Landscape genetics of a tropical rescue pollinator

Pollination services are increasingly threatened by the loss and modification of natural habitats, posing a risk to the maintenance of both native plant biodiversity and agricultural production. In order to safeguard pollination services, it is essential to examine the impacts of habitat degradation on the population dynamics of key pollinators and identify potential “rescue pollinators” capable of persisting in these human-altered landscapes. Using a landscape genetic approach, we assessed the impact of landscape structure on genetic differentiation in the widely-distributed tropical stingless bee Trigona spinipes (Apidae: Meliponini) across agricultural landscape mosaics composed of coffee plantations and Atlantic forest fragments in southeastern Brazil. We genotyped 115 bees at 16 specific and highly polymorphic microsatellite loci, developed using next-generation sequencing. Our results reveal that T. spinipes is capable of dispersing across remarkably long distances, as we did not find genetic differentiation across a 200 km range, nor fine-scale spatial genetic structure. Furthermore, gene flow was not affected by forest cover, land cover, or elevation, indicating that reproductive individuals are able to disperse well through agricultural landscapes and across altitudinal gradients. We also found evidence of a recent population expansion, suggesting that this opportunistic stingless bee is capable of colonizing degraded habitats. Our results thus suggest that T. spinipes can persist in heavily-altered landscapes and can be regarded as a rescue pollinator, potentially compensating for the decline of other native pollinators in degraded tropical landscapes.     

Robust Classification of Small-Molecule Mechanism of Action Using a Minimalist High-Content Microscopy Screen and Multidimensional Phenotypic Trajectory Analysis

Phenotypic screening through high-content automated microscopy is a powerful tool for evaluating the mechanism of action of candidate therapeutics. Despite more than a decade of development, however, high content assays have yielded mixed results, identifying robust phenotypes in only a small subset of compound classes. This has led to a combinatorial explosion of assay techniques, analyzing cellular phenotypes across dozens of assays with hundreds of measurements. Here, using a minimalist three-stain assay and only 23 basic cellular measurements, we developed an analytical approach that leverages informative dimensions extracted by linear discriminant analysis to evaluate similarity between the phenotypic trajectories of different compounds in response to a range of doses. This method enabled us to visualize biologically-interpretable phenotypic tracks populated by compounds of similar mechanism of action, cluster compounds according to phenotypic similarity, and classify novel compounds by comparing them to phenotypically active exemplars. Hierarchical clustering applied to 154 compounds from over a dozen different mechanistic classes demonstrated tight agreement with published compound mechanism classification. Using 11 phenotypically active mechanism classes, classification was performed on all 154 compounds: 78% were correctly identified as belonging to one of the 11 exemplar classes or to a different unspecified class, with accuracy increasing to 89% when less phenotypically active compounds were excluded. Importantly, several apparent clustering and classification failures, including rigosertib and 5-fluoro-2’-deoxycytidine, instead revealed more complex mechanisms or off-target effects verified by more recent publications. These results show that a simple, easily replicated, minimalist high-content assay can reveal subtle variations in the cellular phenotype induced by compounds and can correctly predict mechanism of action, as long as the appropriate analytical tools are used.     

Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.     

Seasonal temperature and precipitation regulate brook trout young‐of‐the‐year abundance and population dynamics

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Canonical and noncanonical inflammasomes in intestinal epithelial cells

Inflammasomes are cytosolic, multimeric protein complexes capable of activating pro‐inflammatory cytokines such as IL‐1β and IL‐18, which play a key role in host defence. Inflammasome components are highly expressed in the intestinal epithelium. In recent years, studies have begun to demonstrate that epithelial‐intrinsic inflammasomes play a critical role in regulating epithelial homeostasis, both by defending the epithelium from pathogenic insult and through the regulation of the mucosal environment. However, the majority of research regarding inflammasome activation has focused on professional immune cells, such as macrophages. Here, we present an overview of the current understanding of inflammasome function in epithelial cells and at mucosal surfaces and, in particular, in the intestine.     

Invasion genetics from eDNA and thousands of larvae: A targeted metabarcoding assay that distinguishes species and population variation of zebra and quagga mussels

Identifying species and population genetic compositions of biological invasions at early life stages and/or from environmental (e)DNA using targeted high‐throughput sequencing (HTS) metabarcode assays offers powerful and cost‐effective means for early detection, analysis of spread patterns, and evaluating population changes. The present study develops, tests, and applies this method with a targeted sequence assay designed to simultaneously identify and distinguish between the closely related invasive Eurasian zebra and quagga mussels (Dreissena polymorpha and D. rostriformis) and their relatives and discern their respective population genetic patterns. Invasions of these dreissenid mussel species have markedly changed freshwater ecosystems throughout North America and Europe, exerting severe ecological and economic damage. Their planktonic early life stages (eggs and larvae) are morphologically indistinguishable, yet each species exerts differential ecological effects, with the quagga often outcompeting the zebra mussel as adults. Our targeted assay analyzes genetic variation from a diagnostic sequence region of the mitochondrial (mt)DNA cytochrome oxidase I (COI) gene, to assess temporal and spatial inter‐ and intra‐specific genetic variability. The assay facilitates analysis of environmental (e)DNA from water, early life stages from thousands of individuals, and simultaneous analysis of 50–100 tagged field‐collected samples. Experiments evaluated its accuracy and performance using: (a) mock laboratory communities containing known DNA quantities per taxon, (b) aquaria with mixed‐species/haplotype compositions of adults, and (c) field‐collected water and plankton versus traditional sampling of adult communities. Results delineated species compositions, relative abundances, and population‐level diversity differences among ecosystems, habitats, time series, and life stages from two allopatric concurrent invasions in the Great Lakes (Lake Erie) and the Hudson River, which had separate founding histories. Findings demonstrate application of this targeted assay and our approach to accurately and simultaneously discern species‐ and population‐level differences across spatial and temporal scales, facilitating early detection and ecological understanding of biological invasions.