Biodiversity of the Peconic River John Buerkel, Brad

Biodiversity of the Peconic River
John Buerkel, Brad Governale, Tyler Haug
Mentor: Mr. Robert Bolen
Eastport South Manor Junior Senior High School
This project was based on the objective of measuring the biodiversity of the
Peconic River. This will then act like a baseline for future studies of the area. We
collected a total 18 invertebrate specimens from the Peconic River and the
surrounding riverbed to barcode, these included, leeches, dragonfly larvae, and
amphipods. Our goal, along gathering a general baseline of biodiversity for the
river, is to determine if any of the species found are directly or indirectly
hazardous to human health, or are indicators of poor health for the river itself.
This required us to use barcoding of the CO1 gene. Through this process we were
able to identify 3 different species, Anax junius (Green Darner), Enallagma civile
(Familiar Bluet) and Lirceolus cocytus, two of which appear commonly in the
habitat of the Peconic River.


Materials & Methods

Out of the 18 samples we sequenced only 3 showed up with the gel electrophoresis. By using
BLAST we found that only a few of them came up with a good bit score. Through this
process of using the CO1 gene and barcoding we were able to identify 3 different species,
Anax junius (Green Darner), Enallagma civile (Familiar Bluet) and Lirceolus cocytus, two of
which are commonly seen in the habitat of the Peconic River. One of the species we
barcoded turned out to be very scarcely documented and appears to be novel.

On Friday, October 20th, 2017, our group went to the Peconic River in New York to collect 20
samples of species. We collected what appear to be dragonfly larvae, leeches, and one unknown
invertebrate that might be an insect. To collect these specimens, we used a seining net and multiple
dip nets to collect all of the species. After collecting all of our specimens we headed back to
Eastport South-Manor Jr-Sr High School and we sorted all of them by individually separating them
into labeled bags that contained 99.9% ethanol. Then, all of the samples were photographed,
measured, and stored in a non-defrosting freezer.
The standard extraction method and amplification of DNA sequences will be provided by Cold
Spring Harbor.
PCR and Gene Amplification: A PCR tube containing a Ready-To-Go PCR Bead was mixed
with 23 uL of primer. Once mixed we added 2uL of the DNA extracted from each sample directly
into the primer mix. We used the (LCO1490/ HC 02198) primer for the invertebrates This mix was
then frozen until we were ready to begin the thermal cycling. We then placed the PCR tube in a
thermal cycler that we programmed to the appropriate protocol. After the thermal cycling we stored
the now amplified DNA in a freezer until we were ready to continue to gel electrophoresis.
The reason we use the process of gel electrophoresis in this lab is because we need to look at
DNA and separate them by size and charge. This permits us to identify the presence of the CO1
gene. If there are bands in some samples but not others, that means they don't have the specific
CO1 sequence we are looking for in their DNA.

Peconic Rivers history has been observed as quite poor as far as water quality
and how healthy it is for the organisms in and around it. In late April 2015 there
were dead turtles, about 100 of them, that started washing ashore near the river.
Then came the dead fish, in numbers no one had seen before. By early June 2015,
tens of thousands of fish carcasses had bobbed to the surface of the Peconic
River(6).In 2015 there was, and still is an amount of nitrogen that is unsafe for the
fish, turtles, whatever takes refuge in Peconic River to live. What is even more
troubling is that some people catch and eat the fish in the river and the fish that
they catch have increased levels of nitrogen in their systems. That is very
dangerous as too much nitrogen can decrease the functionality of the thyroid
gland, and decrease the oxygen carrying capabilities of the blood. This change in
nitrogen levels can also change the ecosystem surrounding the river and create
conditions that would be ideal for many invasive species that can carry bacteria or
viruses that could be harmful to humans. In recording the biodiversity through
DNA barcoding, we can determine if there are any living species that can absorb
the nitrogen and replenish the oxygen into the water. We will also document
biodiversity and study changes in biodiversity over a set period of time.

Figure 1: Anax junius Blast results DNA Subway)
(DNA Subway)

Figure 4: Anax Blast matches and bitscore

Figure 2: Enallagma civile Blast results

Figure 5: Enallagma civile Blast matches and
bitscore (DNA subway)

DNA Subway)

Figure 3: Lirceolus cocytus barcode alignment
results from DNA Subway

Figure 6: Lirceolus cocytus Blast
matches and bitscore (DNA

(1) DNA Barcoding: Potential & Limitations for Botanical Testing. (n.d.). Retrieved September 26,

DNA barcoding is a relatively new process that was developed in 2003 by Paul
D.N. Hebert at the University of Guelph, in Ontario Canada that allows for a short
DNA sequence to be read from any genetic sample (1). This process allows for a
more objective analysis in determining if a specimen can be identified one
particular species. This process is completed by using the gene region
Mitochondrial Cytochrome C Oxidase 1 gene (CO1)(3). This gene region is
being used as the standard barcode region to sequence for almost all animal
groups. This mitochondrial gene sequence can regularly enable discrimination of
closely related species within the animal kingdom (5). One of the most important
elements of DNA barcoding is the construction of an online database that can be
used for further experiments.
Although it has many advantages over the old methods to identify species,
DNA barcoding is not a perfect method and has its set of flaws and disadvantages.
DNA barcoding also makes it difficult to distinguish closely related species
because of widespread hybridization(1). With plants it is even harder because so
many genes are individual to each plant(1).

Our question was, how did the biodiversity of the Peconic river shift from last year to this
year?.We found that, the diversity in this area is dominated by different species of dragonflies. We
also discovered an uncommon species in the area that had yet to be documented on DNA Subway.
This species, Lirceolus cocytus although undocumented on DNA Subway, was found on Blast with
a match of 100%. However the few images of this species found online of this species, did not
match with the pictures so it is possible that this could be a novel
species of a common genus. Figure 6, 7 ,8.

2017, from limitations-for-botanical-testing/2183
(2) New creative uses of DNA 'barcoding'. (2011, November 28). Retrieved September 26, 2017,
(3) What Is DNA Barcoding? (n.d.). Retrieved September 26, 2017, from
(4) Kress, W. J., & Erickson, D. L. (2008, February 26). DNA barcodes: Genes, genomics, and

Figure 7: Blast results that show
match for the species

Figure 8: Known picture of

Figure 9: Image of our
discovered species

We would like to thank the Day in the Life Program, Melissa Parrott with the Central Pine Barrens
Joint Planning and Policy Commission, and Dr. Mel Morris at BNL, for giving us this opportunity
to become stewards of a local river system. Thank you Dr. Sharon Pepenella for you help and

bioinformatics. Retrieved September 26, 2017, from
(5) Hebert, P. D., Ratnasingham, S., & Waard, J. R. (2003, August 07). Barcoding animal life:
cytochrome c oxidase subunit 1 divergences among closely related species. Retrieved
November 14, 2017, from
(6) Semple, K. (2015, June 05). Long Island Sees a Crisis as It Floats to the Surface. Retrieved
November 14, 2017, from
(7) The Problem. (2017, March 10). Retrieved November 16, 2017, from
(8) Environmental Impact Statement for the Hays County regional habitat conservation plan:

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