy of predictions about future communities that are based on current spatial relationships between species and the environment.

Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay.

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop-loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop-loop interactions in hammerhead ribozymes.

Integrating citizen science and environmental DNA metabarcoding to study biodiversity of groundwater amphipods in Switzerland.

Groundwater is the physically largest freshwater ecosystem, yet one of the least explored habitats on earth, both because of accessing difficulties and the scarcity of the organisms inhabiting it. Here, we demonstrate how a two-fold approach provides complementary information on the occurrence and diversity of groundwater amphipods. Firstly, we used a citizen science approach in collaboration with municipal water providers who sampled groundwater organisms in their spring catchment boxes over multiple weeks, followed by DNA barcoding. Secondly, we collected four 10 L water samples at each site, in one sampling event, for environmental DNA (eDNA) metabarcoding. We found that citizen science was very effective in describing the distribution and abundance of groundwater amphipods. Although the single time-point of eDNA sampling did not detect as many amphipods, it allowed the assessment of the entire groundwater community, including microorganisms. By combining both methods, we found different amphipod species co-occurring with distinct sequences from the eDNA-metabarcoding dataset, representing mainly micro-eukaryotic species. We also found a distinct correlation between the diversity of amphipods and the overall biodiversity of groundwater organisms detected by eDNA at each site. We thus suggest that these approaches can be used to get a better understanding of subterranean biodiversity.

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-ß are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-ß was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.