**Work Package 1**
Based on the previous discovery of the reduced nature of the genome of Pr. siddallii, symbiont of Haementeria officinalis, and its conserved presence across different Haementeria species, further sequencing of bacteriomes from species of this clade was performed.
We found that similarly to other long-term vertically transmitted symbionts, the Providencia siddallii ancestor evolved a small gene-dense mobile element-free genome. Most surprisingly, we found that a “rare” genetic code reassignment (11->4) evolved in the symbiont lineage associated to the leech H. acuecueyetzin (fun fact! the specific epithet means “leech” in Nahuatl). This reassignment means that the STOP codon ‘UGA’ is now coding for tryptophan! Moreover, features form the symbiont genomes suggest an early stage of reassignment and an ancestral propensity for the P. siddallii bacteria to undergo such a reassignment, e.g. the ancestral accumulation of ‘UGA’ codons in essential genes. In addition, this work also resulted in the reconstruction of the mitochondrial genome of the leech Haementeria aceuceuyetzin, resulting in its phylogenetic positioning (based on its mitochondrial proteins) within the Haementeria genus. This small side-project also served as a platform for the training of a Mexican student in genome assembly and annotation, insuring a two-way transfer of knowledge with the Mexican partner. This work was a great collaboration with long-lived colleagues Dr. Alejandro Oceguera Figueroa (Biology Institute, UNAM, Mexico) and Dr. Sebastian Kvist (Natural History Museum, Stockholm, Sweeden).
Lastly, we have also uncovered a novel symbiotic association between a yet-undescribed Haementeria sp., where the ancestral Providencia symbiont has beenreplaced by a novel Pluralibacter symbiont, a genus generally known as a soil- and water-dweller, hinting towards its ecological origin. This work is currently being drafted nd expected to be submitted by the end of the year.
**Work Package 2**
In this work package, and through a collaboration with Dr. Sebastian Kvist (Swedish Natural History Museum), we sequenced and analysed genomes of six 'Reichenowia' endosymbionts of Placobdella leeches. This work is currently being drafted and a submission is expected early 2024.
For this work package, the genomes of six 'Reichenowia' symbionts were sequences, and phylogenetic inference and comparative genomics were used to infer the origin and evolution of this symbiont lineage. We surprisingly found that this symbiont lineage actually includes the free-living Gellertiella hungarica, isolated from water from the Gellert baths (Hungary). In addition, 16S rRNA gene identity and ANI values suggest that these strains are all part of the species G. hungarica. However, genomic characteristics are quite different across the strains, suggesting the symbionts of Placobdella leeches have actually either evolved anew from closely related free-living strains, or the symbionts have undergone different histories of genome reduction following the diversification of their hosts. An analysis of 16S rRNA genes belonging to the same species revealed that this symbiont is in fact distributed particularly in water- and soil-environments, while still being a rare taxon. These results open the door for future studies on the evolution of this symbiont lineage, especially in the light of the transition form a free-living organism to an obligate endosymbiont.
**Work Package 3**
In this work package, we aimed to sequence the genome of the historical European medicinal leech (Hirudo medicinalis) and its associated digestive symbionts. The first draft of this genome was produced in collaboration with Dr. Peter trontelj (Slovenia) and Dr. Sebastian Kvist (Canada), and has already been published. We determined the repertoire of anticoagulant proteins from this historical and medically important leech and managed to not only identify 18 well-known anticoagulant enzymes and their previously unknown copy number in the leech’s genome but also a previously unknown set of 23 putative anticoagulants. In all, the large anticoagulant repertoire that H. medicinalis encodes suggests that the potency of the leech’s saliva in counteracting the clotting cascade of blood is dependent on a larger than previously known.
In a second stage, we used a combination of Nanopore long-read and Illumina short-read sequencing to produce high-quality genomes for the medicinal leech and another five leeches. This sequencing resulted not only in a recovery of the leeches' genomes, but also those of a part of their associated bacteria. We have identified most symbionts form the digestive system of Hirudo verbana in its congener Hirudo medicinalis. Current work is aimed at reconstructing these genomes as well as those from closely and distantly related leeches, targeted at analysing the metabolic function and evolutionary conservation (or not) of the core microbiota of these species.
This work has been disseminated in 4 scientific conferences and a press release (for the medicinal leech's genome). In addition, the organisation of a conference and a symposium within a conference was done.
