Hox3 duplication and divergence in the Lepidoptera

Using the Speckled Wood Butterfly Pararge aegeria as the model species, this thesis presents the possible evolutionary significance of a set of duplications found in the Hox cluster of the Lepidoptera, called the Special Homeobox genes. An annotation of this duplicated cluster across a wide number of Lepidoptera was performed in order to assess patterns of duplication and loss across the order. The sequences recovered revealed a large amount of variation associated with the duplicate genes, indicating these are evolving very rapidly in different lineages. Patterns of sequence variation were examined to ascertain whether the observed variation was maintained due to selection at three separate levels of divergence: within the Ditrysia, within the more recently diverged Heliconius genus, and at the intraspecific level by quantifying nucleotide polymorphism within Pararge aegeria. Selective pressures were found to be operating between paralogous and orthologous genes, suggesting these have evolved, in part, under positive selection. The potential function of the duplicates was examined by means of CRISPR/Cas9 geneome editing, but revealed inconclusive results. Genome editing, however, was shown to be largely applicable to P. aegeria, and resulted in consistent mutations associated with wing patterning genes. The potential significance of the duplications for Lepidopeteran biology are discussed, as well as future applications for genome editing techniques in P. aegeria

Historical and current patterns of gene flow in the butterfly Pararge aegeria

Aim. We have investigated the phylogeography and genetic structure of the Speckled Wood butterfly (Pararge aegeria) across its entire distribution range and studied its dispersal both on mainland and across sea straits. The apparent lack of gene flow between Sardinia and Corsica was further investigated by means of mating experiments. Location. Europe and North Africa. Methods. We sampled 345 individuals and sequenced one mitochondrial gene (Cytochrome c Oxidase subunit I, COI) for all samples and two nuclear genes (wingless and zerknullt) for a subset of the specimens. A total of 22 females from Corsica and Sardinia were used to establish a series of crosses to investigate reproductive compatibility and were screened for the presence of Wolbachia. Bayesian inference (BI) and haplotype networks were employed to infer phylogenetic relationships and a Principal Coordinate Analysis (PCoA) was used to represent 4 geographical patterns of genetic diversity. Mating and courtship data were analysed using linear mixed effect models. Results. We detected two main COI lineages separated by the Mediterranean Sea and maintained over relatively short sea straits. While nuclear gene variation was generally in agreement with that of COI, this was not the case in all areas (e.g. Iberian Peninsula and Corsica/Sardinia). Mating experiments revealed no evidence of reproductive isolation between the lineages, nor clear relation to Wolbachia infection status. Main conclusions. We propose that following the post-glacial recolonisation of Europe, the ancestral COI lineage of P. aegeria was maintained in North Africa and Mediterranean islands, while a new lineage colonised from Eastern Europe, replacing and apparently outcompeting the ancestral variant. Several hypotheses are discussed that may explain the local discordance between the nuclear genes and COI, including sex-specific dispersal, selection and differential rates of gene evolution

Vertically transmitted rhabdoviruses are found across three insect families and have dynamic interactions with their hosts

A small number of free-living viruses have been found to be obligately vertically transmitted, but it remains uncertain how widespread vertically transmitted viruses are and how quickly they can spread through host populations. Recent metagenomic studies have found several insects to be infected with sigma viruses (Rhabdoviridae). Here, we report that sigma viruses that infect Mediterranean fruit flies (Ceratitis capitata), Drosophila immigrans, and speckled wood butterflies (Pararge aegeria) are all vertically transmitted. We find patterns of vertical transmission that are consistent with those seen in Drosophila sigma viruses, with high rates of maternal transmission, and lower rates of paternal transmission. This mode of transmission allows them to spread rapidly in populations, and using viral sequence data we found the viruses in D. immigrans and C. capitata had both recently swept through host populations. The viruses were common in nature, with mean prevalences of 12% in C. capitata, 38% in D. immigrans and 74% in P. aegeria. We conclude that vertically transmitted rhabdoviruses may be widespread in a broad range of insect taxa, and that these viruses can have dynamic interactions with their hosts

Early life of Neanderthals

The early onset of weaning in modern humans has been linked to the high nutritional demand of brain development that is intimately connected with infant physiology and growth rate. In Neanderthals, ontogenetic patterns in early life are still debated, with some studies suggesting an accelerated development and others indicating only subtle differences to modern humans. Here we report the onset of weaning and rates of enamel growth using an unprecedented sample set of three late (~70-50 ka) Neanderthals and one Upper Paleolithic modern human from Northeastern-Italy via spatially-resolved chemical/isotopic analyses and histomorphometry of deciduous teeth. Our results reveal that the modern human nursing strategy, with onset of weaning at 5-6 months, was present among these Neanderthals. This evidence, combined with dental development akin to modern humans, highlights their similar metabolic constraints during early life and excludes late weaning as a factor contributing to Neanderthals' demise

Cortex cis -regulatory switches establish scale colour identity and pattern diversity in Heliconius

In Heliconius butterflies, wing colour pattern diversity and scale types are controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes, cortex, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knockouts in multiple Heliconius species and show that cortex is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified cis-regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that cis-regulatory structural variation underlies these mimetic adaptations. Our work shows that cortex functionally controls scale colour fate and that its cis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion

Identification of methylation signatures to assess homologous recombination deficiency in breast cancer

DNA methylation is the most studied epigenetic modification in cancers. Methylation signatures from tissue samples and liquid biopsies can help in the identification of cancer subtypes and are used for prognostication and treatment decision. Homologous recombination deficiency (HRD) is a characteristic of some cancers related to inefficient DNA damage repair and is measured through the analysis of mutations in specific genes and global genomic aberrations. Breast cancers with HRD are more sensitive to certain treatments, therefore HRD evaluation is an important aid in treatment selection. In a publicly available dataset of breast cancer samples with matched genomic and methylation data (TCGA-BRCA), we identified sites with differential methylation between HRD-high and HRD-low samples. Using bioinformatic tools, we found methylation signatures that could identify HRD cancers, and we created different models (gaussian and binomial) that could classify breast cancer samples into HRD classes. We then validated the models in another independent dataset of breast cancers (SCAN-B), demonstrating the feasibility to use methylation signatures to infer HRD status. Given the feasibility of methylation tests in liquid biopsies even with low amount of cancer free DNA, these models could help in identifying breast cancer patients that would benefit from HRD-directed therapies using blood-based test, thus enhancing the ability to recognize those patients in the clinical setting

A long non-coding RNA at the cortex locus controls adaptive colouration in butterflies

Data repository for Livraghi et al (2024). "A long non-coding RNA at the cortex locus controls adaptive colouration in butterflies.

Pararge aegeria Hox3 sequences

A genomics dataset containing the sequences of 5 paralogous genes (zerknullt and four Shx genes) in the butterfly Pararge aegeria (Speckled Wood). Sequenced Individuals originate from a range of populations in Europe and North Africa and the dataset contains information as to where the populations come from. Detailed information on sequencing procedures has been provided