Elucidating the effect of tomato leaf surface microstructure on Botrytis cinerea using synthetic systems.

For some pathogenic fungi, sensing surface topography is part of their infection strategy. Their directional growth and transformation to a new developmental stage is influenced by contact with topographic features, which is referred to as thigmo-response, the exact functionality of which is not fully understood. Research on thigmo-responses is often performed on biomimetically patterned surfaces (BPS). Polydimethylsiloxane (PDMS) is especially suitable for fabrication of BPS. Here, we used synthetic BPS surfaces, mimicking tomato leaf surface, made from PDMS with the pathogenic fungus Botrytis cinerea to study the influence of structural features of the leaf surface on the fungus behavior. As a control, a PDMS surface without microstructure was fabricated to maintain the same chemical properties. Pre-penetration processes of B. cinerea, including the distribution of conidia on the surface, germination, and germ tube growth were observed on both leaf-patterned and flat PDMS. Microstructure affected the location of immediate attachment of conidia. Additionally, the microstructure of the plant host stimulated the development of germ tube in B. cinerea, at a higher rate than that observed on flat surface, suggesting that microstructure plays a role in fungus attachment and development.

Low-Resolution Raman Spectroscopy for the detection of contaminant species in algal bioreactors.

Fouling of aquatic systems by harmful microalgal and cyanobacterial species is an environmental and public health concern. Microalgal bioreactors are engineered ecosystems for the cultivation of algal biomass to meet the increasing demand for alternative protein sources and algae-derived products. Such bioreactors are often open or semi-open ponds or raceways that are prone to contamination by contaminant photosynthetic microorganisms, including harmful cyanobacterial species (HCBs). HCBs affect the quality of products through the accumulation of off-flavours, reducing their acceptance by consumers, and through the production of several different toxins collectively known as cyanotoxins. The density of cultured species within the bioreactor environment creates difficulty in detecting low concentrations of contaminant cells, and there is currently no technology enabling rapid monitoring of contaminations. The present study demonstrates the potential of Low-Resolution Raman Spectroscopy (LRRS) as a tool for rapid detection of low concentrations of HCBs within dense populations of the spirulina (Arthrospira platensis) cultures. An LRRS system adapted for the direct measurement of raw biomass samples was used to assemble a database of Raman spectral signatures, from eight algal and cyanobacterial strains. This dataset was used to develop both quantitative and discriminative chemometric models. The results obtained from the chemometric analyses demonstrate the ability of the LRRS to detect and quantify algal and cyanobacterial species at concentrations as low as 103 cells/mL and to robustly discriminate between species at concentrations of 104 cells/mL. The LRRS and chemometric analyses were further able to detect the presence of low concentrations (103cells/mL) of contaminating species, including the toxic cyanobacterium Microcystis aeruginosa, within dense (>107 cells/mL) spirulina cultures. The results presented provide a first demonstration of the potential of LRRS technology for real-time detection of contaminant species within microalgal bioreactors, and possibly for early detection of developing harmful algal blooms in other aquatic ecosystems.

Evolved attitudes to risk and the demand for equity.

The equity premium puzzle refers to the observation that people invest far less in the stock market than is implied by measures of their risk aversion in other contexts. Here, we argue that light on this puzzle can be shed by the hypothesis that human risk attitudes were at least partly shaped by our evolutionary history. In particular, a simple evolutionary model shows that natural selection will, over the long haul, favor a greater aversion to aggregate than to idiosyncratic risk. We apply this model-via both a static model of portfolio choice and a dynamic model that allows for intertemporal tradeoffs-to show that an aversion to aggregate risk that is derived from biology may help explain the equity premium puzzle. The type of investor favored in our model would indeed invest less in equities than other common observations of risk-taking behavior from outside the stock market would imply, while engaging in reasonable tradeoffs over time.

Pectin Induced Colony Expansion of Soil-Derived Flavobacterium Strains.

The genus Flavobacterium is characterized by the capacity to metabolize complex organic compounds and a unique gliding motility mechanism. Flavobacteria are often abundant in root microbiomes of various plants, but the factors contributing to this high abundance are currently unknown. In this study, we evaluated the effect of various plant-associated poly- and mono-saccharides on colony expansion of two Flavobacterium strains. Both strains were able to spread on pectin and other polysaccharides such as microcrystalline cellulose. However, only pectin (but not pectin monomers), a component of plant cell walls, enhanced colony expansion on solid surfaces in a dose- and substrate-dependent manner. On pectin, flavobacteria exhibited bi-phasic motility, with an initial phase of rapid expansion, followed by growth within the colonized area. Proteomic and gene expression analyses revealed significant induction of carbohydrate metabolism related proteins when flavobacteria were grown on pectin, including selected SusC/D, TonB-dependent glycan transport operons. Our results show a positive correlation between colony expansion and the upregulation of proteins involved in sugar uptake, suggesting an unknown linkage between specific operons encoding for glycan uptake and metabolism and flavobacterial expansion. Furthermore, within the context of flavobacterial-plant interactions, they suggest that pectin may facilitate flavobacterial expansion on plant surfaces in addition to serving as an essential carbon source.

Using high-resolution climate change information in water management: a decision-makers' perspective.

The UK Climate Change Act requires the Environment Agency to report the risks it faces from climate change and actions taken to address these. Derived information from projections is critical to understanding likely impacts in water management. In 2019, the UK published an ensemble of high-resolution model simulations. The UKCP Local (2.2 km) projections can resolve smaller scale physical processes that determine rainfall and other variables at subdaily time-scales with the potential to provide new insights into extreme events, storm runoff and drainage management. However, simulations also need to inform adaptation. The challenge ahead is to identify and provide derived products without the need for further analysis by decision-makers. These include a wider evaluation of uncertainty, narratives about rainfall change across the projections and bias-corrected datasets. Future flood maps, peak rainfall estimates, uplift factors and future design storm profiles also need detailed guidance to support their use. Central government support is justified in the provision of up-to-date impacts information to inform flood risk management, given the large risks and exposure of all sectors. The further development of projections would benefit from greater focus and earlier scoping with industry representatives, operational tool developers and end users. This article is part of a discussion meeting issue 'Intensification of short-duration rainfall extremes and implications for flash flood risks'.

Microscale tracking of coral-vibrio interactions.

To improve our understanding of coral infection and disease, it is important to study host-pathogen interactions at relevant spatio-temporal scales. Here, we provide a dynamic microscopic view of the interaction between a coral pathogen, Vibrio coralliilyticus and its coral host Pocillopora damicornis. This was achieved using a microfluidics-based system facilitating control over flow, light and temperature conditions. Combined with time-resolved biochemical and microbial analyses of the system exudates, this approach provides novel insights into the early phases of a coral infection at unprecedented spatio-temporal resolution. We provide evidence that infection may occur through ingestion of the pathogen by the coral polyps, or following pathogen colonization of small tissue lesions on the coral surface. Pathogen ingestion invariably induced the release of pathogen-laden mucus from the gastrovascular cavity. Despite the high bacterial load used in our experiments, approximately one-third of coral fragments tested did not develop further symptoms. In the remaining two-thirds, mucus spewing was followed by the severing of calicoblastic connective tissues (coenosarc) and subsequently necrosis of most polyps. Despite extensive damage to symptomatic colonies, we frequently observed survival of individual polyps, often accompanied by polyp bail-out. Biochemical and microbial analyses of exudates over the course of symptomatic infections revealed that severing of the coenosarc was followed by an increase in matrix metaloprotease activity, and subsequent increase in both pathogen and total bacterial counts. Combined, these observations provide a detailed description of a coral infection, bringing us a step closer to elucidating the complex interactions underlying coral disease.

Improved Method for Transformation of Vibrio vulnificus by Electroporation.

Vibrio vulnificus, an emergent human pathogen, causes fulminant septicemia with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, the factors to conclusively indicate the virulence potential of V. vulnificus strains remain largely unknown. Understanding the pathogenesis of this bacterium at a molecular level is severely hindered by inefficiencies in transformation, for instance, due to the presence of a periplasmic nuclease, Vvn. Currently, successful transformation of V. vulnificus is nearly impossible due to lack of mobilizable plasmids for the bacterium, requiring (i) very high DNA concentrations, (ii) plasmid linearization, (iii) development of novel V. vulnificus-derived plasmids, or (iv) time-consuming conjugation-based methods. To overcome these limitations, we describe a rapid, efficient, and reproducible electroporation protocol to effectively transform widely available plasmids, with different copy numbers and antibiotic resistances, into phylogenetically distant strains of V. vulnificus. Cells are made competent in high concentrations of sucrose devoid of cations and recovered from electroporation using a high-salinity recovery medium. Compared to existing methods for transformation of V. vulnificus, significantly higher efficiencies are obtained using this improved protocol. Rapid and effective transformations can markedly improve molecular analyses of V. vulnificus leading to a greater understanding of its virulence potential. This is crucial to develop rapid detection methods which have the potential to prevent future outbreaks. The electroporation protocol described here may be particularly useful for optimizing transformation of other nuclease-producing bacteria. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent cells Basic Protocol 2: Transformation of cells by electroporation.

The Population Genetics of Evolutionary Rescue in Diploids: X Chromosomal versus Autosomal Rescue.

Most population genetic theory assumes that populations adapt to an environmental change without a change in population size. However, environmental changes might be so severe that populations decline in size and, without adaptation, become extinct. This "evolutionary rescue" scenario differs from traditional models of adaptation in that rescue involves a race between adaptation and extinction. While most previous work has usually focused on models of evolutionary rescue in haploids, here we consider diploids. In many species, diploidy introduces a novel feature into adaptation: adaptive evolution might occur either on sex chromosomes or on autosomes. Previous studies of nonrescue adaptation revealed that the relative rates of adaptation on the X chromosome versus autosomes depend on the dominance of beneficial mutations, reflecting differences in effective population size and the efficacy of selection. Here, we extend these results to evolutionary rescue and find that, given equal-sized chromosomes, there is greater parameter space in which the X is more likely to contribute to adaptation than the autosomes relative to standard nonrescue models. We also discuss how subtle effects of dominance can increase the chance of evolutionary rescue in diploids when absolute heterozygote fitness is close to 1. These effects do not arise in standard nonrescue models.

Synergistic Inhibition of Mycotoxigenic Fungi and Mycotoxin Production by Combination of Pomegranate Peel Extract and Azole Fungicide.

Fungal plant pathogens cause considerable losses in yield and quality of field crops worldwide. In addition, under specific environmental conditions, many fungi, including such as some Fusarium and Aspergillus spp., are further able to produce mycotoxins while colonizing their host, which accumulate in human and animal tissues, posing a serious threat to consumer health. Extensive use of azole fungicides in crop protection stimulated the emergence of acquired azole resistance in some plant and human fungal pathogens. Combination treatments, which become popular in clinical practice, offer an alternative strategy for managing potentially resistant toxigenic fungi and reducing the required dosage of specific drugs. In the current study we tested the effect of pomegranate peel extract (PPE) on the growth and toxin production of the mycotoxigenic fungi Aspergillus flavus and Fusarium proliferatum, both alone and in combination with the azole fungicide prochloraz (PRZ). Using time-lapse microscopy and quantitative image analysis we demonstrate significant delay of conidial germination and hyphal elongation rate in both fungi following PPE treatment in combination with PRZ. Moreover, PPE treatment reduced aflatoxin production by A. flavus up to 97%, while a combined treatment with sub-inhibitory doses of PPE and PRZ resulted in complete inhibition of toxin production over a 72 h treatment. These findings were supported by qRT-PCR analysis, showing down-regulation of key genes involved in the aflatoxin biosynthetic pathway under combined PPE/PRZ treatment al low concentrations. Our results provide first evidence for synergistic effects between the commercial drug PRZ and natural compound PPE. Future application of these findings may allow to reduce the required dosage of PRZ, and possibly additional azole drugs, to inhibit mycotoxigenic fungi, ultimately reducing potential concerns over exposure to high doses of these potentially harmful fungicides.

N-Halamine Derivatized Nanoparticles with Selective Cyanocidal Activity: Potential for Targeted Elimination of Harmful Cyanobacterial Blooms.

Harmful cyanobacterial blooms (HCBs) are becoming a major challenge for the management of both natural and man-made freshwater lakes and reservoirs. Phytoplankton communities are an essential component of aquatic ecosystems, providing the basis for natural food webs as well as important environmental services. HCBs, driven by a combination of environmental pollution and rising global temperatures, destabilize phytoplankton communities with major impacts on aquatic ecology and trophic interactions. Application of currently available algaecides generally results in unselective elimination of phytoplankton species, disrupting water ecology and environmental services provided by beneficial algae. There is thus a need for selective cyanocidal compounds that can eliminate cyanobacteria while preserving algal members of the phytoplankton community. Here, we demonstrate the efficacy of N-halamine derivatized nanoparticles (Cl NPs) in selectively eliminating cyanobacteria, including the universal bloom-forming species Microcystis aeruginosa, while having minimal effect on co-occurring algal species. We further support these results with the use a simple microfluidic platform in combination with advanced live-imaging microscopy to study the effects of Cl NPs on both laboratory cultures and natural populations of cyanobacteria and algae at single cell resolutions. We note that the Cl NPs used in this work were made of polymethacrylamide, a nonbiodegradable polymer that may be unsuitable for use as a cyanocide in open aquatic environments. Nevertheless, the demonstrated selective action of these Cl NPs suggests a potential for developing alternative, biodegradable carriers with similar properties as future cyanocidal agents that will enable selective elimination of HCBs.

Light-dependent single-cell heterogeneity in the chloroplast redox state regulates cell fate in a marine diatom.

Diatoms are photosynthetic microorganisms of great ecological and biogeochemical importance, forming vast blooms in aquatic ecosystems. However, we are still lacking fundamental understanding of how individual cells sense and respond to diverse stress conditions, and what acclimation strategies are employed during bloom dynamics. We investigated cellular responses to environmental stress at the single-cell level using the redox sensor roGFP targeted to various organelles in the diatom Phaeodactylum tricornutum. We detected cell-to-cell variability using flow cytometry cell sorting and a microfluidics system for live imaging of oxidation dynamics. Chloroplast-targeted roGFP exhibited a light-dependent, bi-stable oxidation pattern in response to H2O2 and high light, revealing distinct subpopulations of sensitive oxidized cells and resilient reduced cells. Early oxidation in the chloroplast preceded commitment to cell death, and can be used for sensing stress cues and regulating cell fate. We propose that light-dependent metabolic heterogeneity regulates diatoms' sensitivity to environmental stressors in the ocean.

Measures of the efficiency of natural selection during gene substitution.

Natural selection is not perfectly efficient: it does not cause the instantaneous substitution of a beneficial mutation. Instead, substitution takes time, reflecting the statistical consequences of fitness differences over some number of generations. In this note, I suggest two measures of the efficiency of natural selection during gene substitution. I compare these measures against both ideal (instantaneous) and failed evolution. I also compare these measures to Haldane's cost of natural selection.

Evolutionary Model of Cluster Divergence of the Emergent Marine Pathogen Vibrio vulnificus: From Genotype to Ecotype.

Vibrio vulnificus, an opportunistic pathogen, is the causative agent of a life-threatening septicemia and a rising problem for aquaculture worldwide. The genetic factors that differentiate its clinical and environmental strains remain enigmatic. Furthermore, clinical strains have emerged from every clade of V. vulnificus In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species from an evolutionary and ecological point of view. Genome comparisons and bioinformatic analyses of 113 V. vulnificus isolates indicate that the population of V. vulnificus is made up of four different clusters. We found evidence that recombination and gene flow between the two largest clusters (cluster 1 [C1] and C2) have drastically decreased to the point where they are diverging independently. Pangenome and phenotypic analyses showed two markedly different lifestyles for these two clusters, indicating commensal (C2) and bloomer (C1) ecotypes, with differences in carbohydrate utilization, defense systems, and chemotaxis, among other characteristics. Nonetheless, we identified frequent intra- and interspecies exchange of mobile genetic elements (e.g., antibiotic resistance plasmids, novel "chromids," or two different and concurrent type VI secretion systems) that provide high levels of genetic diversity in the population. Surprisingly, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together. We propose an evolutionary model of V. vulnificus that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections and emergence.IMPORTANCEVibrio vulnificus is an emergent marine pathogen and is the cause of a deadly septicemia. However, the genetic factors that differentiate its clinical and environmental strains and its several biotypes remain mostly enigmatic. In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species to elucidate the traits that make these strains emerge as a human pathogen. The acquisition of different ecological determinants could have allowed the development of highly divergent clusters with different lifestyles within the same environment. However, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together, posing a potential risk of recombination and of emergence of novel variants. We propose a new evolutionary model that provides a perspective that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections.

A novel application of remote sensing for modelling impacts of tree shading on water quality.

Uncertainty in capturing the effects of riparian tree shade for assessment of algal growth rates and water temperature hinders the predictive capability of models applied for river basin management. Using photogrammetry-derived tree canopy data, we quantified hourly shade along the River Thames (UK) and used it to estimate the reduction in the amount of direct radiation reaching the water surface. In addition we tested the suitability of freely-available LIDAR data to map ground elevation. Following removal of buildings and objects other than trees from the LIDAR dataset, results revealed considerable differences between photogrammetry- and LIDAR-derived methods in variables including mean canopy height (10.5 m and 4.0 m respectively), percentage occupancy of riparian zones by trees (45% and 16% respectively) and mid-summer fractional penetration of direct radiation (65% and 76% respectively). The generated data on daily direct radiation for 2010 were used as input to a river network water quality model (QUESTOR). Impacts of tree shading were assessed in terms of upper quartile levels, revealing substantial differences in indicators such as biochemical oxygen demand (BOD) (1.58-2.19 mg L-1 respectively) and water temperature (20.1 and 21.2 °C respectively) between 'shaded' and 'non-shaded' radiation inputs. Whilst the differences in canopy height and extent derived by the two methods are appreciable they only make small differences to water quality in the Thames. However such differences may prove more critical in smaller rivers. We highlight the importance of accurate estimation of shading in water quality modelling and recommend use of high resolution remotely sensed spatial data to characterise riparian canopies. Our paper illustrates how it is now possible to make better reach scale estimates of shade and make aggregations of these for use at river basin scale. This will allow provision of more effective guidance for riparian management programmes than currently possible. This is important to support adaptation to future warming and maintenance of water quality standards.

Tracking Root Interactions System (TRIS) Experiment and Quality Control.

Soil organisms are diverse taxonomically and functionally. This ecosystem experiences highly complex networks of interactions, but may also present functionally independent entities. Plant roots, a metabolically active hotspot in the soil, take an essential part in shaping the rhizosphere. Tracking the dynamics of root-microbe interactions at high spatial resolution is currently limited due to methodological intricacy. In this study, we developed a novel microfluidics-based device enabling direct imaging of root-bacteria interactions in real time.

Live imaging of root-bacteria interactions in a microfluidics setup.

Plant roots play a dominant role in shaping the rhizosphere, the environment in which interaction with diverse microorganisms occurs. Tracking the dynamics of root-microbe interactions at high spatial resolution is currently limited because of methodological intricacy. Here, we describe a microfluidics-based approach enabling direct imaging of root-bacteria interactions in real time. The microfluidic device, which we termed tracking root interactions system (TRIS), consists of nine independent chambers that can be monitored in parallel. The principal assay reported here monitors behavior of fluorescently labeled Bacillus subtilis as it colonizes the root of Arabidopsis thaliana within the TRIS device. Our results show a distinct chemotactic behavior of B. subtilis toward a particular root segment, which we identify as the root elongation zone, followed by rapid colonization of that same segment over the first 6 h of root-bacteria interaction. Using dual inoculation experiments, we further show active exclusion of Escherichia coli cells from the root surface after B. subtilis colonization, suggesting a possible protection mechanism against root pathogens. Furthermore, we assembled a double-channel TRIS device that allows simultaneous tracking of two root systems in one chamber and performed real-time monitoring of bacterial preference between WT and mutant root genotypes. Thus, the TRIS microfluidics device provides unique insights into the microscale microbial ecology of the complex root microenvironment and is, therefore, likely to enhance the current rate of discoveries in this momentous field of research.

A coral-on-a-chip microfluidic platform enabling live-imaging microscopy of reef-building corals.

Coral reefs, and the unique ecosystems they support, are facing severe threats by human activities and climate change. Our understanding of these threats is hampered by the lack of robust approaches for studying the micro-scale interactions between corals and their environment. Here we present an experimental platform, coral-on-a-chip, combining micropropagation and microfluidics to allow direct microscopic study of live coral polyps. The small and transparent coral micropropagates are ideally suited for live-imaging microscopy, while the microfluidic platform facilitates long-term visualization under controlled environmental conditions. We demonstrate the usefulness of this approach by imaging coral micropropagates at previously unattainable spatio-temporal resolutions, providing new insights into several micro-scale processes including coral calcification, coral-pathogen interaction and the loss of algal symbionts (coral bleaching). Coral-on-a-chip thus provides a powerful method for studying coral physiology in vivo at the micro-scale, opening new vistas in coral biology.

Early activation of microglia and astrocytes in mouse models of spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is an incurable, dominantly inherited neurodegenerative disease of the cerebellum caused by a polyglutamine-repeat expansion in the protein ataxin-1 (ATXN1). While analysis of human autopsy material indicates significant glial pathology in SCA1, previous research has focused on characterizing neuronal dysfunction. In this study, we characterized astrocytic and microglial response in SCA1 using a comprehensive array of mouse models. We have discovered that astrocytes and microglia are activated very early in SCA1 pathogenesis even when mutant ATXN1 expression was limited to Purkinje neurons. Glial activation occurred in the absence of neuronal death, suggesting that glial activation results from signals emanating from dysfunctional neurons. Finally, in all different models examined glial activation closely correlated with disease progression, supporting the development of glial-based biomarkers to follow disease progression.

Canine rabies in Australia: a review of preparedness and research needs.

Australia is unique as a populated continent in that canine rabies is exotic, with only one likely incursion in 1867. This is despite the presence of a widespread free-ranging dog population, which includes the naturalized dingo, feral domestic dogs and dingo-dog cross-breeds. To Australia's immediate north, rabies has recently spread within the Indonesian archipelago, with outbreaks occurring in historically free islands to the east including Bali, Flores, Ambon and the Tanimbar Islands. Australia depends on strict quarantine protocols to prevent importation of a rabid animal, but the risk of illegal animal movements by fishing and recreational vessels circumventing quarantine remains. Predicting where rabies will enter Australia is important, but understanding dog population dynamics and interactions, including contact rates in and around human populations, is essential for rabies preparedness. The interactions among and between Australia's large populations of wild, free-roaming and restrained domestic dogs require quantification for rabies incursions to be detected and controlled. The imminent risk of rabies breaching Australian borders makes the development of disease spread models that will assist in the deployment of cost-effective surveillance, improve preventive strategies and guide disease management protocols vitally important. Here, we critically review Australia's preparedness for rabies, discuss prevailing assumptions and models, identify knowledge deficits in free-roaming dog ecology relating to rabies maintenance and speculate on the likely consequences of endemic rabies for Australia.