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Applied and Environmental Microbiology, July 2008, p. 4346-4353, Vol. 74, No. 14
0099-2240/08/$08.00+0     doi:10.1128/AEM.02132-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, Georgia 31411,¹ Department of Biology, University of Bergen, P.O. Box 7800, N-5020 Bergen, Norway²

Received 17 September 2007/ Accepted 16 May 2008

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.

Published ahead of print on 23 May 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

This article has been cited by other articles:

• Troedsson, C., Lee, R. F., Stokes, V., Walters, T. L., Simonelli, P., Frischer, M. E. (2008). Development of a Denaturing High-Performance Liquid Chromatography Method for Detection of Protist Parasites of Metazoans. Appl. Environ. Microbiol. 74: 4336-4345 [Abstract] [Full Text]