AFSC/RACE/SAP/Shavey: DNA extraction from archived Giemsa-stained blood smears using polymerase chain reaction to detect host and parasitic DNA

Eastern Bering Sea, Alaska; Gulf of Alaska; Southeast Alaska, Washington State Coast, North Atlantic Coast

Note: Dataset migrated by Dan Woodrich (AFSC data management coordinator) on 12/15/2021. Contact: Daniel.woodrich@noaa.gov

A subset of 76 blood smears that were prepared in the field from 1988 to 2004 were chosen based on smear preparation (Giemsa-stained vs. no stain and coverslipped vs no coverslip) and parasite status (parasite presence vs. no parasite). All content was removed from each smear using a pipette tip and sterile blade. DNA was extracted and amplified using crab host and parasite PCR primer sets.

SPNO is a number assigned to each collected crab. Duplicate numbers represent replicate smears taken from sample.

The sex of the crab is identified as: 1) Male, 2) Female, 3) Unknown. Individual crab carapaces were measured (1 mm), excluding spines, and are reported as size. Null or blank fields in data represent no size information reported for crab specimen.

Shell condition class serves as a semi-quantitative index of molt status and time in shell post-molt. Carapace shell condition was assessed for each Chionoecetes crab sampled and assigned to one of six classes according to specific criteria (0 = premolt or molting, 1 = soft and pliable, 2 = new hardshell both firm and clean, 3 = oldshell slightly worn, 4 = oldshell worn, 9 = no shell condition information reported for crab specimen). Collection location was not a factor, so only general location is listed.

Blood smears analyzed were either unstained (none), or fixed with methanol and Giemsa-stained for 30 minutes (Giemsa). Treatment of blood smears are identified as (0 = no cover slip, 1 = cover slip attached using mounting media).

30 fields at 250X were read to determine presence or absence of Hematodinium spp. parasite on blood smear. Results were documented as: (0 = Hematodinium parasite absent on smear, 1 = Hematodinium parasite present on smear). Hematodinium parasite presence on smear was identified as T-rating (0 = No rating due to parasite absence, T1 = Up to 10% Hematodinium parasite present on smear (trophont stage), T2 = 10 - 40% Hematodinium parasite present on smear (trophont stage), T3 = 40 - 60% Hematodinium parasite present on smear (trophont stage), T4 = 60 - 90% Hematodinium parasite present on smear (trophont stage), T4P1 = 60 - 90% Hematodinium parasite present on smear (trophont stage) and up to 10% Hematodium prespore stage, T5 = Above 90% Hematodinium parasite present on smear (trophont stage), T5P1 = Above 90% Hematodinium parasite present on smear (trophont stage) and up to 10% Hematodium prespore stage).

PCR assays: for 16S, COI, 18S and ITS Primer (0 = Did not use primer set on this sample, 1 = Used primer set on this sample). For 16S, COI, 18S and ITS Amplification (0 = No DNA amplification using this primer set, 1 = DNA was amplified using primer set, 9 = no amplification to report due to primer set not used in sample). Primer set descriptions: 16S = 16Sar-L (CgC CTg TTT ATC AAA AAC AT) & 16Sbr-H (CCg gTC TgA ACT CAg ATC ACg T) primer set used in PCR assay, targeting mitochondrial DNA 16S ribosomal RNA gene. COI = LCO1490 (ggT CAA CAA ATC ATA AAg ATA TTg g) & HCO2198 (TAA ACT TCA ggg TgA CCA AAA AAT CA) primer set used in PCR assay, targeting the mitochondrial cytochrome c oxidase subunit I gene. 18S = HF1487 (CCT ggC TCg ATA gAg TTg) & HR1654 (ggC TgC CgT CCg AAT TAT TCA C) primer set used in PCR assay, targeting the 18S ribosomal gene located at the small subunit 3'-end of the ribosomal RNA unit. ITS = HSP7f (AGT CAT CAG CTC GTG CTG A) & HSP9r (TTC ACG GAA TTC TGC AAT TCG) primer set used in PCR assay, targeting the first internal transcribed spacer of the ribosomal RNA unit. Statistical analysis: Blood smears were analyzed using two-sample t-test (0 = smear was not used in statistical analysis, 1 = smear was used in statistical analysis).