Abstract:
Juvenile blue crabs, Callinectes sapidus, were exposed to increasing concentrations of 4-nonylphenol and butylated hydroxytoluene (BHT). Uptake was measured with GC/MS and relative mRNA expression of genes coding for heat shock protein 90 (hsp90) and vitellogenin (vtg) was measured using qPCR. These compounds were chosen based on contaminants detected in megalopae collected at multiple sites in the Northern Gulf of Mexico.
Suggested Citation:
Susan C. Chiasson and Caz M. Taylor. 2017. Uptake of 4-nonylphenol and butylated hydroxytoluene in juvenile blue crabs, and expression of mRNA of genes encoding heat shock protein 90 and vitellogenin in response to different concentrations of 4-nonylphenol and butylated hydroxytoluene. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/N7N29V8B
Purpose:
This dataset was generated to evaluate molecular effects on juvenile blue crabs of contaminant exposure at multiple concentrations of contaminants present in the Northern Gulf of Mexico. This dataset provides information about dose-response effects of compounds that crabs are exposed to in the environment.
Data Parameters and Units:
GCMS_BHT.xlsx -ID =internal sample ID, ppm_treatment (concentration of butylated hydroxytoluene crab exposed to, ppm), ppm_crab_BHT (concentration of butylated hydroxytoluene measured in the hepatopancreas after 24 hours (ppm)) GSMS_NP.xlsx - ID = internal sample ID, ppm_treatment (concentration of 4-nonylphenol crab exposed to, ppm), ppm_crab_NP (concentration of 4-nonylphenol measured in the hepatopancreas after 24 hours (ppm)) qPCR_uptake_1.xlsx - ID = internal sample ID , Ct AK = Arginine kinase (AK) cycle threshold, Ct VT = vitellogenin cycle threshold, Ct HSP = heat shock protein 90 cycle threshold qPCR_update_2.xlsx - ID = internal sample ID, Ct AK = Arginine kinase (AK) cycle threshold, Ct VT = vitellogenin cycle threshold, Ct HSP = heat shock protein 90 cycle threshold RE_update_NP_BHT.csv 0 ID = internal sample ID (Ctrl = control), Replicate = replicate number in treatment, NP.ppm = 4-nonylphenol concentration in treatment (ppm), RE VT = mRNA relative expression gene coding for vitellogenin, BHT.ppm = butylated hydroxytoluene concentration in treatment (ppm) RE HSP = mRNA relative expression for gene coding for heat shock protein 90 Relative expression of hsp90 and vtg, concentrations of 4-nonylphenol and BHT in juvenile blue crabs
Methods:
Juvenile blue crabs were exposed to nonylphenol and BHT at multiple concentrations for 24 hours, after which half of the hepatopancreas was removed for gene expression analysis and the rest of the crab was dried, homogenized, and extracted with methylene chloride to test for accumulation in tissues. GC/MS was used to test for accumulation of compounds and qPCR was used to test for differential gene expression. Collection of juvenile crabs Juvenile blue crabs were collected with dip nets from a dock at Louisiana Universities Marine Consortium (LUMCON) in Cocodrie, LA in August 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 and gradually acclimated to 20 ppt salinity in artificial seawater (Instant Ocean in filtered water) over 72 hours. To prevent cannibalism, crabs were placed in individual boxes with slits for water exchange, and the boxes were placed in 10-gallon aquaria (10 boxes per aquarium). Ammonia levels were measured during the acclimation period to ensure ammonia did not exceed 0.5 ppm, and 75% water changes were done as needed. Crabs were not fed for 24 hours prior to the start of the experiment. Crabs that were most similar in size were used for the experiments, and a similar size variation was selected for each treatment. Experimental conditions Crabs were exposed to NP and BHT under static conditions in 2 l glass jars, with N = 10 for each treatment. Because NP and BHT have limited solubility in water, 0.01% un-denatured ethanol was used as a carrier solvent. A pilot experiment was done to ensure that a 2 l jar was sufficient to hold one crab for 24 hours without ammonia levels exceeding 0.5 ppt. For uptake experiments, juvenile blue crabs were exposed to 0, 0.1 ppm, 1 ppm, 10 ppm, 100 ppm, and 200 ppm concentrations of NP and BHT in 20 ppt artificial seawater for 24 hrs. After 24 hrs crabs were sacrificed, and approximately half of the hepatopancreas was removed, placed in RNAlater, (Life Technologies) and stored at -30 degrees C . The remaining crab tissue was stored at -80 degrees C freezer for GC/MS analysis. To test for effects on gene expression and growth at a single concentration, crabs were exposed to 200 ppb of NP and BHT for 24 hrs. For mRNA analysis, crabs were sacrificed immediately after the exposure period, and the hepatopancreas was removed and stored in RNAlater at -30 degrees C . 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 10x ul buffer volume per 1 mg tissue weight. Tissue was then homogenized and homogenate additive added at 1 ul per 1 mg tissue weight. 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 was added at 1.25x volume to the aqueous aliquot, and washed three times with wash solution. mRNA was recovered with nuclease-free water at 90 degrees C. 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 nuclease-free water (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 vitellogenin 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 against which target gene expression was normalized.
View Metadata