Mitochondrial Genomics: structure, inheritance and phylogenetic utility of the Mitochondrial genome in Bivalvia and Insecta

This PhD Thesis is the result of my research activity in the last three years. My main research interest was centered on the evolution of mitochondrial genome (mtDNA), and on its usefulness as a phylogeographic and phylogenetic marker at different taxonomic levels in different taxa of Metazoa. From a methodological standpoint, my main effort was dedicated to the sequencing of complete mitochondrial genomes, and the approach to whole-genome sequencing was based on the application of Long-PCR and shotgun sequences. Moreover, this research project is a part of a bigger sequencing project of mtDNAs in many different Metazoans’ taxa, and I mostly dedicated myself to sequence and analyze mtDNAs in selected taxa of bivalves and hexapods (Insecta). Sequences of bivalve mtDNAs are particularly limited, and my study contributed to extend the sampling. Moreover, I used the bivalve Musculista senhousia as model taxon to investigate the molecular mechanisms and the evolutionary significance of their aberrant mode of mitochondrial inheritance (Doubly Uniparental Inheritance, see below). In Insects, I focused my attention on the Genus Bacillus (Insecta Phasmida). A detailed phylogenetic analysis was performed in order to assess phylogenetic relationships within the genus, and to investigate the placement of Phasmida in the phylogenetic tree of Insecta. The main goal of this part of my study was to add to the taxonomic coverage of sequenced mtDNAs in basal insects, which were only partially analyzed.

Mitochondrial inheritance and germ line determination in a bivalve species with Doubly Uniparental Inheritance (DUI)

Mitochondria are inherited maternally in most metazoans. However, in some bivalves, two mitochondrial lineages are present: one transmitted through eggs (F), the other through sperm (M). This is called Doubly Uniparental Inheritance (DUI). During male embryo development, spermatozoon mitochondria aggregate and end up in the primordial germ cells, while they are dispersed in female embryos. The molecular mechanisms of segregation patterns are still unknown. In the DUI species Ruditapes philippinarum, I examined sperm mitochondria distribution by MitoTracker, microtubule staining and TEM, and I localized germ line determinants with immunocytochemical analysis. I also analyzed the gonad transcriptome, searching for genes involved in reproduction and sex determination. Moreover, I analyzed an M-type specific open reading frame that could be responsible for maintenance/degradation of M mitochondria during embryo development. These transcripts were also localized in tissues using in situ hybridization. As in Mytilus, two distribution patterns of M mitochondria were detected in R. philippinarum, supporting that they are related to DUI. Moreover, the first division midbody concurs in positioning aggregated M mitochondria on the animal-vegetal axis of the male embryo: in organisms with spiral segmentation this zone is not involved in further cleavages, so aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area where germ plasm is transferred, suggesting their contribution in male germ line formation. The finding of reproduction and ubiquitination transcripts led to formulate a model in which ubiquitination genes stored in female oocytes during gametogenesis would activate sex-gene expression in the early embryonic developmental stages (preformation). Only gametogenetic cells were labeled by in situ hybridization, proving their specific transcription in developing gametes. Other than having a role in sex determination, some ubiquination factors could also be involved in mitochondrial inheritance, and their differential expression could be responsible for the different fate of sperm mitochondria in the two sexes.