Detection and Discovery of Crustacean Parasites in Blue Crabs (Callinectes sapidus) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography

Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence info...

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Bibliographic Details
Published in:Applied and Environmental Microbiology 2008-07, Vol.74 (14), p.4346-4353
Main Authors: Troedsson, Christofer, Lee, Richard F, Walters, Tina, Stokes, Vivica, Brinkley, Karrie, Naegele, Verena, Frischer, Marc E
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biological and medical sciences
Brachyura - parasitology
Callinectes sapidus
Chromatography
Chromatography, High Pressure Liquid - methods
Cloning, Molecular
Conserved sequence
Crustaceans
Dinoflagellida - isolation & purification
DNA Primers
Fundamental and applied biological sciences. Psychology
Genetics
Invertebrate Microbiology
Kinetoplastida - classification
Kinetoplastida - isolation & purification
Microbiology
Molecular Sequence Data
Nucleic Acid Probes
Parasites
Peptide Nucleic Acids
Phylogeny
Polymerase Chain Reaction
Ribonucleic acid
RNA
RNA, Protozoan - isolation & purification
RNA, Ribosomal, 18S - isolation & purification
Sensitivity and Specificity
Sequence Alignment
Species Specificity
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Summary:Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected parasites. However, initial field evaluations of the DHPLC assay suggested that because of PCR-biased amplification of dominant host genes it was not possible to detect relatively rare parasite genes in infected crab tissue. Here, we describe the use of a peptide nucleic acid (PNA) PCR hybridization blocking probe in association with DHPLC (PNA-PCR DHPLC) to overcome inherent PCR bias associated with amplification of rare target genes by use of generic primers. This approach was utilized to detect infection of blue crabs (Callinectes sapidus) by the parasitic dinoflagellate Hematodinium sp. Evaluation of 76 crabs caught in Wassaw Sound, GA, indicated a 97% correspondence between detection of the parasite by use of a specific PCR diagnostic assay and that by use of PNA-PCR DHPLC. During these studies, we discovered one crab with an association with a previously undescribed protist symbiont. Phylogenetic analysis of the amplified symbiont 18S rRNA gene indicated that it is most closely related to the free-living kinetoplastid parasite Procryptobia sorokini. To our knowledge, this is the first report of this parasite group in a decapod crab and of this organism exhibiting a presumably parasitic life history.
ISSN:0099-2240
1098-5336
1098-6596
DOI:10.1128/AEM.02132-07