Biology subjects

Hughes, G.

Publications and source records attributed to Hughes, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Impact of thermal variation on the transcriptome of Plasmodium falciparum infected Anopheles stephensi

Temperature is a key determinant of malaria transmission, influencing both parasite development and mosquito physiology, yet the underlying mechanisms remain poorly understood. Here, we examined how temperature and time modulate gene expression in Anopheles stephensi infected with Plasmodium falciparum. Using RNA-sequencing over 1-19 days post-blood meal and three temperature regimes (20, 24, and 28{degrees}C with diurnal fluctuations of 9{degrees}C), we characterize transcriptome responses to infection with P. falciparum at the site of infection in the midgut, and systemically, in the carcasses. Oocyst prevalence and density declined over the thermal gradient, albeit with distinct, non-linear temporal dynamics in parasite development rates. Although infection contributed minimally to global variation in gene expression relative to temperature and time, infection-associated genes in the midgut showed coordinated transcriptional responses enriched in canonical Plasmodium associated extracellular, proteolytic, immune, and metabolic functions; notably, decline in oocyst infections in the midguts over the thermal gradient was reflected in reduced expression of immune genes known to regulate P. falciparum. Network analysis demonstrated that these genes participate in a significantly interconnected protein-protein interaction network, within which a small number of high betweenness centrality proteins act as bottlenecks linking immune, metabolic, reproductive and behavioral processes. Our results suggest responses to infection may be mediated through coordinated physiological networks rather than large-scale transcriptional changes. Our findings also indicate that differences in thermal conditions may be an important factor when comparing mechanisms of vector- parasite interactions between Plasmodium species. Together, our results highlight the importance of integrating thermal context into mechanistic studies of vector-parasite interactions.

ecology↗

bioRxiv2026

β-hydroxybutyrate modulates enteric pathogen susceptibility through regulation of commensal bacteria and intestinal Th17 responses

T helper 17 (Th17) cells are a critical T lymphocyte subset involved in mucosal immunity and host defense against enteric pathogens. Although ketogenic diets (KD) and the major ketone body {beta}-hydroxybutyrate (BHB) reshape gut microbiota and suppress Th17 responses under defined diet conditions, it remains unclear whether elevation of BHB alone, independent of dietary macronutrient composition and systematic metabolic shift, is sufficient to remodel Th17-inducing commensals and alter host susceptibility to enteric infection. Here, we used 1,3-butanediol (BD), a precursor metabolized to BHB independently of KD, to elevate systemic BHB levels in mice. BD treatment significantly reduced the frequency of ileal Th17 cells, as assessed by flow cytometry for Th17 markers IL-17A and ROR{gamma}t. 16S rRNA gene sequencing revealed that BD altered gut microbial community structure, as indicated by beta-diversity analysis based on Bray-Curtis dissimilarity, and reduced Shannon diversity and evenness. Linear discriminant analysis effect size identified segmented filamentous bacteria (SFB) as significantly decreased in the ileum following BD treatment, and SFB abundance positively correlated with Th17 markers. Microbiota transplantation demonstrated that BD-shaped microbiota was sufficient to suppress Th17 responses in recipient mice, accompanied by reduced SFB abundance. In a Citrobacter rodentium infection model, BD treatment was associated with increased pathogen burden, and fecal C. rodentium levels were negatively correlated with SFB abundance. Together, these results indicate that BD-induced elevation of BHB reshapes commensal microbiota, including decreasing SFB levels, resulting in dampened Th17 responses and increased susceptibility to enteric infection. IMPORTANCEDiet is a key determinant of gut microbial composition and mucosal immune function, yet the microbial mechanisms linking how diet-mediated changes to metabolism regulate immune responses remain incompletely understood. Th17 cells play central roles in both protective mucosal immunity and inflammatory pathology, making them a critical target of immunometabolic regulation. In this study, we show that {beta}-hydroxybutyrate (BHB), generated independently of diet, suppresses intestinal Th17 responses by reshaping the gut microbiota, reducing SFB levels, a potent Th17-inducing murine commensal. We further demonstrate that BHB-associated microbiota changes are linked to increased susceptibility to enteric infection. This work provides a mechanistic framework illustrating how metabolic state can influence host immunity through selective effects on commensal microbes. These findings inform future studies of microbiota-mediated immune regulation.

microbiology↗

bioRxiv2025

Vertical transmission of mosquito microbiota and its effects on offspring development

BackgroundThe mosquitos gut microbiota plays a crucial role in determining its capacity to transmit harmful viruses and parasites. Accordingly, manipulating mosquito gut microbiota is a promising avenue towards reducing mosquito-borne human pathogen transmission. A successful microbial control campaign will require a thorough understanding of how bacteria are transmitted through mosquito populations. Through two parallel but complementary studies using the yellow fever mosquito Aedes aegypti as a focal host species, we surveyed vertical transmission of bacteria from individual mothers to cohorts of offspring maintained in a closed system. ResultsLaboratory- and field-derived mothers deposited bacteria that support offspring development, and the relative abundance of commonly transmitted taxa correlated with offspring fitness. Maternally transmitted bacteria were detected in both larval and adult offspring, and the relative abundances of specific taxa differed between life stages. Microbiota composition in adult offspring closely resembled microbiota composition in mothers, despite dramatic shifts in the relative abundance of specific microbial community members during the larval stage. Variability in microbiota composition in offspring was also greater than variability across the population of egg-laying mothers. Eggs that underwent a period of desiccation before hatching produced larval communities dominated by endospore-forming bacteria that were rare in maternal samples. ConclusionsOverall, our results demonstrate the vertical transmission of mosquito-associated microbiota across generations, including bacterial taxa that could potentially be leveraged for mosquito and mosquito-borne disease control.

microbiology↗

bioRxiv2025

Neural population dynamics of direct electrical stimulation of neocortex

Intracranial electrical stimulation is foundational in neuroscience and clinical neuromodulation, yet how applied electrical fields engage neural tissue and influences perception remains unclear. Using three orthogonal Neuropixels probes in mouse visual areas, we recorded extracellular voltages and spikes with sub-millisecond resolution in three dimensions around a stimulation source. The evoked potential extended asymmetrically and grew sub-linearly with amplitude, influenced by anatomical heterogeneity. Increasing amplitude increased the density, but not spatial extent, of directly responsive neurons, but fewer than 5% of neurons within the evoked potential were activated. Fast-spiking interneurons were recruited nearer the source, whereas pyramidal neurons showed anisotropic activation. Direct responses were polarity- and amplitude-selective. Sparse direct spiking drove large, asymmetric cortical responses and modulated >50% of neurons at higher amplitudes. Perceptually, mice weakly detected single pulses, and performance did not improve with amplitude. Thus, anatomy and spatiotemporal patterning--not spike counts--govern physiological and perceptual impact, informing biomimetic, multi-contact prostheses.

neuroscience↗

bioRxiv2025

Gap scheduling of a PARP inhibitor and nanoparticle TOP1 agent combination avoids synergistic bone marrow toxicity

Although combinations of DNA damage response inhibitors (DDRi) and DNA damaging chemotherapy enhance cytotoxicity in cell-based systems, clinical success has been limited by overlapping bone marrow toxicities. Here, we show that a tumor-targeted nanoparticle camptothecin CRLX101, administered concurrently with DDRi, enhances anti-tumour efficacy but also increases bone marrow toxicity in preclinical models. Using rat bone marrow progenitor cells as biomarkers and leveraging differential repair kinetics of CRLX101-induced DNA damage in tumour and bone marrow, we identified a gap schedule of the PARP inhibitor olaparib and CRLX101 that enhanced efficacy over single agents but demonstrated a reduced combination marrow toxicity. A clinical trial has been designed using the gap schedule identified here and represents a template that can be used to successfully deliver DDRi with tumor-targeted chemotherapy in combination.

cancer biology↗

bioRxiv2025

Impact of temperature on vector competence of Culex pipiens molestus: implications for Usutu virus transmission in temperate regions

IntroductionUsutu virus (USUV) has been detected annually in the southeast of England since 2020. USUV RNA has been identified in wild birds and mosquito populations, and exposure of captive birds to USUV at Zoological Society of London (ZSL) has also been confirmed. Since its first detection in London, USUVs distribution has expanded across the South East, highlighting a need to understand the transmission dynamics of this virus in the UK. The primary vectors of USUV in the UK are likely Culex pipiens mosquitoes. Two biotypes have been identified, the bird-feeding Cx. pipiens pipiens and Cx. pipiens molestus which shows no restriction in host preference. The latter may play an important role in transmitting USUV from birds to humans. MethodsA laboratory colony of Cx. pipiens molestus mosquitoes were orally infected with the London strain of USUV and, incubated at 22 {degrees}C, 20 {degrees}C and 18{degrees} C for up to 28 days. Body samples and mosquito saliva samples were collected and analysed using a quantitative real-time reverse transcription PCR to determine infection and transmission potential, respectively. ResultsUSUV RNA was detected in all sample times at all temperatures assessed; with the highest temperature (22 {degrees}C) showing the greatest proportion of saliva and body positive samples. At this temperature, there was also an eight-fold increase in the relative viral copy number in the mosquito bodies, which was unobserved at other experimental temperatures. When a more sensitive PCR assay was used at the lowest experimental temperature used (18 {degrees}C) USUV RNA was present in the mosquito saliva and body samples for longer and showed a greater proportion of positive samples when compared to 20 {degrees}C. ConclusionThis study has demonstrated that Cx. pipiens molestus may be able to transmit USUV at 22 {degrees}C. Active replication of USUV was identified in the mosquito bodies at 22 {degrees}C but could not be demonstrated at lower temperatures, suggesting that 20 {degrees}C to 22 {degrees}C may be an important temperature threshold in USUV replication and transmission. Utilisation of a more sensitive assay for the lower experimental temperatures revealed that USUV was detectable at 18 {degrees}C. Therefore, when conducting infection studies on temperate mosquito-borne viruses, it is important to consider assay sensitivity.

molecular biology↗

bioRxiv2024

SARS-CoV-2 Survival on Skin and its Transfer from Contaminated Surfaces

Understanding the transmission dynamics of SARS-CoV-2, particularly its transfer from contaminated surfaces (fomites) to human skin, is crucial for mitigating the spread of COVID-19. While extensive research has examined the persistence of SARS-CoV-2 on various surfaces, there is limited understanding of how efficiently it transfers to human skin, and how long it survives on the skin. This study investigates two key aspects of SARS-CoV-2 transmission: (1) the transfer efficiency of SARS-CoV-2 from non-porous (plastic and metal) and porous (cardboard) surfaces to a 3D human skin model (LabSkin), and (2) the survival of SARS-CoV-2 on the skin under different temperature conditions. First, we validated LabSkin as a suitable surrogate for human skin by comparing the transfer efficiency of the bacteriophage Phi 6 from surfaces to LabSkin and to human volunteers fingers. No significant differences were observed, confirming LabSkins suitability for these studies. Subsequently, the transfer of SARS-CoV-2 from surfaces to LabSkin was assessed, showing that plastic and metal surfaces had similar transfer efficiencies ([~]13%), while no transfer occurred from cardboard once the inoculum had dried on the surface. Finally, the survival of SARS-CoV-2 on skin was assessed, showing a rapid decay at higher temperatures, with a half-life ranging from 2.8 to 17.8 hours depending on the temperature. These findings enhance our understanding of viral transmission via fomites and inform public health strategies to reduce the risk of SARS-CoV-2 transmission through surface contact.

microbiology↗

bioRxiv2024

Mosquitoes reared in distinct insectaries within an institution in close spatial proximity possess significantly divergent microbiome

The microbiome affects important aspects of mosquito biology and differences in microbial composition can affect the outcomes of laboratory studies. To determine how the biotic and abiotic conditions in an insectary affect the composition of the bacterial microbiome of mosquitoes we reared mosquitoes from a single cohort of eggs from one genetically homogeneous inbred Aedes aegypti colony, which were split into three batches, and transferred to each of three different insectaries located within the Liverpool School of Tropical Medicine. Using three replicate trays per insectary, we assessed and compared the bacterial microbiome composition as mosquitoes developed from these eggs. We also characterised the microbiome of the mosquitoes food sources, measured environmental conditions over time in each climate-controlled insectary, and recorded development and survival of mosquitoes. While mosquito development was overall similar between all three insectaries, we saw differences in microbiome composition between mosquitoes from each insectary. Furthermore, bacterial input via food sources, potentially followed by selective pressure of temperature stability and range, did affect the microbiome composition. At both adult and larval stages, specific members of the mosquito microbiome were associated with particular insectaries; and the insectary with less stable and cooler conditions resulted in slower pupation rate and higher diversity of the larval microbiome. Tray and cage effects were also seen in all insectaries, with different bacterial taxa implicated between insectaries. These results highlight the necessity of considering the variability and effects of different microbiome composition even in experiments carried out in a laboratory environment starting with eggs from one batch; and highlights the impact of even minor inconsistencies in rearing conditions due to variation of temperature and humidity.

microbiology↗

bioRxiv2024

Data-driven modeling of E. coli transcriptional regulation

The growth of bacterial gene expression datasets has offered unprecedented coverage of achievable transcriptomes, reflecting diverse activity states of the transcription regulatory network. Machine learning methods like Independent Component Analysis (ICA) can decompose gene expression datasets into regulatory modules and condition-specific regulator activities. Here, we present a workflow to utilize inferred regulator activities to construct quantitative models of promoter regulation in E. coli. Resulting models are validated by predicting condition-specific TF effector concentrations and binding site motif strength based on differential gene expression data alone. We show how reconstructed promoter models can capture multi-scale regulation and disentangle regulator interactions, including resolving the apparent paradox where argR expression is positively correlated with its regulon despite being a repressor. We applied the workflow for all regulator-linked components extracted by ICA, demonstrating the scalability of the workflow to capture the E. coli TRN. This work suggests a path toward systematic, quantitative reconstruction of transcription regulatory networks driven by the large-scale databases that are now available for many organisms.

bioinformatics↗

bioRxiv2024

Contrasting patterns of Asaia sp association with Plasmodium falciparum between field collected Anopheles gambiae and Anopheles coluzzii from Cameroon

The presence of bacteria of the genus Asaia in mosquitoes makes them suitable candidates for malaria paratrangenic control. To better understand whether this bacterium could be used for malaria control, we investigated and quantified Asaia influence in An.Lgambiae sl populations naturally infected by the malaria parasite Plasmodium falciparum. Adult Anopheles mosquitoes were collected across two different eco-geographical localities in Cameroon, during the dry and wet seasons. DNA was extracted from the whole individual mosquitoes and Asaia was quantified using real-time Q-PCR by amplification of 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in same mosquitoes. The density of Asaia was quantified in 864 mosquitoes. This included 439 An. gambiae collected from Bankeng and 425 An. coluzzii collected from Gounougou. The prevalence of Asaia in An. gambiae and An. coluzzii was 88.3% and 80.9% respectively, with An. gambiae were more heavily infected by Asaia than An. coluzzii. There was also a significant difference between the infection densities of Asaia per season in the two localities. We also had a significant difference between the infection densities of Asaia according to Plasmodium infection status in the two localities. However, no correlation was observed between the amount of Asaia and P. falciparum infection. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii. Further studies as experimental infection and innate immune response are required to better investigate the correlation between Anopheles mosquitoes, Asaia and Plasmodium. ImportanceMalaria control relies mainly on insecticide-based tools. However, the effectiveness of these tools is threatened by the widespread insecticide resistance in malaria vectors highlighting the need for alternative control approaches. The endosymbiont Asaia has emerged as a promising candidate for paratransgenic control of malaria, but its biology and genetics still need to be further analyzed across Africa. Here, we investigated and quantified Asaia influence in An.Lgambiae sl populations naturally infected by the malaria parasite Plasmodium falciparum. DNA was extracted from the whole individual mosquitoes collected from two localities, and then Asaia was quantified using real-time Q-PCR by amplification of 16S ribosomal RNA gene. We also detected and quantified Plasmodium falciparum infection in same mosquitoes and correlated Plasmodium infection them with the presence of Asaia infection. This study provides evidence that naturally occurring Asaia infection is not correlated to P. falciparum development within An. gambiae and An. coluzzii.

microbiology↗

bioRxiv2023

Interkingdom interactions shape the fungal microbiome of mosquitoes

The mosquito microbiome is an important modulator of vector competence and vectoral capacity. Unlike the extensively studied bacterial microbiome, fungal communities in the mosquito microbiome (mycobiome) remain largely unexplored. To work towards getting an improved understanding of the fungi associated with mosquitoes, we sequenced the mycobiome of three field-collected and laboratory-reared mosquito species (Aedes albopictus, Aedes aegypti, and Culex quinquefasciatus). Our analysis showed both environment and host species were contributing to the diversity of the fungal microbiome of mosquitoes. When comparing species, Ae. albopictus possessed a higher number of diverse fungal taxa than Cx. quinquefasciatus, while strikingly less than 1% of reads from Ae. aegypti samples were fungal. Fungal reads from Ae. aegypti were <1% even after inhibiting host amplification using a PNA blocker, indicating that this species lacked a significant fungal microbiome that was amplified using this sequencing approach. Using a mono-association mosquito infection model, we confirmed that mosquito-derived fungal isolates colonize and for Aedes mosquitoes, support growth and development at comparable rates to their bacterial counterparts. Strikingly, native bacterial taxa isolated from mosquitoes impeded the colonization of symbiotic fungi in Ae. aegypti suggesting interkingdom interactions shape fungal microbiome communities. Collectively, this study adds to our understanding of the fungal microbiome of different mosquito species, that these fungal microbes support growth and development, and highlights that microbial interactions underpin fungal colonization of these medically relevent species.

microbiology↗

bioRxiv2023

Microbiome variability of mosquito lines is consistent over time and across environments

The composition of the microbiome is shaped by both environment and host genetic background in most organisms, but in the mosquito Aedes aegypti the role of host genetics in shaping the microbiome is poorly understood. Previously, we had shown that four lines of Ae. aegypti harbored different microbiomes when reared in the same insectary under identical conditions. To determine whether these lines differed from each other across time and in different environments, we characterized the microbiome of the same four lines of Ae. aegypti reared in the original insectary and at another institution. While it was clear that the environment influenced the microbiomes of these lines, we did still observe distinct differences in the microbiome between lines within each insectary. Clear differences were observed in alpha diversity, beta diversity, and abundance of specific bacterial taxa. To determine if the line specific differences in the microbiome were maintained across environments, pair-wise differential abundances of taxa was compared between insectaries. Lines were most similar to other lines from the same insectary than to the same line reared in a different insectary. Additionally, relatively few differentially abundant taxa identified between pairs of lines were shared across insectaries, indicating that line specific properties of the microbiome are not conserved across environments, or that there were distinct microbiota within each insectary. Overall, these results demonstrate that mosquito line can shape the microbiome across microbially- diverse environments and host by microbe interactions affecting microbiome composition and abundance is dependent on environmentally available bacteria. Author SummaryThe mosquito microbiome plays a critical role in shaping interactions with human pathogens. The factors that contribute to shaping the composition of the mosquito microbiome are of high importance due to its role in pathogen interactions and the successful development of control strategies. In other organisms, both host genetics and environment shape the microbiome composition, but the role of host genetics in shaping the mosquito microbiome is less clear. Previously, we have shown that different lines of Aedes aegypti harbor different microbiomes when reared in the same environment. We were curious to see if these differences could still be detected after further generations in the same insectary and across environments in a different insectary. We found that found that the microbiome differed between these lines in each insectary indicating an element of both host genetic background and environment play a role in establishing the microbiome. Our results indicate that different genetic backgrounds of Ae. aegypti will interact with their environment differently to shape their microbiome, which could potentially influence interactions with human pathogens and/or the effectiveness of control strategies. More broadly, our results are of interest for the ecology of host-microbe interactions.

microbiology↗

bioRxiv2023

Aedes aegypti gut transcriptomes respond differently to microbiome transplants from field-caught or laboratory-reared mosquitoes.

The mosquito microbiome is critical for host development and plays a major role in many aspects of mosquito biology. While the microbiome is commonly dominated by a small number of genera, there is considerable variation in composition among mosquito species, life stages, and geography. How the host controls and is affected by this variation is unclear. Using microbiome transplant experiments, we asked whether there were differences in transcriptional responses when mosquitoes of different species were used as microbiome donors. We used microbiomes from four different donor species spanning the phylogenetic breadth of the Culicidae, collected either from the laboratory or field. We found that when recipients received a microbiome from a donor reared in the laboratory, the response was remarkably similar regardless of donor species. However, when the donor had been collected from the field, far more genes were differentially expressed. We also found that while the transplant procedure did have some effect on the host transcriptome, this is likely to have had a limited effect on mosquito fitness. Overall, our results highlight the possibility that variation in mosquito microbiome communities are associated with variability in host-microbiome interactions and further demonstrate the utility of the microbiome transplantation technique.

microbiology↗

bioRxiv2023

Persistence of SARS-CoV-2 and its surrogate, bacteriophage Phi6, on surfaces and in water

The COVID-19 pandemic has motivated research on the persistence of infectious SARS-CoV-2 in environmental reservoirs such as surfaces and water. Viral persistence data has been collected for SARS-CoV-2 and its surrogates, including bacteriophage Phi6. Despite its wide use, no side-by-side comparisons between Phi6 and SARS-CoV-2 exist. Here, we quantified the persistence of SARS-CoV-2 and Phi6 on surfaces (plastic and metal) and in water and evaluated the influence that the deposition solution has on viral persistence by using four commonly used deposition solutions: two culture media (DMEM and Tryptone Soya Broth (TSB)), Phosphate Buffered Saline (PBS), and human saliva. Phi6 remained infectious in water significantly longer than SARS-CoV-2, having a half-life of 27 hours as compared with 15 hours for SARS-CoV-2. The persistence of viruses on surfaces was significantly influenced by the virus used and the deposition solution, but not by the surface material. Phi6 remained infectious significantly longer than SARS-CoV-2 when the inoculation solution was culture media (DMEM, TSB) and saliva. Using culture media and saliva led to half-lives between 9 hours and 2 weeks for Phi6, as compared to 0.5 to 2 hours for SARS-CoV-2. Using PBS as a deposition solution led to half-lives shorter than 4 hours for both viruses on all surfaces. Our results showed that, although it has been frequently used as a surrogate for coronaviruses, bacteriophage Phi6 is not an adequate surrogate for studies quantifying SARS-CoV-2 persistence, as it over-estimates infectiousness. Additionally, our findings reveal the need of using adequate deposition solutions when evaluating viral persistence on surfaces.

microbiology↗

bioRxiv2022

Bat pluripotent stem cells reveal unique entanglement between host and viruses

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat (Rhinolophus ferrumequinum) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats unique traits.

developmental biology↗

bioRxiv2022

MINUUR: Microbial INsight Using Unmapped Reads

BackgroundOngoing research of the mosquito microbiome aims to uncover novel strategies to reduce pathogen transmission. Sequencing costs, especially for metagenomics, are however still significant. A resource that is increasingly used to gain insights into host-associated microbiomes is the large amount of publicly available genomic data based on whole organisms like mosquitoes, which includes sequencing reads of the host-associated microbes and provides the opportunity to gain additional value of these initially host-focused sequencing projects. MethodsTo analyse non-host reads from existing genomic data, we developed a snakemake workflow called MINUUR (Microbial INsights Using Unmapped Reads). Within MINUUR, reads derived from the host-associated microbiome were extracted and characterised using taxonomic classifications and metagenome assembly followed by binning and quality assessment. We applied this pipeline to five publicly available Aedes aegypti genomic datasets, consisting of 62 samples with a broad range of sequencing depths. ResultsWe demonstrate that MINUUR recovers previously identified phyla and genera and is able to extract bacterial metagenome assembled genomes (MAGs) associated to the microbiome. Of these MAGS, 42 are high-quality representatives with >90% completeness and <5% contamination. These MAGs improve the genomic representation of the mosquito microbiome and can be used to facilitate genomic investigation of key genes of interest. Furthermore, we show that samples with a high number of KRAKEN2 assigned reads produce more MAGs. ConclusionsOur metagenomics workflow, MINUUR, was applied to a range of Aedes aegypti genomic samples to characterise microbiome-associated reads. We confirm the presence of key mosquito-associated symbionts that have previously been identified in other studies and recovered high-quality bacterial MAGs. In addition, MINUUR and its associated documentation are freely available on GitHub (https://github.com/aidanfoo96/MINUUR) and provide researchers with a convenient workflow to investigate microbiome data included in the sequencing data for any applicable host genome of interest.

bioinformatics↗

bioRxiv2022

Vertical and horizontal transmission of cell fusing agent virus in Aedes aegypti

Cell fusing agent virus (CFAV) is an insect specific flavivirus (ISF) found in field and laboratory populations of Aedes aegypti. ISFs have recently demonstrated the ability to block the transmission of arboviruses such as dengue, West Nile and Zika viruses. It is thought that vertical transmission is the main route for ISF infections. This has been observed with CFAV, but there is evidence of horizontal and venereal transmission in other ISFs. Understanding the route of transmission can inform strategies to spread ISFs to wild vector populations as a method of controlling pathogenic arboviruses. We crossed individually reared male and female mosquitoes from both a naturally occurring CFAV-positive Ae. aegypti colony and its negative counterpart to provide information on maternal, paternal, and horizontal transmission. RT-PCR was used to detect CFAV in individual female mosquito pupal exuviae and was 89% sensitive, but only 41% in male mosquito pupal exuviae. This is a possible way to screen individuals for infection without destroying the adults. Female-to-male horizontal transmission was not observed during this study, however there was a 31% transmission rate from mating pairs of CFAV-positive males to negative female mosquitoes. Maternal vertical transmission was observed with a filial infection rate of 93%. The rate of paternal transmission was 85% when the female remained negative, 61% when the female acquired CFAV horizontally, and 76% overall. Maternal and paternal transmission of CFAV could allow the introduction of this virus into wild Ae. aegypti populations through male or female mosquito releases, and thus provides a potential strategy for ISF-derived arbovirus control.

microbiology↗

bioRxiv2021

Integration of whole genome sequencing and transcriptomics reveals a complex picture of insecticide resistance in the major malaria vector Anopheles coluzzii

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

genomics↗