Title Here

Title Here

Is Environmental Pollution Resulting in Biodiversity of Indigenous Species from the North and South Shores of Long Island?
Ashley Hernandez & Kimberly Serna E. Irwin Mentor Freeport High School
Objective

Abstract

As members of the Long Island Community, we have the responsibility to monitor and protect our
environment. The goal of our DNA Barcode project is to compare insects and fungi from the
north shore (Smithtown area) and south shore (Freeport area) of Long Island. Using DNA
Barcoding to study the biodiversity of these regions, we would determine if the identification of

Discussion / Conclusion

DNA barcoding has emerged as the primary method of identifying species of plants, animals, invertebrates

Preliminary results support our hypothesis that locations high in mercury (and possible other

and fungi. The primary goal of our DNA Barcode project is to compare insects and fungi from the north shore

metals) will result in the larger numbers of mismatches following DNA Barcode analysis. Five of

(Smithtown area) and south shore (Freeport area) of Long Island. Using DNA Barcoding, it is hypothesized

our samples had a significant number of mismatches. We also noticed that the insects with the

that the identification of new species can be used as an indication that environmental contamination has direct most mismatches tended to have the highest mercury level in the soil collected with the sample.

new species can be used as an indication of environmental pollution, specifically trace metals.

Introduction

involvement in altering biodiversity on Long Island. Insects and fungi were collected along with soil samples.

For example, one of our samples had 78 mismatches and the mercury level was 0.218 ug Hg/g

Soil was analyzed for mercury using atomic absorbance and used as an indicator of environmental pollution.

and another organism had 4 mismatches and the mercury level was .052 ug Hg/g. We believe

As anticipated, soil samples taken near landfills of the north and south shores showed an increased level of

that these results indicate that high levels of mercury in the environment can push organisms to

mercury concentration. Values ranged between .218 to .371 ug Hg/g compared to the value found in local

adapt to a harsher environment. For example, values near landfills ranged between .218

A "DNA barcode" is a unique pattern of DNA sequence that identifies each living thing.

households that ranged from .032 to .065 ug Hg/g. The environmental Protection Agency set the 5% level in

and .371 ug Hg/g compared to local households that ranged from .032 to .065 ug Hg/g. The

DNA barcoding first came to the attention of the scientific community in 2003 when Paul Heberts

soil at .05 - 08.ug/g. At a 5% level, 95% of the organisms will be affected. In preparation for sequencing, DNA

environmental Protection Agency set the 5% level in soil at .05 to .08 ug/g. At a 5% level, 95%

research group at the University of Guelph published a paper titled "Biological identifications through

extractions were performed followed by the PCR reaction. PCR products were displayed by agarose gel

of the organisms will be affected. In preparation for sequencing, DNA extractions were

DNA barcodes".

electrophoresis and only successful PCR products submitted for sequencing. Eight insects and four

performed followed by the PCR reaction using the primers COl for insects and ITSI for fungi.

They proposed a new system of species identification and discovery using a short section of DNA

mushrooms samples were successfully sequenced. NPQ-008, a mushroom, showed 53 mismatches, with a

PCR products were displayed by agarose gel electrophoresis and only successful PCR

from a standardized region of the genome. That DNA sequence can be used to identify different

soil reading equal to .134 ug Hg / g. NPQ-003, a grasshopper and a worm NPQ-018 from near the Kings

products submitted for sequencing. Eight insects and four mushrooms samples were

species, in the same way a supermarket scanner uses the familiar black stripes of the UPC barcode

Park landfill, showed 26 and 16 mismatches respectively and a soil reading of .240 ug Hg/ g. An insect from

successfully sequenced. Definite conclusions should not be drawn due to low sample number

to identify your purchases.

near the Smithtown landfill with a soil concentration of Hg = .218 ug/g showed 78 mismatches. Legal

and our inability to obtain samples within the landfills. Once the legal concerns have been

DNA barcoding will allow users to efficiently recognize known species and speed the discovery of

considerations prevented the team from taking samples inside the landfills. These legal issues are being

addressed similar insects and mushrooms from landfills will be included to broaden this study.

species yet to be found in nature.

addressed and In the future landfill organisms with soil samples will be analyzed.

Data indicates that mercury is present in larger amounts in soil samples taken from landfills in
Smithtown and Merrick and a wide range of levels was observed in fish and shellfish caught locally.

The Montana Department of Environmental Quality has published an in-depth report on species

proposal included analysis of organisms and levels of mercury in soil as a

genetic analysis in order to distinguish between three morphologically similar species of fungi. DNA

possible indication of biodiversity. Unexpectedly, we were not allowed to take

primers are the key to Barcoding success and are based on the requirement for amplifying specific

samples from any of the local landfills. Attempts to acquire samples resulted in

DNA sequences.

a requirement for us to obtain legal permission. Our school district was notified

As part of the iPlant Collaborative, the Dolan DNA Learning Center and a team of computer

and we are in the process of acquiring this legal permission. In order to

Amanita cylindrispora

Roach

programmers have developed a workflow to make high-level genome analysis broadly available to

Moth

Lumbricus terrestris

How samples were collected:

biology students and teachers. DNA Subway uses key bioinformatics tools and databases to

Collecting procedures are outlined in Cold Spring Harbor Laboratorys DNA Barcoding 101. The sample collection site

assemble gene models, investigate genomes, work with phylogenetic trees and analyze DNA
barcodes. DNA Subway is an appealing and intuitive interface that uses the metaphor of a network of

We are in the process of analyzing samples within the phylogenetic tree. Our

Step 1: Collect Samples

collection and identification using the taxonomists approach. Pota et. al. reported the need to include

Future Work

Procedures

strengthen our hypothesis that soil contamination is pushing biodiversity, more
samples from control and within landfills are necessary.

was approached as how a forensic investigator examines a crime scene. The area to be investigated for samples was
identified initially at a distance of several yards. Then, the site was approached with a measuring tape keeping track of

subway lines to guide users through the maze of analysis steps necessary to annotate and compare

the distance from the starting point. A one square meter form was placed over the area to study. Each sample site

DNA sequences.

and surrounding area was photographed and a GPS reading recorded. Samples were collected by reaching from

The concern over soil contamination stems primarily from health risks, from direct contact with the

outside the collecting area and transferred into an opened zip-lock bag (the quadrant technique). The bag was
labelled with suspected species (if known), and location including GPS coordinates and stored in a portable cooler for

contaminated soil, vapors from the contaminants, and from secondary contamination of water

the trip back to the high school and stored at -20C .

supplies within and underlying the soil. The most common chemicals involved in causing soil

Step 5: Data entered into DNA Subway

contamination are petroleum hydrocarbons, heavy metals, pesticides, solvents. Although lead and
mercury may be found naturally in soil, high concentrations of either metal may cause damage to the
developing brains of young children, which in turn may lead to neurological problems. Humans of any

Nadolnya, Robyn, Holly Gaffa, Jens Carlssonc, David Gauthiera Comparative population genetics of two
invading ticks: Evidence of the ecological mechanisms underlying tick range expansions Infection, Genetics and
Evolution Volume 35, October 2015, Pages 153162
Ratnasingham, S., Hebert, P.D.N (2007). BOLD: The Barcode of Life Data System. Molecular Ecology Notes
7(3): 355-64.

Primers:
Invertebrate COI gene LCO I 490 / HCO2 I 98
LCO1490_F
5-GGTCAACAAATCATAAAGATATTGG-3 (forward primer)
HC02198_R
5-TAAACTTCAGGGTGACCAAAAAATCA-3 (reverse primer)

5-TCCGTAGGTGAACCTGCGG-3 (forward primer)
5-TCCTCCGCTTATTGATATGC-3 (reverse primer)

94C 1 minute
35 cycles of the following profile:
Denaturing Step:
94C
1 minute
Annealing Step:
55C
1 minute
Extending Step:
72C
2 minutes
72C 15 minutes
40C then store at -20C until 2% agarose gel electrophoresis

Hebert P.D.N., Penton E.H., Burns J.M., Janzen D.H., Hallwachs W. (2004). Ten species in one: DNA barcoding
reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci USA.
101(41):14812-7.

Jian Huanga, Qin Xub, Zhen Jun Suna, Gui Lan Tanga, Zi You Sua Identifying earthworms through DNA
Barcodes Volume 51, Issue 4, 19 October 2007, Pages 301309

PCR procedure:
Thaw DNA samples Label the tubes with PCR beads Thaw then add 23 uL primer mix
per tube Dissolve beads by flicking Add 2uL sample DNA, flick to mix Cover with 2030uL mineral oil Amplify then store PCR products at -20C until ready to run 2% agarose
gel

Fungi ITS
ITSI F
ITS4 R

Benson D.A., Cavanaugh M., Clark K., Karsch-Mizrachi I, Lipman D.J., Ostell J., Sayers E.W. (2013).
Nucleic Acids Res. GenBank. 41(D1): D36D42.
Hebert P.D., Cywinska A., Ball S.L., deWaard J.R. (2003). Biological identifications through DNA barcodes.
Proceedings of the Royal Society B: Biological Sciences 270(1512): 313-21.

Hollingsworth P.M. et al (2009). A DNA barcode for land plants.
Proc Natl Acad Sci USA 106(31): 12794-7.

Step 2: DNA Extraction

age may also suffer kidney or liver damage from exposure to excessive mercury in soil.

94C 1 minute
35 cycles of the following profile:
Denaturing Step:
950C
30 seconds
Annealing Step:
500C
30 seconds
Extending Step:
720C
45 seconds
72C 15 minutes
40C then store at -20C until 2% agarose gel electrophoresis

References

Step 4: Gel electrophoresis

Use the Blue Line to determine sequence relationships.
The Blast function allows for a query against DNA data bases and
list the most significant alignments.
Users can analyze up to 100 kb of DNA predicting and annotating
genes (Red Line), prospecting genomes for similar genes and
building gene trees (Yellow Line).

Mushroom samples (all Smithtown locations)
Soil ug Hg /g
Tissue ug Hg /g
NPQ-008
.134
.013
NPQ-010
.060
.040
NPQ013
.052
.032

1
2
PCR# 4-12
NPQ- 001
Insect/invertebrate repeat
3
PCR#4-13
NPQ- 002
Insect/invertebrate repeat
4
PCR#4-14
NPQ-003
Insect/invertebrate repeat
5
PCR#4-15
NPQ-004
Insect/invertebrate repeat
6
PBR322/BstN1 (7uL)
7
8
Lambda DNA 6uL + 2 uL cyber green
(PCR products: 5uL PCR + 2 uL cyber green)

Insect samples
Soil ug Hg /g
NPQ-001
.073
NPQ-002
.110
NPQ-003
.240
NPQ-018
.240
NPQ-014
.218

Mismatches
53
8
4

Siriporn Pota, Sinchai Chatasiri, Jintana Unartngam, Yuichi Yamaoka, Kentaro Hosaka, and Yoshitaka Ono.
Taxonomic identity of a Phakopsora fungus causing the grapevine leaf rust disease in Southeast Asia and
Australasia Mycoscience Volume 56, Issue 2, March 2015, Pages 198204
Stoeckle M. (2003). Taxonomy, DNA, and the Bar Code of Life. BioScience 53(9): 2-3.
Van Den Berg C., Higgins W.E., Dressler R.L., Whitten W.M., Soto-Arenas M.A., Chase M.W. (2009) A
phylogenetic study of Laeliinae (Orchidaceae) based on combined nuclear and plastid DNA sequences. Annals
of Botany 104(3): 417-30.
Zhihua Liu, , Xu Zeng, Dan Yang, Guiyan Chu, Zhengrong Yuan, Shilin Chen Applying DNA barcodes for
identification of plant species in the family Araliaceae
Gene Volume 499, Issue 1, 10 May 2012, Pages 7680

Acknowledgements
Location
Freeport
Freeport
Kings Park
(near landfill)
Smithtown
(near land fill)

Mismatches
14
3
26
16
78

Need mushroom and insect samples from other locations including landfills and south
shore

DNA Barcode Team: Cold Spring Harbor
Outside Advisor: Dr. Cristina Fernandez-Marco
DNA Learning Center Cold Spring Harbor
Mr. E. Irwin FHS research advisor

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