Biology subjects

Stevens, M.

Publications and source records attributed to Stevens, M..

29 records · Page 2Linked to original sources

bioRxiv2021

HAM-ART: An optimised culture-free Hi-C metagenomics pipeline for tracking antimicrobial resistance genes in complex microbial communities.

Shotgun metagenomics is a powerful tool to identify antimicrobial resistance (AMR) genes in microbiomes but has the limitation that extrachromosomal DNA, such as plasmids, cannot be linked with the host bacterial chromosome. Here we present a laboratory and bioinformatics pipeline HAM-ART (Hi-C Assisted Metagenomics for Antimicrobial Resistance Tracking) optimised for the generation of metagenome-assembled genomes including both chromosomal and extrachromosomal AMR genes. We demonstrate the performance of the pipeline in a study comparing 100 pig faecal microbiomes from low- and high-antimicrobial use pig farms (organic and conventional farms). We found significant differences in the distribution of AMR genes between low- and high-antimicrobial use farms including a plasmid-borne lincosamide resistance gene exclusive to high-antimicrobial use farms in three species of Lactobacilli. Author SummaryAntimicrobial resistance (AMR) is one of the biggest global health threats humanity is facing. Understanding the emergence and spread of AMR between different bacterial species is crucial for the development of effective countermeasures. In this paper we describe a user-friendly, affordable and comprehensive (laboratory and bioinformatics) workflow that is able to identify, associate and track AMR genes in bacteria. We demonstrate the efficiency and reliability of the method by comparing 50 faecal microbiomes from pig farms with high-antibiotic use (conventional farms), and 50 faecal microbiomes from pig farms with low-antibiotic use (organic farms). Our method provides a novel approach to resistance gene tracking, that also leads to the generation of high quality metagenomic assembled genomes that includes genes on mobile genetic elements, such as plasmids, that would not otherwise be included in these assembled genomes.

bioinformatics↗

bioRxiv2021

Analysis of wild plant pathogen populations reveals a signal of adaptation in genes evolving for survival in agriculture in the beet rust pathogen (Uromyces beticola)

Improvements in crop resistance to pathogens can reduce yield losses and address global malnourishment today. Gene-for-gene -type interactions can identify new sources of resistance but genetic resistance is often short lived. Ultimately an understanding of how pathogens rapidly adapt will allow us to both increase resistance gene durability and more effectively target chemical treatments. Until recently all agricultural pathogens were living on wild hosts. To understand crop pathogen evolution, we compared genetic diversity in agricultural and wild populations. Wild reservoirs may be the source of emergent pathogen lineages, but here we outline a strategy for comparison of wild and agricultural pathogen populations to highlight genes adapting to agriculture. To address this, we have selected and developed the beet rust system (Beta vulgaris, Uromyces beticola, respectively) as our wild-agricultural model. Our hypothesis is that pathogen adaptation to agricultural crops will be evident as divergence in comparisons of wild and agricultural plant pathogen populations. We sampled isolates in both the wild and agriculture, sequenced and assembled and annotated a large fungal genome and analysed genetic diversity in 42 re-sequenced rust isolates. We found population differentiation between isolates in the wild compared to a predominantly agricultural group. Fungal effector genes are co-evolving with host resistance and are important for successful colonisation. We predicted (and found) that these exhibit a greater signal of diversification and adaptation and more importantly displayed increased wild agricultural divergence. Finding a signal of adaptation in these genes highlights this as an important strategy to identify genes which are key to pathogen success, that analysis of agricultural isolates alone cannot. Author SummaryAs quickly as we develop new strategies for crop defence, pathogens evolve to circumvent them. Novel crop pathogen strains emerge periodically and sweep through the agricultural system. However, because of the (often) clonal nature of these crop pathogens it is difficult to identify the trait that is key to their success. In other words, if there is a trait that is key for success in agriculture, all agricultural isolates will have it (or die without it). What we need is a case and control system where we identify genes important to pathogen success in agricultural by comparing them to pathogens that live in the wild. Here we exemplify this strategy by focussing on genes already known to specifically adapt for the successful colonisation of the host, the fungal effector genes. We find that these genes appear to be evolving quickly and that they are more different between the wild and agriculture than other non-effector genes. These differences between wild and agricultural pathogens suggest we are observing adaptation to agriculture. We do this work in the sugar beet rust system because of its tractability to sample but this understanding about how to identify genetic variation that is key to pathogen success in agriculture is applicable to crop systems where pathogen reservoirs exist as well as other pathogen reservoir systems (e.g. zoonoses).

evolutionary biology↗

bioRxiv2021

The development of ovine gastric and intestinal organoids for studying ruminant host-pathogen interactions

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.

microbiology↗

bioRxiv2021

Global ubiquitylation analysis of mitochondria in primary neurons identifies physiological Parkin targets following activation of PINK1

Autosomal recessive mutations in PINK1 and Parkin cause Parkinsons disease. How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell culture studies with few molecular studies in neurons. Herein we have undertaken a global proteomic-analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin activation. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1-dependent diGLY sites upregulated upon mitochondrial depolarisation in 22 proteins conserved across mouse and human systems. These proteins were exclusively localised at the mitochondrial outer membrane (MOM) including, CISD1, CPT1, ACSL1, and FAM213A. We demonstrate that these proteins can be directly ubiquitylated by Parkin in vitro. We also provide evidence for a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation including SNX3, CAMK2 and CAMK2{beta} indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarisation in neurons. Finally we have developed an online resource to visualise mitochondrial ubiquitin sites in neurons and search for ubiquitin components recruited to mitochondria upon mitochondrial depolarisation, MitoNUb. This analysis will aid in future studies to understand Parkin activation in neuronal subtypes. Our findings also suggest that monitoring ubiquitylation status of the 22 identified MOM proteins may represent robust biomarkers for PINK1 and Parkin activity in vivo.

biochemistry↗

bioRxiv2020

Ecological niche adaptation of a bacterial pathogen associated with reduced zoonotic potential

The emergence of new bacterial pathogens is a continuing challenge for agriculture and food safety. Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of foodborne illness worldwide, with pigs a major zoonotic reservoir. Two variants, S. Typhimurium phage type U288 and monophasic S. Typhimurium (S. 4,[5],12:i:-) ST34 emerged and have accounted for the majority of isolates from pigs in the UK in the past two decades, but have distinct host range and risk to food safety. ST34 accounts for over 50% of all S. Typhimurium infections in people while U288 less than 2%. U288 and ST34 form distinct phylogenetic clusters within S. Typhimurium, defined by approximately 600 SNPs within their 5 Mbp genomes. Evolution of the U288 clade from an LT2-like ancestor was characterised by the acquisition of AMR genes, insertions and deletions in the virulence plasmid pU288-1, and the accumulation of polymorphisms, some of which resulted in truncation of coding sequences. U288 isolates exhibited lower growth rate and viability following desiccation compared to ST34 isolates, characteristics that could affect transmission through the food chain. U288 and ST34 isolates exhibited distinct outcomes of infection in the murine model of colitis, and colonised pigs in a manner that affected the disease symptoms and distribution in organs. U288 infection was more disseminated in the lymph nodes while ST34 were recovered in greater numbers in the intestinal contents. These data are consistent with the evolution of S. Typhimurium U288 adaptation to pigs that may determine their reduced zoonotic potential. ImportanceBacterial pathogens continually evolve to exploit new ecological niches as they emerge due to human activity including agricultural, medical or societal practice. The consequences of the emergence of new pathogens may affect outcome of infection and risk to human or animal health. Genome sequence can resolve the population structure, identify variants that are evolving as they enter a new niche, and pinpoint potential functional divergence. We report a variant S. Typhimurium that adapted to a unique niche distinct to that occupied by a second S. Typhimurium variant circulating in the same pig populations. Adaptation was accompanied by phenotypic and genotypic changes consistent with a more invasive lifestyle and a decreased zoonotic potential observed in the epidemiological record. Our findings suggest that pathogen genotypic variation may be useful in estimating zoonotic potential and threat to livestock welfare.

microbiology↗

bioRxiv2020

A rapid urban biodiversity blitz using aquatic environmental DNA

Background As global biodiversity declines, there’s an increasing need to create an educated and engaged society. Having people from all ages participate in measuring biodiversity where they live helps to create awareness. Recently, the use of environmental DNA (eDNA) for biodiversity surveys has gained momentum. Here, we test whether sampling eDNA and metabarcoding can be used for rapid urban biodiversity surveys for educational purposes.Materials & Methods We sampled 2×1 L of water from each of 15 locations in the city of Trondheim, Norway, including a variety of freshwater, marine and brackish habitats. DNA was extracted, amplified in triplicate for the COI gene and sequenced. The obtained data were analysed on the novel mBRAVE platform, an online open access software and computing resource.Results The water samples were collected in two days by two people and the lab analysis was completed in five days by one person. Overall, we detected the presence of 501 taxa identified as belonging to 435 species, representing 90 orders and 18 phyla. On average, only 5.4% of the taxa were shared among six replicates per site. Based on the observed diversity, three distinct clusters were detected and related to geographic distribution of sites. There were some taxa shared between the habitats, with a substantial presence of terrestrial biota.Discussion Our results match expected patterns of biodiversity in the landscape and show that with minimal sampling effort, hundreds of species can be detected. Thus, using eDNA analysis of water is promising for rapid biodiversity surveys, and it is likely that more detailed results could be obtained by optimising field and lab methods for particular groups of interest. We recommend that rapid eDNA surveys, with openly available services and softwares, can be used to raise awareness in the importance of biodiversity.Competing Interest StatementThe authors have declared no competing interest.View Full Text

scientific communication and education↗

bioRxiv2020

Genetic control of Campylobacter colonisation in broiler chickens: genomic and transcriptomic characterisation

Campylobacter is the leading cause of bacterial foodborne gastroenteritis in many countries. Source attribution studies unequivocally identify the handling or consumption of contaminated poultry meat as the primary risk factor. One potential strategy to control Campylobacter is to select poultry with increased resistance to colonisation. We conducted genomic and transcriptomic analyses of commercial pedigree broilers exposed to Campylobacter to examine persistent colonisation of the caecum as a quantitative trait. 3,000 broilers were genotyped using a 50K single nucleotide polymorphism (SNP) array and imputed to 600K SNPs. Genotypes were analysed for associations with the number of viable Campylobacter in the caeca. Heritability of the trait was modest but significantly greater than zero (h2=0.11 {+/-} 0.03). Genome-wide association analyses confirmed quantitative trait loci (QTL) on chromosomes 14 and 16 previously identified using the progeny of crosses of inbred lines differing in resistance, and detected two additional genome-wide significant QTLs on chromosomes 19 and 26. RNA-Seq analysis of the transcriptome of caecal tonsils from birds at the low and high extremes of C. jejuni colonisation phenotype identified differentially transcribed genes, mainly located within the QTL on chromosome 16 and proximal to the major histocompatibility complex (MHC) locus. We also identified strong cis-QTLs located within the MHC suggesting the presence of cis-acting variation in both MHC class I, class II and BG genes. Multiple other cis-acting variants were identified in association with key immune genes (COPS3, CCL4, CR1L, C4BP, PLGR) in the other QTLs. Pathway and network analysis implicated cooperative functional pathways and networks in colonisation, including those related to antigen presentation, innate and adaptive immune responses, calcium, and renin-angiotensin signalling. While co-selection for enhanced resistance and other breeding goal traits is feasible, the frequency of resistance-associated alleles was high in the population studied and non-genetic factors significantly influence Campylobacter colonisation in poultry. Author summaryCampylobacter infection is estimated to cause 95 million illnesses in people worldwide each year. Human infections mostly involve gastroenteritis, but can have severe complications. The handling or consumption of contaminated poultry meat is a key risk factor for human campylobacteriosis. The bacteria reach high numbers in the intestines of chickens reared for meat (broilers) and are frequently found on carcasses after slaughter. Effective vaccines against Campylobacter are not yet available, and treatments to reduce carcass contamination (e.g. chlorination) are not acceptable in some markets. One alternative is to breed for chickens with improved resistance to Campylobacter colonisation. To test the feasibility of this option in commercial birds, we analysed the genetic make-up of 3,000 pedigree broilers and determined the number of Campylobacter in their gut. There were associations between specific regions of the chicken genome and resistance to Campylobacter. Within some of these regions, expression of certain genes differed between birds at the low and high extremes of Campylobacter colonisation, providing a potential explanation for genetic variation in resistance. Selection of poultry with increased resistance to Campylobacter colonisation may be a complementary strategy to improved biosecurity, management, handling and processing procedures to reduce the burden of Campylobacter on human health.

genomics↗

bioRxiv2019

The conspicuousness of the toxic Heliconius butterflies across time and habitat

Forests are a mosaic of light spectra, and colour signal efficiency might change in different light environments. Local adaptation in Heliconius butterflies is linked to microhabitat use and the colourful wing colour patterns may be adapted for signalling in different light environments. These toxic butterflies exhibit conspicuous colours as a warning to predators that they should be avoided, but also find and choose potential mates based on colour signals. The two selection pressures of predation and mate preference are therefore acting together. Colour conspicuousness should show habitat-specific contrast for the butterflies, which would facilitate detection and species identification. On the other hand, selection for signal stability would be stronger in the avian visual system. In this study we analysed the contrast of two Heliconius mimicry rings in their natural habitats under varying degrees of forest fragmentation and light conditions. We used digital image analyses and mapped the bird and butterfly vision colour space in order to examine whether warning colours have greater contrast and if they transmit a consistent signal across time of the day and habitat in a tropical forest. We tested conspicuousness using opponent colour channels against a natural green background. For avian vision, colours are generally very stable through time and habitat. For butterfly vision, there is some evidence that species are more contrasting in their own habitats, where conspicuousness is higher for red and yellow bands in the border and for white in the forest. Light environment affects Heliconius butterflies warning signal transmission to a higher degree through their own vision, but to a lesser degree through avian predator vision. This work provides insight into the use of colour signals in sexual and natural selection in the light of ecological adaptation.

ecology↗

bioRxiv2018

The chemistry and pharmacology of putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) 5F-PY-PICA, 5F-PY-PINACA, and their analogues

The structural diversity of synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) has increased since the first examples were reported a decade ago. 5F-PY-PICA and 5F-PY-PINACA were identified in 2015 as putative SCRA NPS, although nothing is known of their pharmacology. 5F-PY-PICA, 5F-PY-PINACA, and analogues intended to explore structure-activity relationships within this class of SCRAs were synthesized and characterized by nuclear magnetic resonance spectroscopy and liquid chromatography-quadrupole time-of-flight-mass spectrometry. Using competitive binding experiments and fluorescence-based plate reader membrane potential assays, the affinities and activities of all analogues at cannabinoid type 1 and type 2 receptors (CB1 and CB2) were evaluated. All ligands showed minimal affinity for CB1 (pKi < 5), although several demonstrated moderate CB2 binding (pKi = 5.45-6.99). At 10 M none of the compounds produced an effect > 50% of CP55,950 at CB1, while several compounds showed a slightly higher relative efficacy at CB2. Unlike other SCRA NPS, 5F-PYPICA and 5F-PY-PINACA did not produce cannabimimetic effects in mice at doses up to 10 mg/kg.

pharmacology and toxicology↗

bioRxiv2018

Using camouflage for conservation: colour change in juvenile European lobster

Changes in coloration enable animals to refine their camouflage to match different visual environments. Such plasticity provides ecological benefits and could potentially be exploited to support conservation or stock enhancement efforts. One application could be ensuring that hatchery-reared animals, reared to stock wild populations, are appropriately matched to their environment on release. European lobster (Homarus gammarus) hatcheries aim to restock or enhance local lobster populations by rearing juveniles through their most vulnerable stages, then releasing them into the wild. However, little consideration has been given to their camouflage and the implications of matching individuals to their release site. This study assesses to what extent juvenile lobsters can change coloration to match their background and whether hatchery practices could be altered to enhance lobster camouflage. We test this by switching individuals between black or white backgrounds in the laboratory and monitoring their coloration over time. Our work demonstrates the capacity of juvenile lobsters to change lightness in response to their surroundings. We show that juvenile lobsters are capable of small changes in luminance (perceived lightness) to better match their background over 2-3 weeks. These changes potentially correspond to improved camouflage, based on a model of predator (European pollack, Pollachius pollachius) vision. However, over a longer period (5 weeks), lobsters maintained on either background converged on the same darker coloration, suggesting that lobsters also experience changes in appearance associated with ontogeny. By refining the approaches used here, there is potential for hatcheries to rear lobsters on backgrounds that better match their release site. However, such manipulations should be considered in the context of ontogenetic changes and release timing (which varies between stocking programmes). This study highlights the potential to use colour change in stocking and aquaculture, as well as gaps that could be addressed through further research in this area.

ecology↗

bioRxiv2018

Evolution of correlated complexity in the radically different courtship signals of birds-of-paradise

Ornaments used in courtship often vary wildly among species, reflecting the evolutionary interplay between mate preference functions and the constraints imposed by natural selection. Consequently, understanding the evolutionary dynamics responsible for ornament diversification has been a longstanding challenge in evolutionary biology. However, comparing radically different ornaments across species, as well as different classes of ornaments within species, is a profound challenge to understanding diversification of sexual signals. Using novel methods and a unique natural history dataset, we explore evolutionary patterns of ornament evolution in a group - the birds-of-paradise - exhibiting dramatic phenotypic diversification widely assumed to be driven by sexual selection. Rather than the tradeoff between ornament types originally envisioned by Darwin and Wallace, we found positive correlations among cross-modal (visual/acoustic) signals indicating functional integration of ornamental traits into a composite unit - the courtship phenotype. Furthermore, given the broad theoretical and empirical support for the idea that systemic robustness - functional overlap and interdependency - promotes evolutionary innovation, we posit that birds-of-paradise have radiated extensively through ornamental phenotype space as a consequence of the robustness in the courtship phenotype that we document at a phylogenetic scale. We suggest that the degree of robustness in courtship phenotypes among taxa can provide new insights into the relative influence of sexual and natural selection on phenotypic radiations.\n\nAuthor SummaryAnimals frequently vary widely in ornamentation, even among closely related species. Understanding the patterns that underlie this variation is a significant challenge, requiring comparisons among drastically different traits - like comparing apples to oranges. Here, we use novel analytical approaches to quantify variation in ornamental diversity and richness across the wildly divergent birds-of-paradise, a textbook example of how sexual selection can profoundly shape organismal phenotypes. We find that color and acoustic complexity, along with behavior and acoustic complexity, are positively correlated across evolutionary time-scales. Positive covariation among ornament classes suggests that selection is acting on correlated suites of traits - a composite courtship phenotype - and that this integration may be partially responsible for the extreme variation we see in birds-of-paradise.

evolutionary biology↗