Phylogenies of Aquatic Insects to Give Insight on Environmental Cues and
Evolutionary Relatedness
Department of Ecology, Evolution, and Natural Resources
Abstract
From DNA sequences, accurate insect species
identifications can be made and thus patterns in water quality,
organism distribution, and evolutionary relatedness can be
determined. We have extracted DNA from aquatic insect samples,
amplified the DNA with PCR, and are in the process of sequencing
them. Using DNA barcoding techniques we hypothesize that we
will find globally constant indicator species by identifying patterns
in DNA segments, and furthermore, evaluate the environmental
quality of the regions sampled. These sequences have and will
continue to be submitted to the BOLD (Barcode of Life Data
Systems) website for identification, allowing us to understand
links between species diversity and environmental/regional
factors.
Samples of aquatic organisms have been used from
New Jersey, Eastern Russia, the Philippines, and Vietnam to create
an expansive pool. Individually I have focused on extracting and
sequencing over 200 Vietnam insect specimens. The data is
helping contribute to the knowledge of biodiversity of aquatic
insects worldwide and those results are available for scientists to
access regardless of location.
Background
Insects, the most diverse group of organisms on the
planet, are found across every continent and although their
presence is often unwanted—play important roles in their natural
ecosystems, as well as serve as bio-indicators. Aquatic insects,
primarily the orders Ephemeroptera, Plecoptera, and
Trichoptera—or Mayflies, Stoneflies, and Caddisflies, are known
for their variability in pollution tolerance. Knowing which is
present in a given location is key in recognizing environmental
conditions of a region. Due to their small size, relative obscurity,
and large diversity, it can be extremely hard to correctly identify
them visually.
By DNA sequencing specimens, lineage
development can give insight into evolutionary constraints that
may have led to adaptations in any branching of the tree—and
the Barcode of Life Database can also catalog sequences which
allows for accurate insect ID. Vietnam has large amounts of
economic reliance on its’ water systems through crop growth, but
with increased pollution and outdated water quality monitoring,
bio-indication can be a great tool and give insight into the health
of the water as well as of the crop.
Nicole Reiff, Dr. Karl Kjer
Objective
Use proper DNA Extraction and PCR techniques to determine
connections between evolutionary divergences, regional
differences in diversity, and aquatic ecosystem health
Figure 1: Aquatic Insect Key
Each order of insect is
associated to a quality index
factor that attributes to the
environmental quality these
organisms can be found in.
When visual ID is unknown or
impossible, DNA sequencing
must be used
Methods
Aquatic insects were collected from a variety of locations in New Jersey, Eastern
Russia, Vietnam, and the Philippines, to be sequenced.
Each insect was transferred to a numerically labeled preservation tube, and
digitally scanned for record.
Muscle tissue from each specimen was removed for Polymerase Chain Reaction
(PCR)
Primers, water, and DNA (muscle Tissue) is added to PCR beads, and DNA is
amplified using PCR
Used gel electrophoresis to see if PCR methods were efficient
If efficient, DNA samples were sent to a lab to be sequenced
Used BOLD Website to submit sequences and compare with others
Results/Discussion
While results are not yet complete, there is a pattern of expected
results that we expect to see based on previous work performed in Dr.
Kjers’ lab and other insect phylogeny projects.
•PCR success will be about 60 percent for all insect specimens tested
in the lab
•Approximately 200 individual Vietnam organisms will have been
sequenced, identified, and submitted to the BOLD database
•The specimens will be identified by the CO1 gene sequence with
high certainty from already catalogued sequences on BOLD
•A phylogenetic tree will be created to display the relationships of the
sequenced organisms
•These organisms will be marked by their pollution tolerance level
Conclusions
At the end of this project we will be able to learn a lot about insect
diversity from the different orders sequenced.
 Submissions on the BOLD website will contribute to the databases
growth for future users.
 We will be able to identify methods in our PCR techniques and DNA
extraction methods that had higher success rates than in past trials.
This will allow us to enhance our DNA extraction and PCR
performance.
 Furthermore, with the varied regional diversity of our results, we
will be able to compare evolutionary history across locations and
see if we can identify patterns that cross continents, in terms of
environmental indications or behaviors.
Resources
"CIPRES Outreach." Cyber Infrastructure for Phylogenetic Research.
CIPRES.
Misof, B. , S. Liu, et al. "Phylogenomics resolves the timing and
pattern of insect evolution." Science. 346.6210
"Water quality monitoring systems outdated." Vietnam News 13 02
2012