Download Effects of increased mercury levels on

GCAT-SEEKquence
The Genome Consortium for Active Teaching
NextGen Sequencing Group
NextGen Sequencing Request Form
Complete fields below, save file with your last name at the beginning of
the filename (e.g. newman-GCAT-SEEK Sequence request form.pdf) and
email to Vincent Buonaccorsi <[email protected]>
A. Contact Information
1. Name:
Chris Grant (PI) and Vince Buonaccorsi (co-PI)
2. Department: Biology
3. Institution: Juniata College
4. Phone Number: 814-641-3579
5. Email Address:[email protected]
B. Project Information
1. Title: Effects of increased mercury levels on smallmouth bass Micropterus dolomie gene expression
profiles.
2. Category: Transcriptome
3. Total amount of sequence requested: 1 lane of Illumina sequencing
4. Preferred technology: Illumina
5. Do you have funds for a partial run next Spring? no
C. Describe the background, hypotheses and specific aims (500 words max)
Mercury (Hg) has long been known to have detrimental effects on wildlife and humans through
bioaccumulation and biomagnifications up the food chain (Cleckner et al 1998; Chen et al 2005; Paller et al
2004). Many fish species found in Pennsylvania not only play integral roles in stream ecosystem food webs,
but are also an important source food to humans (Meissner and Moutka 2006). Fish eating advisories due to
mercury have been posted for over 877 stream miles and 28 lakes (28,500 acres) across Pennsylvania (Lynch
et al. 2005). Current USEPA fish eating advisory levels for human consumption are 0.30micro grams of Hg
per gram of fish muscle (U.S. Environmental Protection Agency 2001b). The smallmouth bass (Micropterus
dolomieu) is a top predatory fish found in large streams and rivers that is commonly targeted by anglers in
Pennsylvania and across the Northeast. While data suggests there is regional variation of Hg concentrations
in smallmouth bass (Scudder et al 2009), little is known about how variations in Hg levels are impacting
individuals and populations of fish at the genetic level.
Long term chronic Hg affects have been shown to result in loss of coordination, hormonal imbalances, and
decreased growth and development of fish (Wiener and Spry 1996; Friedman et al 1996; Chan et al 2003).
These chronic effects of increased Hg burden in smallmouth bass could lead to increased susceptibility to
predation, and decreased ability to forage and reproduce. Coupling transcriptome technology with Hg
analysis can determine how smallmouth bass are responding to increased Hg levels at a biochemical level
that has implications for human health as well (Schirmer et al. 2010 and Sanchez et al. 2011). This research
aims to compare two metapopulations of smallmouth bass, one group with Hg levels above the USEPA fish
eating advisory and one group with Hg levels below the advisory.
D. Describe the methods [sample prep, calculation of amount of sequence required, analysis plan]
RNA-seq technology and differential gene expression will be used to compare unique properties of each
gene expression profile, allowing for an understanding of distinct aspects of the modes of action of Hg.
Mercury concentration depends on age and size of fish, so a total of ten similar sized fish (biological
replicates) will be caught per metapopulation. White muscle biopsies (200 mg) will be taken from fish white
muscle and preserved in RNAlater solution. Total RNA will be isolated using RNeasy Plus Mini kit (Qiagen).
Quantification will be performed on a Qubit 2.0 Fluorometer (Invitrogen) and mRNA purity checked with an
Implen Nanodrop Fluorometer. Isolation of mRNA from total RNA will be performed using magnetic porous
glass (MPG) mRNA Purification Kit (PureBiotech). Samples will be sent to the PSU sequencing facility for
further quality check on their Agilent Bioanalyzer. 24 human expression samples can be run on one lane of
the Illumina Hiseq so we anticipate that with a smaller genome size (~1GB), 20 samples for RNA-seq is
feasible. Results will be assembled using NextGENe or CLC Bio software if available, or on the cloud using
Velvet/Oases. Reads will be mapped against the assembled transcripts using NextGENe or Bowtie on the
Galaxy framework. Reads per Kilo-base of transcript per million mapped reads (RPKM) will be estimated to
measure gene expression and differential expression determined using the R-based DEseq package. Identity
of differentially expressed genes will be determined using Blast.
E. Describe the role and number of undergraduates involved in the project, and how they would benefit.
Grant and Buonaccorsi will teach a special topics course on Hg and environmental genomics with class size
20/year. Hands on lab bioinformatic exercises will be developed to help train the students how to perform
RNAseq analysis.
F. I agree to administer the GCAT-SEEK pre- and post-activity assessment test for students and to complete the
faculty post-utilization survey. X yes, ____ no
G. Describe any other broader impact or intellectual merit considerations.
The ultimate goal of this line of research is to use gene expression profiling to identify specific contaminant
exposures causing adverse effects in natural populations. Understanding the effects of increased Hg levels in
smallmouth bass is integral to understanding how to best address and manage Hg for the long term success of
the fish and as a sentinel for human health.
H. References:
References:
Chan, H.M., A.M. Scheuhammer, A. Ferran, C. Loupelle, J. Holloway and S. Weech. Impacts of
Mercury on Freshwater Fish-Eating Wildlife and Humans. 2003. Human and Ecological Risk
Assessment. 9(4):867-883.
Chen, Celia Y., Stemberger, R.S., Kamman, N.C., Mayes, and C.L. Folt (2005) Patterns of Hg
bioaccumulation and Transfer in Aquatic Food Webs Across Multi-lake Studies in the
Northeast US. Ecotoxicology. 14: 135-147.
Cleckner, Lisa B., Garrison, P.J., Hurley, J.P., Olson, M.L., and D.P. Krabbenhoft (1998) Trophic
Transfer of Methyl Mercury in the Northern Florida Everglades. Biogeochemistry.
40:347-361.
Friedman, A.S., C. Watzin, T. Brinck-Johnsen and J.C. Leiter. 1996. Low levels of dietary
methylmercury inhibit growth and gonadal development in juvenile walleye
(Stizostedion vitreum). Aquat. Toxicol. 35:265-278.
Lynch J.A., H.C. Carrick, K.S. Horner, and J.W. Grimm (2005). Mercury deposition in
Pennsylvania: 2005 status report. Report of the Environmental Resources Research
Institute, Penn State University, 113 pp. Lynch J.A., H.C. Carrick, K.S. Horner, and J.W.
Grimm (2005). Mercury deposition in Pennsylvania: 2005 status report. Report of the
Environmental Resources Research Institute, Penn State University, 113 pp.
Meissner, K. and T. Moutka. 2006. The role of trout in stream food webs: integrating evidence
from field surveys and experiments. Journal of Animal Ecology Vol. 75 pp: 421-433.
Paller, M.H., Jagoe, C.H., Bennett, H., Brant, H.A., and J.A. Bowers (2004). Influence of
Methylmercury from Tributary Streams on Mercury Levels in Savannah River Asiatic c
lams. Science of the Total Environment. 325: 209-219.
Sanchez BC, Carter B, Hammers HR, Sepúlveda MS. Transcriptional response of hepatic
largemouth bass (Micropterus salmoides) mRNA upon exposure to environmental
contaminants. J Appl Toxicol. 2011 Mar;31(2):108-16. doi: 10.1002/jat.1553.
Schirmer, K., Fishcher, B.B., Madureira, D.J., Pillai S. Transcriptomics in ecotoxicology.
Anal Bioanal Chem. 2010 June; 397(3): 917–923.
Scudder B.C., Chasar L.C., Wentz D.A., Bauch N.J., Brigham M.E., Moran P.W., and D.P.
Krabbenhoft (2009). Mercury in Fish, Bed Sediment, and Water from Streams Across
the United States, 1998-2005. USGS Scientific Investigations Report 2009-5109.
Wiener, J.G. and D.J. Spry. 1996. Toxicological significance of mercury in freshwater fish. Pp.
297-339 in W.N. Beyer, G.H. Heinz and A.W. Redmon-Norwood (eds.). Environmental
contaminants in wildlife: Interpreting tissue concentrations. Lewis Publ. Boca Raton, FL