Biology subjects

Llewellyn, M.

Publications and source records attributed to Llewellyn, M..

9 recordsLinked to original sources

bioRxiv2026

Spatial Transcriptomics Reveals Region-Specific Host Responses in Amoebic Gill Disease of Atlantic Salmon

Amoebic gill disease (AGD), caused by the parasite Neoparamoeba perurans, is a major welfare and economic burden for the Atlantic salmon aquaculture industry. However, the spatial organisation of immune functions within the gill, the site of infection, remains poorly resolved. Here, we present the first spatial transcriptomic atlas of the Atlantic salmon gill affected by AGD, generated using 10x Genomics Visium on four AGD-positive fish, confirmed by gill scoring and quantitative PCR for parasite load. As no uninfected gills were included, this study maps the anatomical and immunological organisation of AGD-affected gills rather than defining AGD-specific pathology. Unsupervised clustering of spatial gene expression resolved distinct transcriptional domains that grouped into four principal anatomical compartments of the gill, including (i) epithelial and lamellar regions defined by keratin and osmoregulatory gene expression, (ii) gill arch regions enriched for connective tissue and smooth muscle markers, (iii) an erythroid-associated vascular cluster, and (iv) a skeletal muscle cluster with signatures of glycolysis and oxidative phosphorylation. Across these domains, we identified a spatially organised and diffused immune landscape, with pattern recognition receptors, antimicrobial effectors and chemokines concentrated at the filament surface, and immunoglobulin light chain transcripts consistent with B lymphocyte activity enriched toward the gill arch. Integration of the spatial data with a published single-nucleus reference atlas using reference-based deconvolution resolved the cellular identity underlying these domains and revealed frequent co-localisation of immune and structural cell types, suggesting regulated cell to cell communication within the gill-associated lymphoid tissue. Overall, our results show that the Atlantic salmon gill is not a transcriptionally uniform surface but a regionally specialised organ, with immune functions distributed across, and specialised within, distinct anatomical compartments

immunology↗

bioRxiv2026

Single-cell transcriptomics reveals life cycle and cell cycle gene expression dynamics during in vitro growth of two Leishmania species

Leishmania species are transmitted between mammals by sand flies, within which they undergo a complex developmental programme involving transitions between replicative and non-replicative life cycle forms. Recent work using single-cell transcriptomics has revealed the developmental pathways of Leishmania major in the sand fly, but it is unclear how faithfully such development is recapitulated during Leishmania culture in vitro. In addition, no work has revealed how gene expression changes across the cell cycle of replicative Leishmania forms. Using single-cell transcriptomics on unsynchronised cultures of L. major and L. mexicana, we addressed both questions. We show that in vitro L. mexicana promastigotes display the same two developmental endpoints seen in L. major cells recovered from sand flies, metacyclics and haptomonads. In contrast, L. major promastigote development in vitro does not predict the presence of leptomonads or haptomonads, although non-dividing late metacyclics form through an early metacyclic stage as parasites enter stationary phase. Using computational reconstruction of the cell cycle in procyclic promastigote forms of both Leishmania species identified hundreds of genes whose transcript levels vary as the cell cycle progresses, revealing core conserved processes between the two species and with the closely related Trypanosoma brucei. These genes allowed us to examine connections between life cycle and cell cycle progression, revealing that some developmental transitions are strongly linked to a cell cycle phase, whereas others occur asynchronously. This work provides a comprehensive atlas and marker gene list for the interlinked cell and life cycles of two laboratory-cultured Leishmania species.

microbiology↗

bioRxiv2026

Repurposing drugs to treat Amoebic Gill Disease in Atlantic Salmon

Neoparameoba perurans causes Amoebic Gill Disease (AGD), a major parasitic disease of marine-phase Atlantic salmon and rainbow trout worldwide. Treatment options are limited to freshwater baths, which are costly at scale and exhibit only limited long-term efficacy. N. perurans contains an obligate eukaryotic symbiont, Perkinsela-like organism (PLO). PLO belongs to the class Kinetoplastida, which includes medically and veterinary important parasites such as Trypanosoma and Leishmania. As such, we hypothesised that trypanocidal drugs developed against other kinetoplastids might also affect N. perurans, potentially through disruption of its PLO symbiont, and used this hypothesis as a rationale for prioritising a focused panel of candidate compounds for screening. A holographic motility-based cytotoxicity assay was established to identify promising candidates in vitro, followed by controlled host tolerance testing and finally a field efficacy sea trial using naturally AGD-exposed site in the west of Ireland. Several compounds showed activity in vitro, especially miltefosine (EC50 1.84 uM, amoebicidal) and isometamidum (EC50 4.63 uM, amoebostatic). In vivo (two intramuscular injections, two weeks apart), miltefosine (Odds Ratio (OR) 0.62), isometamidum (OR 0.61) and benznidazole (OR 0.64) significantly improved gill score over four weeks, with miltefosine showing the largest effect size. Gill parasitaemia, measured via qPCR, was not reduced. Instead, two compounds increased apparent amoeba loads. This work support trypanocidal as potential AGD treatments in the field, although optimisation of dosing, delivery and mode of action requires further study.

microbiology↗

bioRxiv2026

Asymmetric population structure in Neoparamoeba perurans and its kinetoplastid symbiont

Secondary endosymbioses typically involve photosynthetic gain-of-function, at least initially. For Neoparamoeba perurans, the agent of Amoebic Gill Disease in Atlantic salmon, the evolutionary significance of its non-photosynthetic kinetoplastid endosymbiont, Perkinsela, remains a mystery. While such endosymbionts usually mirror organellar uniparental inheritance, departures from strict vertical transmission can significantly impact holobiont evolution and the spread of traits like drug resistance. However, genomic analysis of N. perurans is hampered by continual traffic of bacteria in and out of the amoeba, limiting our understanding of its population dynamics. Here, we developed a dual-target AmpSeq panel from a draft N. perurans genome, enabling simultaneous genotyping of host and symbiont directly from xenic biological samples. Analysis of 58 North Atlantic isolates revealed a striking asymmetry in population structure: the amoeba host forms a diverse, panmictic population with low linkage disequilibrium, whereas Perkinsela exhibits lower diversity and strong clonality. While cophylogenetic analyses confirm a global signal of vertical fidelity, fine-scale genotyping reveals frequent shuffling of host-symbiont pairs. These results suggest that strict host-symbiont co-inheritance is not absolute, perhaps reflecting a system characterised by long-term vertical stability punctuated by intermittent reassortment. Ultimately, we validate AmpSeq as a robust, scalable tool for decoupling complex host-symbiont trajectories and monitoring multi-partner disease dynamics in aquaculture environments.

genomics↗

bioRxiv2020

Deploying and in vitro gut model to assay the impact of a mannan-oligosaccharide prebiotic, Bio-MOS(R) on the Atlantic salmon (Salmo salar) gut microbiome.

Mannose-oligosaccharide (MOS) pre-biotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and associated metabolism. Bio-MOS is a commercially available pre-biotic currently used in the agri-feed industry. To assay Bio-MOS for potential use as a pre-biotic growth promotor in salmonid aquaculture, we modified an established Atlantic salmon in vitro gut model, SalmoSim, to evaluate its impact on host microbial communities. In biological triplicate, microbial communities were stabilised in SalmoSim followed by twenty-day exposure to the pre-biotic and then an eight day wash out period. Exposure the MOS resulted in a significant increase in formate (p=0.001), propionate (p=0.037) and isovalerate (p=0.024) levels, correlated with increased abundances of several principally anaerobic microbial genera (Fusobacteria, Agarivorans, Pseudoalteromonas, Myroides). 16S rDNA sequencing confirmed a significant shift in microbial community composition in response to supplementation. In conjunction with previous in vivo studies linking enhanced VFA production alongside MOS supplementation to host growth and performance, our data suggest that Bio-MOS may be of value in salmonid production. Furthermore, our data highlight the potential role of in vitro gut models to augment in vivo trials of microbiome modulators.

microbiology↗

bioRxiv2020

Development of a three-compartment in vitro simulator of the Atlantic Salmon GI tract and associated microbial communities: SalmoSim

Atlantic salmon are a species of major economic importance. Intense innovation is underway to improve salmon feeds and feed additives to enhance fish performance, welfare, and the environmental sustainability of the industry. Several gut models targeted at monogastric vertebrates are now in operation. Here we report progress in the development of an Atlantic salmon in vitro gut model, SalmoSim, to simulate three gut compartments (stomach, pyloric caecum and mid gut) and associated microbial communities. The artificial gut model was established in a series of linked bioreactors seeded with biological material derived for adult marine phase salmon. In biological triplicate, the response of the in vitro system to two distinct dietary formulations (fish meal and fish meal free) was compared to a parallel in vivo trial over forty days. 16S rDNA sequencing, qPCR, ammoniacal nitrogen and volatile fatty acid measurements were undertaken to survey microbial community dynamics and function. SalmoSim communities were indistinguishable (p=0.230) from their founding inocula at 20 days and most abundant genera (e.g. Psycrobacter, Staphylococcus, Pseudomonas) proliferated the in vitro system. Real salmon and SalmoSim responded similarly to the introduction of the novel feed, with most taxa (96% Salmon, 97% SalmoSim) unaffected, while a subset of taxa was affected non-identically across both systems. Consistent with a low impact of the novel feed on microbial community function, VFA profiles were not significantly different in SalmoSim pre and post the switch feed. This study represents an important first-step in the development of an in vitro gut system as a tool for the improvement of salmon nutrition.

microbiology↗

bioRxiv2020

Unpicking the mysterious symbiosis of mycoplasmas in salmonids

Lacking a peptidoglycan cell wall, mycoplasmas are the smallest self-replicating life forms. Members of this bacterial genus are known to parasitise a wide array of metazoans including vertebrates. Whilst much research has been significant targeted at parasitic mammalian mycoplasmas, very little is known about their role in other vertebrates. In the current study, we aim to explore the biology and evolution of Mycoplasma in salmonids, including cellular niche, genome size structure and gene content. Using Fluorescence in-situ hybridisation (FISH), mycoplasmas were identified in epithelial tissues across the digestive tract (stomach, pyloric caecum and midgut) during the developmental stages (eggs, parr, subadult) of farmed Atlantic salmon (Salmo salar), showing a high abundance in acidic compartments. With high throughput sequencing from subadults farmed Atlantic salmon, we assembled a nearly complete genome (~0.57 MB) via shotgun-metagenomics. The phylogenetic inference from the recovered genome revealed successful taxonomic proximity to Mycoplasma penetrans (~1.36 Mb) from the recovered genome. Although, no significant correlation between genome size and its phylogeny was observed, we recovered functional signatures, especially, riboflavin encoding genes pathway and sugars transporters, suggesting a symbiotic relationship between Mycoplasma and the host. Though 247 strains of Mycoplasma are available in public databases, to the best of our knowledge, this is the first study to demonstrate ecological and functional association between Mycoplasma and Salmo salar which delineates symbiotic reductive evolution and genome erosion primarily and also serves as a proxy for salmonid health in aquaculture processes (cell lines, in vitro gut models).

evolutionary biology↗

bioRxiv2020

In-situ non-lethal rapid test to accurately detect the presence of the nematode parasite, Anguillicoloides crassus, in European eel, Anguilla anguilla.

Anguillicoloides crassus is an invasive nematode parasite of the European eel, Anguilla anguilla, and one of the primary drivers of eel population collapse. The presence of the parasite has been shown to impact many features of eel physiology and life history. Early detection of the parasite is vital to limit the spread of A. crassus. However, until recently, accurate diagnosis of infection could only be achieved via terminal dissection. To support A. anguilla fisheries management in the context of A. crassus we developed a rapid non-lethal and non-invasive environmental DNA method to detect the presence of the parasite in the swim bladder. Screening of 131 wild eels was undertaken between 2017 and 2019 Ireland and UK to validate the procedure. DNA extractions and PCR were conducted using both a Qiagen Stool kit at Glasgow University and in situ using Whatman qualitative filter paper No. 1 and a miniPCR DNA Discovery System. Primers were specifically designed from the cytochrome oxidase mtDNA gene region. In situ extraction and amplification takes approx. 3h for up to 16 individuals with higher specificity and sensitivity compare to the laboratory Qiagen kit extraction. The local diagnostic procedure demonstrated Positive Predictive Values at 96% and Negative Predictive Values at 87%. Our method will be a powerful tool in the hands of fisheries managers to help protect this iconic but critically endangered species. It will allow a non-invasive monitoring of the A. crassus dispersion across the European waters.

ecology↗

bioRxiv2020

Malaria in Southern Venezuela: The Hottest Hotspot in Latin America

Malaria cases in Latin America reached ~1 million in 2017 and 2018, with 53% and 51% reported from Venezuela, respectively. In this study, we characterized the spatiotemporal dynamics of malaria transmission between 2007-2017 in southern Venezuela, the main endemic area of the country. We found that disease transmission was focal and more prevalent in the southeast of southern Venezuela where two persistent hotspots of Plasmodium vivax (76%) and P. falciparum (18%) linked to deforestation for illegal gold mining accounted for ~60% of the country-wide number of cases. Incidence has increased nearly tenfold in the last decade, showing an explosive epidemic growth due to a significant lack of disease control. We suggest that a source-sink pattern of Plasmodium sp. dispersal account for the re-emergence and progression of malaria transmission in the last 4 years across the country due to the internal migration of infected people to and from the hotspots and other malaria-prone ecosystems. We observe a similar pattern explaining the spillover of cases across international borders affecting neighboring countries. This study provides baseline epidemiological data and guidance for malaria control to further assess the dynamics of cross-border malaria, the role of asymptomatic carriers, drug-resistant evolution, and innovative control efforts in the Latin America region.

ecology↗