• Largemouth bass, Micropterus salmoides (Great Lakes, Hudson
Bay, and Mississippi river drainages)
• Lake trout, Salvelinus namaycush (northern Canada south to
Great Lakes and New England)
Students working with freshly caught fish had the most success
generating CO1 gene sequences from dissected prey items; however, students were able to successfully amplify prey DNA from fish
that had been stored frozen for up to two months.
Lesson 1: Dissecting Lionfish
In teams of three or four, students dissected lionfish and collected
small tissue samples from dissected fish prey items. During dissection, special care must be taken to limit cross-contamination of
prey items and to ensure that prey tissue samples are sufficiently
preserved in 95% ethanol until DNA extraction.
Lesson 2: DNA Extraction
Incubation of prey tissue samples at 95°C for 20 minutes in 500 µL
of a 5% solution of Chelex 100 Chelating Resin (Bio-Rad Laboratories, Hercules, CA) was used to provide rapid, cost-effective extraction of whole genomic DNA from preserved prey tissue (Walsh
et al., 1991). The DNA extraction protocol was designed to be
completed by students in a standard 50-minute class; however,
the extracted DNA is unstable compared to more time-intensive
extraction protocols (e.g., DNeasy Blood and Tissue Kit) and must,
therefore, be used within one week to avoid DNA degradation.
Lesson 3: Polymerase Chain Reaction
Following Ivanova et al. (2007), a “universal” PCR cocktail containing
four M13-tailed oligonucleotide primers (Table 2; Integrated DNA
Technologies, Coralville, IA), MyTaq Red mastermix (Bioline USA,
Taunton, MA), and PCR-grade water was used to amplify a 631 bp
mitochondrial CO1 gene fragment from dissected lionfish prey.
Lesson 4: Agarose Gel Electrophoresis
Students visualized 5 µL of their PCR product on a 1% agarose gel
stained with 1X SYBR Green Staining Solution (Lonza Rockland,
Preparing Samples for DNA Sequencing
A large number of commercial and academic labs offer DNA sequencing services; however, sample preparation requirements and sequencing cost can vary considerably ($1.90 to $8.00 per sample) depending
on the provider, number of samples submitted, and eligibility for academic discounts.
Lesson 5: Prey Identification
Students viewed and edited prey CO1 gene sequences using the free
software FinchTV (Geospiza, http://www.geospiza.com). Edited
CO1 sequences where identified using the online Barcode of Life
Database (BOLD) Identification System (Ratnasingham & Hebert,
Discussion: Explaining &
The objective of the lesson discussion phase is to help students construct evidence-based explanations, improve students’ ability to
communicate complex information in a clear and effective manner,
and empower students to engage in informed decision making.
Some effective strategies to consider include discussion or debate,
an article summarizing findings to the school newspaper, video presentation of work, letters to government officials, or presentations to
the class or a larger student group. The majority of the 44 middle
and high school classes undertaking the lionfish unit combined in-class discussion with a final written summary of research findings
to encourage students to reflect on, and construct their own understanding of, the experience. To provide a more authentic research
experience, students in six classes presented their findings publicly
as a research poster, team oral presentation, or individual oral presentation. Presentation venues included school assemblies, regional
high school science symposia, and a marine science festival.
Understanding & Skills
The objective of the student evaluation phase is to determine students’ conceptual understandings and identify their proficiency
with scientific practices. To date, nine teachers involving 911 students from seven schools have participated in the “Invasive Aliens”
DNA barcoding project. Students extracted and amplified DNA
from 360 lionfish prey. Of these, 60% (217 samples) resulted in
high-quality DNA sequences. The remaining samples were either
contaminated and unusable (37 samples) or failed to sequence
(106 samples). There are a number of reasons why samples might
not sequence, including student error, PCR primer mismatch, and
DNA degradation. Over the course of the project, we were able to
improve student results by sampling only moderately digested prey
items and by using freshly collected lionfish; this suggests that prey
quality is an important factor in sequencing success.
Table 2. PCR primer cocktail to amplify a 631 bp CO1 gene fragment (Ivanova et al., 2007). M13-tails are
Name Primer Sequence