Sources of Hematodiniumsp. infected blue crabs and DNA extractions
Hematodinium sp.-infected blue crabs and ethanol-fixed blue crab hemolymph from Mississippi were kindly provided by colleagues at the Gulf Coast Research Lab (GCRL, Ocean Springs, MS; Noah Zimmerman, Jeff Lotz) and shipped to the Institute of Marine and Environmental Technology (IMET) by overnight courier. Blue crabs infected with Hematodinium sp., obtained from the MD coastal bays, were transported to IMET in coolers and frozen after arrival. Originally identified by the appearance of parasites in fresh hemolymph, all Hematodinium sp. infections were verified by PCR amplification using the primers described in Nagle et al. or Gruebl et al. .
DNA was extracted from hemolymph of confirmed Hematodinium sp.-infected crabs using the Qiagen DNAEasy Blood and Tissue Kit (Qiagen, Valencia, CA, Cat. # 69506). For DNA extractions from field-collected samples of crabs and other invertebrates, the MoBio Tissue Kit was employed (MoBio, Carlsbad, CA, cat. # 2334). DNA was extracted from water and sediment samples using the MoBio Ultraclean Soil kit (cat. #12800), which includes mechanical tissue disruption. DNA was quantified by spectrophotometry (Nanodrop Inc., Wilmington, DE).
Amplification and cloning the ITS1-5.8S-ITS2 region of the ribosomal gene cluster
Using Taq polymerase (ExTaq, Takara Bio, Inc., Otsu, Shiga, Japan), and primers 1487 F and D2C (Table 1), DNA fragments encompassing the ITS1-5.8S-ITS2 region were amplified from genomic DNA preparations of a Mississippi and Maryland H. perezi- infected blue crab. Hematodinium-specific primer 1487 F  and a generic eukaryotic primer D2C  were used to amplify and clone a ~2 kb region of the rRNA gene cluster from DNA extracted from 4 individual H. perezi-infected crabs from both Maryland and Mississippi. Thermocycling conditions were 95°C 5 min, followed by 30 cycles of 94°C, 25 sec; 54°C, 30 sec; 72°C, 3 min. The resulting ~2 kb fragment was ligated to pGEM-T according to manufacturer’s instructions (Promega Corp., Madison, WI) and transformed into E. coli strain JM109 (Promega Corp.). Sequencing was performed on a minimum of 3 clones from each infected crab (Gulf or Atlantic), using a combination of primers originating in the pGEM-T vector sequence and cloned H. perezi DNA (see Table 1: primers 1487 F, M13F, M13R, HemITSF2, and HemITSR2). Sequencing was conducted using Big Dye Terminator reagents (Applied Biosystems, Inc., Carlsbad, CA) and analyzed with the ABI 3130 XL Genetic Analyzer (Applied Biosystems, Inc.). Sequences were aligned using Sequencher (Genecodes Corp, Ann Arbor, MI) and CLC DNA Workbench (CLC Bio, Aarhus, Denmark). From assembled consensus sequences, additional primers were designed to amplify specific segments of the ITS1-5.8S-ITS2 region to provide additional sequence depth . A consensus sequence of 2056 nucleotides was derived from the assembly and has been submitted to GenBank as accession JQ815886.
Development of ITS2-targeted qPCR assay
BLAST analyses of the consensus sequence were used to identify boundaries between SSU, ITS1, 5.8S, ITS2 and LSU regions, as compared to cognate sequences in GenBank . Based on the ~280 bp ITS2 region, PCR primers ITS2For and ITS2Rev (Table 1) were designed (CLC bioinformatics software). A TaqMan probe (HemITS2probe) was designed, carrying 6-FAM and BHQ1 as reporter and quencher dyes, respectively. Using the above primers at 400 nm, probe at 300 nm, Taq-pro complete (Denville Scientific, Metuchen, NJ) with 2.5 mM final MgCl2 and 0.25 mg/ml BSA (Idaho Technologies, Salt Lake City, Utah), thermocycling was carried using the following conditions: initial heating to 95°C, 5 min; followed by 95°C, 15 sec; 58°C, 30 sec, 45 cycles. For routine analyses, thermocycling was performed on 1 μl of DNA (10 to 50 ng) using an Applied Biosystems Fast7500 thermocycler (Indian River Inlet samples) or Bio-Rad iCycler iQ Optical Model (Chincoteague Bay samples). Cross-checking with a dilution series of the same preparation of plasmid-borne target DNA confirmed that the assay performed with the same sensitivity on both iCycler and Fast7500 thermocyclers.
Sensitivity and Specificity of the ITS2 assay
The sensitivity of the ITS2 assay was investigated by conducting qPCR on a serial dilution of plasmid pES103, which carries the 2 kb amplicon produced from primers 1487F and D2C, encompassing the 3’ end of the SSU gene as well as complete ITS1, 5.8S, ITS2 and partial LSU genes. An amplicon of the ITS2 region, produced by PCR amplification of pES103 using primers HemITS2for and HemITS2rev, was produced and cloned in pGEM-T to create plasmid pES146. Based on spectrophotometric measurements of plasmid concentration, a series of samples from 13 to 1.3 × 10E + 06 copies were prepared in nuclease free water. Quantitative PCR was conducted on 1 μl of each dilution in triplicate and the results plotted as Ct versus copy number. Slope and R2 of the standard curve were calculated using the ABI software (Applied Biosystems 7500 Fast Real-Time PCR system).
Validation of the qPCR assay using H. perezicell surrogates
Because H. perezi cells were not easily obtainable, it was necessary to develop an H. perezi surrogate to use in the validation of the assay. This cell surrogate consisted of E. coli strain JM109 carrying the plasmid pES103 (ITS1-5.8S-ITS2 regions). The H. perezi surrogate was grown in LB + AMP liquid broth medium overnight. Based on ODA600 of 1.9 = 1 × 109 cells ml-1 we made a 10 fold serial dilution of the overnight culture that was calculated to have from 3 to 3 × 105 bacteria/microliter to spike into sediment. The bacteria cell number was also checked by plating overnight culture on LB + AMP plates. DNA extraction of the H. perezi surrogate was done using the MoBio Tissue and Cells DNA extraction kit per manufacturer’s instructions. For spiked sediment, the serially diluted H. perezi surrogate was added to sediment from Baltimore Harbor (and shown to be negative for H. perezi by ITS2 qPCR). DNA was then extracted from each spiked sediment sample using the MoBio Soil DNA Extraction kit per manufacturer’s instructions. We used the qPCR assay to compare slopes and limits of detection using 1 μl of DNA.
It has been reported that using linearized target DNA may amplify more than one cycle earlier than circular cDNA for a given amount of input DNA, resulting in a miscalculation of gene copy number in unknown samples . To address this issue we compared the slopes and limits of detection of plasmid pES146 in the linear and circular conformations. We found that the linearized target and intact plasmid gave rise to similar Ct numbers compared to those from use of circular DNA (data not shown).
Environmental sample collection and DNA extractions
Sampling locations and seasons
Sampling was conducted at 13 sites in the Indian River Inlet during 2008 and 2009 (Table 2 and Figure 3). In 2008 (07.01.08, 07.29.08, 12.09.08) single samples were collected, while in 2009 (07.15.09), Indian River Inlet sediment samples were taken in triplicate. At all dates, single water samples were taken. Sampling was conducted at 18 sites in and near Chincoteague Bay (Figure 4) between April and November of 2010, in conjunction with the National Park Service water quality monitoring program. The only exception was September, when only three sites were sampled: Verrazano Bridge (Site 2), Newport Bay (Site 3), and Public Landing (Site 5). Maps of sampling were generated with The Generic Mapping Tools v. 4.5.9  using hydrography data retrieved with National Map 2.0 Viewer (U.S. Geological Survey; http://viewer.nationalmap.gov).
Estuarine invertebrates were collected 07.29.08 in Indian River Inlet near sites 1, 2, 5, and 7 at low tide by hand or using long handled nets. In the field, individual animals were placed in labeled ziplock bags and kept on ice until transport to the lab, where they were stored at −20°C. DNA was extracted using the MoBio Cell and Tissue Kit. From snails (I. obsoleta), DNA was extracted from a cross section of the viscera that included the stomach or intestine. From grass shrimp (P. pugio), DNA was extracted from a cross section of the abdomen that included the intestine. From crab species (C. sapidus, C. maenas, O. ocellatus), DNA was extracted from walking leg or back fin muscle tissue.
The top 3–5 cm of sediment was collected within Indian River Inlet, DE and Chincoteague Bay, MD and VA. In shallow water (DE), sediment was collected directly with a 50 ml conical tube. In water over 1.5 m deep, a petit ponar grab was used to obtain sediment, and the top layer collected with a 50 ml conical tube. All samples were maintained on ice for transport to the laboratory. Sediment samples were mixed with an equal volume of 15 ppt sterile artificial seawater (Crystal Sea, Marinemix, Baltimore, MD) and 0.5 ml aliquots were frozen at −20°C for up to 6 months until DNA extractions were performed.
DNA was extracted from Indian River Inlet sediment using the MoBio UltraClean Soil DNA kit following manufacturer’s recommendations, with the exception that DNA was eluted from the purification matrix in two aliquots of nuclease free water rather than a single elution. DNA preparations were divided into two aliquots and stored at −80°C. DNA was extracted from MD and VA coastal bays sediment using the Sureprep (Fisher Bioreagents) soil DNA isolation kit.
In Indian River Inlet, surface water samples were collected using 1, 2, and 4 L bottles. Water was maintained at 0–4°C until further processing. For 2008 samples, 60 ml aliquots were filtered onto 2.5 cm diameter 1 μM filters (Millipore) using a Swinnex cartridge. DNA was extracted from filters using the MoBio UltraClean Soil DNA kit. For 2009 samples, water was centrifuged at 500 × g for 30 min to sediment particles, and the entire pellet was extracted with the MoBio UltraClean Soil DNA as described above. In Chincoteague Bay, water was sampled using a 30 cm diameter plankton net, towed at ~3 knots for 3 min, resulting in an effective sampling volume of ca. 25 m3. The collected material was saved in 500 ml seawater, refrigerated, and transported to the laboratory where 200 μl was used for DNA extractions using the Illustra tissue and cells GenomicPrep™ DNA kit (GE Healthcare, Piscataway, NJ).