Biology subjects

Canestro, C.

Publications and source records attributed to Canestro, C..

10 recordsLinked to original sources

bioRxiv2026

A cold-adapted Cas9 nuclease from the deep ocean

The abundance of CRISPR systems in bacteria and archaea fuels the search for Cas nucleases that perform where established enzymes fail. Deep Ocean 1 Cas9 (DO1Cas9) is to our knowledge the first Cas9 nuclease characterized from the deep ocean, recovered from a metagenomic sample at 1,500 m depth in the Atlantic Ocean. DO1Cas9 shows cold-adapted structural flexibility, thermal susceptibility, salt-dependent stability, higher specificity than SpCas9 and cleaves double-stranded DNA efficiently under cold conditions where mesophilic SpCas9 retains only residual activity. DO1Cas9 edits genomes in diverse organisms such as Caenorhabditis elegans, zebrafish, the marine tunicate Oikopleura dioica, and the malaria parasite Plasmodium falciparum. In the marine bacterium Marinomonas mediterranea, DO1Cas9 programmed against an invading phage reduced plaquing efficiency about 10-fold more than SpCas9. These results expand the CRISPR-Cas9 toolbox with a nuclease for cold-chain editing and marine biotechnology.

genetics↗

bioRxiv2026

Developmental genetic response of the zooplanktonic tunicate Oikopleura dioica to marine noise pollution.

BackgroundAnthropogenic noise is an emerging threat to marine ecosystems, yet its effects on marine invertebrates, particularly zooplanktonic species, remain poorly understood. Despite increasing evidence of behavioral and physiological impacts in invertebrates, the effects of noise on embryonic development and the molecular mechanisms underlying acoustic responses remain largely unexplored. Here, to address this gap, we investigated the impact of high-intensity underwater noise exposure on embryogenesis of the appendicularian tunicate Oikopleura dioica, a cosmopolitan zooplanktonic tunicate that plays important ecological roles in marine trophic webs and carbon cycling. Under lab-controlled conditions, we examined the effects of experimental noise exposure on early embryogenesis at both morphological and transcriptomic levels using RNA-seq in 8-cell (8c) and early tailbud (ETB) stages. ResultsNoise exposure produced no significant increase in embryo malformations compared to non-exposed controls, indicating substantial phenotypic resilience under laboratory conditions. Interestingly, transcriptomic analyses revealed a rapid molecular response of 70 differentially expressed genes (DEG) already detectable after only 30 minutes of exposure at the 8-cell stage, which became markedly amplified with 700 DEGs by the ETB stage. Together, differential expression, GO enrichment, and co-expression network analyses identified coordinated regulation of processes associated with membrane homeostasis, pyrimidine/CTP metabolism, extracellular matrix organization, cytoskeletal architecture, RNA regulation, translational control, proteostasis, mitochondrial metabolism, and developmental pathways. Importantly, both developmental stages precede the formation of differentiated mechanosensory structures, suggesting that the observed responses are unlikely to reflect conventional sound perception. ConclusionsThese findings provide the first molecular characterization of noise effects during O. dioica embryogenesis and reveal an unexpected molecular sensitivity of O. dioica embryos to underwater noise despite preserved morphological development. The transcriptional signatures support a mechanobiological framework in which acoustic exposure may directly perturb cellular mechanical homeostasis through membrane-and cytoskeleton-associated processes, triggering compensatory stress-adaptation responses involving proteostasis, RNA regulation, and metabolic reprogramming. Together, these findings establish O. dioica as a valuable emerging model for investigating the developmental and evolutionary consequences of acoustic pollution in marine ecosystems.

developmental biology↗

bioRxiv2025

Downstream effects of the ''Less, but More'' Fgf signaling in Oikopleura dioica: Fgf receptor expansion and RTK pathway simplification.

Fibroblast growth factor (FGF) signaling is central to chordate development and has been extensively remodeled in tunicates. Recent findings show that appendicularians have massively lost all ancestral chordate Fgf subfamilies except two, the Fgf9/16/20 and Fgf11/12/13/14 subfamilies, which in contrast have undergone a burst of lineage-specific duplications and diversification into novel paralogs, in an evolutionary scenario that we have named "Less, but More". Here, we investigate the downstream effects of the Fgf losses and duplications ion Fgf receptors (FgfRs) and intracellular RTK components in the appendicularian Oikopleura dioica. We show that the single ancestral FgfR gene has expanded into three paralogs (FgfRa-c), which are conserved across cryptic O. dioica species, yet highly divergent from other chordates. Despite strong sequence divergence, structural modeling indicates preservation of canonical FgfR architecture. Expression analyses reveal distinct spatiotemporal patterns: FgfRa and FgfRb are maternally supplied and enriched in mesodermal derivatives, whereas FgfRc is restricted to neural and epithelial tissues. Genome surveys of downstream RTK pathways show conservation of core RAS/MAPK, PLC{gamma}/PKC, and PI3K/AKT cascades, but with losses of classical Ras genes and several adaptors, suggesting a lineage-specific simplification of transduction complexes. Transduction gene expression shifts from broad maternal ubiquity to tissue-specific domains, particularly in brain, notochord, muscle, and gonadal primordia throughout embryonic and larval development. Appendicularians appear as the only non-vertebrate chordate lineage that recapitulate the vertebrate-like FgfR expansion following Fgf ligands diversification. Downstream components, however, evolved more conservatively, tending toward simplification, reinforcing the view that appendicularians generate signaling innovation despite extensive gene loss.

evolutionary biology↗

bioRxiv2025

Evolution of Tunicate lifestyles shaped by Myosin heavy chain gene duplications, losses, and the diversification of tail muscle cell identities in Oikopleura dioica

Tunicates offer a suitable system to study the evolutionary mechanisms underlying lifestyle transitions, ranging from the biphasic lifestyle of ascidians alternating swimming larva with sessile adults, to the fully free-swimming appendicularians. The appendicularian Oikopleura dioica, despite having only ten pairs of tail muscle cells, roughly half than in ascidian larvae, exhibits a remarkably rich repertoire of behaviours required for house inflation, swimming, nodding, and the rhythmic water pumping through the house. This apparent paradox suggests that functional diversification might have arose not from increased cell number, but from molecular specialization within a minimal musculature. To address this question, here, we reconstruct the evolution of the Myosin class II heavy-chain (Myh) gene family across tunicates and generated a developmental expression atlas of all O. dioica Myh genes. Phylogenetic analysis reveals that the cardio-paraxial Myh subfamily duplicated into two tunicate-specific subfamilies, followed by independent bursts of paralogue duplications in ascidians and appendicularians. In O. dioica, two distinct Myh-Scp-Tb paralogs show cardiac expression, and surprisingly, combinations of different Myh-Scp-Tb paralogues define multiple muscle cell identities along the tail. The innovation of this anteroposterior muscle regionalization associated to the Myh-Scp-Tb expansion provides a plausible mechanism for fine-scale modulation of contractile properties within a minimal musculature, helping to resolve the paradox of "fewer cells, but more contractile properties" in appendicularian tail movements. Conversely, the loss of the Myh-Sj/bw gene, which in ascidians is expressed with post-metamorphic body-wall and siphon muscles, is consistent with a pattern of regressive evolution associated to the absence of trunk muscles in O. dioica. This finding supports the view that the appendicularian lifestyle is a secondary derived condition, and that the last common ancestor of tunicates likely possessed an ascidian-like biphasic lifestyle. Together, our findings indicate that both gene-family duplication and gene loss have shaped the evolution of appendicularian tail muscle, enabling the emergence of complex tail-driven behaviours, and offering new insights into the ancestral lifestyle of tunicates.

evolutionary biology↗

bioRxiv2025

CORAL: Accurate annotation of compact genomes using long-read RNA-seq, demonstrated in Oikopleura dioica

The burst of high-quality genome assemblies has created an urgent need to improving genome annotation tools, especially for problematic species such as those with compact, fast-evolving genomes in which standard annotation tools underperform when facing short intergenic regions, overlapping UTRs, non-canonical splicing and widespread operons. We present CORAL (Compact-genome Oriented RNA-based Annotation using Long-reads), a long-read RNA-seq-based workflow for de novo annotation of compact eukaryotic genomes, together with GAMBA (Gene Aggregation tool for Multicistronic Block Annotation), a Rust-based module that detects polycistronic transcriptional units directly from GTF annotations and is integrated within CORAL. Using the exceptionally compact chordate genome of Oikopleura dioica as a case study, CORAL increases the number of annotated genes, improves gene-model completeness and reduces chimeric predictions relative to existing annotations, while GAMBA recovers thousands of operons, most of which are independently supported by splice-leader data. Together, CORAL and GAMBA provide an accurate, scalable framework for annotating compact, structurally atypical genomes using long-read transcriptomic data alone. Availability and ImplementationCORAL and GAMBA are open source and available at https://github.com/EvoDevoGenomics-UB/CORAL and https://github.com/nurie05/gamba-tool.

bioinformatics↗

bioRxiv2024

Less, but more: new insights from appendicularians on chordate Fgf evolution and the divergence of tunicate lifestyles.

The impact of gene loss on the divergence of taxa and the generation of evolutionary innovations is a fundamental aspect of Evolutionary Biology that remains unclear. Here, using the evolution of the Fibroblast Growth Factors (FGFs) in appendicularians as a case study, we investigate how gene losses have influenced the evolution of chordates, especially the divergence among tunicates. Our work reveals an unprecedented case of massive losses of all Fgf gene subfamilies, except for the Fgf9/16/20 and Fgf11/12/13/14, which in turn suffered two bursts of gene duplications. Phylogenetic inferences and genomic analyses of gene synteny conservation, gene architecture, alternative splicing and protein 3D-structure have allowed us to reconstruct the history of appendicularian Fgf genes in the context of chordate evolution, providing compelling evidence supporting the paracrine secreting functions and the intracellular functions of the Fgf9/16/20 and Fgf11/12/13/14 subfamilies, respectively. Exhaustive analysis of developmental Fgf expression in Oikopleura dioica as a model for appendicularians reveals a paradigmatic case of what could be referred as "less, but more", providing a conceptual evolutionary framework characterized by four associated evolutionary patterns: conservation of ancestral Fgf expression domains; function shuffling between paralogs upon gene loss; innovation of new expression domains after the bursts of Fgf duplications; and the extinction of Fgf functions linked to gene losses. The findings of this work allow us to formulate novel hypotheses about the potential impact of losses and duplications of Fgf genes on the transition from an ancestral ascidian-like biphasic lifestyle to a fully free-living style of appendicularians. These hypotheses include the massive co-option of Fgf genes for the patterning of the oikoblast responsible of the house architecture, and for the development of the tail fin; the recruitment of Fgf11/12/13/14 genes into the evolution of a new mouth, and their role modulating neuronal excitability; the evolutionary innovation of an "anterior tail" FGF signaling mesodermal source upon the loss of retinoic acid signaling; and the potential link between the loss of Fgf7/10/22 and Fgf8/17/18 and the loss of drastic metamorphosis, mesenchymal cells and lack of tail absorption in appendicularians, in contrast to ascidians.

evolutionary biology↗

bioRxiv2024

Developmental toxicity of pre-production plastic pellets affects a large swathe of invertebrate taxa

Microplastics pose risks to marine organisms through ingestion, entanglement, and as carriers of toxic additives and environmental pollutants. Plastic pre-production pellet leachates have been shown to affect the development of sea urchins and, to some extent, mussels. The extent of those developmental effects on other animal phyla remains unknown. Here, we test the toxicity of environmental mixed nurdle samples and new PVC pellets for the embryonic development or asexual reproduction by regeneration of animals from all the major animal superphyla (Lophotrochozoa, Ecdysozoa, Deuterostomia and Cnidaria). Our results show diverse, concentration-dependent impacts in all the species sampled for new pellets, and for molluscs and deuterostomes for environmental samples. Embryo axial formation, cell specification and, specially, morphogenesis seem to be the main processes affected by plastic leachate exposure. Our study serves as a proof of principle for the potentially catastrophic effects that increasing plastic concentrations in the oceans and other ecosystems can have across animal populations from all major animal superphyla.

developmental biology↗

bioRxiv2023

Extreme genome scrambling in cryptic Oikopleura dioica species

Genes are not randomly distributed throughout chromosomes. How gene order evolves and how selective constraints act to preserve or vary gene order, both at the macrosyntenic level of whole chromosomes or microsyntenic level of gene blocks, are central questions of evolutionary biology and genomics that remain largely unsolved. Here, after sequencing several genomes of the appendicularian tunicate Oikopleura dioica from different locations around the globe, we show an unprecedented amount of genome scrambling in animals with no obvious morphological differences, consistent with cryptic speciation. Our assemblies suggest that all members of this clade possess a common 3-chromosome karyotype, and that different species largely preserve gene content, despite the presence of thousands of rearrangements in gene order. The movements of genes are largely restricted to chromosome arms and sex-specific regions, which appear to be the primary unit of macrosynteny conservation, and examples of these within-arm movements can be seen in the Hox and Fgf gene families. Our approach employing whole-genome alignments demonstrates that segments containing protein-coding elements tend to be preserved at the microsyntenic scale, consistent with strong purifying selection, with appreciably less preservation of non-coding elements. Unexpectedly, scrambling did not preserve operon structure across species, suggesting an absence of selective pressure to maintain operon structure. As well, genome scrambling does not occur uniformly across all chromosomes, as short chromosome arms possess shorter genes, smaller operons, more breakpoints, and elevated dN/dS values compared to long chromosome arms. Estimation of divergence times among the cryptic O. dioica lineages yielded an estimated breakpoint accumulation rate of 6 to 25 breakpoints per megabase per million years, which is an order of magnitude higher than the rates for other ascidian tunicates or Drosophila species. Therefore, O. dioica appears to be an attractive animal system to unravel the mechanisms that underlie gene order and synteny conservation, as well as exploring the limits of genome scrambling without an apparent impact on phenotypic evolution.

genomics↗

bioRxiv2022

The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe

Appendicularian tunicates are some of the most abundant mesozooplankton organisms with key roles in marine trophic webs and global carbon flux. Like most appendicularians with cosmopolitan distributions, Oikopleura dioica Fol, 1872 is considered a single species worldwide based on morphological features that distinguish them from other appendicularians. Despite their abundance however, there are still only [~]70 described appendicularian species, compared with over 2,800 ascidian tunicates. Here we perform a molecular phylogenetic, morphological, and reproductive assessment of O. dioica specimens collected from the Ryukyu Archipelago, mainland Japan, and Europe. The specimens are morphologically very similar, with only detailed examination of the oikoplastic epithelium and quantitative measurements revealing minor distinguishing characteristics. Phylogenetic analyses of the ribosomal gene loci and mitochondrial cytochrome oxidase I (COI) gene strongly indicate that they form three separate genetic clades despite their morphological similarities. Finally, in vitro crosses between the Ryukyu and mainland Japanese specimens show total prezygotic reproductive isolation. Our results reveal that the current taxonomic O. dioica classification likely hides multiple cryptic species, highlighting the genetic diversity and complexity of their population structures. Cryptic organisms are often hidden under a single species name because their morphological similarities make them difficult to disinguish and their correct identification is fundamental to understanding Earths biodiversity. O. dioica is an attractive model to understand how morphological conservation can be maintained despite pronounced genetic divergence.

evolutionary biology↗

bioRxiv2021

Cardiopharyngeal deconstruction and ancestral tunicate sessility

A key problem in understanding chordate evolution has been the origin of sessility of ascidians, and whether the appendicularian free-living style represents a primitive or derived condition of tunicates. To address this problem, we performed comprehensive developmental and genomic comparative analyses of the cardiopharyngeal gene regulatory network (GRN) between appendicularians and ascidians. Our results reveal that the cardiopharyngeal GRN has suffered a process of evolutionary deconstruction with massive ancestral losses of genes (Mesp, Ets1/2, Gata4/5/6, Mek1/2, Tbx1/10, and RA- and FGF-signaling related genes) and subfunctions (e.g. FoxF, Islet, Ebf, Mrf, Dach and Bmp signaling). These losses have led to the deconstruction of two modules of the cardiopharyngeal GRN that in ascidians are related to early and late multipotent state cells involved in lineage fate determination towards first and secondary heart fields, and siphon muscle. Our results allow us to propose an evolutionary scenario, in which the evolutionary deconstruction of the cardiopharyngeal GRN has had an adaptive impact on the acceleration of the developmental cardiac program, the redesign of the cardiac architecture into an open-wide laminar structure, and the loss of pharyngeal muscle. Our findings, therefore, provide evidence supporting that the ancestral tunicate had a sessile ascidian-like lifestyle, and points to the deconstruction of the cardiopharyngeal GRN in appendicularians as a key event that facilitated the evolution of their pelagic free-living style connected to the innovation of the house.

evolutionary biology↗