Biology subjects

Llewellyn, M. S.

Publications and source records attributed to Llewellyn, M. S..

8 recordsLinked to original sources

bioRxiv2024

Understanding the Transfer and Persistence of Antimicrobial Resistance in Aquaculture Using a Model Teleost Gut System

The development, progression, and dissemination of antimicrobial resistance (AMR) is determined by interlinked human, animal, and environmental drivers, posing severe risks to human health. Conjugative plasmid transfer drives the rapid dissemination of AMR among bacteria. Besides antibiotic judicious use and implementation of antibiotic stewardship programs, mitigating antibiotic resistance spread requires an understanding of the dynamics of AMR transfer among microbial communities, as well as the role of various microbial taxa as potential reservoirs that promote long term AMR persistence. Here, we employed Hi-C, a high-throughput, culture-free technique, combined with qPCR, to monitor carriage and transfer of a multidrug-resistant plasmid within an Atlantic salmon in vitro gut model during florfenicol treatment, a benzenesulfonyl antibiotic widely deployed in fin-fish aquaculture. Microbial communities from the pyloric ceaca of three healthy adult farmed salmon were inoculated into three bioreactors developed for the SalmoSim gut system. The model system was then inoculated with an Escherichia coli strain ATCC 25922 carrying plasmid pM07-1 and treated with florfenicol at a concentration of 150 mg/L fish feed media for five days prior to a washout/recovery phase. Hi-C and metagenomic sequencing identified numerous transfer events, including to gram-negative and gram-positive taxa and, crucially, continuing transfer and persistence of the plasmid once florfenicol treatment had been withdrawn. Our findings highlight the role of commensal teleost gut flora as a reservoir for AMR, and our system provides a model to study how different treatment regimes and interventions may be deployed to mitigate AMR persistence.

microbiology↗

bioRxiv2023

High resolution longitudinal molecular and morphological tracking of planktonic threats to salmon aquaculture.

Marine-phase salmonid aquaculture is a major component of the coastal economies of Northern Europe, North America and Chile and is under threat from numerous challenges to gill health, many of which originate from the phyto- and zooplankton. Associated losses are growing as a proportion of production year on year. A first step towards mitigating losses is to characterize the biological drivers of poor gill health. Numerous planktonic species have been implicated, including toxic and siliceous microalgae, hydrozoans and scyphozoans; however, rigorous longitudinal surveys of planktonic diversity and gill health have been lacking. In the current study, we present and assess an exhaustive identification approach combining both morphological and molecular methods (environmental DNA metabarcoding) approaches in combination with robust statistical models to identify the planktonic drivers of complex gill disease (CGD) and fish mortality. We undertook longitudinal molecular and microscopic evaluation at two marine aquaculture facilities on the west coast of Scotland using daily data collected during the 2021 growing season (March-October). Examining these two different sites, one sheltered and one exposed to the open sea, we identified new, important, and unexpected planktonic drivers (e.g. doliolids and appendicularians) of CGD and mortality and confirmed the significance of some established threats (e.g. hydrozoans and diatoms). We also explored delayed or lagged effects of planktonic abundances on gill health and undertook a comparison of environmental DNA metabarcoding and microscopy in their ability to identify and quantify planktonic species. Our data highlight the diversity of planktonic threats to salmonid aquaculture as well as the importance of using both molecular and morphological approaches to detect those. Despite our study relying on two farm sites only, our results evidence the role of the different planktonic players on salmon gill disease; there is now an urgent need to expand systematic longitudinal molecular and morphological approach across multiple sites and over multiple years. The resultant catalogue of main biological drivers will enable early warning systems, new treatments and, ultimately, a sustainable platform for future salmonid aquaculture in the marine environment.

zoology↗

bioRxiv2021

The genomic basis of domestic colonisation and dispersal in Chagas disease vectors

The biology of vector adaptation to the human habitat remains poorly understood for many arthropod-borne diseases but underpins effective and sustainable disease control. We adopted a landscape genomics approach to investigate gene flow, signatures of local adaptation, and drivers of population structure among multiple linked wild and domestic population pairs in Rhodnius ecuadoriensis, an important vector of Chagas Disease. Evidence of high triatomine gene flow (FST) between wild and domestic ecotopes at sites throughout the study area indicate insecticide-based control will be hindered by constant re-infestation of houses. Genome scans revealed genetic loci with strong signal of local adaptation to the domestic setting, which we mapped to annotated regions in the Rhodnius prolixus genome. Our landscape genomic mixed effects models showed Rhodnius ecuadoriensis population structure and connectivity is driven by landscape elevation at a regional scale. Our ecologically- and spatially-explicit vector dispersal model enables targeted vector control and recommends spatially discrete, periodic interventions to local authorities as more efficacious than current, haphazard approaches. In tandem, evidence for parallel genomic adaptation to colonisation of the domestic environment at multiple sites sheds new light on the evolutionary basis of adaptation to the human host in arthropod vectors.

genomics↗

bioRxiv2020

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly from vector/host material but are often too complex and expensive for resource-poor settings where infectious diseases prevail. This study proposes a simple, cost-effective genome-wide locus sequence typing (GLST) tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation at under 4 USD per sample. Approximately 100 libraries can be sequenced together in one Illumina MiSeq run. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/l T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate DTUs. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research.

genetics↗

bioRxiv2019

Remarkable genetic diversity of Trypanosoma cruzi and Trypanosoma rangeli in two localities of southern Ecuador identified via deep sequencing of mini-exon gene amplicons

Trypanosoma cruzi, causative agent of Chagas disease, and T. rangeli are kinetoplastid parasites endemic to Latin America. Although closely-related to T. cruzi and capable of infecting humans, T. rangeli is non-pathogenic. Both parasite species are transmitted by triatomine bugs, and the presence of T. rangeli constitutes a confounding factor in the study of Chagas prevalence and transmission dynamics. T. cruzi possesses high molecular heterogeneity: six discrete typing units (DTUs) are currently recognized. In Ecuador, TcI predominates while other DTUs are seldom reported. Here, infection by T. cruzi and/or T. rangeli in triatomine bugs from two communities of southern Ecuador was evaluated via PCR product size polymorphism of kinetoplast-minicircle sequences and the non-transcribed spacer region of the mini-exon gene. Additionally, mini-exon amplicons were deep-sequenced to analyze single nucleotide polymorphisms and mixed infections. As a control, mini-exon products from several monoclonal reference strains were included. T. cruzi genetic diversity was significantly greater in adult vectors than in nymphal stage V vectors. Among infected triatomines, deep sequencing revealed one T. rangeli infection (3%), 9 T. cruzi infections (27.3%) and 23 T. cruzi / T. rangeli mixed infections (69.7%), suggesting that T. rangeli prevalence has been largely underestimated in the region. Furthermore, deep-sequencing detected TcIV sequences in six samples (first TcIV record in southern Ecuador). Our data indicate that amplicon size analysis alone is not reliable for parasite identification/typing in mixed infections containing both T. cruzi and T. rangeli, or when multiple T. cruzi DTUs are present. Additionally, our analysis showed extensive overlap among the parasite populations present in the two studied localities (ca. 28 km apart); suggesting active parasite dispersal over the study area. Our results highlight the value of amplicon sequencing methodologies to clarify the population dynamics of kinetoplastid parasites in endemic regions and inform control campaigns in southern Ecuador.

genetics↗

bioRxiv2018

Parallel sexual and parasexual population genomic structure in Trypanosoma cruzi

Genetic exchange and hybridization in parasitic organisms is fundamental to the exploitation of new hosts and host populations. Variable mating frequency often coincides with strong metapopulation structure, where patchy selection or demography may favor different reproductive modes. Evidence for genetic exchange in Trypanosoma cruzi over the last 30 years has been limited and inconclusive. The reproductive modes of other medically important trypanosomatids are better established, although little is known about their variability on a spatio-temporal scale. Targeting a contemporary focus of T. cruzi transmission in southern Ecuador, we present compelling evidence from 45 sequenced genomes that T. cruzi (discrete typing unit I) maintains sexual populations alongside others that represent clonal bursts of parasexual origin. Strains from one site exhibit genome-wide Hardy-Weinberg equilibrium and intra-chromosomal linkage decay consistent with meiotic reproduction. Strains collected from adjacent areas (>6 km) show excess heterozygosity, near-identical haplo-segments, common mitochondrial sequences and levels of aneuploidy incompatible with Mendelian sex. Certain individuals exhibit trisomy in as many as fifteen chromosomes. Others present fewer, yet shared, aneuploidies reminiscent of mitotic genome erosion and parasexual genetic exchange. Genomic and intra-genomic phylogenetics as well as haplotype co-ancestry analyses indicate a clear break in gene-flow between these distinct populations, despite the fact that they occasionally co-occur in vectors and hosts. We propose biological explanations for the fine-scale disconnectivity we observe and discuss the epidemiological consequences of flexible reproductive modes and their genomic architecture for this medically important parasite.

evolutionary biology↗

bioRxiv2017

Environmental DNA: a new low-cost monitoring tool for pathogens in salmonid aquaculture.

Sequencing of environmental DNA (eDNA-seq) is an emergent new monitoring tool that promises to facilitate the accurate and cost effective detection of species in environmental samples. eDNA monitoring is likely to have a major impact on the ability of salmonid aquaculture industry producers and their regulators to detect the presence and abundance of pathogens and other biological threats in the surrounding environment. However, for eDNA-seq to develop into a useful bio-monitoring tool it is necessary to (a) validate that sequence datasets derived from amplification of meta-barcoding markers reflect the true species identity and abundances in biological samples, and (b) establish a low-cost sequencing method to enable the bulk processing of environmental samples. In this study, we employed an elaborate experimental design whereby different combinations of five biological agents were crossed at three abundance levels and exposed to pre-filtered and normal seawater, prior to coarse filtering and then eDNA ultrafiltration of the resultant material. We then benchmarked the low-cost, scalable, Ion Torrent sequencing method against the current gold-standard Illumina platform for eDNAseq detection in aquaculture. Based on amplicon-seq of the 18S SSU rDNA v9 region, we found that Illumina and Ion Torrent were equally good in identifying the two parasite species (Lepeophtheirus salmonis and Paramoeba perurans), whereas the microalgae species Prymnesium parvum, Pseudo-nitzschia seriata and P. delicatissima could be assigned correctly only to the genus level. Illumina and Ion Torrent were also equally able to reflect community composition in our samples, whereas Ion Torrent was more sensitive in detecting species richness when the medium was unfiltered seawater. Both methods were able to reflect the correct abundances of 4 out of 5 species in samples from unfiltered seawater, despite the significant amount of background noise from both bacteria and eukaryotes. Our findings indicate that eDNA-seq offers significant potential in the monitoring of species harmful to aquaculture and for this purpose, the low-cost Ion Torrent sequencing is equally as accurate as Illumina.

genetics↗

bioRxiv2017

Genetic fingerprinting of salmon louse (Lepeophtheirus salmonis) populations in the North-East Atlantic using a random forest classification approach

Caligid sea lice represent a significant threat to salmonid aquaculture worldwide. Lepeophtheirus salmonis is the predominant species that occurs in the Northern Hemisphere. Dispersal of sea lice between marine aquaculture sites and geographic regions is thought to occur rapidly via planktonic transport of larvae. Population genetic analyses have consistently shown minimal population genetic structure in North Atlantic L. salmonis, frustrating efforts to track louse populations, improve targeted control measures and understand local adaption to environmental conditions. The aim of this study was to test the power of reduced representation library sequencing (IIb-RAD sequencing) coupled with random forest machine learning algorithms to define markers for fine-scale discrimination of louse populations. We identified 1286 robustly supported SNPs among four L. salmonis populations from Ireland (N=2, 27 individuals), Scotland (N=1, 11 individuals) and North Norway (N=1, 12 individuals). Weak global structure (FSC = 0.018, p<0.0001) and only one significant pairwise FST comparison was observed (Scotland vs Kenmare Bay, (FST = 0.018, p<0.0001)) using all 1286 SNPs. The application of a random forest machine-learning algorithm identified 98 discriminatory SNPs that dramatically improved population assignment (DAPC assignment probability = 1), increased global Fsc = 0.098, (p<0.0001) and resulted in pairwise comparisons that all showed highly significant Fst-values (range = 0.081 - 0.096, p<0.0001). Out of 19 SNPs found to be under directional selection between populations, 12 corresponded to the discriminatory SNPs identified using random forest. Taken together our data suggest that L. salmonis SNP diversity exists with which it is possible to discriminate differences between nearby populations given suitable marker selection approaches, and that such differences might have an adaptive basis. We discuss these data in light of sea lice adaption to anthropogenic and environmental pressures as well as novel approaches to track and predict sea louse dispersal.

ecology↗