Coupling feeding activity, growth rates and molecular data shows dietetic needs of Ciona robusta (Ascidiacea, Phlebobranchia) in automatic culture plants.
ABSTRACT: The sea squirt Ciona robusta is a model organism characterized by a transparent body, exhibiting peculiar physiologic and evolutionary characters. In vitro fertilization and breeding of sea squirts is possible, in order to preserve consistent genetic pools. However, some aspects of its biology, as the feeding efficiency according to diet quantity and quality, are still scarcely known. Here we test the effects of three experimental diets on survival and growth, to detect physiological and molecular responses to various types of alimentary suspended particles and the effects of feed concentrations. We also aimed at determining rearing conditions able to limit handling operations, save artificial seawater and control water pollution. Molecular analyses of growth-related genes were performed to detect stressful effects due to feed quality and quantity. A strong effect of doses was highlighted, but water pollution may represent a major concern. A compound diet containing both live algae and non-live particles of a correct size is indispensable to assure development, low stress and high survival rates. Overall, our findings suggest protocols for an easier rearing of Ciona robusta in the laboratory, increasing the potentialities of these organisms as models for research.
Project description:Human-mediated dispersal interplays with natural processes and complicates understanding of the biogeographical history of species. This is exemplified by two invasive tunicates, Ciona robusta (formerly Ciona intestinalis type A) and C. intestinalis (formerly Ciona intestinalis type B), globally distributed and sympatric in Europe. By gathering new mitochondrial sequences that were merged with published datasets, we analysed genetic patterns in different regions, with a focus on 1) their sympatric range and 2) allopatric populations in N and S America and southern Europe. In the sympatric range, the two species display contrasting genetic diversity patterns, with low polymorphism in C. robusta supporting the prevalent view of its recent introduction. In the E Pacific, several genetic traits support the non-native status of C. robusta. However, in the NE Pacific, this appraisal requires a complex scenario of introduction and should be further examined supported by extensive sampling efforts in the NW Pacific (putative native range). For C. intestinalis, Bayesian analysis suggested a natural amphi-North Atlantic distribution, casting doubt on its non-native status in the NW Atlantic. This study shows that both natural and human-mediated dispersal have influenced genetic patterns at broad scales; this interaction lessens our ability to confidently ascertain native vs. non-native status of populations, particularly of those species that are globally distributed.
Project description:Molecular studies sometimes reveal evolutionary divergence within accepted species. Such findings can initiate taxonomic revision, as exemplified in the formerly recognized species Ciona intestinalis. While an increasing number of studies have examined the ecology, reproductive barriers and genetics of C. intestinalis and C. robusta, there are still much uncertainties regarding other species of this genus. Using experimental crosses and mitochondrial data, we investigated the evolutionary relationships among four native and introduced Ciona spp., found in sympatry in the Mediterranean Sea or English Channel. Outcome of 62 bi-parental reciprocal crosses between C. intestinalis, C. robusta, C. roulei and C. edwardsi showed that C. edwardsi is reproductively isolated from the other taxa, which is in agreement with its distinct location in the phylogenetic tree. Conversely, hybrids are easily obtained in both direction when crossing C. intestinalis and C. roulei, reinforcing the hypothesis of two genetically differentiated lineages but likely being from a same species. Altogether, this study sheds light on the evolutionary relationship in this complex genus. It also calls for further investigation notably based on genome-wide investigation to better describe the evolutionary history within the genus Ciona, a challenging task in a changing world where biological introductions are shuffling species distribution.
Project description:Whole-genome sequence data from samples of natural populations provide fertile grounds for analyses of intraspecific variation and tests of population genetic theory. We show that the urochordate Ciona savignyi, one of the species of ocean-dwelling broadcast spawners commonly known as sea squirts, exhibits the highest rates of single-nucleotide and structural polymorphism ever comprehensively quantified in a multicellular organism. We demonstrate that the cause for the extreme heterozygosity is a large effective population size, and, consistent with prediction by the neutral theory, we find evidence of strong purifying selection. These results constitute in-depth insight into the dynamics of highly polymorphic genomes and provide important empirical support of population genetic theory as it pertains to population size, heterozygosity, and natural selection.
Project description:BACKGROUND:Adaptive evolution is one of the crucial mechanisms for organisms to survive and thrive in new environments. Recent studies suggest that adaptive evolution could rapidly occur in species to respond to novel environments or environmental challenges during range expansion. However, for environmental adaptation, many studies successfully detected phenotypic features associated with local environments, but did not provide ample genetic evidence on microevolutionary dynamics. It is therefore crucial to thoroughly investigate the genetic basis of rapid microevolution in response to environmental changes, in particular on what genes and associated variation are responsible for environmental challenges. Here, we genotyped genome-wide gene-associated microsatellites to detect genetic signatures of rapid microevolution of a marine tunicate invader, Ciona robusta, during recent range expansion to the harsh environment in the Red Sea. RESULTS:The Red Sea population was significantly differentiated from the other global populations. The genome-wide scan, as well as multiple analytical methods, successfully identified a set of adaptive genes. Interestingly, the allele frequency largely varied at several adaptive loci in the Red Sea population, and we found significant correlations between allele frequency and local environmental factors at these adaptive loci. Furthermore, a set of genes were annotated to get involved in local temperature and salinity adaptation, and the identified adaptive genes may largely contribute to the invasion success to harsh environments. CONCLUSIONS:All the evidence obtained in this study clearly showed that environment-driven selection had left detectable signatures in the genome of Ciona robusta within a few generations. Such a rapid microevolutionary process is largely responsible for the harsh environmental adaptation and therefore contributes to invasion success in different aquatic ecosystems with largely varied environmental factors.
Project description:BACKGROUND:Previously published work has demonstrated that the LPS injection of Ciona robusta leads to the overexpression of a truncated form of an immune-related mRNA (C8short) by means of Ciona robusta (CR) alternative polyadenylation (APA) (CR-APA). METHODS:The 3D structure of the C8short-derived Ciona robusta chemo-attractive peptide (CrCP) was evaluated by homology modeling. The biological activity of the CrCP was studied in vitro using a primary human dermal cell line (HuDe). Real-Time PCR was used to investigate the expression levels of genes involved in cell motility. NF-?B signaling was studied by western blotting. RESULTS:In silico modeling showed that CrCP displayed structural characteristics already reported for a short domain of the vertebrate CRK gene, suggesting its possible involvement in cell migration mechanisms. In vitro assays demonstrated that CrCP was capable of inducing the motility of HuDe cells in both wound healing and chemo-attractive experiments. qPCR demonstrated the capability of CrCP to modulate the expression of the matrix metalloproteinase-7 (MMP-7) and E-cadherin genes. Finally, western blot analysis demonstrated that treatment with CrCP induced activation of the NF-?B signaling pathway. CONCLUSION:Our results describe the characterization of the 3D structure and chemo-attractive activity of an LPS-induced CrCP peptide from Ciona robusta.
Project description:The tunicate Ciona robusta is an emerging model system to study the evolution of the nervous system. Due to their small embryos and compact genomes, tunicates, like Ciona robusta, have great potential to comprehend genetic circuitry underlying cell specific gene repertoire, among different neuronal cells. Their simple larvae possess a sensory vesicle comprising two pigmented sensory organs, the ocellus and the otolith. We focused here on Klhl21/30, a gene belonging to Kelch family, that, in Ciona robusta, starts to be expressed in pigmented cell precursors, becoming specifically maintained in the otolith precursor during embryogenesis. Evolutionary analyses demonstrated the conservation of Klhl21/30 in all the chordates. Cis-regulatory analyses and CRISPR/Cas9 mutagenesis of potential upstream factors, revealed that Klhl21/30 expression is controlled by the combined action of three transcription factors, Mitf, Dmrt, and Msx, which are downstream of FGF signaling. The central role of Mitf is consistent with its function as a fundamental regulator of vertebrate pigment cell development. Moreover, our results unraveled a new function for Dmrt and Msx as transcriptional co-activators in the context of the Ciona otolith.
Project description:This study aimed to determine whether isotopic ratio mass spectrometry (IRMS) can discriminate farmed European sea bass according to different farming systems and geographic origins. Dicentrarchus labrax of commercial size from three different rearing systems (concrete tank inland, sea cages, and extensive methods in valleys or salt works) were collected at the trading period (autumn-winter). For each farming type, different locations spread over Italy were monitored. Once the fish were harvested, the muscle and feed were sampled. For both muscle and feed, ?13C and ?15N were measured by continuous flow elemental analyzer isotope ratio mass spectrometry (CF-EA-IRMS) with the goal of discriminating samples based on the rearing system. Additional ?2H and ?18O measurements of fish samples were performed by continuous flow total combustion elemental analyzer isotope ratio mass spectrometry (CF-TC/EA-IRMS) to track the geographical origin. The measurements of ?13C and ?15N made it possible to discriminate cultured sea bass from different farming systems (extensive vs. intensive) reared at different geographical sites in Italy. Additional information was obtained from ?18O and ?2H, which enabled the geographical areas of origin of the sea bass farmed extensively and intensively (in cages) to be distinguished.
Project description:Tunicates occupy the evolutionary position at the boundary of invertebrates and vertebrates. It exhibits adaptation to broad environmental conditions and is distributed globally. Despite hundreds of years of embryogenesis studies, the genetic basis of the invasive habits of ascidians remains largely unknown. The leathery sea squirt, Styela clava, is an important invasive species. We used the chromosomal-level genome and transcriptome of S. clava to explore its genomic- and molecular-network-based mechanisms of adaptation to environments. Compared with Ciona intestinalis type A (C. robusta), the size of the S. clava genome was expanded by 2-fold, although the gene number was comparable. An increase in transposon number and variation in dominant types were identified as potential expansion mechanisms. In the S. clava genome, the number of genes encoding the heat-shock protein 70 family and members of the complement system was expanded significantly, and cold-shock protein genes were transferred horizontally into the S. clava genome from bacteria. The expanded gene families potentially play roles in the adaptation of S. clava to its environments. The loss of key genes in the galactan synthesis pathway might explain the distinct tunic structure and hardness compared with the ascidian Ciona species. We demonstrated further that the integrated thyroid hormone pathway participated in the regulation of larval metamorphosis that provides S. clava with two opportunities for adapting to their environment. Thus, our report of the chromosomal-level leathery sea squirt genome provides a comprehensive genomic basis for the understanding of environmental adaptation in tunicates.
Project description:Reproductive isolation is central to speciation, but interspecific crosses between two closely related species can produce viable and fertile hybrids. Two different species of tunicates in the same ascidian genus, Ciona robusta and Ciona intestinalis, can produce hybrids. However, wild sympatric populations display limited gene flow, suggesting the existence of obstacles to interspecific reproduction that remain unknown. Here, we took advantage of a closed culture system to cross C. robusta with C. intestinalis and established F1 and F2 hybrids. We monitored post-embryonic development, survival, and sexual maturation to characterize the genetic basis of simple traits, and further probe the physiological mechanisms underlying reproductive isolation. Partial viability of first and second generation hybrids suggested that both pre- and postzygotic mechanisms contributed to genomic incompatibilities in hybrids. We observed asymmetric fitness, whereby the C. intestinalis maternal lines fared more poorly in our system, pointing to maternal origins of species-specific sensitivity. We discuss the possibility that asymmetrical second generation inviability and infertility emerge from interspecific incompatibilities between the nuclear and mitochondrial genomes, or other maternal effect genes. This work paves the way to quantitative genetic approaches to study the mechanisms underlying genomic incompatibilities and other complex traits in the genome-enabled Ciona model.
Project description:The tadpole-type larva of Ciona has emerged as an intriguing model system for the study of neurodevelopment. The Ciona intestinalis connectome has been recently mapped, revealing the smallest central nervous system (CNS) known in any chordate, with only 177 neurons. This minimal CNS is highly reminiscent of larger CNS of vertebrates, sharing many conserved developmental processes, anatomical compartments, neuron subtypes, and even specific neural circuits. Thus, the Ciona tadpole offers a unique opportunity to understand the development and wiring of a chordate CNS at single-cell resolution. Here we report the use of single-cell RNAseq to profile the transcriptomes of single cells isolated by fluorescence-activated cell sorting (FACS) from the whole brain of Ciona robusta (formerly intestinalis Type A) larvae. We have also compared these profiles to bulk RNAseq data from specific subsets of brain cells isolated by FACS using cell type-specific reporter plasmid expression. Taken together, these datasets have begun to reveal the compartment- and cell-specific gene expression patterns that define the organization of the Ciona larval brain.