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Infection with the parasitic dinoflagellate Hematodinium sp. can be devastating to blue crab Callinectes sapidus populations. Morbidity and mortality appear to depend on the burden of parasitic organisms. Heavily infected crabs become lethargic and, if not preyed upon, succumb to overwhelming infection. We report on the transmission of Hematodinium sp. into blue crabs that were fed pieces of infected tissues and examined for evidence of infection at time periods from 1 to 48 h and for the general state of their health after 4 d. During the first 16 h after feeding, Hematodinium sp. was found in the gut, followed by large increases in hemolymph hemocytes and the appearance of hemocytic nodules in tissues. By 16 h, the hemocytic nodules appeared poorly circumscribed and disorganized. No nodules were seen in a heavily infected crab after 24 h. By the end of the 48 h after feeding, 73% (11 of 15) of the crabs had shown evidence of infection with Hematodinium sp. Those crabs with infection intensities (Hematodinium sp. as percent of cells in hemolymph) higher than 20% were dead within 4 d.

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.

Increasingly, diseases of marine organisms are recognized as significant biotic factors affecting ecosystem health. However, the responsible disease agents are often unknown and the discovery and description of novel parasites most often rely on morphological descriptions made by highly trained specialists. Here, we describe a new approach for parasite discovery, utilizing denaturing high-performance liquid chromatography (DHPLC) reverse-phase ion-pairing technology. Systematic investigations of major DHPLC variables, including temperature, gradient conditions, and target amplicon characteristics were conducted to develop a mechanistic understanding of DNA fragment separation by DHPLC. As a model system, 18S rRNA genes from the blue crab (Callinectes sapidus) and a parasitic dinoflagellate Hematodinium sp. were used. Binding of 18S rRNA gene PCR amplicons to the DNA separation column in the presence of triethylammonium acetate (TEAA) was inversely correlated with temperature and could be predicted based on the estimated DNA helicity of the PCR amplicon. Amplicons of up to 498 bp were resolved as single chromatographic peaks if they had high (>95%) DNA helicity. Amplicons that differed by as few as 2 bp could be resolved. Separation of 18S rRNA gene PCR amplicons was optimized by simultaneous manipulation of both temperature and solvent gradients. The optimal conditions included targeting regions of high DNA helicity (>95%), temperatures in the range of 57 to 63 degrees C, and a linear acetonitrile gradient from 13.75 to 17.5% acetonitrile in 0.1 M TEAA (55 to 70% buffer B) over a 9-min period. Under these conditions, amplicons from a variety of parasites and their hosts can be separated and detected by DHPLC.

The 18S rRNA gene from Hematodinum sp., a parasitic dinoflagellate that infects blue crabs, was amplified, cloned, and sequenced. The sequence showed a high similarity (95% at the nucleotide level) to sequences obtained from other dinoflagellate species, including both free-living and symbiotic species. Sequence similarity was much lower when compared with parasites of other marine invertebrates with similar life histories and with the 18S rRNA gene from the blue crab. Based on comparison of sequence alignments between Hematodinium, other dinoflagellate species, protozoan pathogens of oysters, and blue crab 18S rRNA gene sequences, 2 sets of PCR primers that specifically amplified fragments of the Hematodinium 18S rRNA gene were developed and tested. One of these primer sets (Hemat-F-1487 and Hemat-R-1654) amplified a 187 bp fragment that could be used routinely as a diagnostic test for the presence of Hematodinium in hemolymph from blue crabs. This fragment was consistently amplified from genomic DNA extracted from hemolymph of Hematodinium infected blue crabs. Comparison between the PCR technique and standard histological examination indicated that the PCR technique was reliable and provided 1000 times more sensitivity than the histological methods. The sensitivity of the PCR diagnostic was estimated to be one parasite cell among 300,000 crab hemocytes. Preliminary studies using the PCR diagnostic technique suggest that Hematodinium sp. is absent in crabs collected from waters with low salinity (5 to 10 ppt), but common in crabs from higher salinity environments in estuarine waters from southeastern Georgia (USA).