Biology subjects

Darby, A.

Publications and source records attributed to Darby, A..

11 recordsLinked to original sources

bioRxiv2026

An improved Trypanosoma vivax genome assembly confirms the distinct organisation of its variant antigen repertoire

Trypanosoma vivax is a unicellular parasite spread by biting flies and an important livestock pathogen across sub-Saharan Africa and South America. Alongside its better known relative, T. brucei, it causes animal trypanosomosis. An accurate genome sequence is essential to functional genetics as well as the design of novel diagnostics, drugs and vaccines. However, the existing T. vivax genome sequence is fragmentary, making it difficult to examine genomic features such as the Variant Surface Glycoprotein (VSG) genes that are the basis for antigenic variation, a key phenotype that underpins immune evasion by African trypanosomes. Our aim was to produce a T. vivax genome sequence with improved contiguity to properly characterise the VSG genes. We present a new core genome assembly in 23 scaffolds, forming approximately 10 chromosomes. Many scaffolds are articulated with extensive sub-telomeric domains that, like T. brucei, contain VSG gene arrays but in T. vivax these are near-perfect repeats of intact genes. Using transcriptomic and proteomic analysis of VSG expression during bloodstream infection we show that the active VSG is expressed from a sub-telomeric array while other arrays are silent. We find no evidence for a VSG expression site at the telomeres or elsewhere. Therefore, although monoallelic expression and antigenic switching occur in T. vivax, the familiar regulatory mechanism of T. brucei does not exist in T. vivax. This improved T. vivax genome assembly demonstrates that T. brucei is a poor guide for T. vivax genetics generally and for regulation of antigenic variation specifically.

genomics↗

bioRxiv2025

Genome assembly of five Tephritid species for the enhancement of the Sterile Insect Technique

Tephritidae insect pests account for extensive crop damage and yield losses globally. Modern, sustainable pest management approaches are species-specific and, therefore, high-quality genome assemblies are required for their application. Here, we present chromosome-level assemblies for five members of the Tephritidae family: Anastrepha fraterculus, Anastrepha ludens, Bactrocera dorsalis, Bactrocera zonata and Zeugodacus cucurbitae. The assemblies used long read sequencing polished with short read sequencing and scaffolded using Hi-C (chromatin conformation capture) sequencing. Prior to scaffolding the assembly deduplication was performed to separate a primary assembly and an alternate assembly, and each was then scaffolded independently. The scaffolded assemblies reached N50 length in the range of 60Mb to 120Mb. The scaffolded assemblies were verified with BUSCO and completeness was in the range 97% to 98.5% and had very low duplicated, fragmented and missing orthologs.

ecology↗

bioRxiv2024

MUTE drives asymmetric divisions to form stomatal subsidiary cells in Crassulaceae succulents

Amongst the evolutionary innovations of many succulents is a photosynthetic lifestyle, where stomatal gas exchange is decoupled from light-dependent carbon fixation. Stomatal complexes in the emerging succulent model Kalanchoe laxiflora consist of two guard cells surrounded by three anisocytic subsidiary cells (SCs). Here, we show that these SCs shuttle ions and thus likely support stomatal movements. Furthermore, gene editing, reporter lines and protein overexpression implicate the stomatal transcription factor MUTE in facilitating additional rounds of asymmetric divisions that form SCs in succulents. This is opposite to the role of MUTE in Arabidopsis thaliana, where it stops rather than induces asymmetric divisions, but reminiscent of MUTEs SC-related function in grasses. Together, our work deciphers an intricate genetic mechanism that generates innovative stomatal morphology in Crassulaceae succulents.

plant biology↗

bioRxiv2023

Arsenophonus symbiosis with louse flies: multiple origins, coevolutionary dynamics, and metabolic significance

Arsenophonus is a widespread insect symbiont with life strategies that vary from parasitism to obligate mutualism. In insects living exclusively on vertebrate blood, mutualistic Arsenophonus strains are presumed to provide B vitamins missing in the insect host diet. Hippoboscidae, obligate blood-feeders related to tsetse flies, were previously suggested to have acquired Arsenophonus symbionts in several independent events. Based on comparative genomic analyzes of eleven Hippoboscidae-associated strains, nine of them newly assembled, we reveal a wide range of their genomic characteristics and phylogenetic affiliations. Phylogenetic patterns and genomic traits split the strains into two different types. Seven strains display characteristics of obligate mutualists with significantly reduced genomes and long phylogenetic branches. The remaining four genomes cluster on short branches, and their genomes resemble those of free-living bacteria or facultative symbionts. Both phylogenetic positions and genomic traits indicate that evolutionary history of the Hippoboscidae-Arsenophonus associations is a mixture of short-term coevolutions with at least four independent origins. The comparative approach to a reconstruction of B vitamin pathways across the available Arsenophonus genomes produced two kinds of patterns. On one hand, it indicated the different importance of individual B vitamins in the host-symbiont interaction. While some (riboflavin, pantothenate, folate) seem to be synthesized by all Hippoboscidae-associated obligate symbionts, pathways for others (thiamine, nicotinamide, cobalamin) are mostly missing. On the other hand, the broad comparison produced patterns which can serve as bases for further assessments of the pathways completeness and functionality. ImportanceInsects that live exclusively on vertebrate blood utilize symbiotic bacteria as a source of essential compounds, e.g. B vitamins. In louse flies, the most frequent symbiont originated in genus Arsenophonus, known from a wide range of insects. Here, we analyze genomic traits, phylogenetic origins, and metabolic capacities of eleven Arsenophonus strains associated with louse flies. We show that in louse flies Arsenophonus established symbiosis in at least four independent events, reaching different stages of symbiogenesis. This allowed for comparative genomic analysis, including convergence of metabolic capacities. The significance of the results is two-fold. First, based on a comparison of independently originated Arsenophonus symbioses, it determines the importance of individual B vitamins for the insect host. This expands our theoretical insight into insect-bacteria symbiosis. The second outcome is of methodological significance. We show that the comparative approach reveals artifacts that would be difficult to identify based on a single-genome analysis.

evolutionary biology↗

bioRxiv2022

Genomic Analysis of Two Phlebotomine Sand Fly Vectors of Leishmania from the New and Old World

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the devastating kinetoplastid parasites of the genus Leishmania, the causative agents of diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. As no approved efficacious vaccine exists, available drugs are expensive and/or toxic, and resistance is emerging, management of sand fly populations to break transmission is currently the most effective disease control strategy. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two of the most important sand fly species: Phlebotomus papatasi, a cutaneous leishmaniasis vector, (distributed in the Middle East and North Africa) and Lutzomyia longipalpis, a visceral leishmaniasis vector (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites. Author SummaryThe leishmaniases are a group of neglected tropical diseases caused by protist parasites from the Genus Leishmania. Different Leishmania species present a wide clinical profile, ranging from mild, often self-resolving cutaneous lesions that can lead to protective immunity, to severe metastatic mucosal disease, to visceral disease that is ultimately fatal. Leishmania parasites are transmitted by the bites of sand flies, and as no approved vaccine exists, available drugs are toxic and/or expensive and resistance is emerging, new dual control strategies to combat these diseases must be developed, combining interventions on human infections and integrated sand fly population management. Effective vector control requires a good understanding of the biology of sand flies. To this end, we sequenced and annotated the genomes of two sand fly species that are important leishmaniasis vectors from the Old and New Worlds. These genomes allow us to better understand, at the genetic level, processes important in the vector biology of these species, such as finding hosts, blood-feeding, immunity, and detoxification. These genomic resources highlight the driving forces of evolution of two major Leishmania vectors and provide foundations for future research on how to better prevent leishmaniasis by control of the sand fly vectors.

genomics↗

bioRxiv2022

Re-inventing pathogen passage for social microbes

Passage experiments that sequentially infect hosts with parasites have long been used to manipulate virulence. However, in many invertebrate pathogens passage has been applied naively without a full theoretical understanding of how best to select for increased virulence. This has led to very mixed results. Understanding the evolution of virulence is complex because selection on parasites occurs across multiple spatial scales with potentially different conflicts operating on parasites with different life-histories. For example, in social microbes, strong selection on replication rate within hosts can lead to cheating and loss of virulence, because investment in public goods virulence reduces replication rate. In contrast, selection acting at a between host scale maintains virulence by selecting on parasite population size. In this study we tested how different scales of selection and varying mutation supply affect evolution of virulence against resistant hosts in the specialist insect pathogen Bacillus thuringiensis., aiming to optimize methods for strain improvement against a difficult to kill insect target. We show that selection for infectivity using competition between sub-populations in a metapopulation prevents social cheating, acts to retain key virulence plasmids and facilitates increased virulence. Increased virulence was associated with reduced efficiency of sporulation, and loss of function in putative regulatory genes but not with altered expression of known virulence factors. Selection in a metapopulation provides a broadly applicable tool for improving the efficacy of biocontrol agents. Moreover, a structured host population can facilitate artificial selection on infectivity, while selection on life history traits such as faster replication or larger population sizes can reduce virulence can reduce virulence in social microbes.

evolutionary biology↗

bioRxiv2021

Rapid selection of P323L in the SARS-CoV-2 polymerase (NSP12) in humans and non-human primate models and confers a large plaque phenotype

The mutational landscape of SARS-CoV-2 varies at both the dominant viral genome sequence and minor genomic variant population. An early change associated with transmissibility was the D614G substitution in the spike protein. This appeared to be accompanied by a P323L substitution in the viral polymerase (NSP12), but this latter change was not under strong selective pressure. Investigation of P323L/D614G changes in the human population showed rapid emergence during the containment phase and early surge phase of wave 1 in the UK. This rapid substitution was from minor genomic variants to become part of the dominant viral genome sequence. A rapid emergence of 323L but not 614G was observed in a non-human primate model of COVID-19 using a starting virus with P323 and D614 in the dominant genome sequence and 323L and 614G in the minor variant population. In cell culture, a recombinant virus with 323L in NSP12 had a larger plaque size than the same recombinant virus with P323. These data suggest that it may be possible to predict the emergence of a new variant based on tracking the distribution and frequency of minor variant genomes at a population level, rather than just focusing on providing information on the dominant viral genome sequence e.g., consensus level reporting. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

microbiology↗

bioRxiv2021

Identification and quantification of SARS-CoV-2 leader subgenomic mRNA gene junctions in nasopharyngeal samples shows phasic transcription in animal models of COVID-19 and aberrant pattens in humans

IntroductionSARS-CoV-2 has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgRNAs has a unique 5 sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS-junction), that can be identified using sequencing. ResultsHigh resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS-junctions and be used as a proxy to quantify sgmRNAs for understanding virus biology. This was tested on published datasets and clinical samples from patients and longitudinal samples from animal models with COVID-19. DiscussionLeTRS identified known leader-TRS-junctions and identified novel species that were common across different species. The data indicated multi-phasic abundance of sgmRNAs in two different animal models, with spikes in sgmRNA abundance reflected in human samples, and therefore has implications for transmission models and nucleic acid-based diagnostics.

microbiology↗

bioRxiv2020

Amplicon and metagenomic analysis of MERS-CoV and the microbiome in patients with severe Middle East respiratory syndrome (MERS)

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional spill overs into humans. The severity of infection in humans is influenced by numerous factors and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are co-incident in the Middle East and a rapid way is required of sequencing MERS-CoV to derive genotype information for molecular epidemiology. Additionally, complicating factors in MERS-CoV infections are co-infections that require clinical management. The ability to rapidly characterise these infections would be advantageous. To rapidly sequence MERS-CoV, we developed an amplicon-based approach coupled to Oxford Nanopore long read length sequencing. The advantage of this approach is that insertions and deletions can be identified - which are the major drivers of genotype change in coronaviruses. This and a metagenomic approach were evaluated on clinical samples from patients with MERS. The data illustrated that whole genome or near whole genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants including deletion mutants. Whereas, the metagenomic analysis provided information of the background microbiome.

molecular biology↗

bioRxiv2019

Insect-symbiont gene expression in the midgut bacteriocytes of a blood-sucking parasite

BackgroundAnimals interact with a diverse array of both beneficial and detrimental microorganisms. These interactions sometimes spark obligate symbioses where the host depends on beneficial bacteria for survival and reproduction. In insects, these obligate symbioses in many cases allow feeding on nutritionally unbalanced diets such as plant sap and vertebrate blood. It is, however, still not clear how are these obligate intracellular symbioses maintained at the cellular level for up to several hundred million years. Exact mechanisms driving host-symbiont interactions are only understood for a handful of model species and data on blood-feeding hosts with intracellular bacteria are particularly scarce.\n\nResultsHere, we analyzed interactions at the symbiotic interfaces of an obligately blood-sucking parasite of sheep, the louse fly Melophagus ovinus. We assembled a reference transcriptome from one male and one female individual and used RNA-Seq with five biological replicates to compare expression in the midgut cells housing bacteria to the rest of the gut (foregut-hindgut). We focused on nutritional and immunity interactions between the insect host and its obligate symbiont Arsenophonus melophagi, and also generated lower-coverage data for three facultative bacterial symbionts (Sodalis melophagi, Bartonella melophagi, and Wolbachia sp.) and one facultative eukaryote Trypanosoma melophagium. We found strong evidence for the importance of zinc in the system likely caused by symbionts using zinc-dependent proteases when acquiring amino acids, and for likely different immunity mechanisms controlling the symbionts than in closely related tsetse flies.\n\nConclusionsOur results show that cellular and nutritional interactions between this blood-sucking insect and its symbionts are less intimate than what was previously found in some (but not all) plant-sap sucking insects such as aphids, psyllids, whiteflies, and mealybugs. This finding is likely interconnected to several features observed in symbionts in blood-sucking arthropods, particularly their midgut intracellular localization (as opposed to being localized in truly specialized bacteriocytes), intracytoplasmic presence (as opposed to having an outermost host-derived symbiosomal membrane), less severe genome reduction, and relatively recent associations caused by frequent evolutionary losses and replacements.\n\nData depositionRaw RNA-Seq data were made available through the European Nucleotide Archive (ENA) database under the study accession number PRJEB30632. All assemblies and additional large supplementary files are available on FigShare [https://doi.org/10.6084/m9.figshare.6146777.v1]. All commands used for data analyses are available on Github [https://github.com/filip-husnik/melophagus].

genomics↗

bioRxiv2018

Comparative Genomics of Staphylococcus Reveals Determinants of Speciation and Diversification of Antimicrobial Defense.

The bacterial genus Staphylococcus comprises diverse species with most being described as colonizers of human and animal skin. A relational analysis of features that discriminate its species and contribute to niche adaptation and survival remains to be fully described. In this study, an interspecies, whole-genome comparative analysis of 21 Staphylococcus species was performed based on their orthologues. Three well-defined multi-species groups were identified: group A (including aureus/epidermidis); group B (including saprophyticus/xylosus) and group C (including pseudintermedius/delphini). The machine learning algorithm Random Forest was applied to identify variable orthologues that drive formation of the Staphylococcus species groups A-C. Orthologues driving staphylococcal infrageneric diversity comprised regulatory, metabolic and antimicrobial resistance proteins. Notably, the BraSR (NsaRS) two-component system (TCS) and its associated BraDE transporters that regulate antimicrobial resistance distinguish group A Staphylococcus species from others in the genus that lack the BraSR TCS. Divergence of BraSR and GraSR antimicrobial peptide survival TCS and their associated transporters was observed across the staphylococci, likely reflecting niche specific evolution of these TCS/transporters and their specificities for AMPs. Experimental evolution, with selection for resistance to the lantibiotic nisin, revealed multiple routes to resistance and differences in the selection outcomes of the BraSR- positive species S. hominis and S. aureus. Selection supported a role for GraSR in nisin survival responses of the BraSR-negative group B species S. saprophyticus. Our study reveals diversification of antimicrobial-sensing TCS across the staphylococci and hints at differential relationships between GraSR and BraSR in those species positive for both TCS.\n\nImportanceThe genus Staphylococcus includes species that are commensals and opportunist pathogens of humans and animals. Identifying the features that discriminate species of staphylococci is relevant to understanding niche selection and the structure of their microbiomes. Moreover, the determinants that structure the community are relevant for strategies to modify the frequency of individual species associated with dysbiosis and disease. In this study, we identify orthologous proteins that discriminate genomes of staphylococci. In particular, species restriction of a major antimicrobial survival system, BraSR (NsaRS), to a group of staphylococci dominated by those that can colonize human skin. The diversity of antimicrobial sensing loci was revealed by comparative analysis and experimental evolution with selection for nisin resistance identified the potential for variation in antimicrobial sensing in BraRS-encoding staphylococci. This study provides insights into staphylococcal species diversity.

microbiology↗