Large-Scale Infection of the Ascidian Ciona Intestinalis by the Gregarine Lankesteria Ascidiae in an Inland Culture System

Large-Scale Infection of the Ascidian Ciona Intestinalis by the Gregarine Lankesteria Ascidiae in an Inland Culture System

Vol. 101: 185–195, 2012 DISEASES OF AQUATIC ORGANISMS Published November 19 doi: 10.3354/dao02534 Dis Aquat Org Large-scale infection of the ascidian Ciona intestinalis by the gregarine Lankesteria ascidiae in an inland culture system Kaoru Mita1, Narudo Kawai1,2, Sonja Rueckert1,3, Yasunori Sasakura1,* 1Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka 415-0025, Japan 2Present address: Department of Biology, Research and Education Center for Natural Sciences, Keio University, 4-1-1 Hiyoshi, Kohoku-ku, Yokohama 223-8521, Japan 3Present address: School of Life, Sport & Social Sciences, Edinburgh Napier University, Sighthill Campus, Sighthill Court, Edinburgh EH11 4BN, UK ABSTRACT: An important way to keep transgenic and mutant lines of the ascidian Ciona intesti- nalis, a model system for e.g. genetic functions, in laboratories is via culturing systems. Here we report a disease of C. intestinalis observed in an inland culturing system. The disease, called ‘long feces syndrome,’ is expressed in affected animals by the following characteristic symptoms of the digestive system: (1) excretion of long and thin feces, (2) pale color of the stomach, and (3) conges- tion of the digestive tube by digested material. Severely diseased animals usually die within a week after the first symptoms occur, implying a high risk of this disease for ascidian culturing sys- tems. The digestive tubes of the diseased animals are occupied by the gregarine apicomplexan parasite Lankesteria ascidiae, suggesting that large-scale infection by this parasite is the cause of long feces syndrome. KEY WORDS: Parasite · Apicomplexan · Digestive tube · Stomach · Congestion · Long feces syndrome · Disease Resale or republication not permitted without written consent of the publisher INTRODUCTION mental techniques, gene functions of C. intestinalis have been extensively studied. Genetic approaches The ascidian Ciona intestinalis (Linnaeus, 1767) have recently been developed in this ascidian as provides an excellent model system to understand exemplified by the creation of transgenic lines with the genetic functions in a simplified chordate body transposable elements, enhancer detection, and plan (Satoh 2003). Rapid embryogenesis, a tadpole mutagenesis (Sasakura et al. 2003, 2005, Awazu et al. larva consisting of a countable number (~2600) of 2004, Sasakura 2007). These are powerful methods cells, and quick metamorphosis are advantages for for understanding the functions of genes. The rela- studying the molecular and cellular mechanisms tively short generation time of this ascidian, approxi- underlying these events. In 2002, the draft genome of mately 2 to 3 mo for the reproductive stage, helps to this species was determined with about 160 mega carry out genetic approaches. An inland culture sys- base pairs (Mbp) per haploid, encoding 15 852 pro- tem is necessary to conduct C. intestinalis genetics, tein-coding genes (Dehal et al. 2002). Basic tech- as it allows for the maintenance of genetically modi- niques of molecular biology and embryology for ana- fied C. intestinalis in laboratories isolated from the lyzing gene functions have been developed in this ocean. For this reason, it is becoming increasingly ascidian (reviewed by Lemaire 2011). With the help important to develop a functioning and maintainable of the genome information and established experi- culture system for this ascidian. *Corresponding author. © Inter-Research 2012 · www.int-res.com Email: [email protected] 186 Dis Aquat Org 101: 185–195, 2012 The Shimoda Marine Research Center (SMRC) of Feces addition experiment the University of Tsukuba (Japan) has established an inland culturing system of Ciona intestinalis similar Several hundred fertilized eggs of wild-type Ciona to that described by Joly et al. (2007). The seawater intestinalis and a ~1 cm long feces string from a dis- used for culturing is obtained from the nearby sea at eased animal were added to a 9 cm petri dish. In around 3 m depth without filtration, and the diatom total, we added feces strings of 6 diseased animals to Chaetoceros calcitrans (Paulsen) Takano, 1968 is petri dishes with fertilized eggs from 4 different used as feed. With this system we maintain trans- clutches. In addition, we had 1 negative control for genic, mutant, and inbred lines of C. intestinalis in each clutch with no added feces strings. Two days culture. after C. intestinalis completed settlement, the sea - From late 2010, we frequently observed the excre- water was discarded and new seawater was added to tion of long feces and occasional clogging of the all petri dishes. Seawater was exchanged every other digestive tubes in Ciona intestinalis by digested day. Nine days after fertilization, juveniles were material. We named this health irregularity ‘long stripped off the dishes. After washing once with new feces syndrome’ after one of its symptoms. We found seawater, 60 to 100 juveniles for each dish were col- that the digestive tubes of diseased animals are occu- lected in a 1.5 ml test tube (2 batches from each dish), pied by a gregarine apicomplexan, namely Lank - and their DNA was isolated according to the method esteria ascidiae (Lankester, 1872) Mingazzini, 1891. described in the following section. This species is the type species of the genus Lank - esteria (Levine 1977, Rueckert & Leander 2008) and infects ascidian hosts along with other Lankesteria DNA isolation, PCR amplification, cloning, species. and sequencing In this study, we recorded the symptoms of the dis- ease. Lankesteria ascidiae was isolated and docu- DNA of Ciona intestinalis juveniles and feces was mented by means of light microscopy (LM) and scan- isolated with a Wizard Genome purification kit (Pro- ning electron microscopy (SEM) as well as small mega). After precipitation with isopropanol, DNA subunit (SSU) rDNA sequencing. We conclude that was dissolved in 20 µl water, and 1 µl of the DNA large-scale infection of Ciona intestinalis by the gre- solution was subjected to PCR analysis with ExTaq garine apicomplexan L. ascidiae is the cause of long DNA polymerase (Takara Bio). The PCR program feces syndrome. was set to 40 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C, followed by 10 min at 72°C for final extension. The nucleotide sequences of the primers MATERIALS AND METHODS were 5’-ACT GAG AAC TTC CTG CTT GCT C-3’ and 5’-GAG CTT GAG AAA TGG CTA CCA C-3’. Culture conditions DNA from Lankesteria ascidiae was extracted from 50 individual trophozoites that were manually iso- The inland culture system of Ciona intestinalis is lated from dissected hosts. They were washed 3 managed according to the suggestions of Joly et al. times in filtered and autoclaved seawater, and de - (2007). Two major adaptations are (1) exclusive use posited into a 1.5 ml tube. Genomic DNA was ex - of the diatom Chaetoceros calcitrans as feed, and tracted from the cells using the MasterPure complete (2) seawater exchange 3 times a week. The SMRC DNA and RNA purification kit (Epicentre). SSU is equipped with 110 pairs of 10 to 30 l tanks, and rDNA sequences were PCR amplified using puReTaq currently, over 100 transgenic lines are cultured in ready-to-go PCR beads (GE Healthcare) and the fol- total. In the initial step of culturing C. intestinalis, lowing eukaryotic PCR primers: F1 5’-GCG CTA we stock about 200 juveniles (about 10 d after fer- CCT GGT TGA TCC TGC C-3’ and R1 5’-GAT CCT tilization) in a tank. The number of animals is TCT GCA GGT TCA CCT AC-3’ (Leander et al. brought down during the successive days according 2003). The PCR program was set to an initial 4 cycles to the growth of animals (on average 30 animals of 4 min, 30 s at 94°C, 1 min at 45°C, and 1 min 45 s after 3 mo). Wild or cultured wild-type C. intesti- at 72°C, followed by 34 cycles of 30 s at 94°C, 1 min nalis in this study had been collected from Ona- at 50°C, and 1 min 45 s at 72°C, and then 10 min at gawa Bay (Mi yagi), Maizuru Bay (Kyoto), Sagami 72°C for final extension. PCR products of L. ascidiae Bay (Kanagawa), Tosa Bay (Kochi), and Mukai - corresponding to the expected size (~1800 bp) were shima (Hiro shima). gel isolated and cloned into the pSC-A-amp/kan vec- Mita et al.: Gregarines in an ascidian culture system 187 tor using the StrataClone PCR cloning kit (Agilent were taken with a system microscope (Olympus Technologies). Nine cloned plasmids, for each PCR BX50) connected to a digital camera (Olympus product, were digested with EcoRI, and inserts were DP70) or Carl Zeiss AxioImager Z1 connected to a screened for size using gel electrophoresis. Two iden- digital camera (Carl Zeiss Axiocam MRm). Individ- tical clones were se quenced with ABI Big-dye reac- ual tro pho zoites of L. ascidiae (n = 20) were pre- tion mix using vector primers and internal primers pared for SEM using the OsO4 vapor protocol oriented in both directions. The GenBank accession described previously (Rueckert & Leander 2008, number of the SSU rDNA se quence of L. ascidiae is 2009). Isolated cells were deposited directly into the JX187607. The SSU rDNA sequences were identified threaded hole of a Swinnex filter holder, containing by BLAST analysis, and molecular phylogenetic a 5 µm polycarbonate membrane filter (Millipore), analyses were performed as described below. that was submerged in 10 ml of seawater within a small canister (2 cm diameter and 3.5 cm tall). A piece of Whatman filter paper was mounted on the Molecular phylogenetic analyses inside base of a beaker (4 cm diameter and 5 cm tall) that was slightly larger than the canister. The The SSU rDNA sequence from Lankesteria asci diae Whatman filter paper was saturated with 4% OsO4, was incorporated into an 88-sequence alignment rep- and the beaker was turned over the canister.

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