Nakashima K, Kimura S, Ogawa Y, Watanabe S, Soma S, Kaneko T, Yamada L, Sawada H, Tung CH, Lu TM, Yu JK, Villar-Briones A, Kikuchi S, Satoh N.

Nat Commun. 2018 Aug 24;9(1):3402. doi:10.1038/s41467-018-05884-0

Chitin-based barrier immunity and its loss predated mucus-colonization by indigenous gut microbiota.

The gut cells of Ciona robusta are protected by a membrane made of cellulose and chitin, that bacteria can not cross. The three most abundant proteins in this membrane are Ci-MACPF1, related to perforins, variable region-containing chitin-binding protein (VCBP), and Ci-GFM1, a gel-forming mucin. Thus, the tunicate gut has features related to insects (chitin barrier) and vertebrates (mucin barrier).

Hitoshi Michibata

Zoological Science, 13(4), 489-502, (1 August 1996) doi:10.2108/zsj.13.489

The Mechanism of Accumulation of Vanadium by Ascidians: Some Progress towards an Understanding of this Unusual Phenomenon

Vanadium detected by neutron activation that causes γ-ray emission at 1,434 keV. Blood cells of Ascidia gemmata contained ~350 mM ov vanadium. Other Phlebobranchia contained 20–60 mM, but C. intestinalis contained only 0.6. Stolidobranchia contained much lower amounts. Vanadium-containing cells (vanadocytes) were identified as signet cells. Sulfonic acid might be the counter ion. Vanadocyte pH is very low (1.9–4.2). There exist other low-pH cells that do not contain vanadium. Accumulation of vanadium may involve a mechanism using a vacuolar-type H+-ATPase. Vanadium contents of the embryo are low but increase during embryogenesis.

Ohta N, Kaplan N, Ng JT, Gravez BJ, Christiaen L.

G3 (Bethesda). 2020 Aug 5;10(8):2697-2711. doi:10.1534/g3.120.401427

Asymmetric Fitness of Second-Generation Interspecific Hybrids Between Ciona robusta and Ciona intestinalis.

Comprehensive introduction on intestinalis-robusta taxonomic situation. Conclusions weakened by the the absence of homotypic F2 C. intestinalis animals caused by the failure of C. intestinalis to reproduce in the culture system. The viability and fertility hybrids born from intestinalis oocytes or robusta (sperm) x intestinalis (oocytes) oocytes is reduced.

“Wild-type Ciona robusta (C. intestinalis type A) and Ciona intestinalis (C. intestinalis type adults were collected in San Diego (CA) and Woods Hole (MA)”. “Sea water (Bio-Actif Salt, Tropic Marin) was controlled by bio-balls (Biomate, Lifegard Aquatics) seeded with bacteria (BioDigest, Prodibio)”. “We obtained hundreds of swimming larvae from each cross [..] this contrasts with previous studies, which suggested that C. robusta oocytes were largely refractory to fertilization by C. intestinalis sperm”. “there were no significant differences in the survival rate between F1 RxI and IxR hybrids”. “By 50 dpf, half of the C. robusta individuals were producing sperm, whereas that proportion dropped significantly for the other groups of animals.”

”For both RxI and IxR hybrids, the majority of animals had [orange pigment organ] at the tip of the sperm duct, in agreement with a previous report (Sato et al. 2014), thus indicating that [orange pigment organ] formation is a dominant trait.”

”C. intestinalis has yellow and orange pigmentation around the tip of siphons that is lacking in C. robusta [...] the majority of RxI and IxR hybrids displayed a bright red pigmentation at the rim of oral and atrial siphons, also consistent with a previous report (Sato et al. 2014). The observation that siphon pigmentation displays an overdominant phenotype in hybrids is consistent with its lack of reliability for taxonomic purposes.“

“the sperm of F1 RxI hybrid appeared less potent to fertilize C. robusta eggs than that of F1 IxR hybrids, which is reminiscent of previously reported difficulties in using C. robusta eggs in interspecific fertilizations.” “Both BC1 (RxI)xR and (IxR)xR hybrids had lower survival rates than F2 C. robusta animals, while an ANOVA did not show significant differences in survival rate on 28 and 50 dpf between (RxI)xR and (IxR)xR hybrids.”

“We obtained sperm from 7 and 10 individuals, and eggs from 7 and 11 F1 RxI and IxR mature animals, respectively, and used them for within-type fertilizations. Fertilization rates were significantly higher for IxR hybrids than for RxI hybrids”

“Finally, F2 IxR hybrids grew and matured to produce sperm and eggs (Table 5 and Supplemental table S3). The sperm and eggs could fertilize each other to produce F3 IxR hybrids, which survived at least 28 dpf, after which we stopped observations.”

“simple quantitative traits, such as body size, showed an increased variability in F2 hybrids as expected for polygenic traits following allele segregation.”

Francesco Mastrototaro, Federica Montesanto, Marika Salonna, Frédérique Viard, Giovanni Chimienti, Egidio Trainito, Carmela Gissi

Zoological Journal of the Linnean Society, Volume 190, Issue 4, December 2020, Pages 1193–1216, doi:10.1093/zoolinnean/zlaa042

An integrative taxonomic framework for the study of the genus Ciona (Ascidiacea) and description of a new species, Ciona intermedia

“The percentage identity between C. intermedia and other Ciona species, including C. roulei and C. edwardsi, is lower than 94.28% for cox1, 90.14% for x3n1 [“cox2 and cob”] and 91.89% for x2cb [ cox3, trnK, nad1, and two non-coding spacers].”

Satou Y, Sato A, Yasuo H, Mihirogi Y, Bishop J, Fujie M, Kawamitsu M, Hisata K, Satoh N.

Genome Biol Evol. 2021 Jun 8;13(6):evab068. doi:10.1093/gbe/evab068

Chromosomal Inversion Polymorphisms in Two Sympatric Ascidian Lineages.

Chromosoma assembly of Ciona intestinalis finds ~20 inversions compared to C. robusta. Some inversions are heterozygous.

“Chromosomal-level assemblies for the two type-B individuals, sampled at Roscoff (France; specimen R) and Plymouth (England; specimen P)” “For each specimen, we built contigs from long Nanopore reads with the NECAT assembler and polished obtained contigs with Illumina reads using the Nextpolish software [and removed overlaping contigs] using the purge_dups software [...]. Both of these contig sets were 140 Mb in length (table 1).” “Although the heterozygosity rate of type-A animals has been estimated to be 1.1–1.2% (Dehal et al. 2002; Satou et al. 2012), those of specimens R and P were 3.0% and 3.6%, respectively.”

“Exons are highly conserved between type-A and type-B animals, whereas intergenic regions are highly varied between the two types.” [Supplemental material shows 97% similarity in aligned regions between R and P and 94% similarity between R/P and A]

“To identify translocations, we searched the genomic sequences for blocks containing three or more genes that were not found in the positions expected from the type-A genome. We found two small interchromosomal translocations between the genomes of specimen P and type A, [but] we did not find such translocations between the genomes of specimen R and type A.”

“Similarly, to identify inversions, we searched the genomic sequences for blocks containing three or more genes that were mapped in the reverse direction to the order in the type-A genome. We found 21 and 20 inversions in the genomes of specimens R and P, respectively (supplementary fig. S7, Supplementary Material online). Among them, 15 sites were common, and the remaining 11 sites were specific to specimen R or P (supplementary fig. S10 and tables S3 and S4, Supplementary Material online). Thus, our data indicate that there are structural variations not only between type-A and type-B animals but also between specimens R and P.”

“We found R-derived haplotype contigs that were structurally different from the chromosome of specimen R, but the same as both the specimen-P and the type-A chromosomes. [In “an inversion in chromosome 7 [that] contained the largest number of genes over an ∼900-kb region”] genomic PCR using four primers flanking the junctional sites demonstrated that specimen R was indeed heterozygous” “We also confirmed experimentally an inversion on chromosome 3 [...]. A PCR analysis demonstrated that specimen P was indeed heterozygous in this region.” “we manually inspected genomic alignments of the two type-B specimens against the type-A genome, and found nine additional inversions.” “To understand how prevalent the inversions we found were in type-B populations, we collected eight wild specimens at Roscoff and Plymouth, RO1–RO4 and PL1–PL4, respectively, and performed genomic PCR [that demonstrated that] type-B animals indeed have inversion polymorphisms.” “The inversions we found in the present study are relatively small (3 kb ∼ 873 kb)” “our data highlight the likely importance of inversions in speciation of Ciona, an invertebrate with mating governed by interactions of aquatic gametes”.

Berná L, Alvarez-Valin F, D'Onofrio G.

Comp Funct Genomics. 2009;2009:875901. doi:10.1155/2009/875901

How fast is the sessile ciona?

“Tajima’s test results [...] for the great majority of the alignments, Ciona genes evolve faster than those of all vertebrate groups[...].” “On the average, Ciona evolves 50% faster than all vertebrates, with the exception of O. anatinus and M. domestica.” “The divergence between C. intestinalis and C. savignyi was reestimated and found to took place ~184 (±15) My ago.”

Exon 3 of the NUMB Gene Emerged in the Chordate Lineage Coopting the NUMB Protein to the Regulation of MDM2.

Confalonieri S, Colaluca IN, Basile A, Pece S, Di Fiore PP.

G3 (Bethesda). 2019 Oct 7;9(10):3359-3367. doi: 10.1534/g3.119.400494.

Ciona NUMB can inhibit ubiquitination of P53 by MDM2. This and a phylogenetic analysis demonstrates that PTB-long isoform encoded by Exon3 is a Chordate invention. O. dioica has 2 NUMB genes, more similar to NUMB than to vertebrate-specific NUMBL. Their intron/exon structures are different from any other organism.

Zoolog Sci. 2005 May;22(5):511-6 doi:10.2108/zsj.22.511

Shoguchi E, Kawashima T, Nishida-Umehara C, Matsuda Y, Satoh N.

Molecular cytogenetic characterization of Ciona intestinalis chromosomes.

Ciona intestinalis has 10 metacentric pairs and 8 submetacentric or subtelomeric pairs of chromosomes.

Genome Biology and Evolution, 2019 Oct 17, evz228, doi:10.1093/gbe/evz228

Yutaka Satou, Ryohei Nakmura, Yu Deli, Reiko Yoshida, Mayuko Hamada, Manabu Fujie, Kanako Hisata, Hiroyuki Takeda, Noriyuki Satoh

A nearly-complete genome of Ciona intestinalis type A (C. robusta) reveals the contribution of inversion to chromosomal evolution in the genus Ciona.

More complete assembly (HT, standing for Hoya T-strain) where most protein-coding genes are included in chromosomal contigs. Finds and discusses inversions within Ciona robusta genomes and between Ciona robusta and savigniy.

Dehal P, Satou Y, Campbell RK, Chapman J, Degnan B, De Tomaso A, Davidson B, Di Gregorio A, Gelpke M, Goodstein DM, Harafuji N, Hastings KE, Ho I, Hotta K, Huang W, Kawashima T, Lemaire P, Martinez D, Meinertzhagen IA, Necula S, Nonaka M, Putnam N, Rash S, Saiga H, Satake M, Terry A, Yamada L, Wang HG, Awazu S, Azumi K, Boore J, Branno M, Chin-Bow S, DeSantis R, Doyle S, Francino P, Keys DN, Haga S, Hayashi H, Hino K, Imai KS, Inaba K, Kano S, Kobayashi K, Kobayashi M, Lee BI, Makabe KW, Manohar C, Matassi G, Medina M, Mochizuki Y, Mount S, Morishita T, Miura S, Nakayama A, Nishizaka S, Nomoto H, Ohta F, Oishi K, Rigoutsos I, Sano M, Sasaki A, Sasakura Y, Shoguchi E, Shin-i T, Spagnuolo A, Stainier D, Suzuki MM, Tassy O, Takatori N, Tokuoka M, Yagi K, Yoshizaki F, Wada S, Zhang C, Hyatt PD, Larimer F, Detter C, Doggett N, Glavina T, Hawkins T, Richardson P, Lucas S, Kohara Y, Levine M, Satoh N, Rokhsar DS.

Science. 2002 Dec 13;298(5601):2157-67 doi: 10.1126/science.1080049

The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins.

The genome of C. intestinalis contains at least one cellulose synthans and multiple endoglycanases.

Zoolog Sci. 2004 Feb;21(2):153-7 doi:10.2108/zsj.21.153

Shoguchi E1, Ikuta T, Yoshizaki F, Satou Y, Satoh N, Asano K, Saiga H, Nishikata T.

Fluorescent in situ hybridization to ascidian chromosomes.

“Embryos at three different developmental stages (32-cell embryos, 64-cell embryos and gastrulae) were used to produce metaphase spreads, although treatment of 32-cell embryos and 64-cell embryos with 0.05% colchicine (Sigma) in seawater caused aggregated and disorganized chromosomes. After treatment with colchicine for 20–30 min, embryos were transferred to a 1.5-ml microfuge tube, to which cold methanol:glacial acetic acid (3:1) fixative was added (chilled on ice). The fixative was changed twice over a period of 1 hr and once after overnight (embryos kept at 4°C). The specimens were then stored in fixative at 4°C.”

Genome Res. 2006 Feb;16(2):297-303 doi:10.1101/gr.4156606

Shoguchi E, Kawashima T, Satou Y, Hamaguchi M, Sin-I T, Kohara Y, Putnam N, Rokhsar DS, Satoh N.

Chromosomal mapping of 170 BAC clones in the ascidian Ciona intestinalis.

The short arm of chromosome 4, 5 and 6 contains rDNA clusters.

J Mol Evol. 2003 Nov;57(5):574-87 doi: 10.1007/s00239-003-2511-9

Yokobori S, Watanabe Y, Oshima T.

Mitochondrial genome of Ciona savignyi (Urochordata, Ascidiacea, Enterogona): comparison of gene arrangement and tRNA genes with Halocynthia roretzi mitochondrial genome.

The DDBJ accession number of complete nucleotide sequence of the Ciona mtDNA is AB079784.

“The atp8 gene is absent.” “The Ciona mt genome uses the same codon table as the Halocynthia mt genome: AUA specifies Met, UGA specifies Trp, and AGA/AGG specify Gly.” “To predict the genetic code used in the Ciona mt genome, the well-conserved cox1 sequence was compared between Ciona and several other metazoans.” “The Ciona mtDNA is extremely (77.3%) AT-rich.”

Brunetti, R. , Gissi, C. , Pennati, R. , Caicci, F. , Gasparini, F. and Manni, L.

J Zoolog Syst Evol Res, 2015, 53: 186-193. doi:10.1111/jzs.12101

Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis.

Discriminates C. robusta from C. intestinalis by the presence of tubercular prominences in the tunic.

Mitogenomics reveals two cryptic species in Ciona intestinalis.

Trends Genet. 2007 Sep;23(9):419-22 doi:10.1016/j.tig.2007.07.001

Iannelli F, Pesole G, Sordino P, Gissi C.

Mitochondrial gene order differs between C. robusta and C. intestinalis.

Mol Biol Evol. 2013 Jul;30(7):1574-87. doi:10.1093/molbev/mst066

Roux C, Tsagkogeorga G, Bierne N, Galtier N.

Crossing the species barrier: genomic hotspots of introgression between two highly divergent Ciona intestinalis species.

Speciation during the Pliocene (≈ 3.8 Ma) and then recent introgression 15,000 years ago.

Bouchemousse S, Liautard-Haag C, Bierne N, Viard F.

Mol Ecol. 2016 Nov;25(21):5527-5542. doi:10.1111/mec.13854

Distinguishing contemporary hybridization from past introgression with postgenomic ancestry-informative SNPs in strongly differentiated Ciona species.

The only hybrids detected are F1 and have a C. int mitochondrial haplotype.

Caputi L, Andreakis N, Mastrototaro F, Cirino P, Vassillo M, Sordino P.

Proc Natl Acad Sci U S A. 2007 May 29;104(22):9364-9 doi:10.1073/pnas.0610158104

Cryptic speciation in a model invertebrate chordate.

Only C. robusta female gametes could be fertilised by sperm from either C. rob or C. int. Hybrids were infertile. Allospecific crosses between two sympatric strains from Plymouth could not develop beyond a few cell cleavages.

Mol Phylogenet Evol. 2010 Aug;56(2):718-26. doi:10.1016/j.ympev.2010.03.042

Nydam ML and Harrison RG

Polymorphism and divergence within the ascidian genus Ciona.

Sequencing of nuclear genes show intragenic recombination in C. robusta and C. intestinalis, buy supports monophyly of these two species. The data also supports paraphyly of C. intestinalis with respcet to Ciona roulei.

Nydam ML and Harrison RG.

Mar Biol (2007) 151: 1839. doi:10.1007/s00227-007-0617-0

Genealogical relationships within and among shallow-water Ciona species (Ascidiacea)

Mitochondrial CO1 sequencing shows that Type A and B Ciona intestinalis are as distant as separate species.

Suzuki MM, Nishikawa T and Bird A.

J Mol Evol. 2005 Nov;61(5):627-35. Epub 2005 Oct 4.

Genomic approaches reveal unexpected genetic divergence within Ciona intestinalis.

C. robusta sperm can fertilise C. intestinalis eggs. In 3 reciprocal crosses, C. int sperm was less efficient (40~70%) and in 2 crosses, it was ver inefficient (~5 %). Hybrids developed to young adults in laboratory. Reproductive isolation estimated to have started 30 MY ago. Ideal temperature for breeding differs of 5 °C.

Sci Rep. 2018 Jan 24;8(1):1480. doi:10.1038/s41598-018-19811-2

Malfant M, Darras S and Viard F.

Coupling molecular data and experimental crosses sheds light about species delineation: a case study with the genus Ciona.

C. robusta sperm can fertilise C. intestinalis eggs, but fertilisation rates in the converse crosses are much lower. Phylogenetic analysis of mitochondrial CO1 sequences suggests that there are no hybrids in wild populations.

Mol Reprod Dev. 2000 Jan;55(1):109-16 doi:10.1002/(SICI)1098-2795(200001)55:1%3C109::AID-MRD15%3E3.0.CO;2-B

Byrd J and Lambert CC.

Mechanism of the block to hybridization and selfing between the sympatric ascidians Ciona intestinalis and Ciona savignyi.

C. intestinalis and C. savignyi can cross-fertilise after removal of the vitelline enveloppe.

Berná L and Alvarez-Valin F.

Genome Biol Evol. 2014 Jul 8;6(7):1724-38. doi:10.1093/gbe/evu122

Evolutionary genomics of fast evolving tunicates.

Parallel review about genome compaction in Ciona and Oikopleura.

Matthysse AG, Deschet K, Williams M, Marry M, White AR, Smith WC.

A functional cellulose synthase from ascidian epidermis.

Proc Natl Acad Sci U S A. 2004 Jan 27;101(4):986-91.

The CesA mRNA is trans-spliced in Ciona savignyi. C. sav CesA can rescue mutant A. tumefaciens bacteria.

Genes Dev. 2018 Sep 18. doi:10.1101/gad.317669.118

Horie T, Horie R, Chen K, Cao C, Nakagawa M, Kusakabe TG, Satoh N, Sasakura Y, Levine M.

Regulatory cocktail for dopaminergic neurons in a protovertebrate identified by whole-embryo single-cell transcriptomics.

Ptf1a and Meis can reprogram the CNS of Ciona intestinalis into a dopaminergic cell type.

Yokomori R, Shimai K, Nishitsuji K, Suzuki Y, Kusakabe TG, Nakai K.

Genome Res. 2016 Jan;26(1):140-50. doi:10.1101/gr.184648.114

Genome-wide identification and characterization of transcription start sites and promoters in the tunicate Ciona intestinalis.

TSS-Seq libraries made of 200 µg of total RNA, sequenced on Illumina GA with a read length of 36, which is sufficient to go through the 16-nt splice leader.

Nucleic Acids Res. 2006 Jul 5;34(11):3378-88 doi:10.1093/nar/gkl418

Satou Y, Hamaguchi M, Takeuchi K, Hastings KE, Satoh N.

Genomic overview of mRNA 5'-leader trans-splicing in the ascidian Ciona intestinalis.

Analysis of oligo-capped cDNAs reports a single splice leader (16 nt-long) in 27% of the cDNAs.