Suggested Citation:
Susan C. Chiasson and Caz M. Taylor. 2017. Expression of hsp90 and vtg in the Juvenile Blue Crab, Callinectes sapidus, Following Exposure to Oil and Dispersed Oil. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7348HRT
Data Parameters and Units:
RE_oil_disp.csv - ID = internal Sample ID, Treatment = treatment crab was exposed to (oil (500 ppb WAF), oil_disp (oil concentration 500 ppb WAF, dispersant concentration 25 ppm)), RE VT Relative expression of vitellogenin, RE HSP = Relative expression heat shock protein 90 qPCR_oil_disp.xlsx = treatment (oil (500 ppb WAF), oil_disp (oil concentration 500 ppb WAF, dispersant concentration 25ppb)), ID = internal sample ID, CT Ak = control gene (Arginine kinase) cycle threshold, Ct HSP90 = heat shock protein 90 cycle threshold, Ct VT = vitellogenin cycle threshold
Methods:
Collection of juvenile crabs Juvenile blue crabs were collected with dip nets from a dock at Louisiana Universities Marine Consortium (LUMCON) in Cocodrie, LA at night in July 2015. Crabs were placed in 5-gallon containers and aerated during transport to laboratory facilities at Tulane University in New Orleans. Upon arrival at the laboratory, crabs were fed freeze-dried shrimp (Omega One) and gradually acclimated to 20 ppt salinity in artificial seawater (Instant Ocean in deionized water) over 72 hours. Crabs were placed in individual containers covered with mesh to prevent cannibalism, and the containers were placed in 10-gallon aquaria (10 boxes per aquarium). Ammonia levels were measured during the acclimation period to ensure ammonia did not exceed 0.5ppm, and 75% water changes were done as needed. Crabs were not fed for 24 hrs prior to the start of the experiment. Out of 85 crabs that were collected, 60 crabs that were most similar in size were used for the experiments. Experimental conditions A Water Accommodated Fraction (WAF) and a Chemically Enhanced Water Accommodated Fraction (CEWAF) of South Louisiana Crude oil (MC252 surrogate) were prepared using standard protocols. The WAF and CEWAF were both made with 20 ppt artificial seawater to match the salinity crabs were acclimated to, and 150 mg of crude oil was added to 1.5 l water, making the nominal crude oil concentration in both 100 ppm. Corexit EC9500 dispersant was then added to the CEWAF to create a 1:20 dispersant:oil ratio. The WAF and CEWAF were stirred for 24 hrs, after which they were further diluted to 500 ppb to mimic the highest reported oil concentration in the NGOM during the spill. The concentration of Total Petroleum Hydrocarbons measured in the WAF was 177 ug/l and in the CEWAF 6220 ug /l, and GC/MS analysis was conducted using EPA method 3510 C. Toxicity test were conducted using according to CROSERF guidelines for Static Exposure. Crabs were exposed to oil, oil/dispersant, or 20 ppt artificial seawater (control) for both acute (mRNA expression) response experiments with sample sizes, N = 15 for each gene expression experiments. Crabs were assigned to the different treatments so that the size variation of crabs in each treatment was uniform. The exposure was done in 2 l glass jars for 24 hrs. Based on our earlier pilot experiment, we determined that a 2 l jar was sufficient to hold one crab for 24 h without ammonia levels exceeding 0.5 ppm. The crabs were not fed during the exposure. For mRNA expression analysis, crabs were sacrificed immediately after the 24 h exposure period, and the hepatopancreas was removed and stored in a -30 degrees C freezer in RNAlater(Invitrogen). Prior to dissection, crabs were anesthetized by placing them on ice. RNA extraction and quantitative PCR RNA was extracted with the Ambion mirVANA kit (Life Technologies #AM1560). Hepatopancreatic tissue was weighed and lysis buffer was added at 10 x buffer volume per 1 mg tissue. Tissue was then homogenized and homogenate additive added at 1 ul per 1 mg tissue. Tissue samples were put on ice and an equal volume of acid-phenol:chloroform as lysis buffer was added. Samples were centrifuged for 5 min at 10,000 g, and the aqueous layer was recovered. 100 % ethanol (Pharmco-Aaper; #111ACS200) was added at 1.25x volume to the aqueous aliquot, and washed three times with wash solution. mRNA was recovered with nuclease-free water (NFW) at 90 degrees C. The Superscript III First Strand Synthesis kit (Invitrogen, #18080051) was used to generate first-strand cDNA. For 1 ug RNA, 1 ul random hexamers, 1 ug 10 mM dNTP were added, and NFW was added for a total volume of 10 uL. Extracts were incubated at 65 degrees C for 5 minutes, and put on ice for 1 min. A 10 uL mastermix consisting of 2 ul 10X RT buffer, 4 ul 25 mM MgCl, 2 ul 0.1M DTT, 1 ul RNase OUT (40U/ul), and 1 ul Superscript III RT was added to each sample and incubated for 10 min at 25 degrees C, 50 min at 50 degrees C, and finally 5 min at 85 degrees C to stop the reaction. Quantitative PCR was done using the Applied Biosystems 7500 Real-Time PCR system with SYBR green dye (Applied Biosystems, #4368708). Amplification of all cDNA samples was done in triplicate, and gene expression was quantified with the delta delta CT method. Gene-specific primers TaqvitF (5′-TGTACAGCTGAAAGGCGTGG-3′) and TaqvitR (5′-CATGGGCCGAGAACAGTCA-3′) for vtg and HSP90F2 (5’-CACCGACAACATCAAGCTGTAC-3’) and HSP90R2 (5’-ACACCACGCACAAAGTTGAG-3’) for hsp90 were purchased from Life Technologies (Thermo Fisher Scientific). Arginine kinase (ak) abundance, TaqAKF (5′-ACCACAAGGGTTTCAAGCAG-3′) and TaqAKR (5′-GGTGGAGGAAACCTTGGACT-3′) was used as the control gene to normalize target gene expression against.
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