The evolution and genetics of vector competence in mosquito disease vectors

Vector competence is a complex characteristic which governs an insect's ability to acquire, support the development and transmit a parasite from one host to another. It influences variation in disease transmission among mosquito populations, hence affecting disease epidemiology. In this project, I have studied some aspects of ecological interactions and genetic factors in a step towards understanding how these affect variation in disease transmission and exploiting these in future disease control programmes. Mosquito gut bacteria affect the development of parasites ingested by mosquitoes. As different bacterial species have different effects, dissimilarities in gut composition could be an important cause of variation in vector populations. The first study investigates the gut microbiome of mosquitoes collected from Kenya. Using 454 pyrosequencing of 16S rRNA, I provide a comprehensive catalogue of the gut composition of 8 species of mosquitoes (Chapter 2). I show that while there is greater variation within host species (fixation index= 0.64), different mosquito species tend to have rather similar gut bacteria. An individual mosquito gut has a low diversity of bacteria with, the microbiota being dominated by a single Operational Taxonomic Unit. This suggests that gut bacteria may be one factor influencing within-species variation in disease transmission, and a minor factor in between-species variation. Wolbachia endosymbionts are able to reduce the intensity and development of RNA viruses and metazoan parasites in their insect hosts, blocking the transmission of such parasites. This makes Wolbachia a likely candidate for control programmes. I extend the investigation of naturally-occurring bacteria to Wolbachia (Chapter 3). Using the gut samples used in Chapter 2, I amplify the Wolbachia surface protein gene to identify Wolbachia infections. I identify Wolbachia in Aedes bromeliae, a vector of yellow fever, and its close relative Aedes metallicus and in Mansonia uniformis and Mansonia africana, which are competent vectors of human bancroftian filariasis. Aedes bromeliae showed the highest prevalence (75%) suggesting that this strain of Wolbachia may be manipulating the host reproduction by cytoplasmic incompatibility. Using a multi locus typing system and accounting for effects of recombination in the construction of bacterial phylogeny, I show that these mosquito Wolbachia strains cluster into supergroups A and B of Wolbachia. The phylogeny also shows significant recombination events indicating horizontal transfer events between taxa. These Wolbachia strains, isolated from the disease vectors, may be reducing parasite intensity and transmission, and could be a better choice for transinfecting other mosquito vectors rather than distantly related strains. Previous studies show that high frequency of susceptibility to Brugia pahangi exists among populations of Aedes aegypti from East Africa, providing an excellent resource for investigating variation in a natural population. I test the frequency of susceptibility of peri-domestic subpopulations of Aedes aegypti collected from Kenya to Brugia malayi (Chapter 4). The results are consistent with previous data with up to 30% of individuals being susceptible. The number of susceptible individuals varied significantly between populations (Fisher's exact: p= 0.03). These populations now provide the resource to identify polymorphisms associated with susceptibility to Brugia and also enable comparison with results obtained from laboratory strains. In Chapter 5, I continue with efforts to identify and map quantitative trait loci (QTL) associated with Brugia susceptibility in Aedes aegypti. However, with the Aedes genome still highly fragmented with many supercontigs having no chromosomal assignments, mapping the gene to a definitive locus is almost impossible. Using an improved DNA-based mapping technology, Restricted-site Associated DNA tags (RADtags), I make novel assignments of 79 supercontigs to the 3 chromosomes of Aedes aegypti. These new assignments account for 122Mb of the genome, increasing the percentage genome mapped to /approx 40%. The technique also identifies potential scaffold misassemblies and misassignments of supercontigs to chromosomes. I also use the same method to prepare libraries for sequencing which will provide more markers and allow mapping and identification of candidate genes which can be evaluated for involvement in susceptibility to Brugia infections. Aedes aegypti and Anopheles gambiae share similarities in their immune proteins, but little is known about the functions of immune proteins in Aedes aegypti. To be able to make functional comparisons between mosquito vectors, I inoculate Sephadex beads into a laboratory strain of Aedes aegypti to investigate the expression of pathogen recognition genes (Chapter 6). Thioester-containing proteins (TEPs) show significant up-regulation (p= 0.03-0.0002) with up to 7-fold increase in gene expression of TEP20 in immune-challenged individuals compared to non-challenged controls. TEP20 is an orthologue of Anopheles gambiae TEP1, emphasising the evolutionary function of TEPs in immune activation. As TEP1 is an important determinant of vectorial capacity in Anopheles gambiae, this indicates that TEPs may also be an important factor influencing variation in susceptibility to pathogens in Aedes aegypti. Generally, this project has contributed to three broad areas of factors that influence variability in diseases transmission by mosquitoes: ecological interactions with bacteria, host genetic background and immune system. The results, resources and techniques used in this thesis can be widely used in further studies in these areas and extended to other mosquito vectors and natural populations

The epidemiology of lymphatic filariasis in Ghana, explained by the possible existence of two strains of Wuchereria bancrofti

Introduction Lymphatic filariasis is a debilitating disease caused by the filarial worm Wuchereria bancrofti. It is earmarked for elimination by the year 2020 through the Global Program for the Elimination of LF (GPELF). In Ghana, mass treatment has been on-going since the year 2000. Earlier studies have revealed differing epidemiology of LF in the North and South of Ghana. This study was therefore aimed at understanding the possible impacts of W. bancrofti diversity on the epidemiology and control of LF in Ghana. Methods The Mitochondrial, Cytochrome C Oxidase I gene of W. bancrofti samples was sequenced and analyzed. The test sequences were grouped into infrapopulations, and pairwise differences (Π) and mutation rates (θ) were computed. The amount of variance within and among populations was also computed using the AMOVA. The evolutionary history was inferred using the Maximum Parsimony method. Results Seven samples from the South and 15 samples from the North were sequenced, and submitted to GenBank with accession numbers GQ479497- GQ479518. The results revealed higher mutation frequencies in the southern population, compared to the northern population. Haplotype analyses revealed a total of 11 haplotypes (Hap) in all the 22 DNA sequences, with high genetic variation and polymorphisms within the southern samples. Conclusion This study showed that there is considerable genetic variability within W. bancrofti populations in Ghana, differences that might explain the observed epidemiology of LF. Further studies are however required for an in-depth understanding of LF epidemiology and control

Host-switching by a vertically transmitted rhabdovirus in Drosophila

A diverse range of endosymbionts are found within the cells of animals. As these endosymbionts are normally vertically transmitted, we might expect their evolutionary history to be dominated by host-fidelity and cospeciation with the host. However, studies of bacterial endosymbionts have shown that while this is true for some mutualists, parasites often move horizontally between host lineages over evolutionary timescales. For the first time, to our knowledge, we have investigated whether this is also the case for vertically transmitted viruses. Here, we describe four new sigma viruses, a group of vertically transmitted rhabdoviruses previously known in Drosophila. Using sequence data from these new viruses, and the previously described sigma viruses, we show that they have switched between hosts during their evolutionary history. Our results suggest that sigma virus infections may be short-lived in a given host lineage, so that their long-term persistence relies on rare horizontal transmission events between hosts

Data from: Deep sequencing reveals extensive variation in the gut microbiota of wild mosquitoes from Kenya.

The mosquito midgut is a hostile environment that vector-borne parasites must survive in order to be transmitted. Commensal bacteria in the midgut can reduce the ability of mosquitoes to transmit disease, either by having direct anti-parasite effects or by stimulating basal immune responses of the insect host. As different bacteria have different effects on parasite development, the composition of the bacterial community in the mosquito gut is likely to affect the probability of disease transmission. We investigated the diversity of mosquito gut bacteria in the field using 454 pyrosequencing of 16S rRNA to build up a comprehensive picture of the diversity of gut bacteria in 8 mosquito species. We found that mosquito gut typically has a very simple gut microbiota that is dominated by a single bacterial taxon. Although different mosquito species share remarkably similar gut bacteria, individuals in a population are extremely variable and can have little overlap in the bacterial taxa present in their guts. This may be an important factor in causing differences in disease transmission rates within mosquito populations

OTUs

Multiple alignment of filtered and annotated bacterial 16S based OTUs

Identification of <em>Wolbachia</em> Strains in Mosquito Disease Vectors

<div><p><em>Wolbachia</em> bacteria are common endosymbionts of insects, and some strains are known to protect their hosts against RNA viruses and other parasites. This has led to the suggestion that releasing <em>Wolbachia-</em>infected mosquitoes could prevent the transmission of arboviruses and other human parasites. We have identified <em>Wolbachia</em> in Kenyan populations of the yellow fever vector <em>Aedes bromeliae</em> and its relative <em>Aedes metallicus,</em> and in <em>Mansonia uniformis</em> and <em>Mansonia africana,</em> which are vectors of lymphatic filariasis. These <em>Wolbachia</em> strains cluster together on the bacterial phylogeny, and belong to bacterial clades that have recombined with other unrelated strains. These new <em>Wolbachia</em> strains may be affecting disease transmission rates of infected mosquito species, and could be transferred into other mosquito vectors as part of control programs.</p> </div

Prevalence of <i>Wolbachia</i> in mosquitoes from Kenya.

<p>The prevalence is shown with the 95% confidence interval in parentheses.</p

Examples of two misassembled scaffolds.

<p>Individual contigs are shown as gray rectangles. Contigs with markers in this study are indicated with an * above the scaffold and are labeled with their position on the genetic map. The new scaffolds created by splitting misassemblies (identified in this study only) are shown with solid lines. New scaffolds with suffixes ‘a,’ ‘b,’or ‘c’ contain markers that allow placement on the genetic map. New scaffolds with suffixes ‘m’ or ‘n’ fall between conflicting markers and therefore contain a misassembly and cannot be placed on the genetic map. A) Supercontig 1.1 with markers from the previously published integrated map <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652-Timoshevskiy1" target="_blank">[20]</a> shown below. The mapping of markers to two different chromosomes indicates a misassembly within the scaffold, which is supported by both markers sets. B) Supercontig 1.48 with synteny with <i>An. gambiae</i> shown below the scaffold and the different colors indicating different chromosome arms. Both our markers and syntenic breaks with <i>An. gambiae</i> indicate that this scaffold is misassembled in at least two instances.</p

Summary of our assembly of the genome onto a genetic map.

1<p>scaffolds which have been ordered along the chromosome (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd-0002652-g002" target="_blank">Figure 2</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652.s006" target="_blank">Table S2</a>).</p>2<p>number of mapped scaffolds after splitting misassemblies.</p>3<p>scaffolds assigned to the chromosome only (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002652#pntd.0002652.s006" target="_blank">Table S2</a>).</p

Assembly of the Genome of the Disease Vector <i>Aedes aegypti</i> onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission

<div><p>The mosquito <i>Aedes aegypti</i> transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled — there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of <i>Ae. aegypti</i>, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes <i>Ae. aegypti</i> resistant to the human parasite <i>Brugia malayi</i>, and generated a list of candidate genes that could be affecting the trait.</p></div