North Pacific Research Board
Core Research Program
Semi-Annual Progress Report
Project number: 1705
Project title: Effects of temperature change and Hematodinium sp. infection (Bitter Crab Disease) on Tanner crab (Chionoecetes bairdi)
Principal Investigator(s): Dr. Pamela C Jensen & Dr. Steven Roberts
Reporting period: Sept 1, 2017 to July 31, 2018
Submission date: July 31, 2018
Accomplishments and results
Approximately 400 male Tanner crabs, Chionoecetes bairdi, were collected during the Alaska Department of Fish & Game fall survey and transported to the Alaska Fisheries Science Center (AFSC) Auke Bay Laboratories (ABL) in Juneau. Crabs were placed in insulated tanks at approximately 7°C, the temperature of the bottom water where they were collected. Crab biometric data was recorded, and hemolymph samples drawn for preparation of blood smears and preservation in ethanol. Crabs were held at 7°C for 9 days while ethanol-preserved DNA was extracted & Hematodinium sp. infection status determined for each crab with a conventional PCR assay for Hematodinium sp. (Jensen et al. 2010). On Day 9, based on the PCR results, 180 crabs were equally distributed among 9 tanks (10 Hematodinium-positive and 10 Hematodinium-negative crabs per tank) and the surplus crabs were removed from the tanks. Hemolymph samples were taken from the 180 experimental crabs and preserved in RNAlater Stabilization Solution. After the hemolymph draws on Day 9, over the following 2 days, tank temperatures in 3 ‘cold’ tanks and 3 ‘warm’ tanks were adjusted to the experimental treatment temperatures of 4°C and 10°C, respectively, while 3 tanks remained at ‘ambient’ temperature of 7°C; these temperatures were maintained for the duration of the experiment. On Day 11, hemolymph was drawn and preserved in RNAlater. On Day 27, the final hemolymph samples were drawn and preserved in RNAlater and the experiment terminated. RNAlater-preserved samples were transported to the Roberts’ laboratory at the University of Washington for processing.
Figure 1. A, experimental treatment tanks at ABL; B, Grace Crandall, the graduate student on the project, taking a hemolymph sample for preservation in RNAlater from a Tanner crab.
We anticipated high initial mortality of the crabs due to capture and transport stress, reflected by the number of surplus crabs collected, and indeed, lost ~100 crabs before the experiment began. Throughout the experiment, survival in the cold and ambient treatments was higher than anticipated, with 92% of the crabs in each treatment surviving to the end of the experiment. However, in the warm treatment, mortality was high with 50% of the crabs dying between Days 9 and 11, and all but 1 crab per treatment tank succumbing by the end of the experiment.
qPCR assay for Hematodinium infection intensity
In Seattle, DNA was extracted from an aliquot of the Day 27 RNAlater preserved hemolymph. That DNA and DNA extracted from Day 1 ethanol-preserved hemolymph processed in ABL was subjected to a qPCR assay for Hematodinium sp. (Crosson et al., in prep) in order to quantify Hematodinium infection intensity (DNA copy number of Hematodinium). The results will be used to corroborate RNA-Seq analysis results and to investigate the relationship between infection intensity and gene expression levels in the crabs. The qPCR assay, which is more sensitive than the conventional PCR assay, revealed some Hematodinium positive crabs among the crabs that were negative for Hematodinium via the conventional Hematodinium PCR assay. We are using this additional information to inform our sample selection for transcriptome sequencing and analysis.
Hemolymph samples from 51 crabs (3 sample dates for most crabs, 2 sample dates for some warm treatment crabs: 138 total samples) were processed using a RNA isolation protocol adapted from RNAzol RT. After the completion of the protocol, the isolated RNA was resuspended in 50ul of 0.1% DEPC-treated water, quantified, and stored in -80˚ until sample pooling and/or sequencing. We are currently performing quality assessments on samples for library construction and sequencing that we plan to have underway by the end of this summer.
Based on the timeline in the proposal we are slightly behind schedule in constructing RNA-seq libraries. Due to the high mortality in one treatment group (see below), we decided to evaluate more samples for sequencing than initially planned and to submit samples for sequencing in series, rather than all at the same time. This will allow us to fine tune the sequencing to maximize information from all the crabs, but especially the treatment group that suffered high mortality. We expect to be back on schedule for transcriptome analysis by the end of the year.
Crosson LM, Wight N, White VC, Vadopalas B, Morado JF, Freidman CS Validation of a real-time PCR assay to detect and quantify Hematodinium sp. in Alaskan Tanner crab, Chionoecetes bairdi.in prep.
Jensen PC, Califf K, Lowe V, Hauser L, Morado JF (2010) Molecular detection of Hematodinium sp. in Northeast Pacific Chionoecetes spp. and evidence of two species in the Northern Hemisphere. Diseases of Aquatic Organisms 89:155-166