The in vitro life cycle of Hematodinium species has been characterized by culturing methods [17, 24]. These, along with other studies [3, 13], have supported a hypothesis for the mode of disease transmission in nature to entail infective dinospores, as a small portion of cultured dinospores develop into filamentous trophonts . As the filamentous trophont is routinely observed in infected crustaceans from field studies [4, 8, 11], it is possible that released dinospores from these hosts may develop into trophonts in the water column and/or an intermediate vector. Alternatively, the dinospore may itself be the infectious stage, or may require a preparatory cyst phase. In order to explore these life cycle questions in a coastal bay ecosystem, it was important to first search for the chronological manifestations of Hematodinium sp. within the study site.
Based on a preliminary PCR-based screen, Hematodinium sp. was equally distributed in both water and sediment samples collected in 2010 and 2011 (Table 1). In most cases we detected parasite DNA in only water or sediment, but not both, for positive sites. The only exceptions were at Newport Bay, MD in June of 2010, and Sinnickson, VA in July, August, and October of 2010.
That Hematodinium sp. was detectable in sediment during all months was expected, as we anticipated that both free-living forms, in addition to parasites from degraded crab tissue, would be present. By contrast it was predicted that the preponderance of detections in water samples would be between June-November, as these months have traditionally been those in which blue crabs have their highest prevalence and intensity of infection [16, 18], and are thus most likely to be releasing dinospores. Surprisingly, 17.4% (4/23) of our water column detections occurred in April 2010 when little or no dinospores were anticipated to be present in the water column. To our knowledge this is the earliest environmental identification of Hematodinium sp. in this ecosystem. Previous work in a Virginia coastal ecosystem detected Hematodinium sp. in the water column, but in the month of November 2007 . It should be noted that a study conducted in a Georgia estuary system in 1999 and 2000 also tested for Hematodinium sp. in the environment prior to disease in blue crabs. It was not detectable in surface waters in March or April, but was detectable in May when blue crabs began to manifest disease .
To further investigate the temporal manifestation of Hematodinium sp. in water we analyzed samples acquired from Sinnickson, VA (site 10), which in 2010 was a ‘hotspot” of environmental detection. Using primers targeting the 18 S rRNA of dinoflagellates, the presence of Hematodinium sp. was confirmed within the ecological context of other resident species (Table 2). Although not strictly quantitative, the distribution of Hematodinium sp. clones in these months was intriguing, particularly its relative abundance in April. This month yielded 15/16 (94%) of sequences that matched Hematodinium sp., and coincides with the peak abundance of 20–40 mm carapace width juveniles (MDDNR personal communication,). It is also known that, in this ecosystem, juveniles have the highest disease prevalence . Thus the presence of Hematodinium sp. in the water column at this stage may point to an important means of disease acquisition, as it is known with Chionoecetes opilio that actively molting crabs acquire infection . It should be noted that the sampling method used in this study was capable of harvesting free-living Hematodinium sp., potentially in association with zooplankton. It has been suggested that macrozooplankton, such as amphipods or crustacean larvae, may harbor parasites [16, 21]. In April, amphipods as vectors are a reasonable supposition. However, blue crab larval vectors during this month are unlikely, as release of larvae from females does not typically occur until May in this ecosystem.
The vast majority of Hematodinium sp. infections in the Chesapeake Bay region occur in the predominant crustacean species, C. sapidus, which has been classified as a clade A host species . A recent study from Delmarva Peninsula waters has suggested that a single species is responsible for all infections . We thus examined the population structure of Hematodinium sp. from sites that showed a high environmental presence, to determine if these reservoirs maintained one genetically homogenous species or other potential sub-species. All 131 clones from our libraries were >98.5% identical, with only four clones containing two nucleotide substitutions (Figure 4). Based on this particular ribosomal marker, our results suggest that the diversity of Hematodinium sp. in the Maryland Coastal Bays is low.
The consensus ITS-1 sequence in our clones is identical to ITS-1 sequences recently reported for five xalternate host species from Delmarva waters (Accession #: JN368194, JN368172, JN368154, JN368162, and JN368158) which are the: skeleton shrimp (Caprella geometrica), atlantic mud crab (Panopeus herbstii), longnosed spider rab (Libnia dubia), depressed mud crab Eurypanopeus depressus, and flat-clawed hermit crab (Pagurus pollicaris), respectively. In addition it is identical with the C. sapidus ITS-1 from an isolate in 2006 (Accession: DQ925229). Our data thus supports the previously suggested hypothesis that a single species of Hematodinium is responsible for infections in the Delmarva ecosystem . ITS sequences in ribosomal genes are predicted to show the greatest variation as they are removed during ribosomal processing. Since the ITS1 consensus sequence we observed is identical to those in alternative hosts, and has not diverged significantly since 2006, two additional implications can be drawn. A) The Hematodinium sp. that infects blue crabs is not likely to have recently received new pathogen species into this ecosystem, and B) the time that it spends associated with alternative hosts is likely brief, since more variations would be predicted if its infectivity was limited to individual host species.