Parasite-stage E. lineata were collected from a rock jetty at Woods Hole, MA from July through October of 2009 and 2010. Tissue from parasite-stage E. lineata was collected by excising the endoparasitic anemones from M. leidyi with forceps and a scalpal. The excised anemones were immediately transferred to TRIzol and homogenized with a 7mL glass dounce. Parasites were excised from ctenophores and allowed to develop at room temperature. Anemones were selected by eye that represented a similar transitional stage of development and processed with TRIzol for Total RNA isolation. For the parasite-to-planula transition stage, anemones were selected that met the following criteria (1) reduction in pharynx size, (2) movement, and (3) change in body shape that was intermediate between the parasitic (elongate/vermiform) and planula (shortened/ovoid) ontogenies. Planula stage anemones were allowed to develop for 2-4 days post host excision, and selected by eye for individuals representing a similar stage of planula development. The criteria for planula stage identification was (1) pharynx absent, (2) vigorous swimming, (3) shortened/ovoid bauplan. Excised parasitic anemones were allowed to develop from free-swimming planulae until they began showing signs of metamorphosis into polyps, such as (1) cessation of swimming, (2) pharynx formation, and (3) tentacle eruption. Adult E. lineata tissue was obtained from excised parasitic anemones that successfully metamorphosized into adult polyps capable of feeding and were maintained in laboratory conditions (33ppt artificial salt water) until RNA isolation.
Total RNA was isolated from pooled specimens for each of the five developmental stage. mRNA was isolated from each pool of Total RNA (Poly(A) Purist mRNA isolation kit, Ambion) and used to generate cDNA libraries with the mRNA Sample Preparation kit, (Illumina). cDNA libraries were sequenced on an Genome Analyzer IIx (Illumina).
Sequencing with Illumina GAIIx 40bp paired-end reads produced a total of 376,243,854 sequencing reads which passed the Illumina GAIIx quality filiter. Assembly was then performed with the short read de novo assembler Velvet (version 1.1.05). Subsequent assembly into transcripts was performed with Oases (version 0.1.22). Transcripts were assembled into contigs by using multiple-kmer approach to assemble many different transcript sets for each life stage. The long assembled reads were taken from each life stage and were merged together using both Velvet and Oases a final time in order to generate the final reference transcriptome. This transcriptome was then annotated utilizing Blast2GO.
|Edwardsiella lineata in the Latest Literature|
Edwardsiella lineata Genomics & Transcriptomics Database
Created by: Tristan Lubinski, Brian Granger, and Sarah McAnulty
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