Echinostoma paraensei: differential gene transcription in the sporocyst stage.
The sporocyst stage of trematode development plays the crucial role of establishing a successful infection in the molluscan intermediate host. Due to the small size and presence of this stage within the tissues of the host, much of our current knowledge of sporocyst biology relies on cultured specimens. To gain insight into the transcriptional patterns of early sporocysts, suppression subtractive hybridization was employed to identify 69 unique expressed sequence tags likely to be upregulated in cultured sporocysts of Echinostoma paraensei, a trematode parasite of the planorbid snail, Biomphalaria glabrata. Upwards of 70% of the unique sequences were not identified by homology to known genes. However, one transcript may encode an inhibitor of nitric oxide synthase, indicating a possible role in protection against host defense mechanisms. An array containing the majority of the sequenced clones was probed with in vivo-derived cDNA, confirming for the first time in vivo expression of putative sporocyst genes. However, qPCR quantification demonstrated significant reductions in transcription rates in cultured versus in vivo sporocysts for three of six transcripts tested. Additionally, five of the six tested transcripts demonstrated significant variation in expression over the entire life cycle, with the significant upregulation occurring during early intramolluscan development or in the free-living stages immediately preceding snail penetration, confirming the efficacy of the SSH technique.
Nowak
Thomas S
TS
Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA. stasiu@unm.edu
Loker
Eric S
ES
D000818:Animals; D001700:Biomphalaria; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004449:Echinostoma / Q000235:genetics* / Q000254:growth & development; D020224:Expressed Sequence Tags; D018507:Gene Expression Regulation, Developmental; D020411:Oligonucleotide Array Sequence Analysis; D033661:Oocysts / Q000235:genetics / Q000254:growth & development; D016133:Polymerase Chain Reaction / Q000379:methods; D018510:RNA, Helminth / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic / Q000502:physiology*
D018076:DNA, Complementary; D018510:RNA, Helminth; D012333:RNA, Messenger