Search

Mosquitoes bring global health problems by transmitting parasites and viruses such as malaria and dengue. Unfortunately, current insecticide-based control strategies are only moderately effective because of high cost and resistance. Thus, scalable, sustainable, and cost-effective strategies are needed for mosquito-borne disease control. Symbiont-based and genome engineering-based approaches provide new tools that show promise for meeting these criteria, enabling modification or suppression approaches. Symbiotic bacteria like Wolbachia are maternally inherited and manipulate mosquito host reproduction to enhance their vertical transmission. Genome engineering-based gene drive methods, in which mosquitoes are genetically altered to spread drive alleles throughout wild populations, are also proving to be a potentially powerful approach in the laboratory. Here, we review the latest developments in both symbionts and gene drive-based methods. We describe some notable similarities, as well as distinctions and obstacles, relating to these promising technologies.

Background Diverse environmental exposures and risk factors have been implicated in the transmission of Salmonella Typhi, but the dominant transmission pathways through the environment to susceptible humans remain unknown. Here, we use spatial, bacterial genomic, and hydrological data to refine our view of typhoid transmission in an endemic setting. Methods A total of 546 patients presenting to Queen Elizabeth Central Hospital in Blantyre, Malawi, with blood culture–confirmed typhoid fever between April 2015 and January 2017 were recruited to a cohort study. The households of a subset of these patients were geolocated, and 256 S. Typhi isolates were whole-genome sequenced. Pairwise single-nucleotide variant distances were incorporated into a geostatistical modeling framework using multidimensional scaling. Results Typhoid fever was not evenly distributed across Blantyre, with estimated minimum incidence ranging across the city from 100 cases per 100 000 population per year. Pairwise single-nucleotide variant distance and physical household distances were significantly correlated (P = .001). We evaluated the ability of river catchment to explain the spatial patterns of genomics observed, finding that it significantly improved the fit of the model (P = .003). We also found spatial correlation at a smaller spatial scale, of households living Conclusions These findings reinforce the emerging view that hydrological systems play a key role in the transmission of typhoid fever. By combining genomic and spatial data, we show how multifaceted data can be used to identify high incidence areas, explain the connections between them, and inform targeted environmental surveillance, all of which will be critical to shape local and regional typhoid control strategies.

Background Patterns of methylation influence lifespan, but methylation and lifespan may also depend on diet, or differ between genotypes. Prior to this study, interactions between diet and genotype have not been explored together to determine their influence on methylation. The invertebrate Daphnia magna is an excellent choice for testing the epigenetic response to the environment: parthenogenetic offspring are identical to their siblings (making for powerful genetic comparisons), they are relatively short lived and have well-characterised inter-strain life-history trait differences. We performed a survival analysis in response to caloric restriction and then undertook a 47-replicate experiment testing the DNA methylation response to ageing and caloric restriction of two strains of D. magna. Results Methylated cytosines (CpGs) were most prevalent in exons two to five of gene bodies. One strain exhibited a significantly increased lifespan in response to caloric restriction, but there was no effect of food-level CpG methylation status. Inter-strain differences dominated the methylation experiment with over 15,000 differently methylated CpGs. One gene, Me31b, was hypermethylated extensively in one strain and is a key regulator of embryonic expression. Sixty-one CpGs were differentially methylated between young and old individuals, including multiple CpGs within the histone H3 gene, which were hypermethylated in old individuals. Across all age-related CpGs, we identified a set that are highly correlated with chronological age. Conclusions Methylated cytosines are concentrated in early exons of gene sequences indicative of a directed, non-random, process despite the low overall DNA methylation percentage in this species. We identify no effect of caloric restriction on DNA methylation, contrary to our previous results, and established impacts of caloric restriction on phenotype and gene expression. We propose our approach here is more robust in invertebrates given genome-wide CpG distributions. For both strain and ageing, a single gene emerges as differentially methylated that for each factor could have widespread phenotypic effects. Our data showed the potential for an epigenetic clock at a subset of age positions, which is exciting but requires confirmation.

Members of the Klebsiella pneumoniae species complex, particularly K. pneumoniae subsp. pneumoniae are antimicrobial resistance (AMR) associated pathogens of global importance, and polyvalent vaccines targeting Klebsiella O-antigens are in development. Whole-genome sequencing has provided insight into O-antigen distribution in the K. pneumoniae species complex, as well as population structure and virulence determinants, but genomes from sub-Saharan Africa are underrepresented in global sequencing efforts. We therefore carried out a genomic analysis of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae species complex isolates colonizing adults in Blantyre, Malawi. We placed these isolates in a global genomic context, and compared colonizing to invasive isolates from the main public hospital in Blantyre. In total, 203 isolates from stool and rectal swabs from adults were whole-genome sequenced and compared to a publicly available multicounty collection and previously sequenced Malawian and Kenyan isolates from blood or sterile sites. We inferred phylogenetic relationships and analysed the diversity of genetic loci linked to AMR, virulence, capsule and LPS O-antigen (O-types). We find that the diversity of Malawian K. pneumoniae subsp. pneumoniae isolates represents the species’ population structure, but shows distinct local signatures concerning clonal expansions. Siderophore and hypermucoidy genes were more frequent in invasive versus colonizing isolates (present in 13 % vs 1 %) but still generally lacking in most invasive isolates. O-antigen population structure and distribution was similar in invasive and colonizing isolates, with O4 more common (14%) than in previously published studies (2–5 %). We conclude that host factors, pathogen opportunity or alternate virulence loci not linked to invasive disease elsewhere are likely to be the major determinants of invasive disease in Malawi. Distinct ST and O-type distributions in Malawi highlight the need to sample locations where the burden of invasive Klebsiella disease is greatest to robustly define secular trends in Klebsiella diversity to assist in the development of a useful vaccine. Colonizing and invasive isolates in Blantyre are similar, hence O-typing of colonizing Klebsiella isolates may be a rapid and cost-effective approach to describe global diversity and guide vaccine development.

Caloric restriction (CR) produces clear phenotypic effects within and between generations of the model crustacean Daphnia magna. We have previously established that micro RNAs and cytosine methylation change in response to CR in this organism, and we demonstrate here that CR has a dramatic effect on gene expression. Over 6000 genes were differentially expressed between CR and well-fed D. magna, with a bias towards up-regulation of genes under caloric restriction. We identified a highly expressed haemoglobin gene that responds to CR by changing isoform proportions. Specifically, a transcript containing three erythrocruorin domains was strongly down-regulated under CR in favour of transcripts containing fewer or no such domains. This change in the haemoglobin mix is similar to the response to hypoxia in Daphnia, which is mediated through the transcription factor hypoxia-inducible factor 1, and ultimately the mTOR signalling pathway. This is the first report of a role for haemoglobin in the response to CR. We also observed high absolute expression of super-oxide dismutase (SOD) in normally-fed individuals, which contrasts with observations of high SOD levels under in CR in other taxa. However, key differentially expressed genes, like SOD, were not targeted by differentially expressed micro-RNAs. Whether the link between Haemoglobin and CR is the case in other organisms, or is related to the aquatic lifestyle, remains to be tested. It suggests that one response to CR may be to simply transport less oxygen and lower respiration.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.

Macrophage migration inhibitory factor (MIF) has been recognized as a major player in the pathogenesis of atherosclerosis. This study determined the association between polymorphisms of MIF gene and acute coronary syndrome (ACS). The polymorphism of MIF gene (rs755622, rs1007888 and rs2096525) was analyzed in 1153 healthy controls and 699 ACS cases in Chinese Han population. Plasma MIF level was also measured in part of ACS patients (139/19.9%) and healthy controls (129/11.2%) randomly. Most participants including healthy controls and ACS patients carried rs755622 GG (63.1% vs. 56.7%) and CG genotypes (33.1% vs. 38.9%) and G allele of rs755622 (79.6% vs. 76.1%, respectively), while CC genotype (3.8% vs. 4.4%) and C allele (20.4% vs. 23.9%) carriers were the lowest. Multivariate logistic regression analysis showed that carriers with rs755622 C allele had a higher risk of ACS compared to other genotypes (AOR = 1.278, 95% CI: 1.042–1.567). In addition, CC genotype carriers had the highest plasma levels of MIF than other genotype carriers. The MIF level in ACS patients with CC genotype was significantly higher than ACS patients carrying GG genotype and healthy controls carrying 3 different genotypes of MIF gene rs755622. Our findings indicate that MIF gene rs755622 variant C allele is associated with increased risk of ACS. Identification of this MIF gene polymorphism may help for predicting the risk of ACS.

Mobile genetic elements (MGEs) are often associated with antimicrobial resistance genes (ARGs). They are responsible for intracellular transposition between different replicons and intercellular conjugation, and are therefore important agents of ARG dissemination. Detection and characterisation of functional MGEs, especially in clinical isolates, would increase our understanding of the underlying pathways of transposition and recombination, and allow us to determine interventional strategies to interrupt this process.\ud Entrapment vectors can be used to capture active MGEs as they contain a positive selection genetic system conferring a selectable phenotype upon insertion of an MGE within certain regions of that system. Previously, we developed the pBACpAK entrapment vector that results in a tetracycline-resistant phenotype when MGEs translocate and disrupt the cI repressor gene. We have previously used pBACpAK to capture MGEs in clinical Escherichia coli isolates following transformation with pBACpAK.\ud In this study, we aimed to extend the utilisation of pBACpAK to other bacterial species. We utilised an MGE free recipient E. coli strain containing pBACpAK to capture MGEs on conjugative, ARG containing plasmids following conjugation from clinical Enterobacteriaceae donors.\ud Following the conjugative transfer of multiple conjugative plasmids, and screening for tetracycline resistance in these transconjugants, we captured several IS elements and novel transposons (Tn7350 and Tn7351), we detected the de novo formation of novel putative composite transposons where the pBACpAK located tet(A) is flanked by ISKpn25 from the transferred conjugative plasmid and we also detected the ISKpn14 mediated integration of an entire 119kb, blaNDM-1 containing conjugative plasmid from Klebsiella pneumoniae.\ud Importance\ud By analysing transposition activity within our MGE free recipient, we can gain insights into the interaction and evolution of multidrug resistance conferring MGEs following conjugation, including the movement of multiple ISs, the formation of composite transposons and the cointegration and, or recombination between different replicons in the same cell. This combination of recipient and entrapment vector will allow for fine-scale experimental studies into factors affecting intracellular transposition and MGE formation in, and from, ARG encoding MGEs from multiple species of clinically relevant Enterobacteriaceae.

In 2011 mecC, a new mecA gene homologue, was described in a bovine isolate in the UK. Since then, mecC-positive methicillin-resistant Staphylococcus aureus (mecC-MRSA) has also been found in wild animals. An especially high prevalence of mecC-MRSA has been reported among hedgehogs in Sweden (64%) and Denmark (61%). Based on these findings we aimed to survey the hedgehog population for mecC-MRSA in Hungary.\ud \ud Altogether 200 hedgehogs were screened for Staphylococcus aureus using a culture-based method. The antibiotic susceptibility of the isolates to nine drugs was determined, their genetic relatedness was established by PFGE and spa-typing, and virulence genes were identified by PCR. Whole genome sequencing was performed for the single mecC-MRSA isolate found.\ud \ud Of the 200 animals, 13 were carriers of S. aureus (6.5%). Among these, one isolate was mecA positive and one was mecC positive. The isolates were susceptible to non-beta-lactam antibiotics. Toxin genes were not found, but the majority carried genes responsible for adhesion and biofilm production. The mecC-MRSA isolate was a single-locus variant of ST130, had a new spa type (t19701) and belonged to SCCmec type XI. It carried a recently described, novel exfoliative toxin (etE).\ud \ud This is the first report of mecC-MRSA in Hungary and the first survey of staphylococcus carriage among wild animals in the country. The mecC prevalence was much lower than in Northern European countries and rather similar to other countries in our region. MecC-MRSA could potentially emerge as a novel human pathogen, especially where close contact occurs between humans and animals.

Simple Summary Malaria is a huge public health burden that affects predominantly sub-Saharan Africa and is transmitted by Anopheles mosquitoes. As a measure for population control, a method called gene drive has been recently developed, which relies on genetic engineering to introduce specific genetic traits into mosquito populations. Gene drives are designed to insert at specific target sites in the mosquito genome. The efficacy of gene drives greatly depends on the selection of appropriate target sites that are functionally or structurally constrained and less likely to tolerate mutations that can hinder the spread of the desired trait in the population. The aim of this study was to perform a genome-wide analysis of highly conserved genomic regions in Anopheles gambiae and introduce a measure of conservation that could indicate sites of functional or structural constraint. The results of this analysis are gathered in a publicly available dataset that can support gene drive target selection and can offer further insights in the nature of conserved genomic regions. Abstract The increase in molecular tools for the genetic engineering of insect pests and disease vectors, such as Anopheles mosquitoes that transmit malaria, has led to an unprecedented investigation of the genomic landscape of these organisms. The understanding of genome variability in wild mosquito populations is of primary importance for vector control strategies. This is particularly the case for gene drive systems, which look to introduce genetic traits into a population by targeting specific genomic regions. Gene drive targets with functional or structural constraints are highly desirable as they are less likely to tolerate mutations that prevent targeting by the gene drive and consequent failure of the technology. In this study we describe a bioinformatic pipeline that allows the analysis of whole genome data for the identification of highly conserved regions that can point at potential functional or structural constraints. The analysis was conducted across the genomes of 22 insect species separated by more than hundred million years of evolution and includes the observed genomic variation within field caught samples of Anopheles gambiae and Anopheles coluzzii, the two most dominant malaria vectors. This study offers insight into the level of conservation at a genome-wide scale as well as at per base-pair resolution. The results of this analysis are gathered in a data storage system that allows for flexible extraction and bioinformatic manipulation. Furthermore, it represents a valuable resource that could provide insight into population structure and dynamics of the species in the complex and benefit the development and implementation of genetic strategies to tackle malaria.

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes.

RNA editing is a source of molecular diversity that regulates the functional repertoire of animal transcriptomes. Multiple studies in Drosophila have revealed that conserved editing events can be a 10 source of evolutionary ad aptations, and there is a solid body of evidence linking editing and the fine tuning of neural genes, which are often targeted by insecticides used in vector control. Yet, 12 despite these suggestive connections, genome wide analyses of editing in insect vect ors are conspicuously lacking. Future advances will require complementing the growing wealth of vector 14 genomes with targeted transcriptome analyses. Here, we review recent investigations of the genetic\ud footprints of adaptive RNA editing in insects and prov ide an overview of new methodologies 16 applicable to studies of RNA editing in insect vectors.

Introduction\ud \ud Control strategies for human infections are often investigated using individual‐based models (IBMs) to quantify their impact in terms of mortality, morbidity and impact on transmission. Genetic selection can be incorporated into the IBMs to track the spread of mutations whose origin and spread are driven by the intervention and which subsequently undermine the control strategy; typical examples are mutations which encode drug resistance or diagnosis‐ or vaccine‐escape phenotypes.\ud \ud Methods and results\ud \ud We simulated the spread of malaria drug resistance using the IBM OpenMalaria to investigate how the finite sizes of IBMs require strategies to optimally incorporate genetic selection. We make four recommendations. Firstly, calculate and report the selection coefficients, s, of the advantageous allele as the key genetic parameter. Secondly, use these values of “s” to calculate the wait time until a mutation successfully establishes itself in the pathogen population. Thirdly, identify the inherent limits of the IBM to robustly estimate small selection coefficients. Fourthly, optimize computational efficacy: when “s” is small, fewer replicates of larger IBMs may be more efficient than a larger number of replicates of smaller size.\ud \ud Discussion\ud \ud The OpenMalaria IBM of malaria was an exemplar and the same principles apply to IBMs of other diseases.

Galls are plant tissues whose development is induced by another organism for the inducer's benefit. 30,000 arthropod species induce galls, and in most cases the inducing effectors and target plant systems are unknown. Cynipid gall wasps are a speciose monophyletic radiation that induce structurally complex galls on oaks and other plants. We used a model system comprising the gall wasp Biorhiza pallida and the oak Quercus robur to characterise inducer and host plant gene expression at defined stages through the development of galled and ungalled plant tissues, and tested alternative hypotheses for the origin and type of galling effectors and plant metabolic pathways involved. Oak gene expression patterns diverged markedly during development of galled and normal buds. Young galls showed elevated expression of oak genes similar to legume root nodule Nod factor-induced early nodulin (ENOD) genes and developmental parallels with oak buds. In contrast, mature galls showed substantially different patterns of gene expression to mature leaves. While most oak transcripts could be functionally annotated, many gall wasp transcripts of interest were novel. We found no evidence in the gall wasp for involvement of third-party symbionts in gall induction, for effector delivery using virus-like-particles, or for gallwasp expression of genes coding for plant hormones. Many differentially and highly expressed genes in young larvae encoded secretory peptides, which we hypothesise are effector proteins exported to plant tissues. Specifically, we propose that host arabinogalactan proteins and gall wasp chitinases interact in young galls to generate a somatic embryogenesis-like process in oak tissues surrounding the gall wasp larvae. Gall wasp larvae also expressed genes encoding multiple plant cell wall degrading enzymes (PCWDEs). These have functional orthologues in other gall inducing cynipids but not in figitid parasitoid sister groups, suggesting that they may be evolutionary innovations associated with cynipid gall induction., [Author summary] Plant galls are induced by organisms that manipulate host plant development to produce novel structures. The organisms involved range from mutualistic (such as nitrogen fixing bacteria) to parasitic. In the case of parasites, the gall benefits only the gall-inducing partner. A wide range of organisms can induce galls, but the processes involved are understood only for some bacterial and fungal galls. Cynipid gall wasps induce diverse and structurally complex galls, particularly on oaks (Quercus). We used transcriptome and genome sequencing for one gall wasp and its host oak to identify genes active in gall development. On the plant side, when compared to normally developing bud tissues, young gall tissues showed elevated expression of loci similar to those found in nitrogen-fixing root nodules of leguminous plants. On the wasp side, we found no evidence for involvement of viruses or microorganisms carried by the insects in gall induction or delivery of inducing stimuli. We found that gall wasps express many genes whose products may be secreted to the host, including enzymes that degrade plant cell walls. Genome comparisons between galling and non-galling relatives showed cell wall-degrading enzymes are restricted to gall inducers, and hence potentially key components of a gall inducing lifestyle., This study was supported by a NERC studentship awarded to Jack Hearn, a NERC NBAF grant (NBAF375 2009-2010) awarded to Graham Stone, and NERC grant NE/J010499. Jose-Luis Nieves Aldrey was supported by project CGL2015-66571-P (MINECO, Spain).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Polymorphisms in genetic copy number can influence gene expression, coding sequence, and zygosity, making them powerful actors in the evolutionary process. Copy number variants (CNVs) are however understudied, being more difficult to detect than single-nucleotide polymorphisms. We take advantage of the intense selective pressures on the major malaria vector Anopheles gambiae, caused by the widespread use of insecticides for malaria control, to investigate the role of CNVs in the evolution of insecticide resistance. Using the whole-genome sequencing data from 1142 samples in the An. gambiae 1000 genomes project, we identified 250 gene-containing CNVs, encompassing a total of 267 genes of which 28 were in gene families linked to metabolic insecticide resistance, representing significant enrichment of these families. The five major gene clusters for metabolic resistance all contained CNVs, with 44 different CNVs being found across these clusters and multiple CNVs frequently covering the same genes. These 44 CNVs are widespread (45% of individuals carry at least one of them) and have been spreading through positive selection, indicated by their high local frequencies and extended haplotype homozygosity. Our results demonstrate the importance of CNVs in the response to selection, highlighting the urgent need to identify the contribution of each CNV to insecticide resistance and to track their spread as the use of insecticides in malaria endemic countries intensifies and as the operational deployment of next-generation bed nets targeting metabolic resistance gathers pace. Our detailed descriptions of CNVs found across the species range provide the tools to do so.

BACKGROUND\ud Culex quinquefasciatus has a widespread distribution across tropical and sub-tropical regions, and plays an important role in the transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF) and multiple arboviruses. Increased resistance to insecticides threatens the efficacy and sustainability of insecticide-based anti-vector interventions which mitigate the burden of mosquito transmitted diseases in endemic regions. In C. quinquefasciatus two non-synonymous voltage gated sodium channel (Vgsc) variants, both resulting in a leucine to phenylalanine change at codon 1014, are associated with resistance to pyrethroids and DDT. This tri-allelic variation has compromised the ability to perform high-throughput single-assay screening. To facilitate the detection and monitoring of the Vgsc-1014 locus in field-caught mosquitoes, an Engineered-Tail Allele-Specific-PCR (ETAS-PCR) diagnostic assay was developed and applied to wild mosquitoes from Brazil, Tanzania and Uganda.\ud RESULTS\ud This new cost-effective, single-tube assay was compared to two, well-established, genotyping approaches, pyrosequencing and TaqMan. The ETAS-PCR assay showed high specificity for discriminating the three alleles at Vgsc-L1014F, with genotyping results strongly correlated with pyrosequencing and TaqMan results (98.64% and 100% respectively).\ud CONCLUSIONS\ud Our results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.

In hereditary hemochromatosis, iron overload is associated with homozygosity for the p.C282Y mutation. A second mutation, p.H63D, occurs at significant frequencies in Europe, North Africa, the Middle East and Asia. Early studies in Sri Lanka indicated that the variant had arisen independently, suggesting that it had been the subject of selective pressure. However, its role in iron absorption is unclear. In a survey of 7526 Sri Lankan secondary school students, we determined hemoglobin genotype and measured red cell indices, serum ferritin, transferrin receptor, iron zinc protoporphyrin and hepcidin. These variables were compared according to the presence or absence of the p.H63D variant in a subset of 1313 students for whom DNA samples were available. Students were classified as having low red cell indices if they had an MCV

Background The degradation of epigenetic control with age is associated with progressive diseases of ageing, including cancers, immunodeficiency and diabetes. Reduced caloric intake slows the effects of ageing and age-related disease in vertebrates and invertebrates, a process potentially mediated by the impact of caloric restriction on epigenetic factors such as DNA methylation. We used whole genome bisulphite sequencing to study how DNA methylation patterns change with diet in a small invertebrate, the crustacean Daphnia magna. Daphnia show the classic response of longer life under caloric restriction (CR), and they reproduce clonally, which permits the study of epigenetic changes in the absence of genetic variation. Results Global cytosine followed by guanine (CpG) methylation was 0.7–0.9%, and there was no difference in overall methylation levels between normal and calorie restricted replicates. However, 333 differentially methylated regions (DMRs) were evident between the normally fed and CR replicates post-filtering. Of these 65% were hypomethylated in the CR group, and 35% were hypermethylated in the CR group. Conclusions Our results demonstrate an effect of CR on the genome-wide methylation profile. This adds to a growing body of research in Daphnia magna that demonstrate an epigenomic response to environmental stimuli. Specifically, gene Ontology (GO) term enrichment of genes associated with hyper and hypo-methylated DMRs showed significant enrichment for methylation and acyl-CoA dehydrogenase activity, which are linked to current understanding of their roles in CR in invertebrate model organisms. Electronic supplementary material The online version of this article (10.1186/s12864-019-5578-4) contains supplementary material, which is available to authorized users.

Background: Tn916 and Tn1545 are paradigms of a large family of related, broad host range, conjugative transposons that are widely distributed in bacteria and contribute to the spread of antibiotic resistance genes (ARGs). Variation in the copy number (CN) of Tn916-Tn1545 elements and the circularization ratio (CR) may play an important role in propagation of ARGs carried by these elements. Objectives and Design: In this study, the CN and CR of Tn916-Tn1545 elements in oral streptococci were determined using droplet digital PCR (ddPCR). In addition, we investigated the influence of tetracycline on the CR of Tn916-Tn1545 elements. Results: The ddPCR assay designed in this study is a reliable way to rapidly determine CN and CR of Tn916-Tn1545 elements. Conclusions: Our data also suggest that Tn916-Tn1545 elements are generally stable without selective pressure in the clinical oral Streptococcus strains investigated in this study.

Much of what is known about the molecular evolution of vertebrate vision comes from studies of mammals, birds and fish. Reptiles (especially snakes) have barely been sampled in previous studies despite their exceptional diversity of retinal photoreceptor complements. Here, we analyze opsin gene sequences and ocular media transmission for up to 69 species to investigate snake visual evolution. Most snakes express three visual opsin genes (rh1, sws1, and lws). These opsin genes (especially rh1 and sws1) have undergone much evolutionary change, including modifications of amino acid residues at sites of known importance for spectral tuning, with several tuning site combinations unknown elsewhere among verte- brates. These changes are particularly common among dipsadine and colubrine “higher” snakes. All three opsin genes are inferred to be under purifying selection, though dN/dS varies with respect to some lineages, ecologies, and retinal anatomy. Positive selection was inferred at multiple sites in all three opsins, these being concentrated in transmembrane domains and thus likely to have a substantial effect on spectral tuning and other aspects of opsin function. Snake lenses vary substantially in their spectral transmission. Snakes active at night and some of those active by day have very transmissive lenses, whereas some primarily diurnal species cut out shorter wavelengths (including UVA). In terms of retinal anatomy, lens transmission, visual pigment spectral tuning and opsin gene evolution the visual system of snakes is exceptionally diverse compared with all other extant tetrapod orders.

Copy number variation (CNV) in insect genomes is a rich source of potentially adaptive polymorphism which may help overcome the constraints of purifying selection on conserved genes and/or permit elevated transcription. Classic studies of amplified esterases and acetylcholinesterase duplication in Culex pipiens quantified evolutionary dynamics of CNV driven by insecticidal selection. A more complex and potentially medically impactful form of CNV is found in Anopheles gambiae, with both heterogeneous duplications and homogeneous amplifications strongly linked with insecticide resistance. Metabolic gene amplification, revealed by shotgun sequencing, appears common in Aedes aegypti, but poorly understood in other mosquito species. Many methodologies have been used to detect CNV in mosquitoes, but relatively few can detect both duplications and amplifications, and contrasting methods should be combined. Genome scans for CNV have been rare to date in mosquitoes, but offer immense potential to determine the overall role of CNV as a component of resistance mechanisms.sequencing, appears common in Aedes\ud aegypti, but poorly understood in other mosquito species. Many methodologies have been used to detect CNV in\ud mosquitoes, but relatively few can detect both duplications and amplifications, and contrasting methods should be\ud combined. Genome scans for CNV have been rare to date in mosquitoes, but offer immense potential to determine the\ud overall role of CNV as a component of resistance mechanisms.

Tuberculosis (TB) remains one of the most threatening diseases and Nigeria has one of the world’s largest burdens. This study performed high-throughput spoligotyping directly on sputum smears to describe the Mycobacterium tuberculosis strains circulating among new TB patients in Nigeria. Methods All State TB control programmes in Nigeria were requested to submit 25–50 smear-positive Ziehl-Neelsen (ZN) stained slides for screening during 2013–2014. DNA was extracted from 929 slides for spoligotyping and drug-resistance analysis using microbead-based flow-cytometry suspension arrays. Results Spoligotyping results were obtained for 549 (59.1%) of 929 samples. Lineage 4 Cameroon sublineage (L4.6.2) represented half of the patterns, Mycobacterium africanum (L5 and L6) represented one fifth of the patterns, and all other lineages, including other L4 sublineages, represented one third of the patterns. Sublineage L4.6.2 was mostly identified in the north of the country whereas L5 was mostly observed in the south and L6 was scattered. The spatial distribution of genotypes had genetic geographic gradients. We did not obtain results enabling the detection of drug-resistance mutations. Conclusion/Significance We present the first national snapshot of the M. tuberculosis spoligotypes circulating in Nigeria based on ZN slides. Spoligotyping data can be obtained in a rapid and high-throughput manner with DNA extracted from ZN-stained slides, which may potentially improve our understanding of the genetic epidemiology of TB., Author summary Using a classical genotyping method designated as “spoligotyping”, which targets polymorphic repetitive DNA loci, we present a general snapshot of the genetic diversity of Mycobacterium tuberculosis complex causing tuberculosis in Nigeria. Our results were obtained on a collection of 549 DNAs, extracted from Ziehl-Neelsen stained sputum smears gathered and representative from 36 Nigerian states and during 2013–2014. We show the ubiquitous presence on the Nigerian territory of a sublineage of Lineage 4, designated as L4.6.2 or “Cameroon” Lineage, which represents almost 50% of all patterns, more prevalent where Mycobacterium africanum west African 1 (Lineage 5) is absent. We also show that Lineage 5 is geographically linked to the south-east of the country, where it is the most prevalent, and represents approximately 20% of all the samples. The last third is linked to all other L4 sublineages and to L6 (M. africanum west African 2). Our results confirm the strong phylogeographical structure of Mycobacterium tuberculosis complex in Nigeria and are suggestive of a long-term coevolution history between Homo sapiens sapiens and Mycobacterium tuberculosis complex.

Venoms contain variable mixtures of bioactive proteins. New work shows that parasitoid wasp venom toxins evolve by the co-option of genes rather than the canonical process of gene duplication. These findings suggest co-option may be an underappreciated process underpinning protein neofunctionalization.

Variable exposure to anti-tuberculosis (TB) drugs, partially driven by genetic factors, may be associated with poor clinical outcomes. Previous studies have suggested an influence of the SLCO1B1 locus on the plasma area under the concentration-time curve (AUC) of rifampicin. We evaluated the contribution of Single Nucleotide Polymorphisms (SNPs) in SLCO1B1 and other candidate genes (AADAC, CES-1) to inter-individual pharmacokinetic variability in Malawi. 174 adults with pulmonary TB underwent sampling of plasma rifampicin concentrations at 2- and 6-hours post-dose. Data from a prior cohort of 47 intensively sampled, similar patients from the same setting were available to support population pharmacokinetic model development in NONMEM v7.2©, using a two-stage strategy to improve information during the absorption phase. Contrary to recent studies in South Africa and Uganda, SNPs in SLCO1B1 did not explain variability in AUC0-∞ of rifampicin. No pharmacokinetic associations were identified with AADAC or CES-1 SNPs, which were rare in the Malawian population. Pharmacogenetic determinants of rifampicin exposure may vary between African populations. SLCO1B1 and other novel candidate genes, as well as non-genetic sources of inter-individual variability, should be further explored in geographically diverse, adequately powered cohorts.

Background\ud \ud Dengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance.\ud \ud Methodology/Principal Findings\ud \ud Genome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise pyrethroids, DDT and bendiocarb.\ud \ud Conclusion/significance\ud \ud The predominant over-expression of cytochrome P450s suggests that synergist-based (PBO) control tools could be utilised to improve control of this major dengue vector across Malaysia.

Resistance to pyrethroids, the sole insecticide class recommended for treating bed nets, threatens the control of major malaria vectors, including Anopheles funestus Effective management of resistance requires an understanding of the dynamics and mechanisms driving resistance. Here, using genome-wide transcription and genetic diversity analyses, we show that a shift in the molecular basis of pyrethroid resistance in southern African populations of this species is associated with a restricted gene flow. Across the most highly endemic and densely populated regions in Malawi, An. funestus is resistant to pyrethroids, carbamates, and organochlorides. Genome-wide microarray-based transcription analysis identified overexpression of cytochrome P450 genes as the main mechanism driving this resistance. The most up-regulated genes include cytochrome P450s (CYP) CYP6P9a, CYP6P9b and CYP6M7. However, a significant shift in the overexpression profile of these genes was detected across a south/north transect, with CYP6P9a and CYP6P9b more highly overexpressed in the southern resistance front and CYP6M7 predominant in the northern front. A genome-wide genetic structure analysis of southern African populations of An. funestus from Zambia, Malawi, and Mozambique revealed a restriction of gene flow between populations, in line with the geographical variation observed in the transcriptomic analysis. Genetic polymorphism analysis of the three key resistance genes, CYP6P9a, CYP6P9b, and CYP6M7, support barriers to gene flow that are shaping the underlying molecular basis of pyrethroid resistance across southern Africa. This barrier to gene flow is likely to impact the design and implementation of resistance management strategies in the region.

Pathogenic Salmonella strains that cause gastroenteritis are able to colonize and replicate within the intestines of multiple host species. In general, these strains have retained an ability to form the rdar morphotype, a resistant biofilm physiology hypothesized to be important for Salmonella transmission. In contrast, Salmonella strains that are host-adapted or even host-restricted like Salmonella enterica serovar Typhi, tend to cause systemic infections and have lost the ability to form the rdar morphotype. Here, we investigated the rdar morphotype and CsgD-regulated biofilm formation in two non-typhoidal Salmonella (NTS) strains that caused invasive disease in Malawian children, S. Typhimurium D23580 and S. Enteritidis D7795, and compared them to a panel of NTS strains associated with gastroenteritis, as well as S. Typhi strains. Sequence comparisons combined with luciferase reporter technology identified key SNPs in the promoter region of csgD that either shut off biofilm formation completely (D7795) or reduced transcription of this key biofilm regulator (D23580). Phylogenetic analysis showed that these SNPs are conserved throughout the African clades of invasive isolates, dating as far back as 80 years ago. S. Typhi isolates were negative for the rdar morphotype due to truncation of eight amino acids from the C-terminus of CsgD. We present new evidence in support of parallel evolution between lineages of nontyphoidal Salmonella associated with invasive disease in Africa and the archetypal host-restricted invasive serovar; S. Typhi. We hypothesize that the African invasive isolates are becoming human-adapted and ‘niche specialized’ with less reliance on environmental survival, as compared to gastroenteritis-causing isolates., Author summary African clades of nontyphoidal Salmonella cause invasive disease on a daily basis and thousands of deaths each year. Although it is generally accepted that the transmission route for these organisms is fecal-oral, we know very little about their behaviour in the environment between hosts. In this paper, we have identified both a genotype and a phenotype that suggest environmental niche specialization that is distinct from lineages of Salmonella Typhimurium and Salmonella Enteritidis associated with industrialized food supply chains in resource-rich settings. We also compared with strains of Salmonella Typhi, which cause systemic typhoid fever infections exclusively in humans. In each invasive lineage, regulatory or structural gene mutations leading to loss or impairment of biofilm were identified, all associated with curli and cellulose production, the two main structures that comprise the biofilm matrix. This suggests that similar evolutionary pressures are acting on invasive Salmonella isolates. Public health strategies aimed at reducing the burden of invasive Salmonella disease must prevent transmission to vulnerable adults and children via water sanitation and hygiene practices–a process that starts with identification of environmental reservoirs. The results of our study will raise the profile of this neglected aspect of invasive salmonellosis and will challenge researchers and clinicians to search in new places for potential environmental reservoirs of these pathogens.

Understanding why organisms senesce is a fundamental question in biology. One common explanation is that senescence results from an increase in macromolecular damage with age. The tremendous variation in lifespan between genetically identical queen and worker ants, ranging over an order of magnitude, provides a unique system to study how investment into processes of somatic maintenance and macromolecular repair influence lifespan. Here we use RNAseq to compare patterns of expression of genes involved in DNA and protein repair of age-matched queens and workers. There was no difference between queens and workers in 1-day-old individuals, but the level of expression of these genes increased with age and this up-regulation was greater in queens than in workers, resulting in significantly queen-biased expression in 2-month-old individuals in both legs and brains. Overall, these differences are consistent with the hypothesis that higher longevity is associated with increased investment into somatic repair.

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val109Ile, Asp335Glu and Asn384Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.

Background The liver fluke Fasciola hepatica is a major pathogen of livestock worldwide, causing huge economic losses to agriculture, as well as 2.4 million human infections annually. Results Here we provide a draft genome for F. hepatica, which we find to be among the largest known pathogen genomes at 1.3 Gb. This size cannot be explained by genome duplication or expansion of a single repeat element, and remains a paradox given the burden it may impose on egg production necessary to transmit infection. Despite the potential for inbreeding by facultative self-fertilisation, substantial levels of polymorphism were found, which highlights the evolutionary potential for rapid adaptation to changes in host availability, climate change or to drug or vaccine interventions. Non-synonymous polymorphisms were elevated in genes shared with parasitic taxa, which may be particularly relevant for the ability of the parasite to adapt to a broad range of definitive mammalian and intermediate molluscan hosts. Large-scale transcriptional changes, particularly within expanded protease and tubulin families, were found as the parasite migrated from the gut, across the peritoneum and through the liver to mature in the bile ducts. We identify novel members of anti-oxidant and detoxification pathways and defined their differential expression through infection, which may explain the stage-specific efficacy of different anthelmintic drugs. Conclusions The genome analysis described here provides new insights into the evolution of this important pathogen, its adaptation to the host environment and external selection pressures. This analysis also provides a platform for research into novel drugs and vaccines. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0632-2) contains supplementary material, which is available to authorized users.

The pneumococcal pilus has been shown to be an important determinant of adhesion and virulence in mouse models of colonization, pneumonia, and bacteremia. A pilus is capable of inducing protective immunity, supporting its inclusion in next-generation pneumococcal protein vaccine formulations. Whether this vaccine target is common among pneumococci in sub-Saharan Africa is uncertain. To define the prevalence and genetic diversity of type I and II pili among invasive pneumococci in Malawi prior to the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into routine childhood immunization, we examined 188 Streptococcus pneumoniae isolates collected between 2002 and 2008 (17% serotype 1). In this region of high disease burden, we found a low frequency of invasive piliated pneumococci (14%) and pilus gene sequence diversity similar to that seen previously in multiple global pneumococcal lineages. All common serotypes with pilus were covered by PCV13 and so we predict that pilus prevalence will be reduced in the Malawian pneumococcal population after PCV13 introduction.

Human African trypanosomiasis is caused by two subspecies of Trypanosoma brucei. Trypanosoma brucei rhodesiense is found in East Africa and frequently causes acute disease, while Trypanosoma brucei gambiense is found in West Africa and is associated with chronic disease. Samples taken from a single focus of a Ugandan outbreak of T. b. rhodesiense in the 1980s were associated with either chronic or acute disease. We sequenced the whole genomes of two of these isolates, which showed that they are genetically distinct from each other. Analysis of single nucleotide polymorphism markers in a panel of 31 Ugandan isolates plus 32 controls revealed a mixture of East African and West African haplotypes, and some of these haplotypes were associated with the different virulence phenotypes. It has been shown recently that T. b. brucei and T. b. rhodesiense populations undergo genetic exchange in natural populations. Our analysis showed that these strains from the Ugandan epidemic were intermediate between the reference genome sequences of T. b. gambiense and T. b. brucei and contained haplotypes that were present in both subspecies. This suggests that the human-infective subspecies of T. brucei are not genetically isolated, and our data are consistent with genomic introgression between East African and West African T. b. brucei subspecies. This has implications for the control of the parasite, the spread of drug resistance, and understanding the variation in virulence and the emergence of human infectivity., IMPORTANCE We present a genetic study of the acute form of “sleeping sickness” caused by the protozoan parasite Trypanosoma brucei rhodesiense from a single outbreak in Uganda. This represents an advance in our understanding of the relationship between the T. b. rhodesiense and Trypanosoma brucei gambiense subspecies that have previously been considered geographically distinct. Our data suggest that introgression of West African-derived T. brucei haplotypes may be associated with differences in disease presentation in the East African disease. These findings are not only of scientific interest but also important for parasite control, as they suggest that the human-infective T. brucei subspecies are not genetically isolated.

Background Schistosomiasis in one of the most prevalent parasitic diseases, affecting millions of people and animals in developing countries. Amongst the human-infective species S. haematobium is one of the most widespread causing urogenital schistosomiasis, a major human health problem across Africa, however in terms of research this human pathogen has been severely neglected. Methodology/Principal Findings To elucidate the genetic diversity of Schistosoma haematobium, a DNA ‘barcoding’ study was performed on parasite material collected from 41 localities representing 18 countries across Africa and the Indian Ocean Islands. Surprisingly low sequence variation was found within the mitochondrial cytochrome oxidase subunit I (cox1) and the NADH-dehydrogenase subunit 1 snad1). The 61 haplotypes found within 1978 individual samples split into two distinct groups; one (Group 1) that is predominately made up of parasites from the African mainland and the other (Group 2) that is made up of samples exclusively from the Indian Ocean Islands and the neighbouring African coastal regions. Within Group 1 there was a dominance of one particular haplotype (H1) representing 1574 (80%) of the samples analyzed. Population genetic diversity increased in samples collected from the East African coastal regions and the data suggest that there has been movement of parasites between these areas and the Indian Ocean Islands. Conclusions/Significance The high occurrence of the haplotype (H1) suggests that at some point in the recent evolutionary history of S. haematobium in Africa the population may have passed through a genetic ‘bottleneck’ followed by a population expansion. This study provides novel and extremely interesting insights into the population genetics of S. haematobium on a large geographic scale, which may have consequence for control and monitoring of urogenital schistosomiasis., Author Summary Schistosomiasis is a disease caused by parasitic blood flukes of the genus Schistosoma. Species that infect humans are prevalent in developing countries, having a major impact on public health and well-being as well as an impediment to socioeconomic development. More people are infected with Schistosoma haematobium than with all the other schistosome species combined, however mainly due to the inability to maintain S. haematobium in the laboratory system empirical studies on this parasite are minimal. The genetic variation of this Schistosoma species on a wide geographical scale has never been investigated. In this study, we have used a DNA ?barcoding? approach to document the genetic variation and population structure of S. haematobium sampled from 18 countries across Africa and the Indian ocean Islands. The study revealed a distinct genetic separation of S. haematobium from the Indian Ocean Islands and the closely neighbouring coastal regions from S. haematobium found throughout the African mainland, the latter of which exhibited extremely low levels of mitochondrial diversity within and between populations of parasites sampled. The data from this study provides a novel insight into the population genetics of S. haematobium and will have an impact on future research strategies.

Background Pyrethroids are increasingly used to block the transmission of diseases spread by Aedes aegypti such as dengue and yellow fever. However, insecticide resistance poses a serious threat, thus there is an urgent need to identify the genes and proteins associated with pyrethroid resistance in order to produce effective counter measures. In Ae. aegypti, overexpression of P450s such as the CYP9J32 gene have been linked with pyrethroid resistance. Our aim was to confirm the role of CYP9J32 and other P450s in insecticide metabolism in order to identify potential diagnostic resistance markers. Methodology/Principal Findings We have expressed CYP9J32 in Escherichia coli and show that the enzyme can metabolize the pyrethroids permethrin and deltamethrin. In addition, three other Ae. aegypti P450s (CYP9J24, CYP9J26, CYP9J28) were found capable of pyrethroid metabolism, albeit with lower activity. Both Ae. aegypti and Anopheles gambiae P450s (CYP's 6M2, 6Z2, 6P3) were screened against fluorogenic and luminescent substrates to identify potential diagnostic probes for P450 activity. Luciferin-PPXE was preferentially metabolised by the three major pyrethroid metabolisers (CYP9J32, CYP6M2 and CYP6P3), identifying a potential diagnostic substrate for these P450s. Conclusions/Significance P450s have been identified with the potential to confer pyrethroid resistance in Ae.aegypti. It is recommended that over expression of these enzymes should be monitored as indicators of resistance where pyrethroids are used., Author Summary Dengue fever, transmitted by Aedes mosquito vectors, is a major public health problem in over 100 countries. Prevention of the disease relies heavily on the use of insecticides such as pyrethroids. Unfortunately, mosquitoes are becoming resistant to these insecticides so it is urgent to identify the genes involved to develop an effective monitoring tool. In insects, P450s are critically involved in the metabolism and detoxification of insecticides. Several P450s have been found over expressed in pyrethroid resistant Ae. aegypti, although their functions are unknown. Here, we have expressed these enzymes in bacteria and examined their ability to metabolise pyrethroids and other compounds. From this we have identified a set of four P450s (CYPs 9J24, 9J26, 9J28 and 9J32) that are involved in pyrethroid metabolism, thus they may be considered to be key markers of resistance. To try and track their expression in field populations, we have developed diagnostic assays to monitor their activity, which will aid the development of new tools to monitor resistance and aid disease control.

Functional genetic analysis in Anopheles gambiae would be greatly improved by the development of a binary expression system, which would allow the more rapid and flexible characterisation of genes influencing disease transmission, including those involved in insecticide resistance, parasite interaction, host and mate seeking behaviour. The Gal4-UAS system, widely used in Drosophila melanogaster functional genetics, has been significantly modified to achieve robust application in several different species. Towards this end, previous work generated a series of modified Gal4 constructs that were up to 20 fold more active than the native gene in An. gambiae cells. To examine the Gal4-UAS system in vivo, transgenic An. gambiae driver lines carrying a modified Gal4 gene under the control of the carboxypeptidase promoter, and responder lines carrying UAS regulated luciferase and eYFP reporter genes have been created. Crossing of the Gal4 and UAS lines resulted in progeny that expressed both reporters in the expected midgut specific pattern. Although there was minor variation in reporter gene activity between the different crosses examined, the tissue specific expression pattern was consistent regardless of the genomic location of the transgene cassettes. The results show that the modified Gal4-UAS system can be used to successfully activate expression of transgenes in a robust and tissue specific manner in Anopheles gambiae. The midgut driver and dual reporter responder constructs are the first to be developed and tested successfully in transgenic An. gambiae and provide the basis for further advancement of the system in this and other insect species.

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.

Understanding the endogenous factors that drive the population dynamics of malaria mosquitoes will facilitate more accurate predictions about vector control effectiveness and our ability to destabilize the growth of either low- or high-density insect populations. We assessed whether variation in phenotypic traits predict the dynamics of Anopheles gambiae sensu lato mosquitoes, the most important vectors of human malaria. Anopheles gambiae dynamics were monitored over a six-month period of seasonal growth and decline. The population exhibited density-dependent feedback, with the carrying capacity being modified by rainfall (97% w AIC c support). The individual phenotypic expression of the maternal ( p = 0.0001) and current ( p = 0.040) body size positively influenced population growth. Our field-based evidence uniquely demonstrates that individual fitness can have population-level impacts and, furthermore, can mitigate the impact of exogenous drivers (e.g. rainfall) in species whose reproduction depends upon it. Once frontline interventions have suppressed mosquito densities, attempts to eliminate malaria with supplementary vector control tools may be attenuated by increased population growth and individual fitness.

Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control., Author Summary Aedes mosquitoes transmit many human viral pathogens including dengue, yellow fever and chikungunya. Most of these pathogens have no specific treatment or vaccine and hence their control is reliant on controlling the mosquito vectors, which usually involves the use of insecticides. In order to prevent the alarming prospect of mosquito control failure due to the rapid selection and spread of insecticide resistance in several mosquito populations worldwide, it is essential that effective resistance management strategies are implemented and adhered to. The development of simple diagnostic tests for the early identification and monitoring of resistance is an important prerequisite for this task. Here, we describe the development of a simple colorimetric test for the detection of GSTE2-2/DDTase-based resistance in individual mosquitoes. The novel assay combines the most desirable features of specificity and sensitivity with the low cost and ease of use required for a routine test in endemic countries. It can have direct application in routine vector monitoring as a resistance indicator and help improve the sustainability of insecticide based control strategies.

Background Knock-down resistance (kdr) to DDT and pyrethroids in the major Afrotropical vector species, Anopheles gambiae sensu stricto, is associated with two alternative point mutations at amino acid position 1014 of the voltage-gated sodium channel gene, resulting in either a leucine-phenylalanine (L1014F), or a leucine-serine (L1014S) substitution. In An. gambiae S-form populations, the former mutation appears to be widespread in west Africa and has been recently reported from Uganda, while the latter, originally recorded in Kenya, has been recently found in Gabon, Cameroon and Equatorial Guinea. In M-form populations surveyed to date, only the L1014F mutation has been found, although less widespread and at lower frequencies than in sympatric S-form populations. Methods Anopheles gambiae M- and S-form specimens from 19 sites from 11 west and west-central African countries were identified to molecular form and genotyped at the kdr locus either by Hot Oligonucleotide Ligation Assay (HOLA) or allele-specific PCR (AS-PCR). Results The kdr genotype was determined for about 1,000 An. gambiae specimens. The L1014F allele was found at frequencies ranging from 6% to 100% in all S-form samples (N = 628), with the exception of two samples from Angola, where it was absent, and coexisted with the L1014S allele in samples from Cameroon, Gabon and north-western Angola. The L1014F allele was present in M-form samples (N = 354) from Benin, Nigeria, and Cameroon, where both M- and S-forms were sympatric. Conclusion The results represent the most comprehensive effort to analyse the overall distribution of the L1014F and L1014S mutations in An. gambiae molecular forms, and will serve as baseline data for resistance monitoring. The overall picture shows that the emergence and spread of kdr alleles in An. gambiae is a dynamic process and that there is marked intra- and inter-form heterogeneity in resistance allele frequencies. Further studies are needed to determine: i) the importance of selection pressure exerted by both agricultural and public health use of pyrethroid insecticides, ii) the phenotypic effects, particularly when the two mutations co-occur; and iii) the epidemiological importance of kdr for both pyrethroid- and DDT-based malaria control operations, particularly if/when the two insecticides are to be used in concert.

Background: Pseudogenes reveal ancestral gene functions. Some obligate intracellular bacteria, such as Mycobacterium leprae and Rickettsia spp., carry substantial fractions of pseudogenes. Until recently, horizontal gene transfers were considered to be rare events in obligate host-associated bacteria.\ud Results: We present a visualization tool that displays the relationships and positions of degraded and partially overlapping gene sequences in multiple genomes. With this tool we explore the origin and deterioration patterns of the Rickettsia pseudogenes and find that variably present genes and pseudogenes tend to have been acquired more recently, are more divergent in sequence, and exhibit a different functional profile compared with genes conserved across all species. Overall, the origin of only one-quarter of the variable genes and pseudogenes can be traced back to the common ancestor of Rickettsia and the outgroup genera Orientia and Wolbachia. These sequences contain only a few disruptive mutations and show a broad functional distribution profile, much like the core genes. The remaining genes and pseudogenes are extensively degraded or solely present in a single species. Their functional profile was heavily biased toward the mobile gene pool and genes for components of the cell wall and the lipopolysaccharide.\ud Conclusion: Reductive evolution of the vertically inherited genomic core accounts for 25% of the predicted genes in the variable segments of the Rickettsia genomes, whereas 75% stems from the flux of the mobile gene pool along with genes for cell surface structures. Thus, most of the variably present genes and pseudogenes in Rickettsia have arisen from recent acquisitions.

Background Parasites incur periodic mutations which must ultimately be eliminated to maintain their genetic integrity. Methods It is hypothesised that these mutations are eliminated not by the conventional mechanisms of competition between parasites in different hosts but primarily by competition between parasites within the same infection. Results This process is enhanced by the production of a large number of parasites within individual infections, and this may significantly contribute to parasitic virulence. Conclusions Several features of the most virulent human malaria parasite Plasmodium falciparum can usefully be re-interpreted in this light and lend support to this interpretation. More generally, it constitutes a novel explanation for the evolution of virulence in a wider range of microparasites.

Recrudescing Plasmodium falciparum parasitacmia is attributed to the switching of PfEMP1, a variant antigen family encoded by the var gene repertoire, and the host's immune response. We have developed a mathematical model which incorporates var gene switching, and variant specific, non-variant specific and non-specific immunity. By conducting a sensitivity analysis of the model we have defined the parameter limits which produce chronic and recrudescing infections. We explore 3 switching mechanisms: ordered, random and uncoupled switching. We show that if var genes switch on and off independently at variable rates through the repertoire a chronic clinical infection is predicted. The fastest switching-on rate that produces a chronic infection is 0.03% per generation. The model predicts that non-variant specific immunity plays an important role in reducing disease severity. This work illustrates the complex relationship between the malaria parasite and its host and shows that var gene switching at rates substantially slower than 2% are essential for parasite survival.

Sepsis, defined as a life-threatening organ dysfunction triggered by infection, carries a high mortality. Recent improvements in outcome high-income settings have been driven by prompt antimicrobial therapy and fluid resuscitation but mortality remains disproportionately high in low-resource settings like the nations of sub-Saharan Africa (sSA). Sepsis therapy here often consists of empiric, prolonged courses of broad-spectrum antimicrobials, especially third generation cephalosporins like ceftriaxone, which may be driving the rise of ceftriaxone-resistant extended-spectrum �-lactamase producing Enterobacteriaceae (ESBLE).\ud However the aetiology of sepsis in sSA is far from clear, and in this thesis I hypothesise that it may be possible to improve outcomes in sepsis whilst reducing selection pressure for ESBL-E, with novel, targeted, antimicrobial strategies tailored to the pathogens that are truly causing sepsis here.\ud \ud To that end, I present findings from a clinical cohort study of sepsis in Blantyre, Malawi, with two aims: first, a description of the presentation and outcomes of sepsis in Blantyre, with a focus on aetiology and an analysis of the determinants of mortality; and secondly, a description of the gut mucosal carriage of ESBL-E in sepsis survivors (as well as antibiotic unexposed inpatient and community controls) as they pass through the hospital to identify determinants of carriage. An expanded package of diagnostic tests was used to define sepsis aetiology, and serial stool sampling with selective culture for ESBL-E used to define ESBL-E carriage. I use whole-genome sequencing of cultured ESBL E. coli to track bacteria and mobile genetic elements within participants over time, and continuous time Markov models to provide insight into the drivers of carriage.\ud \ud I find that the majority of participants with sepsis are young, and HIV-infected. Disseminated tuberculosis (TB) dominates as a cause of sepsis, and there is an association of receipt of antituberculous chemotherapy with survival that suggests an expanded role for TB therapy in these very unwell patients may be beneficial. Sepsis mortality seems to have improved compared to historic cohorts, but post 28-day mortality in HIV-infected individuals is significant.\ud \ud At baseline gut mucosal ESBL-E carriage is common, with cultured ESBL-E present in the stool of 49% of participants with sepsis on the day of admission. There is further rapid increase in colonisation prevalence following admission and antibacterial exposure. Associations of baseline colonisation - household crowding and unprotected water sources - suggest both within-household and environmental routes of transmission are important. Genomic analysis suggest unrestricted mixing of ESBL E. coli at multiple spatial levels and rapid turnover within the individual, perhaps suggestive of frequent re-exposure. By using the genetic environment of ESBL genes as a proxy for mobile genetic elements (which are difficult to assemble from short read sequencing) I show that, within individuals, the E.coli strain-mobile genetic element combination is conserved over time whereas the strain or mobile genetic element alone is not; this suggests that the unit of transmission of ESBL gene to study participants is the bacterium, rather than mobile genetic element.\ud \ud Longitudinal modelling provides further insight into ESBL-E carriage dynamics: hospitalisation and antibacterial exposure act synergistically to bring about rapid and prolonged carriage driven, in part, by a significant post-antibiotic effect. This effect means that antibacterials act to prolong carriage long after antibacterial exposure stops. In terms of ESBL-E carriage, short courses of antibacterials have a similar effect to longer courses, such that the data generated in this study do not support my hypothesis and it may not be possible to reduce ESBL-E carriage by truncating courses of ceftriaxone. Nevertheless, the post-antibiotic effect deserves further scrutiny to understand the mechanism and as a\ud potential therapeutic target. In addition, the modelling approach suggests cotrimoxazole preventative therapy (CPT) may be a significant driver of long-term ESBL-E carriage, and I suggest that a more nuanced approach to its deployment may be necessary in an era of increasing Gram-negative resistance.

The effects of reproductive compensation on the population genetics of sex-linked recessive lethal mutations are investigated. Simple equations are presented which describe these effects, and so complement existing population genetic theory. More importantly, this type of mutation is responsible for several severe human genetic diseases such as Duchenne muscular dystrophy. It is argued that the applications of three modern reproductive technologies - effective family planning, in utero diagnosis with termination, and embryo sexing - will lead to reproductive compensation. The adoption of any of these technologies may rapidly elevate the frequencies of those mutations which are lethal in childhood. This increase is large, in the order of 33 % upwards, and occurs rapidly over two to five generations. It also depends on the source of mutations, the effect being larger if most mutations are paternal. In utero diagnosis and/or embryo sexing increase the frequency of the mutation, but simultaneously decrease disease incidence by preventing the birth of affected offspring. In contrast, effective family planning may rapidly increase both mutation frequency and disease incidence.