The learning objectives for this set of lecture modules and lab exercises are:

• To help the student to master the fundamental concepts of biology with application to a unifying theme (salmon ecology) and application to a real-world project connected to their local community.
• To develop an appreciation for the power of the scientific method and an understanding of how it is applied to specific questions or issues.
• To provide a context for understanding the usefulness and application of techniques of molecular biology by using these techniques to analyze field samples collected by the entire class.
• To explore sustainability through discussions of environmental impacts of salmon farming, toxicology and bioaccumulation of pollutants in salmon.

After completing these exercises, the student should be able to:

1. Apply the scientific method to answer real-world questions.
2. Understand the fundamental concepts of cellular chemistry, molecular and cellular biology, and how to apply these methods to bioregional environmental applications.
3. Communicate the results of their own experiments, verbally and in writing, both informally and using standard scientific formats.

Each laboratory exercise has a specific objective, including:

1. To understand the principles of gel electrophoresis, PCR amplification, DNA sequencing, and phylogenetic analysis.
2. To learn how to apply DNA analysis to an environmental issue.

Students will demonstrate their abilities by performing a complete phylogenetic analysis (including salmon sample collection, DNA isolation, PCR, sequencing, and sequence analysis). They will answer questions informally (in weekly lab reports) and formally, after completing the project, in a group oral presentation.

Timeframe

This exercise is intended for the molecular and cellular biology quarter of a three-quarter introductory biology sequence. A sample class schedule has been included in the Instructor Lab Manual attached materials describing the exercise. The phylogenetic analysis from DNA extraction to sequence analysis requires a minimum of four (2-½ hour) lab sessions to complete, and therefore works best as the centerpiece of a quarter-long project culminating in a final paper or oral presentation.

There are other methods that could be applied to answering this question to allow the exercise to be completed in a shorter time, which might make this exercise adaptable for a one quarter non-majors biology survey course or a two quarter introductory biology sequence. For example, kits are available from various biology education supply companies to perform protein gel isozyme analysis of myosin, myoglobin, and actin to distinguish between different salmon species. We are currently experimenting with a method using spectrophotometry to quantify salmon pigment levels (typically higher in wild salmon), and chromatography to distinguish between artificial pigments in farmed salmon and natural pigments in wild salmon, both of which could be performed in a single lab session. This exercise could also be easily expanded for a more advanced genetics or phylogenetic analysis courses, by adding a molecular marker (DNA fingerprint) test in addition to the phylogenetic analysis, and by sequencing multiple genes such as Cox I in addition to the internal transcribed spacer (ITS).

Possible Use in Other Courses: It would also be useful to link this exercise to another course dealing with ethics, environmental law, consumer protection, food safety, or pollution and public policy.

It can be challenging to find laboratory exercises that allow students to apply newly learned molecular biology techniques to real world questions. In particular, phylogenetic analysis is a key concept in biology that students often grapple with because it is difficult to relate it to a hands-on exercise. This project allows students to test the species identification of salmon from local stores, with the goal to detect any potential mislabeling that may be occurring. Several recent studies have revealed that salmon mislabeling is rampant, particularly during winter months when fresh wild salmon runs are not occurring, leading to strong temptation to substitute the cheaper farmed Atlantic salmon for Pacific salmon species such as Sockeye or King salmon. This project engages students through an issue that is directly pertinent to their lives: consumer fraud (they are often outraged when their local store is revealed to mislabel salmon), but also invokes wider issues such as sustainable fishing practices, environmental impacts of farming salmon, and the human health implications of toxins that tend to accumulate preferentially in farmed salmon and lower concentrations of omega-3 fatty acids. Consumers intending to minimize their exposure to toxins and reduce environmental impacts through their choice to pay extra for wild salmon may be thwarted by mislabeling, so the impact of potential "cheating" goes beyond the financial level.

Students independently request wild Pacific salmon samples from their local stores and bring these tissue samples in to the lab for analysis. In a five week laboratory sequence, each student performs DNA isolations, PCR using salmon-specific primers, gel electrophoresis, and sets up sequencing reactions which are processed at a separate sequencing facility (approximately $6/sample). Sequences are analyzed using phylogenetic analysis software (available free on the Web) and the results are summarized in an oral presentation. The potential exists to expand this exercise by using protein gel isozyme analysis of myosin, myoglobin, and actin to distinguish between different salmon species, spectrophotometry to quantify salmon pigment levels (typically higher in wild salmon), and chromatography to distinguish between artificial pigments in farmed salmon and natural pigments in wild salmon.

Because salmon is such a Pacific Northwest icon, the exercise resonates with and strengthens the students' sense of connection to their local region (the bioregion). The exercises connect to the students' local communities by requiring them to bring in salmon samples from their own local stores. There is ample opportunity to involve community groups in using the results of the investigation to challenge local stores or local suppliers that are selling mislabeled salmon.

The laboratory exercises are most effective when integrated into a multi-quarter sequence of introductory biology. Salmon become a unifying theme for the whole sequence and a connection point for a series of learning modules relating to multiple topics throughout the year. For example, during the ecology section, topics such as bioaccumulation of pollutants, the environmental impacts of farmed vs. native fish, and conservation biology of salmon can be introduced. During the evolution section, topics such as phylogenetic trees and population genetics of salmon can be discussed. During the animal physiology section, muscle physiology (actin/myosin), toxicology, and osmoregulation changes from saltwater to freshwater can be incorporated. During the cell and molecular biology sections of the biology sequence, the labs provide a hands-on introduction to basic methods in molecular biology.

Anonymous comments from students participating in this exercise at University of Washington Tacoma in Winter 2007 included: "I liked the salmon labs because we were using real world issues to learn about a complicated process."

Description of the Activity

A complete Student Laboratory Manual, including pre-lab and post-lab questions, introduction and context, and detailed instructions, is attached below. An Instructor Manual provides additional information required to set up each lab. Example of student work and a rubric is also included below.

Student Laboratory Manual (Microsoft Word 355kB Oct26 11)
Instructor Manual (Microsoft Word 98kB Oct26 11)
Student Work Example (Microsoft Word 24kB Oct26 11)
Rubric (Microsoft Word 44kB Oct26 11)

Detailed instructions for preparation and materials required for each lab are provided in the attached instructor packet.

It is important to stress accountability and documentation of the sample collection. The potential for using this information to accuse local stores or suppliers of consumer fraud reinforces to the students the importance of keeping track of the identity of their samples and thoroughly documenting where the samples came from. All samples should be stored for confirmation of results.

We encourage other local educators to use any portions of this exercise that are appropriate to their courses. We hope to gather results from all local participants for eventual publication.

Each lab exercise includes a set of pre-lab questions, to be completed before the lab, which gauge student understanding of concepts presented in the lab handout. During the lab exercise, students present their results to each other as they proceed through the lab (for example, displaying a gel and explaining what the presence of the band means), which provides a checkpoint to allow students to help other students and to provide additional information if the whole group is missing key concepts. At the end of each lab exercise, students answer and turn in another set of questions (as a group or individually, depending on the nature of the lab exercise). After completion of the sequence of lab activities, the results are discussed and analyzed in a computer lab so that all students can participate in the analysis and final discussion of results. Finally, students work in small groups independently to give a 15 minute oral presentation on the class results, with each group choosing a different emphasis for their talk.

Several relevant scientific articles, newspaper articles, and websites are included in the introductory material provided in the attached student laboratory manual. Additional articles are included in the attached instructor manual.