Direct visualization of mucus production by the cold-water coral <i>Lophelia pertusa</i> with digital holographic microscopy

<i>Lophelia pertusa</i> is the dominant reef-building organism of cold-water coral reefs, and is known to produce significant amounts of mucus, which could involve an important metabolic cost. Mucus is involved in particle removal and feeding processes, yet the triggers and dynamics of mucus production are currently still poorly described because the existing tools to study these processes are not appropriate. Using a novel microscopic technique—digital holographic microscopy (DHM)–we studied the mucus release of <i>L</i>. <i>pertusa</i> under various experimental conditions. DHM technology permits µm-scale observations and allows the visualization of transparent mucoid substances in real time without staining. Fragments of <i>L</i>. <i>pertusa</i> were first maintained in flow-through chambers without stressors and imaged with DHM, then exposed to various stressors (suspended particles, particulate food and air exposure) and re-imaged. Under non-stressed conditions no release of mucus was observed, whilst mucus strings and sheaths were produced in response to suspended particles (activated charcoal and drill cuttings sediment) i.e. in a stressed condition. Mucus strings and so-called ‘string balls’ were also observed in response to exposure to particulate food (brine shrimp <i>Artemia salina</i>). Upon air-exposure, mucus production was clearly visible once the fragments were returned to the flow chamber. Distinct optical properties such as optical path length difference (OPD) were measured with DHM in response to the various stimuli suggesting that different mucus types are produced by <i>L</i>. <i>pertusa</i>. Mucus produced to reject particles is similar in refractive index to the surrounding seawater, suggesting that the energy content of this mucus is low. In contrast, mucus produced in response to either food particle addition or air exposure had a higher refractive index, suggesting a higher metabolic investment in the production of these mucoid substances. This paper shows for the first time the potential of DHM technology for the detection, characterization and quantification of mucus production through OPD measurements in <i>L</i>. <i>pertusa</i>

Imaging-in-flow: digital holographic microscopy as a novel tool to detect and classify nanoplanktonic organisms

Traditional taxonomic identification of planktonic organisms is based on light microscopy, which is both time-consuming and tedious. In response, novel ways of automated (machine) identification, such as flow cytometry, have been investigated over the last two decades. To improve the taxonomic resolution of particle analysis, recent developments have focused on "imaging-in-flow," i.e., the ability to acquire microscopic images of planktonic cells in a flow-through mode. Imaging-in-flow systems are traditionally based on classical brightfield microscopy and are faced with a number of issues that decrease the classification performance and accuracy (e. g., projection variance of cells, migration of cells out of the focus plane). Here, we demonstrate that a combination of digital holographic microscopy (DHM) with imaging-in-flow can improve the detection and classification of planktonic organisms. In addition to light intensity information, DHM provides quantitative phase information, which generates an additional and independent set of features that can be used in classification algorithms. Moreover, the capability of digitally refocusing greatly increases the depth of field, enables a more accurate focusing of cells, and reduces the effects of position variance. Nanoplanktonic organisms similar in shape were successfully classified from images captured with an off-axis DHM with partial coherence. Textural features based on DHM phase information proved more efficient in separating the three tested phytoplankton species compared with shape-based features or textural features based on light intensity. An overall classification score of 92.4% demonstrates the potential of holographic-based imaging-in-flow for similar looking organisms in the nanoplankton range

Long-distance electron transport occurs globally in marine sediments

Recently, long filamentous bacteria have been reported conducting electrons over centimetre distances in marine sediments. These so-called cable bacteria perform an electrogenic form of sulfur oxidation, whereby long-distance electron transport links sulfide oxidation in deeper sediment horizons to oxygen reduction in the upper millimetres of the sediment. Electrogenic sulfur oxidation exerts a strong impact on the local sediment biogeochemistry, but it is currently unknown how prevalent the process is within the seafloor. Here we provide a state-of-the-art assessment of its global distribution by combining new field observations with previous reports from the literature. This synthesis demonstrates that electrogenic sulfur oxidation, and hence microbial long-distance electron transport, is a widespread phenomenon in the present-day seafloor. The process is found in coastal sediments within different climate zones (off the Netherlands, Greenland, the USA, Australia) and thrives on a range of different coastal habitats (estuaries, salt marshes, mangroves, coastal hypoxic basins, intertidal flats). The combination of a widespread occurrence and a strong local geochemical imprint suggests that electrogenic sulfur oxidation could be an important, and hitherto overlooked, component of the marine cycle of carbon, sulfur and other elements

Multigene Editing Via CRISPR/Cas9 Guided By a Single-sgRNA Seed in Arabidopsis

We report that a single-sgRNA seed is capable of guiding CRISPR/Cas9 to simultaneously edit multiple genes AtRPL10A, AtRPL10B and AtRPL10C in Arabidopsis. Our results also demonstrate that it is possible to use CRISPR/Cas9 technology to create AtRPL10 triple mutants which otherwise cannot be generated by conventional genetic crossing. Compared to other conventional multiplex CRISPR/Cas systems, a single sgRNA seed has the advantage of reducing off-target gene-editing. Such a single sgRNA seed-induced gene editing system might be also applicable to modify other homologous genes or even less-homologous sequences for multiple gene-editing in plants and other organisms. [Abstract copyright: This article is protected by copyright. All rights reserved.

Red Deer Browsing Decelerates Shrub Regrowth Despite Increasing Wolf Presence

Semi-natural open habitats in Europe have been shaped by traditional land use practices, such as extensive mowing or livestock grazing. However, socio-economic transformations have led to the abandonment of many grassland and heathland areas and conservation management is now required to maintain these biodiverse habitats. Grazing by wild red deer (Cervus elaphus) can be a convenient alternative to laborious mechanical management or livestock grazing. Yet it remains unclear if free-ranging ungulates can counteract shrub growth sufficiently to maintain open habitats—especially with natural predators, i.e. wolves (Canis lupus), recolonizing Europe. To assess red deer effects on shrub regrowth after clearance, we installed a cohort of open and fenced plots (17 pairs) in 2016, when wolf presence in our study area (Grafenwöhr military training area, DE) was negligible. When wolf presence had become frequent in 2020, we set up a second cohort (41 paired plots). For both cohorts, the vegetation in the shrub and herb layer was significantly higher in fenced than open plots already after one year. Shrub height increased continuously and dead herbaceous biomass accumulated under red deer exclusion. Consequently, wild red deer can slow down shrub succession in open habitats even under increasing predation pressure. Regardless of wolf presence, a wildlife management that enables red deer to forage in open landscapes could thus reduce the required frequency of conservation management interventions

Crystal Structure of Staphylococcus aureus Cas9

Summary The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer adjacent mot if (PAM) and complementarity to the guide RNA. Recently, we harnessed Staphylococcus aureus Cas9 (SaCas9), which is significantly smaller than Streptococcus pyogenes Cas9 (SpCas9), to facilitate efficient in vivo genome editing. Here, we report the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and the 5′-TTGGGT-3′ PAM, at 2.6 and 2.7 Å resolutions, respectively. The structures revealed the mechanism of the relaxed recognition of the 5′-NNGRRT-3′ PAM by SaCas9. A structural comparison of SaCas9 with SpCas9 highlighted both structural conservation and divergence, explaining their distinct PAM specificities and orthologous sgRNA recognition. Finally, we applied the structural information about this minimal Cas9 to rationally design compact transcriptional activators and inducible nucleases, to further expand the CRISPR-Cas9 genome editing toolbox.National Institute of General Medical Sciences (U.S.) (Grant T32GM007753)National Institutes of Health (U.S.) (Award 1DP1-MH100706

Natural occurrence of microbial sulphur oxidation by long-range electron transport in the seafloor

Recently, a novel mode of sulphur oxidation was described in marine sediments, in which sulphide oxidation in deeper anoxic layers was electrically coupled to oxygen reduction at the sediment surface. Subsequent experimental evidence identified that long filamentous bacteria belonging to the family Desulfobulbaceae likely mediated the electron transport across the centimetre-scale distances. Such long-range electron transfer challenges some long-held views in microbial ecology and could have profound implications for sulphur cycling in marine sediments. But, so far, this process of electrogenic sulphur oxidation has been documented only in laboratory experiments and so its imprint on the seafloor remains unknown. Here we show that the geochemical signature of electrogenic sulphur oxidation occurs in a variety of coastal sediment environments, including a salt marsh, a seasonally hypoxic basin, and a subtidal coastal mud plain. In all cases, electrogenic sulphur oxidation was detected together with an abundance of Desulfobulbaceae filaments. Complementary laboratory experiments in intertidal sands demonstrated that mechanical disturbance by bioturbating fauna destroys the electrogenic sulphur oxidation signal. A survey of published geochemical data and 16S rRNA gene sequences identified that electrogenic sulphide oxidation is likely present in a variety of marine sediments with high sulphide generation and restricted bioturbation, such as mangrove swamps, aquaculture areas, seasonally hypoxic basins, cold sulphide seeps and possibly hydrothermal vent environments. This study shows for the first time that electrogenic sulphur oxidation occurs in a wide range of marine sediments and that bioturbation may exert a dominant control on its natural distribution