Biology subjects

Marshall, H.

Publications and source records attributed to Marshall, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Stronger positive and purifying selection on X-linked genes in a species with paternal genome elimination

The faster-X effect predicts that X chromosomes may evolve more rapidly than autosomes due to selection on expressed recessive alleles. Quantifying the effect empirically usually requires numerous assumptions due to the impact of diplodiploid transmission dynamics, and so the relative contribution of dominance effects to sex chromosome evolution remains unclear. Springtails present a novel opportunity to ameliorate this. Male globular springtails have hemizygous X chromosomes, diploid autosomes, and eliminate their paternal genome during spermatogenesis. This leads to the autosomes matching the X chromosomes haplodiploid transmission. Hence, theoretically the X chromosomes and autosomes only differ in the fixation probability of male-beneficial alleles. We generated whole-genome resequencing and RNA-Seq data for the globular springtail Allacma fusca, to quantify the faster-X effect, leveraging this natural experiment. We find that after accounting for the deleterious distribution of fitness effects, and despite surprisingly depleted neutral X-linked diversity, the sex chromosomes show signals of both stronger purifying and positive selection, supporting the faster-X effect. Furthermore, we provide the first direct evidence for the loss of DNA methylation in Collembola. These results, obtained from a system where many of the confounding sex-autosome differences that typically obscure faster-X patterns are absent, support hemizygosity as a genuine driver of sex chromosome evolution and suggest that sex-linked genes could contribute disproportionately to adaptive potential.

evolutionary biology↗

bioRxiv2026

Loss of PKN2 drives fibroblast reprogramming and extracellular matrix remodelling in pulmonary fibrosis

Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease characterised by aberrant fibroblast function, extracellular matrix (ECM) remodelling and defective tissue repair. Protein kinase N2 (PKN2) is associated with accelerated forced vital capacity decline in IPF, but its functional role in pulmonary fibrosis remains unknown. We hypothesised that PKN2 regulates fibroblast phenotype and tissue repair. Methods PKN2 expression was assessed in human lung tissue, induced sputum and primary airway and parenchymal fibroblasts from non-fibrotic controls and patients with interstitial lung disease (ILD). DNA methylation was profiled using the Illumina HumanMethylationEPIC array. PKN2 function was investigated by siRNA-mediated depletion in primary human lung fibroblasts using transcriptomic, proteomic and functional analyses. Tissue repair was assessed following pharmacological PKN inhibition in zebrafish. Results PKN2 expression was reduced in ILD lung tissue and primary airway and parenchymal fibroblasts and further suppressed by TGF-{beta}1. Differential methylation was identified across the PKN2 locus in both fibroblast populations. Integrated transcriptomic and proteomic profiling following PKN2 depletion revealed coordinated remodelling of ECM, cell adhesion, non-canonical WNT and VEGF pathways, including dysregulation of COL1A1, WNT, VEGF and MMP1. PKN2 loss increased VEGF and MMP-1 secretion and accelerated fibroblast wound closure. PKN inhibition altered epithelial organisation and collagen fibre alignment during zebrafish wound repair. Conclusion PKN2 loss drives fibroblast reprogramming and aberrant ECM remodelling, establishing PKN2 as an important regulator of pulmonary fibroblast homeostasis and tissue repair.

cell biology↗

bioRxiv2026

A comprehensive atlas of somatic mutation rates and mutational signatures in normal human cells

Over the course of a lifetime, somatic mutations accrue in normal human cells, causing variation in cell phenotype and engendering somatic evolution with outcomes ranging from the adaptive immune system to cancer. To inform understanding of somatic evolution in the human body we report the mutation rates and mutational signatures of 53 normal cell types. Most show evidence of linear mutation accumulation over time with single base substitution mutation rates ranging from ~3.5/year/diploid genome in spermatogonia and sperm, to ~20/year in postmitotic neurons, ~50/year in mitotically active colorectal epithelial cells, ~60/year in kidney proximal tubule cells and hepatocytes, 100s/year in sun-exposed skin epidermal cells and 10-50/year in the remainder. Certain cell types, including skin epidermis, cardiac myocytes, bladder urothelium, kidney proximal tubule cells, and hepatocytes, show substantial variability in mutation burdens around the linear age trend, indicating the influence of additional factors which differ between individuals and modulate mutation accumulation, including exogenous mutagen exposures. At least 18 single-base substitution and nine small insertion and deletion mutational signatures are present, some in all cell types, some in a subset and others in a single cell type. Known exogenous mutagen exposures and endogenous mutational processes account for some mutational signatures, but the origins and mechanisms underlying many are uncertain. This comprehensive survey of mutagenesis provides a foundation for understanding somatic evolution of human cell populations in health and disease.

genomics↗

bioRxiv2026

Glyphosate, a herbicide, and fosfomycin, an antibiotic in clinical use- evidence of common selectable genotypes

The emergence of antimicrobial resistance (AMR) is increasingly linked to metabolic adaptation, yet the evolutionary routes underlying cross-resistance between structurally related compounds remain poorly understood. Here, whole genome sequencing (WGS) was used to analyse Klebsiella pneumoniae mutants evolved under sub-lethal glyphosate (GLP) or fosfomycin (FOS) exposure to determine how these stresses shape resistance and physiology. Sub-lethal GLP exposure increased FOS resistance, demonstrating cross-resistance between the two phosphonates. FOS-evolved mutants achieved high-level resistance through the accumulation of multiple mutations affecting the antibiotic target MurA, transport systems, and global metabolic regulation, producing a layered FOS resistance phenotype. In contrast, GLP-evolved mutants acquired similar functional classes of mutations but exhibited lower baseline FOS resistance, suggesting trade-offs between resistance and metabolic fitness. Further, analysis of FOS-evolved and GLP-evolved mutants across known bacterial GLP resistance mechanisms demonstrated a strong overlap. Comparative genomic analysis revealed a small, recurrent set of genes under selection in both evolutionary trajectories, with identical genomic loci repeatedly targeted, consistent with convergent evolution. Many of these changes were linked to central metabolism, redox balance, and cell surface regulation. For some isolates, a hypermutator phenotype was necessary to offset the potentially lethal effects of primary-target mutations through compensatory genomic adaptation. In conclusion, GLP and FOS select for shared adaptive networks that couple metabolic rewiring with AMR, revealing cross-resistance as an emergent property of global physiological reprogramming and providing mechanistic insight into ecological models of co-selection in environmental systems. Importance statementGlyphosate is a herbicide in current use worldwide. Its impact on the susceptibility of bacteria to antibiotics, remains to be described. This manuscript details a critical phenotypic and genomic analysis of shared resistance mechanisms between glyphosate and the antibiotic fosfomycin. Using Klebsiella pneumoniae, a zoonotic pathogen, this manuscript demonstrates that evolutionary adaptation to either compound results in a substantial overlap in gene mutations. Crucially, this manuscript shows that exposure to sub-lethal concentrations of glyphosate can increase resistance to fosfomycin. These findings reveal a link between agricultural chemical use and the emergence of cross-resistance to fosfomycin. By highlighting how environmental factors drive antimicrobial resistance, this study underscores an urgent need for revision of food safety and regulatory frameworks to protect One Health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/725383v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@d44420org.highwire.dtl.DTLVardef@1c4a5d1org.highwire.dtl.DTLVardef@1624052org.highwire.dtl.DTLVardef@16d08d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

Identification of a miRNA signature for schizophrenia in plasma-derived extracellular vesicles

Background and Hypothesis: Extracellular vesicles (EVs) are phospholipid bilayer vesicles released from cells containing proteins, lipids and nucleic acids derived from the parent cell. Alterations in miRNA expression within blood-derived EVs have been proposed as potential biomarkers of disease. Specifically, identification of differentially expressed miRNAs in patients with schizophrenia (SZ) compared to healthy individuals could be used as a "miRNA signature" to aid in diagnosis and treatment. We therefore aimed to identify differentially expressed miRNAs in plasma-derived EVs between people living with SZ and healthy controls and to correlate miRNA levels with SZ-relevant clinical measures. Study Design: Plasma-derived EVs were isolated from a cohort of 33 individuals with SZ and 34 controls. Expression of 84 miRNAs was examined using a RT-qPCR panel. Study Results: Three miRNAs (hsa-miR-30e-5p, hsa-miR-103a-3p, hsa-miR-200b-3p) were differentially expressed between controls and patients. Gene ontology analysis of putative target genes shared between these miRNAs revealed enrichment of biological process terms related to neurogenesis. Analysis of miRNA expression compared to clinical measures showed that hsa-miR-103a-3p expression was associated with working memory and negatively correlated with white matter integrity in the combined patient-control group. Conclusions: We have identified a miRNA signature for SZ in plasma-derived EVs and shown for the first time that hsa-miR-30e-5p expression is significantly increased in plasma-derived EVs in SZ. The genetic links between differentially expressed miRNAs and neurogenesis, along with the correlations of hsa-miR-103a-3p with working memory and white matter integrity may underlie the functional importance of altered expression of the identified miRNAs in SZ.

genetics↗

bioRxiv2026

Ultra-deep duplex sequencing reveals unique features of somatic evolution in the normal tissues of a family with Li-Fraumeni syndrome

Li-Fraumeni Syndrome (LFS) is caused by germline pathogenic variants in TP53 which predispose carriers to early onset cancer across multiple tissues. While genomically profiling those cancers has revealed factors contributing to their formation, little is understood about how LFS impacts clonal evolution in healthy tissues preceding cancer. Here, we use ultra-deep duplex sequencing (mean [~]15,000x depth) to investigate somatic mutation and selection in a family carrying the germline TP53 p.R181H pathogenic variant and a cohort of non-carrier controls. In blood samples, the germline variant is associated with more mutations in a panel designed to capture genomewide mutagenesis, and with reduced positive selection on somatic TP53 mutations, despite confounding by chemotherapy treatment in one individual. DNMT3A and TET2 mutations appear positively selected and GATA2 mutations negatively selected across the cohort, independent of the p.R181H status. Extensive multi-tissue sampling of 22 non-cancerous and 6 cancerous samples was also performed at autopsy in one individual with LFS who succumbed to esophageal cancer. Cross-tissue analysis reveals excess mutations in sun-exposed skin, esophagus and chronically-inflamed stomach tissue, and concordant mutations in the p.R248 hotspot of TP53 across most (18/28) tissue samples. Most somatic TP53 mutations in LFS that can be assessed for phase arose on the chromosomal copy lacking the p.R181H variant. Our study reveals how the germline p.R181H variant reshapes baseline somatic mutation and selection in normal tissues and highlights the importance of understanding early somatic evolution in LFS prior to cancer development and treatment.

cancer biology↗

bioRxiv2025

A spatiotemporal cancer cell trajectory underlies glioblastoma heterogeneity

Cancer cells display highly heterogeneous and plastic states in glioblastoma, an incurable brain tumour. However, how these malignant states arise and whether they follow defined cellular trajectories across tumours is poorly understood. Here, we generated a deep single cell and spatial multi-omic atlas of human glioblastoma that pairs transcriptomic, epigenomic and genomic profiling of 12 tumours across multiple regions. We identify that glioblastoma heterogeneity is driven by spatially-patterned transitions of cancer cells from developmental-like states towards those defined by a glial injury response and hypoxia. This cellular trajectory regionalises tumours into distinct tissue niches and manifests in a molecularly conserved manner across tumours as well as genetically distinct tumour subclones. Moreover, using a new deep learning framework to map cancer cell states jointly with clones in situ, we show that tumour subclones are finely spatially intermixed through glioblastoma tissue niches. Finally, we show that this cancer cell trajectory is intimately linked to myeloid heterogeneity and unfolds across regionalised myeloid signalling environments. Our findings define a stereotyped trajectory of cancer cells in glioblastoma and unify glioblastoma tumour heterogeneity into a tractable cellular and tissue framework.

cancer biology↗

bioRxiv2025

Clinical isolates of Enterobacter species can persist in human macrophages without bacterial replication and minimal cellular cytotoxicity

BackgroundThe Enterobacter cloacae complex (Ecc) encompasses opportunistic Gram-negative bacteria demonstrating considerable phenotypic and genotypic diversity. Bloodstream, respiratory and urinary tract infections by Ecc bacteria are associated with morbidity and mortality worldwide. These infections are often difficult to treat since Ecc bacteria are resistant to penicillins, quinolones, aminoglycosides, and third-generation cephalosporins. Resistance also extends to carbapenems, leaving only polymyxins, such as colistin, as a last resort antibiotic for treatment. However, colistin resistance in Ecc isolates is also unexpectedly frequent. Despite extensive information on antibiotic resistance by Enterobacter species, much less is known about their infection biology. There are few reports on the survival and persistence of selected Enterobacter species in macrophages and epithelial cells, but how Enterobacter isolates interact with innate immune host cells upon engulfment remains unexplored. In this study, we have investigated the intracellular trafficking of a subset of antimicrobial resistant Ecc clinical isolates, including colistin-resistant strains, within human macrophages, and determined the macrophage response to the intracellular infection. MethodsPhagocytosis of 11 clinical Ecc isolates, including E. cloacae, E. bugandensis, E. kobei, E. xiangfangensis, E. roggenkampii, E. hoffmannii, and E. ludwigii was investigated in THP-1 and human monocyte derived macrophages (HMDMs). Confocal fluorescence microscopy was used to ascertain intracellular trafficking via co-localisation of cell markers with fluorescent bacteria. Intracellular bacterial replication was assessed by bacterial enumeration in cell lysates after killing extracellular bacteria and by a fluorescence dilution approach to follow the synthesis of the bacterial cell wall over time. Macrophage cell cytotoxicity was investigated by quantifying the release of lactate dehydrogenase during infection with all isolates. Two prototypic isolates, the E. cloacae ATCC13047 type strain and the E. bugandensis 104107, were used to explore in more detail the response of macrophages to the intracellular infection by determining cleavage of the proinflammatory markers caspase-1, gasdermin D and pro-interleukin-1{beta}. FindingsWe found that Ecc isolates do not replicate in human macrophages but survive within a modified late phagolysosome compartment. Survival occurred in all species investigated and did not correlate with colistin resistance, lipopolysaccharide modifications, or bacterial pathogenicity in the Galleria mellonella infection model. All isolates induced macrophage cell cytotoxicity at significantly lower levels than controls treated with lipopolysaccharide and nigericin treatment (to induce a proinflammatory response). Low cytotoxicity also correlated with absence of cleavage of proinflammatory markers in infected macrophages. InterpretationEcc species can survive without replication inside human macrophages with minimal effects on cell viability and inflammation. These observations could have implications in the clinical outcome of patients that cannot readily clear infecting Ecc bacteria. This can potentially lead to prolonged intracellular survival and infection relapse. FundingBiotechnology and Biological Sciences Research Council grants BB/T005807/1 and BB/S006281/1. Research In contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed with the terms "Enterobacter" AND "macrophage", "Enterobacter" AND "monocytes", and "Enterobacter" AND "intracellular" for original articles published in English up to July 9, 2024. The search excluded terms "sakazakii" and "aerogenes" since E. aerogenes and E. sakazakii have been moved to the genus Klebsiella and Cronobacter, respectively. Of the 55, 15, and 181 studies we identified, respectively, only one reported testing Enterobacter cloacae phagocytosis. Another study reported intracellular bacterial communities in uroepithelial cells, which serve as a reservoir during urinary tract infection. One other study reported the isolation of E. hormaechei from human atherosclerotic tissue and described studies in THP-1 monocytic macrophages. A few earlier studies also reported Enterobacter cytotoxins affecting immune cells, and an E. cloacae polysaccharide capable of inducing apoptosis in epithelial cells. These studies did not investigate mechanisms and there have been no more recent follow-ups; importantly, it remains unclear if the strains employed in these studies were properly identified as Enterobacter species. Therefore, despite isolated observations of survival of Enterobacter isolates surviving in immune and epithelial cells, there is an overall knowledge gap in our understanding of these pathogens concerning intracellular survival compartments, kinetics of survival, and induction of macrophage cytotoxicity and inflammatory responses. Added value of this studyOur study is the first to investigate in detail how clinical isolates of various Enterobacter species can survive intracellularly in human macrophages. All isolates display multidrug antimicrobial resistance, including some with colistin resistance, and can survive intracellularly in human macrophages. Our data demonstrate that intracellular Enterobacter resides in vacuoles for up to 44 hours without replication. Colocalization experiments with various fluid phase and membrane cellular markers revealed that bacteria-containing vacuoles are modified late phagolysosomes, which do not accumulate the autophagosome marker LC3B. Intracellular bacterial survival did not associate to any Enterobacter species tested, the presence of colistin resistance, lipopolysaccharide modifications, or virulence in the Galleria mellonella infection model. Moreover, intracellular infection caused minimal cytotoxicity in macrophages without evidence of macrophage proinflammatory cell death by pyroptosis. Implications of all the available evidenceOur findings underscore the capacity of Enterobacter species, traditionally viewed as extracellular bacteria, to hideout in macrophages without inducing a significant inflammatory response. These properties may further complicate the treatment of antibiotic-resistant Enterobacter infections in susceptible populations such as the elderly and neonates. These findings open a door to the development of host-directed therapeutics to enhance bacterial clearance by macrophage-mediated killing.

microbiology↗

bioRxiv2024

Paternal genome elimination creates contrasting evolutionary trajectories in male and female citrus mealybugs

Most studies of sex-biased genes explore their evolution in familiar chromosomal sex determination systems, leaving the evolution of sex differences under alternative reproductive systems unknown. Here we explore the system of paternal genome elimination employed by mealybugs (Hempitera: Pseudococcidae) which have no sex chromosomes. Instead, all chromosomes are autosomal and inherited in two copies, but sex is determined by the ploidy of expression. Females express both parental alleles, but males reliably silence their paternally inherited chromosomes, creating genome-wide haploid expression in males and diploid expression in females. Additionally, sons do not express alleles directly inherited from their fathers, potentially disrupting the evolution of male-benefitting traits. To understand how these dynamics impact molecular evolution, we generated sex-specific RNAseq, a new gene annotation, and whole-genome population sequencing of the citrus mealybug, Planococcus citri. We found that genes expressed primarily in females hold more variation and evolve more quickly than those expressed in males or both sexes. Conversely, more adaptation occurs in genes expressed mainly in males than those expressed in females. Put together, paternal genome elimination appears to slow change on the male side but, by increasing selective scrutiny, increase the amount of adaptation in these genes. These results expand our understanding of evolution in a non-mendelian genetic system and the data we generated should prove useful for future research on this pest insect.

evolutionary biology↗

bioRxiv2024

Molecular correlates of swarming behaviour in Aedes aegypti males.

Mosquitoes are the deadliest vectors of disease. They impose a huge health burden on human populations spreading parasites as disparate as protozoans (malaria), viruses (yellow fever and more), and nematodes (filariasis), which cause life-threatening conditions. In recent years, mating has been proposed as a putative target for population control. Mosquitoes mate mid-air in swarms initiated by males and triggered by a combination of internal and external stimuli. As the number of females in a swarm is limited, there is intense competition among males. Indeed, males retune their physiology for this demanding behaviour but the underlying genetic changes are largely unknown. Interestingly, recent evidence indicates that the upregulation of circadian clock genes may be involved in the swarming of malaria mosquitoes of the genus Anopheles. Here, we use whole-head RNA-seq to identify gene expression changes in Aedes aegypti males who are engaged in swarming in a laboratory setting. Our results suggest that swarming males tend to lower some housekeeping functions while increasing the remodelling of the cytoskeleton and neuronal connectivity; the transcription of circadian clock genes is unaffected.

genetics↗

bioRxiv2023

A role for DNA methylation in bumblebee morphogenesis hints at female-specific developmental erasure.

Epigenetic mechanisms, such as DNA methylation, are crucial factors in animal development. In some mammals almost all DNA methylation is erased during embryo development and re-established in a sex- and cell-specific manner. This erasure and re-establishment is thought to primarily be a vertebrate-specific trait. Insects are particularly interesting in terms of development as many species often undergo remarkable morphological changes en route to maturity, i.e. morphogenesis. However, little is known about the role of epigenetic mechanisms in this process across species. We have used whole genome bisulfite sequencing to track genome-wide DNA methylation changes through the development of an economically and environmentally important pollinator species, the bumblebee Bombus terrestris. We find overall levels of DNA methylation vary throughout development and we find developmentally relevant differentially methylated genes throughout. Intriguingly, we have identified a depletion of DNA methylation in ovaries and an enrichment in sperm. We suggest this could represent a sex-specific DNA methylation erasure event. To our knowledge this is the first suggestion of possible developmental DNA methylation erasure in an insect species. This study lays the required groundwork for functional experimental work to determine if there is a causal-nature to the DNA methylation differences identified. Additionally, the application of single-cell methylation sequencing to this system will enable more accurate identification of when/if DNA methylation is erased during development.

developmental biology↗

bioRxiv2023

Lack of paternal silencing and ecotype-specific expression in head and body lice hybrids.

Paternal genome elimination (PGE) is a non-Mendelian inheritance system in which males develop from fertilised eggs but their paternally-inherited chromosomes are eliminated before or during spermatogenesis. Therefore, PGE males only transmit their maternally inherited set of chromosomes to their offspring. PGE has been described in numerous arthropod species, many of which are pests or parasites, posing a severe economic burden on crop production and/or with implications for human health. In order to understand how PGE has evolved on the molecular level, to potentially develop novel control strategies, we need to examine species which display basal forms of PGE. The human louse, Pediculus humanus, represents an ideal model system to understand the molecular underpinnings of PGE. In this study we analysed parent-of-origin allele specific expression patterns in male offspring of crosses between head and body lice ecotypes. We have shown that hybrid adult males of P. humanus display biparental gene expression, which constitutes the first known case of a species with PGE in which genetic activity of paternal chromosomes in the soma is not affected by embryonic heterochromatinization or (partial or complete) elimination. We have also identified maternally-biased genes (potentially imprinted genes) which may be involved in the elimination of paternal chromosomes during spermatogenesis. Finally, we have identified genes which show ecotype-specific expression bias. Given the low genetic diversity between ecotypes this is suggestive for a role of epigenetic processes in ecotype differences. These findings have implications for models of pediculicide resistance in human lice and for the development of novel epigenetic-mediated control strategies.

evolutionary biology↗

bioRxiv2022

Sex-specific transcription and DNA methylation landscapes of the Asian citrus psyllid, a vector of huanglongbing pathogens.

The relationship of DNA methylation and sex-biased gene expression is of high interest, it allows research into mechanisms of sexual dimorphism and the development of potential novel strategies for insect pest control. The Asian citrus psyllid, Diaphorina citri Kuwayama, is a major vector for the causative agents of Huanglongbing (HLB), which presents an unparalleled challenge to citrus production worldwide. Here, we identify the X chromosome of D. citri and investigate differences in the transcription and DNA methylation landscapes between adult virgin males and females. We find a large number of male-biased genes on the autosomes and a depletion of such on the X chromosome. We have also characterised the methylome of D. citri, finding low genome-wide levels, which is unusual for an hemipteran species, as well as evidence for both promoter and TE methylation. Overall, DNA methylation profiles are similar between the sexes but with a small number of differentially methylated genes found to be involved in sex differentiation. There also appears to be no direct relationship between differential DNA methylation and differential gene expression. Our findings lay the groundwork for the development of novel epigenetic-based pest control methods, and given the similarity of the D. citri methylome to other insect species, these methods could be applicable across agricultural insect pests.

evolutionary biology↗

bioRxiv2021

Parent of origin DNA methylation as a potential mechanism for genomic imprinting in bees.

Social insects display extreme phenotypic differences between sexes and castes even though the underlying genome can be almost identical. Epigenetic processes have been proposed as a possible mechanism for mediating these phenotypic differences. Using whole genome bisulfite sequencing of queens, males and reproductive female workers we have characterised the sex- and caste-specific methylome of the bumblebee Bombus terrestris. We have identified a potential role for DNA methylation in histone modification processes which may influence sex and caste phenotypic differences. We also find differentially methylated genes generally show low levels of DNA methylation which may suggest a separate function for lowly methylated genes in mediating transcriptional plasticity. Unlike highly methylated genes which are usually involved in housekeeping functions. We also examined the relationship between the underlying genome and the methylome using whole genome re-sequencing of the same queens and males. We find DNA methylation is enriched at zero-fold degenerate sites. We suggest DNA methylation may be acting as a mutagen at these sites thereby providing substrate for selection via changes in gene transcription mediated by the underlying genotype. However, we did not see any relationship between DNA methylation and rates of positive selection in our samples. In order to fully assess a possible role for DNA methylation in adaptive processes a specifically designed study using natural population data is needed.

evolutionary biology↗

bioRxiv2020

Sex-specific expression and DNA methylation in a species with extreme sexual dimorphism and paternal genome elimination

Sexual dimorphism is exhibited in many species across the tree of life with many phenotypic differences mediated by differential expression and alternative splicing of genes present in both sexes. However, the mechanisms that regulate these sex-specific expression and splicing patterns remain poorly understood. The mealybug, Planococcus citri, displays extreme sexual dimorphism and exhibits an unusual instance of sex-specific genomic imprinting, Paternal Genome Elimination (PGE), in which the paternal chromosomes in males are highly condensed and eliminated from the sperm. P. citri also has no sex chromosomes and as such both sexual dimorphism and PGE are predicted to be under epigenetic control. We recently showed that P. citri females display a highly unusual DNA methylation profile for an insect species, with the presence of promoter methylation associated with lower levels of gene expression. In this study we therefore decided to explore genome-wide differences in DNA methylation between male and female P. citri using whole genome bisulfite sequencing. We have identified extreme differences in genome-wide levels and patterns between the sexes. Males display overall higher levels of DNA methylation which manifests as more uniform low-levels across the genome. Whereas females display more targeted high levels of methylation. We suggest these unique sex-specific differences are due to chromosomal differences caused by PGE and may be linked to possible ploidy compensation. Using RNA-Seq we identified extensive sex-specific gene expression and alternative splicing. We found cis-acting DNA methylation is not directly associated with differentially expressed or differentially spliced genes, indicating a broader role for chromosome-wide trans-acting DNA methylation in this species.

evolutionary biology↗

bioRxiv2020

Bumblebee worker castes show differences in allele-specific DNA methylation and allele-specific expression.

Allele-specific expression is when one allele of a gene shows higher levels of expression compared to the other allele, in a diploid organism. Genomic imprinting is an extreme example of this, where some genes exhibit allele-specific expression in a parent-of-origin manner. Recent work has identified potentially imprinted genes in species of Hymenoptera. However, the molecular mechanism which drives this allelic expression bias remains unknown. In mammals DNA methylation is often associated with imprinted genes. DNA methylation systems have been described in species of Hymenoptera, providing a candidate imprinting mechanism. Using previously generated RNA-Seq and whole genome bisulfite sequencing from reproductive and sterile bumblebee (Bombus terrestris) workers we have identified genome-wide allele-specific expression and allele-specific DNA methylation. The majority of genes displaying allele-specific expression are common between reproductive castes and the proportion of allele-specific expression bias generally varies between colonies. We have also identified genome-wide allele-specific DNA methylation patterns in both castes. There is no significant overlap between genes showing allele-specific expression and allele-specific methylation. These results indicate that DNA methylation does not directly drive genome-wide allele-specific expression in this species. Only a small number of the genes identified may be imprinted and it may be these genes which are associated with allele-specific DNA methylation. Future work utilising reciprocal crosses to identify parent-of-origin DNA methylation will further clarify the role of DNA methylation in parent-of-origin allele-specific expression.

evolutionary biology↗

bioRxiv2020

Genome-wide search for parent-of-origin allele specific expression in Bombus terrestris.

Genomic imprinting is the differential expression of alleles in diploid individuals, with the expression being dependent upon the sex of the parent from which it was inherited. Haigs kinship theory hypothesizes that genomic imprinting is due to an evolutionary conflict of interest between alleles from the mother and father. In social insects, it has been suggested that genomic imprinting should be widespread. One recent study identified parent-of-origin expression in honeybees and found evidence supporting the kinship theory. However, little is known about genomic imprinting in insects and multiple theoretical predictions must be tested to avoid single-study confirmation bias. We, therefore, tested for parent-of-origin expression in a primitively eusocial bee. We found equal numbers of maternally and paternally biased expressed alleles. The most highly biased alleles were maternally expressed, offering support for the kinship theory. We also found low conservation of potentially imprinted genes with the honeybee, suggesting rapid evolution of genomic imprinting in Hymenoptera. Impact summaryGenomic imprinting is the differential expression of alleles in diploid individuals, with the expression being dependent upon the sex of the parent from which it was inherited. Genomic imprinting is an evolutionary paradox. Natural selection is expected to favour expression of both alleles in order to protect against recessive mutations that render a gene ineffective. What then is the benefit of silencing one copy of a gene, making the organism functionally haploid at that locus? Several explanations for the evolution of genomic imprinting have been proposed. Haigs kinship theory is the most developed and best supported. Haigs theory is based on the fact that maternally (matrigene) and paternally (patrigene) inherited genes in the same organism can have different interests. For example, in a species with multiple paternity, a patrigene has a lower probability of being present in siblings that are progeny of the same mother than does a matrigene. As a result, a patrigene will be selected to value the survival of the organism it is in more highly, compared to the survival of siblings. This is not the case for a matrigene. Kinship theory is central to our evolutionary understanding of imprinting effects in human health and plant breeding. Despite this, it still lacks a robust, independent test. Colonies of social bees consist of diploid females (queens and workers) and haploid males created from unfertilised eggs. This along with their social structures allows for novel predictions of Haigs theory. In this paper, we find parent of origin allele specific expression in the important pollinator, the buff-tailed bumblebee. We also find, as predicted by Haigs theory, a balanced number of genes showing matrigenic or patrigenic bias with the most extreme bias been found in matrigenically biased genes.

evolutionary biology↗

bioRxiv2019

Investigating the biochemical properties of Streptococcal polysaccharide capsules using atomic force microscopy

In common with many bacterial pathogens, Streptococcus pneumoniae has a polysaccharide capsule, which facilitates immune evasion and is a key virulence determinant. However, recent data has shown that the closely related Streptococcus mitis can also express polysaccharide capsules including those with an identical chemical structure to S. pneumoniae capsular serotypes. We have used atomic force microscopy (AFM) techniques to investigate the biophysical properties of S. mitis and S. pneumoniae strains expressing the same capsular serotypes that might relate to their differences in virulence potential. When comparing S. mitis and S. pneumoniae strains with identical capsule serotypes S. mitis strains were more susceptible to neutrophil killing and imaging using electron microscopy and AFM demonstrated significant morphological differences. Force-volume mapping using AFM showed distinct force-curve profiles for the centre and edge areas of encapsulated S. pneumoniae and S. mitis strains. This \"edge effect\" was not observed in the unencapsulated streptococcal strains and in an unencapsulated Staphylococcus aureus strain, and therefore was a direct representation of the mechanical properties of the bacterial capsule. When two strains of S. mitis and S. pneumoniae expressed an identical capsular serotype, they presented also similar biomechanical characteristics. This would infer a potential relationship between capsule biochemistry and nanomechanics, independent of the bacterial strains. Overall, AFM was an effective tool to explore the biophysical properties of bacterial capsules of living bacteria by reproducibly quantifying the elastic and adhesive properties of bacterial cell surfaces. Using AFM to investigate capsule differences over a wider range of strains and capsular serotypes of streptococci and correlate the data with phenotypic differences will elucidate how the biophysical properties of the capsule can influence its biological role during infection.

microbiology↗