Evolution of an alternative genetic code in theProvidenciasymbiont of the haematophagous leechHaementeria acuecueyetzin

Strict blood-feeding animals are confronted with a strong B vitamin deficiency. Leeches from the Glossiphoniidae family, notably species ofPlacobdella, Placobdelloides, andHaementeriaare regarded as strict blood-feeders, and similarly to blood-feeding insects, have evolved specialised organs called bacteriomes to harbour symbiotic bacteria. Leeches of theHaementeriagenus are found exclusively in the Americas, with most species being South-American and only three found in North America. In these leeches, two pairs of globular bacteriomes attached to the oesophagus harbour intracellularly’CandidatusProvidencia siddallii’ bacteria. Previous work analysing a draft genome of theProvidenciasymbiont of the Mexican leechHaementeria officinalisshowed that, in this species, the bacteria hold a reduced genome with complete biosynthetic pathways for B vitamins. In this work, we aimed to expand our knowledge on the diversity and evolution ofProvidenciasymbionts ofHaementeriaspecies. For this purpose, we sequenced the genomes of theProvidenciasymbionts of the Mexican leechesHaementeria acuecueyetzinandHaementeria lopezi, as well as re-sequenced and closed the symbiont genome ofH. officinalis. We found that all genomes are highly syntenic, mirroring ancient insect endosymbionts and suggesting a conserved gene order at the start of theProvidencia-Haementeriaassociation. Additionally, we found B vitamin pathways to be conserved among these symbionts, pointing to a common biosynthetic machinery and low variation in this trait. Lastly and most notably, we found that the symbiont ofH. acuecueyetzinhas evolved an alternative genetic code, affecting a portion of its proteome and showing evidence of a rather recent and likely intermediate stage of genetic code reassignment.