Periodic Reporting for period 1 - Finding VENomS (Venom Evolution in Nemerteans: Connecting Functional Morphology, Gene Expression and Proteome through Spatial Omics)
Période du rapport: 2019-10-01 au 2021-09-30
Animal venoms are key adaptations that have evolved independently in many taxa to assist in defence, predation and competition. Venoms are some of the most complex biochemical secretions known in nature, but despite this complexity, there is a high degree of convergence in toxin structure and targets, making venomous organisms great model systems to investigate areas as diverse as molecular evolution, functional convergence and drug discovery. However, the processes underlying toxin and venom evolution remain poorly understood, particularly in invertebrates including ribbon worms (Nemertea), active predators that use toxins for defense and predation. This project investigated venom evolution in Nemertea using an integrative evolutionary venomics approach. It leveraged a transcriptomic-proteomic approach referred to as proteogenomics, combining RNA-seq differential gene expression analyses (DGE) and tandem mass spectrometry-based proteomics (MS/MS) to determine venom composition, and integrated this information with expression and functional morphology data derived from spatial omics, both Spatial Transcriptomics (ST) and spatial proteomics (MALDI imaging), and transmission electron microscopy (TEM). This project has advanced our understanding of ribbon worm venom systems, and shed new light into the true diversity of animal venoms and their evolution. It has led to the generation of fundamental molecular resources (e.g. over 200GB of RNAseq data) and to the development of novel technologies (i.e. spatial transcriptomics and MALDI-IMS) for the investigation of the spatial distribution of genes and proteins within ribbon worm tissues. These resources have allowed the identification of more than 100 putative novel toxins used for defence and predation in different lineages of ribbon worms.
The specific work carried out to achieve the objectives of the project is summarized below:
Work Package 1: Sample collection
A field expedition was carried out in which a total of 28 replicated samples of the species Lineus longissimus, Lineus lacteus, Cerebratulus marginatus, Paranemertes peregrina and Amphiporus formidabilis were collected. Samples of the hoplonemerteans Antarctonemertes valida and Antarctonemertes riesgoae were already available. These samples were preserved in RNAlater for RNA sequencing, flash frozen for proteomics and ST, and in glutaraldehyde and ethanol for TEM and SEM respectively.
Work package 2: Proteogenomics
For all species, two tissues (i.e. proboscis and posterior end of body) from three individuals per species (biological replicates) have been sequenced to generate RNAseq data for Differential Gene Expression (DGE) analyses. The fellow generated cDNA libraries for each replicate, that were sequenced with Illumina and assembled with Trinity v2.4.0 into a reference transcriptome. The individual libraries were mapped to the reference transcriptome to calculate expression levels and perform, DGE analyses with edgeR. In addition, mass spectrometry-based proteomic data has been generated for L. longissimus and A. valida, producing a catalogue of ca. 100 candidate toxins validated through proteotranscriptomics.
Work package 3: Spatial Omics
The heteronemerteans L. longissimus and C. marginatus, and the hoplonemertean A. valida have been analyzed following a spatial omics approach, including ST and spatial proteomics. MALDI-IMS color maps have been created for tissue sections including the proboscis of L. longissimus and A. valida, revealing the spatial distribution of venom peptides. The fellow did a research stay with Dr Stefania Giacomello at SciLifeLab (Stockholm, Sweden) where she optimized ST for ribbon worms. She analysed 3 replicates of L. longissimus and C. marginatus obtaining 3.5 and 6 million UMIs per replicate, respectively. This data was used to resolve mRNA spatial distribution and identify putative novel ribbon worm toxins, including a novel paralytic predatory toxin.
Work package 4: Morphology
Samples of L. longissimus proboscis have been imaged with TEM to investigate the structure of the venom secreting system. A complete proboscis, and anterior and posterior sections of a proboscis placed in ethanol to induce venom discharge, were fixed with glutaraldehyde and osmium tetroxide, dehydrated with acetone and embedded in resin for ultrathin sectioning and TEM imaging at MNCN-CSIC. Samples of L. longissimus, C. marginatus, and A. valida have also been processed.
Work package 5: Evolution
Homologs of the putative toxins identified have been searched across Nemertea, and molecular evolution analyses have been completed. The putative toxins identified in A. valida were used to search nemertean transcriptomes, identifying 32 orthogroups with sequences form other nemertean species. Of the 32 orthologous toxin clusters, 9 are common to all species analyzed including representatives of Palaeonemertea, Pilidiophora and Hoplonemertea, 2 are shared by Pilidiophora and Hoplonemertea, and 21 are lineage-specific, including only hoplonemertean sequences. This suggests that venom evolution is divergent across lineages, with a high proportion of lineage-specific toxins tuned to particular predatory habits.
Work Package 1: Sample collection
A field expedition was carried out in which a total of 28 replicated samples of the species Lineus longissimus, Lineus lacteus, Cerebratulus marginatus, Paranemertes peregrina and Amphiporus formidabilis were collected. Samples of the hoplonemerteans Antarctonemertes valida and Antarctonemertes riesgoae were already available. These samples were preserved in RNAlater for RNA sequencing, flash frozen for proteomics and ST, and in glutaraldehyde and ethanol for TEM and SEM respectively.
Work package 2: Proteogenomics
For all species, two tissues (i.e. proboscis and posterior end of body) from three individuals per species (biological replicates) have been sequenced to generate RNAseq data for Differential Gene Expression (DGE) analyses. The fellow generated cDNA libraries for each replicate, that were sequenced with Illumina and assembled with Trinity v2.4.0 into a reference transcriptome. The individual libraries were mapped to the reference transcriptome to calculate expression levels and perform, DGE analyses with edgeR. In addition, mass spectrometry-based proteomic data has been generated for L. longissimus and A. valida, producing a catalogue of ca. 100 candidate toxins validated through proteotranscriptomics.
Work package 3: Spatial Omics
The heteronemerteans L. longissimus and C. marginatus, and the hoplonemertean A. valida have been analyzed following a spatial omics approach, including ST and spatial proteomics. MALDI-IMS color maps have been created for tissue sections including the proboscis of L. longissimus and A. valida, revealing the spatial distribution of venom peptides. The fellow did a research stay with Dr Stefania Giacomello at SciLifeLab (Stockholm, Sweden) where she optimized ST for ribbon worms. She analysed 3 replicates of L. longissimus and C. marginatus obtaining 3.5 and 6 million UMIs per replicate, respectively. This data was used to resolve mRNA spatial distribution and identify putative novel ribbon worm toxins, including a novel paralytic predatory toxin.
Work package 4: Morphology
Samples of L. longissimus proboscis have been imaged with TEM to investigate the structure of the venom secreting system. A complete proboscis, and anterior and posterior sections of a proboscis placed in ethanol to induce venom discharge, were fixed with glutaraldehyde and osmium tetroxide, dehydrated with acetone and embedded in resin for ultrathin sectioning and TEM imaging at MNCN-CSIC. Samples of L. longissimus, C. marginatus, and A. valida have also been processed.
Work package 5: Evolution
Homologs of the putative toxins identified have been searched across Nemertea, and molecular evolution analyses have been completed. The putative toxins identified in A. valida were used to search nemertean transcriptomes, identifying 32 orthogroups with sequences form other nemertean species. Of the 32 orthologous toxin clusters, 9 are common to all species analyzed including representatives of Palaeonemertea, Pilidiophora and Hoplonemertea, 2 are shared by Pilidiophora and Hoplonemertea, and 21 are lineage-specific, including only hoplonemertean sequences. This suggests that venom evolution is divergent across lineages, with a high proportion of lineage-specific toxins tuned to particular predatory habits.
Finding VENomS has generated fundamental molecular resources from ribbon worms for the investigation of the composition and function of their toxic secretions. These findings have been useful to understand the evolution of nemertean toxins, which is essential to advance knowledge on neglected animals venom systems and shed light into the true diversity of animal venoms. The results of this project are of interest to scientists, from evolutionary biologists to biomedical scientists, as well as to the general public. This work has also laid the foundation to expand the use of Spatial Transcriptomics to a wider diversity of organisms, beyond classical model systems. In fact, within the frame of Finding VENomS, ST has already been applied to two ribbon worm species (Nemertea), the bloodworm Glycera tridactyla (Annelida), the cone snail Conus ventricosus (Mollusca) and several species of sponges (Porifera).
The project led to 4 publications available at the time of writing, 1 manuscript under review and 4 more in advanced stages of writing. During the project, the fellow has presented communications in several scientific conferences, including an invited talk at the 20th World Congress of the International Society of Toxinology and a communication at the 1ST International Congress of the European Venom Network that was awarded with the Early Career Researcher’s Award for Best Oral Presentation. The fellow Dr Aida Verdes, has also participated in dissemination activities for the general public, including a ‘Nature Live Lates’ talk at the Natural History Museum and the ‘European Researchers Night’ at the Museo Nacional de Ciencias Naturales. This fellowship also led to important training and career development opportunities for Dr Aida Verdes, including additional bioinformatic skills, increased wet lab experience, opportunities to teach and communicate her science, and formal training in soft skills that allowed her to secure an EMBO Short-Term fellowship and a prestigious Juan de la Cierva Incorporación 3-year postdoctoral fellowship from the Spanish Government, with additional funding to continue her venomics research and advance her scientific career.
The project led to 4 publications available at the time of writing, 1 manuscript under review and 4 more in advanced stages of writing. During the project, the fellow has presented communications in several scientific conferences, including an invited talk at the 20th World Congress of the International Society of Toxinology and a communication at the 1ST International Congress of the European Venom Network that was awarded with the Early Career Researcher’s Award for Best Oral Presentation. The fellow Dr Aida Verdes, has also participated in dissemination activities for the general public, including a ‘Nature Live Lates’ talk at the Natural History Museum and the ‘European Researchers Night’ at the Museo Nacional de Ciencias Naturales. This fellowship also led to important training and career development opportunities for Dr Aida Verdes, including additional bioinformatic skills, increased wet lab experience, opportunities to teach and communicate her science, and formal training in soft skills that allowed her to secure an EMBO Short-Term fellowship and a prestigious Juan de la Cierva Incorporación 3-year postdoctoral fellowship from the Spanish Government, with additional funding to continue her venomics research and advance her scientific career.