Final Report Summary - CARISYM (Evolution and ecophysiology of Cand. Riegeria galateiae, the thiotrophic alphaprotebacterial symbiont of a catenulid flatworm)
Flatworms of the enigmatic genus Paracatenula (Catenulida, Plathyhelminthes) live in shallow water marine sediments. They are part of the interstitial meiofauna at the oxic-anoxic interface and they occur in all tropical to warm temperate oceans. Adult Paracatenula lack a mouth and a gut. Instead, a parenchymatic “trophosome” containing intracellular symbionts fills most of the body. The symbionts are thiotrophic chemosynthetic Alphaproteobacteria (Cand. Riegeria) that are generally large bacteria (3-10µm in size) and have several light refractile storage vesicles that cause a whitish appearance of the trophosome region. The host and symbiont phylogenies based on 16S rRNA (symbiont) and 18S+28S rRNA (host) gene analyses are completely congruent, which indicates strict vertical transmission of the symbionts. Paracatenula flatworms have high regeneration capabilities and can reproduce asexually via fragmentation. Fragments of worms without a rostrum (head) can completely regenerate a new head within two weeks.
During our sampling campaigns in the Caribbean and the Mediterranean we were able to collect material of four different species of Paracatenula that cover the complete known diversity of the genus. Initial test cultivation experiments with the Caribbean species Paracatenula galateia had low recovery rates after up to five day treatments. We newly developed thermally controlled incubation setups and we successfully recovered all treated animals of the Mediterranean species Paracatenula sp. ‘standrea’, which is an up to 15mm long and highly abundant species found in the bay of St’Andrea, Elba, Italy. We have collected a transmission electron microscopic and light microscopic dataset and the manuscript for the formal description of this species is currently in preparation with our collaboration partners at the University of Vienna and will be submitted to Zootaxa.
Together with collaborators at the Max-Plank Genome Centre in Cologne, Germany we developed a library preparation protocol that enables the sequencing of single worm metagenomic libraries based on as little as 1ng of template DNA. The samples that were deeply sequenced on illumina HiSeq and MiSeq platforms yielded closed (2 species) and almost closed (2 species) draft genomes for the Cand. Riegeria symbionts of the four different Paracatenula species. The draft genomes of Cand. Riegeria range from 1.3 to 1.5Mb Mb in size, have a GC content of 49-50%% and encode 1254 to 1520 proteins, 37 to 39 tRNAs and a single rRNA operon. In contrast to previous 16 rRNA gene based analyses, a phylogenomic analysis based on 172 single copy genes (34k amino-acid alignment) shows that the 4 Cand. Riegeria symbionts form a distinct clade within the Rhodospirillales, most closely related to a group of several Rhodospirillacae genera including Rhodospirillum. The genomic data shows that all Cand. Riegeria have the potential for chemolithoautotrophy based on the oxidation of reduced sulfur including sulfide and thiosulfate as well as for the fixation of carbon dioxide via ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Different forms of RuBisCO (type I or II) are encoded by the symbionts of sympatric hosts, suggesting a niche differentiation between different species in regards to the optimal levels of oxygen via the symbiont. A manuscript on the phylogenomic placement and comparative genomic analysis of the symbionts is under preparation and will be submitted to the ISME journal in 2015.
We assessed what role the symbionts play in different physiological states of the host and especially during host regeneration using incubation experiments and expression profiling with RNA-Seq in Paracatenula sp. ‘standrea’. We developed a novel protocol for the RNA-Seq library construction to be able to work with the limited pg to low ng range total RNA amounts that can be achieved from single Paracatenula individuals and small e.g. head regenerating fragments. Compared to the expression levels of the symbionts genes in untreated animals, 10 of 1184 predicted symbiont genes are differentially expressed by the symbionts of animals that only need to perform wound healing processes 5 days after amputation. In stark contrast to this, 188 symbiont genes are differentially expressed in animals that are regenerating their head including the brain 5 days after amputation. Several of the significantly up-regulated genes are involved in pathways for essential amino acid synthesis (e.g. threonine or leucine) or cofactors (e.g. ubiquinone) and suggest a concerted effort of the Paracatenula holobiont to cope with the costly process of head and brain regeneration. A manuscript on the host regeneration dependent expression modulation of the Cand. Riegeria symbionts is currently in preparation and will be submitted to Nature in 2015.
Our experiments to trace the autotrophic fixation of CO2 in the Cand. Riegeria symbionts and the carbon transfer from Cand. Riegeria to the host using radioactively labeled bicarbonate showed peak levels of carbon fixation within the first few hours. Preliminary nanoSims experiments could not track the carbon fixation on a single cell level, but microautoradiography based experiments surprisingly showed that only very few cells in a given region of the worm were active during the labeling period. The master student that conducted these experiments within the framework of this project has now started a PhD project in our research group to continue to work on the carbon-fixation and carbon transfer in Paracatenula using microautoradiography and GC-MS based metabolomics. A manuscript that acknowledges the initial funding through this project will be submitted in 2015 or 2016.
As a spin-off-project from the field trips to the Caribbean and the Mediterranean several thiotrophic symbionts harbouring nematodes of the subfamily Stilbonematinae that co-occur with Paracatenula were newly discovered in the Caribbean. Two manuscripts with 3 Nematode species descriptions and a manuscripts detailing the molecular details of the conspicuous division modes of large filamentous Stilbonematinae symbionts were published with collaboration partners of the University of Vienna and the University of Amsterdam.
The multiple processed NGS-libraries and the contamination problems inherent with low-template concentration libraries led to the development of a high-performance software solution to screen for the phylogenetic composition of NGS data using the rRNA SSU gene. This software phyloFlash will be made available to the public and a manuscript describing the software will be submitted to the journal Bioinformatics in 2014
During our sampling campaigns in the Caribbean and the Mediterranean we were able to collect material of four different species of Paracatenula that cover the complete known diversity of the genus. Initial test cultivation experiments with the Caribbean species Paracatenula galateia had low recovery rates after up to five day treatments. We newly developed thermally controlled incubation setups and we successfully recovered all treated animals of the Mediterranean species Paracatenula sp. ‘standrea’, which is an up to 15mm long and highly abundant species found in the bay of St’Andrea, Elba, Italy. We have collected a transmission electron microscopic and light microscopic dataset and the manuscript for the formal description of this species is currently in preparation with our collaboration partners at the University of Vienna and will be submitted to Zootaxa.
Together with collaborators at the Max-Plank Genome Centre in Cologne, Germany we developed a library preparation protocol that enables the sequencing of single worm metagenomic libraries based on as little as 1ng of template DNA. The samples that were deeply sequenced on illumina HiSeq and MiSeq platforms yielded closed (2 species) and almost closed (2 species) draft genomes for the Cand. Riegeria symbionts of the four different Paracatenula species. The draft genomes of Cand. Riegeria range from 1.3 to 1.5Mb Mb in size, have a GC content of 49-50%% and encode 1254 to 1520 proteins, 37 to 39 tRNAs and a single rRNA operon. In contrast to previous 16 rRNA gene based analyses, a phylogenomic analysis based on 172 single copy genes (34k amino-acid alignment) shows that the 4 Cand. Riegeria symbionts form a distinct clade within the Rhodospirillales, most closely related to a group of several Rhodospirillacae genera including Rhodospirillum. The genomic data shows that all Cand. Riegeria have the potential for chemolithoautotrophy based on the oxidation of reduced sulfur including sulfide and thiosulfate as well as for the fixation of carbon dioxide via ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Different forms of RuBisCO (type I or II) are encoded by the symbionts of sympatric hosts, suggesting a niche differentiation between different species in regards to the optimal levels of oxygen via the symbiont. A manuscript on the phylogenomic placement and comparative genomic analysis of the symbionts is under preparation and will be submitted to the ISME journal in 2015.
We assessed what role the symbionts play in different physiological states of the host and especially during host regeneration using incubation experiments and expression profiling with RNA-Seq in Paracatenula sp. ‘standrea’. We developed a novel protocol for the RNA-Seq library construction to be able to work with the limited pg to low ng range total RNA amounts that can be achieved from single Paracatenula individuals and small e.g. head regenerating fragments. Compared to the expression levels of the symbionts genes in untreated animals, 10 of 1184 predicted symbiont genes are differentially expressed by the symbionts of animals that only need to perform wound healing processes 5 days after amputation. In stark contrast to this, 188 symbiont genes are differentially expressed in animals that are regenerating their head including the brain 5 days after amputation. Several of the significantly up-regulated genes are involved in pathways for essential amino acid synthesis (e.g. threonine or leucine) or cofactors (e.g. ubiquinone) and suggest a concerted effort of the Paracatenula holobiont to cope with the costly process of head and brain regeneration. A manuscript on the host regeneration dependent expression modulation of the Cand. Riegeria symbionts is currently in preparation and will be submitted to Nature in 2015.
Our experiments to trace the autotrophic fixation of CO2 in the Cand. Riegeria symbionts and the carbon transfer from Cand. Riegeria to the host using radioactively labeled bicarbonate showed peak levels of carbon fixation within the first few hours. Preliminary nanoSims experiments could not track the carbon fixation on a single cell level, but microautoradiography based experiments surprisingly showed that only very few cells in a given region of the worm were active during the labeling period. The master student that conducted these experiments within the framework of this project has now started a PhD project in our research group to continue to work on the carbon-fixation and carbon transfer in Paracatenula using microautoradiography and GC-MS based metabolomics. A manuscript that acknowledges the initial funding through this project will be submitted in 2015 or 2016.
As a spin-off-project from the field trips to the Caribbean and the Mediterranean several thiotrophic symbionts harbouring nematodes of the subfamily Stilbonematinae that co-occur with Paracatenula were newly discovered in the Caribbean. Two manuscripts with 3 Nematode species descriptions and a manuscripts detailing the molecular details of the conspicuous division modes of large filamentous Stilbonematinae symbionts were published with collaboration partners of the University of Vienna and the University of Amsterdam.
The multiple processed NGS-libraries and the contamination problems inherent with low-template concentration libraries led to the development of a high-performance software solution to screen for the phylogenetic composition of NGS data using the rRNA SSU gene. This software phyloFlash will be made available to the public and a manuscript describing the software will be submitted to the journal Bioinformatics in 2014