Exploring population history and gall induction in cynipid gall wasps using genomics and transcriptomics

Cynipid gall wasps have fascinating biology that has piqued the interest of naturalists throughout history. They induce morphologically complex, sometimes spectacular, gall structures on plants in which the larval stages develop. Gall wasps have therefore evolved an intimate association with their hosts - both metabolically, and in terms of their population histories. Gall wasps must both interact physiologically with their hosts to induce galls, and track their host plants through space and time. My thesis centres on two uses of genomic data in understanding the biology of the oak apple gall wasp Biorhiza pallida. I provide a comprehensive investigation into patterns of oak and gall wasp gene expression associated with gall induction, and a population genomic reconstruction of the population history of this species across the Western Palaearctic. While advances in sequencing technology and reduced costs have made these aims possible, analysis of the massive resulting datasets generated creates new challenges. Firstly, in reconstructing the population history of B. pallida, I describe the use of shotgun sequencing and an informatic pipeline to generate alignments of several thousand loci for three B. pallida individuals sampled from putative glacial refugia across the Western Palaearctic in Iberia, the Balkans and Iran. This dataset was analysed using a new maximum likelihood method capable of estimating population splitting and admixture among refugia across very large numbers of loci. The results showed an ancient divide between Iberia and the other two refugia, followed by very recent admixture between easternmost and westernmost regions. This suggests that gall wasps have migrated westwards along the North African coast as well as through mainland Europe. Second, I compare the gene expression profiles of gall wasp and oak tissues sampled from each of three stages of gall development, leading to new insights into potential mechanisms of gall wasp-oak interaction. A highly expressed gall wasp protein was identified that is hypothesised to stimulate somatic embryogenesis-like development of the gall through interaction with oak tissue glycoproteins. Highly expressed oak genes include those coding for nodulin-like proteins similar to those involved in legume nodule formation. Finally, analysis of the gall wasp genome has revealed potential, but as yet unconfirmed, horizontal gene transfer events into gall wasp genomes. Genes discovered in three gall wasp genomes and expressed in three transcriptomes encode plant cell wall degrading enzymes. They are not of hymenopteran origin, and are most homologous to genes of plant pathogenic bacteria. These genes could be involved in several aspects of gall wasp biology, including feeding and developmental manipulation of host plant tissue

DNA methylation differs extensively between strains of the same geographical origin and changes with age in Daphnia magna

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

Low coverage genomic data resolve the population divergence and gene flow history of an Australian rain forest fig wasp

Population divergence and gene flow are key processes in evolution and ecology. Model‐based analysis of genome‐wide datasets allows discrimination between alternative scenarios for these processes even in non‐model taxa. We used two complementary approaches (one based on the blockwise site frequency spectrum (bSFS), the second on the Pairwise Sequentially Markovian Coalescent (PSMC)) to infer the divergence history of a fig wasp, Pleistodontes nigriventris. Pleistodontes nigriventris and its fig tree mutualist Ficus watkinsiana are restricted to rain forest patches along the eastern coast of Australia, and are separated into northern and southern populations by two dry forest corridors (the Burdekin and St. Lawrence Gaps). We generated whole genome sequence data for two haploid males per population and used the bSFS approach to infer the timing of divergence between northern and southern populations of P. nigriventris, and to discriminate between alternative isolation with migration (IM) and instantaneous admixture (ADM) models of post divergence gene flow. Pleistodontes nigriventris has low genetic diversity (π = 0.0008), to our knowledge one of the lowest estimates reported for a sexually reproducing arthropod. We find strongest support for an ADM model in which the two populations diverged ca . 196kya in the late Pleistocene, with almost 25% of northern lineages introduced from the south during an admixture event ca . 57kya. This divergence history is highly concordant with individual population demographies inferred from each pair of haploid males using PSMC. Our analysis illustrates the inferences possible with genome‐level data for small population samples of tiny, non‐model organisms and adds to a growing body of knowledge on the population structure of Australian rain forest taxa.Output Status: Forthcoming/Available Onlin

An Africa-wide genomic evolution of insecticide resistance in the malaria vector Anopheles funestus involves selective sweeps, copy number variations, gene conversion and transposons

Insecticide resistance in malaria vectors threatens to reverse recent gains in malaria control. Deciphering patterns of gene flow and resistance evolution in malaria vectors is crucial to improve control strategies and preventing malaria resurgence. A genome-wide survey of Anopheles funestus genetic diversity Africa-wide revealed evidences of a major division between southern Africa and elsewhere, associated with different population histories. Three genomic regions exhibited strong signatures of selective sweep, each spanning major resistance loci (CYP6P9a/b, GSTe2 and CYP9K1). However, a sharp regional contrast was observed between populations correlating with gene flow barriers. Signatures of complex molecular evolution of resistance were detected with evidence of copy number variation, transposon insertion and a gene conversion between CYP6P9a/b paralog genes. Temporal analyses of samples before and after bed net scale up suggest that these genomic changes are driven by this control intervention. Multiple independent selective sweeps at the same locus in different parts of Africa suggests that local evolution of resistance in malaria vectors may be a greater threat than trans-regional spread of resistance haplotypes

From inquilines to gall inducers:Genomic signature of a life-style transition in synergus gall wasps

Gall wasps (Hymenoptera: Cynipidae) induce complex galls on oaks, roses and other plants, but the mechanism of gall induction is still unknown. Here we take a comparative genomic approach to revealing the genetic basis of gall induction. We focus on Synergus itoensis, a species that induces galls inside oak acorns. Previous studies suggested that this species evolved the ability to initiate gall formation recently, as it is deeply nested within the genus Synergus, whose members are mostly inquilines that develop inside the galls of other species. We compared the genome of S. itoensis to that of three related Synergus inquilines to identify genomic changes associated with the origin of gall induction. We used a novel Bayesian selection analysis, which accounts for branch-specific and gene-specific selection effects, to search for signatures of selection in 7,600 single-copy orthologous genes shared by the four Synergus species. We found that the terminal branch leading to S. itoensis had more genes with a significantly elevated dN/dS ratio (Positive Signature Genes, PSGs) than the other terminal branches in the tree; the S. itoensis branch also had more genes with a significantly decreased dN/dS ratio. Gene set enrichment analysis showed that the PSG set of S. itoensis, unlike those of the inquiline species, is enriched in several biological process Gene Ontology terms, the most prominent of which is “Ovarian Follicle Cell Development”. Our results indicate that the origin of gall induction is associated with distinct genomic changes, and provide a good starting point for further characterization of the genes involved

Biomechanical testing of implant free wedge shaped bone block fixation for bone patellar tendon bone anterior cruciate ligament reconstruction in a bovine model

Charles A Willis-Owen, Trevor C Hearn, Gregory C Keene, John J Cost

Listeria monocytogenes isolates from ready to eat plant produce are diverse and have virulence potential.

Listeria monocytogenes is sporadically detected on a range of ready to eat fresh produce lines, such as spinach and rocket, and is a threat to public health. However, little is known about the diversity of L. monocytogenes present on fresh produce and their potential pathogenicity. In this work, fifteen Listeria monocytogenes isolates from the UK fresh produce supply chain were characterised using whole genome sequencing (WGS). Additionally, isolates were characterised based on their ability to form biofilm. Whole genome sequencing data was used to determine the sequence type of isolates based on multi-locus sequence typing (MLST), construct a core single nucleotide polymorphism (SNP) phylogeny and determine the presence of virulence and resistance associated genes. MLST revealed 9 distinct sequence types (STs) spanning 2 lineages (I & II) with one isolate belonging to the ST6 subtype, strains from which have been recently implicated in two large, food-associated L. monocytogenes outbreaks in South Africa and across Europe. Although most of the 15 isolates were different, comparison of core genome SNPs showed 4 pairs of ‘indistinguishable’ strains (< 5 SNPs difference). Virulence profiling revealed that some isolates completely lacked the Listeria pathogenicity island-3 (LIPI-3) amongst other virulence factors. Investigation of the inlA gene showed that no strains in this study contained a premature stop codon (PMSC), an indicator of attenuated virulence. Assessment of biofilm production showed that isolates found in the fresh produce supply chain differ in their ability to form biofilm. This trait is considered important for L. monocytogenes to persist in environments associated with food production and processing. Overall the work indicates that a genetically diverse range of L. monocytogenes strains is present in the UK fresh produce supply chain and the virulence profiles found suggests that at least some of the strains are capable of causing human illness. Interestingly, the presence of some genetically indistinguishable isolates within the 15 isolates examined suggests that cross-contamination in the fresh produce environment does occur. These findings have useful implications in terms of food safety and for informing microbial surveillance programmes in the UK fresh produce supply chain

The duplicated P450s CYP6P9a/b drive carbamates and pyrethroids cross-resistance in the major African malaria vector Anopheles funestus

Cross-resistance to insecticides in multiple resistant malaria vectors is hampering resistance management. Understanding its underlying molecular basis is critical to implementation of suitable insecticide-based interventions. Here, we established that the tandemly duplicated cytochrome P450s, CYP6P9a/b are driving carbamate and pyrethroid cross-resistance in Southern African populations of the major malaria vector Anopheles funestus. Transcriptome sequencing revealed that cytochrome P450s are the most over-expressed genes in bendiocarb and permethrin-resistant An. funestus. The CYP6P9a and CYP6P9b genes are overexpressed in resistant An. funestus from Southern Africa (Malawi) versus susceptible An. funestus (Fold change (FC) is 53.4 and 17 respectively), while the CYP6P4a and CYP6P4b genes are overexpressed in resistant An. funestus in Ghana, West Africa, (FC is 41.1 and 17.2 respectively). Other up-regulated genes in resistant An. funestus include several additional cytochrome P450s (e.g. CYP9J5, CYP6P2, CYP6P5), glutathione-S transferases, ATP-binding cassette transporters, digestive enzymes, microRNA and transcription factors (FC&lt;7). Targeted enrichment sequencing strongly linked a known major pyrethroid resistance locus (rp1) to carbamate resistance centering around CYP6P9a/b. In bendiocarb resistant An. funestus, this locus exhibits a reduced nucleotide diversity, significant p-values when comparing allele frequencies, and the most non-synonymous substitutions. Recombinant enzyme metabolism assays showed that both CYP6P9a/b metabolize carbamates. Transgenic expression of CYP6P9a/b in Drosophila melanogaster revealed that flies expressing both genes were significantly more resistant to carbamates than controls. Furthermore, a strong correlation was observed between carbamate resistance and CYP6P9a genotypes with homozygote resistant An. funestus (CYP6P9a and the 6.5kb enhancer structural variant) exhibiting a greater ability to withstand bendiocarb/propoxur exposure than homozygote CYP6P9a_susceptible (e.g Odds ratio = 20.8, P&lt;0.0001 for bendiocarb) and heterozygotes (OR = 9.7, P&lt;0.0001). Double homozygote resistant genotype (RR/RR) were even more able to survive than any other genotype combination showing an additive effect. This study highlights the risk that pyrethroid resistance escalation poses to the efficacy of other classes of insecticides. Available metabolic resistance DNA-based diagnostic assays should be used by control programs to monitor cross-resistance between insecticides before implementing new interventions