Lab Project

Aim

Most of the molecular labs (as oppoosed to computer labs) in this class have to do with a single project, the identification of rockfish fillets purchased at Seattle markets. This project has several aims:

• To demonstrate the application of molecular markers to a very practical problem in management and conservation
• To integrate laboratory procedures, lecture materials and literature into a single project
• To provide you with an opportunity to exercise your scientific communication skills by writing a project report.

Approach

We will use a 'scaffolding' approach to writing the project report, that is, in each lab we will talk about a specific section of the report, and about what and how to write. These sections are purposefully a fairly small part of the final grade - nevertheless, take them seriously, because they will help you to get a higher grade on the final report.

Background

Enforcement of fisheries regulations is often complicated by the difficulty of identifying the source species of fish products such as fish fillets. While whole fish may be identifiable, products such as fish fillets, shark fins, or whale meat usually are deprived of any morphological diagnostic characters. The potential of molecular makers in the identification of such products has been realized by a number of agencies, for example the US Food & Drug Administration (Regulatory Fish Encyclopedia).

The problem is particular pertinent in groups of closely related species, such as rockfishes (Sebastes sp.). The genus Sebastes consists worldwide of over 100 species, and on the North American west coast over 60 can be found (see rockfish report of the Pacific Marine Conservation Council). Rockfishes can be extremely long lived (some over 100 years), and may mature very late, making them particularly vulnerable to depletion by overfishing. Indeed, many stocks have undergone significant reductions in abundance over the last few decades, requiring urgent protection from overexploitation and leading to petitions for listing as endangered species under the ESA (Endangered Species Act, see NMFS Status Review). Other stocks are doing reasonably well, and could continue to be exploited, as long as bycatch of overexploted species is not too high. Such bycatch is often difficult to quantify because of the problematic species identification of fish products. Rockfish identification is primarily based on general shape, color and spines, none of which are available in fillets.

Another major issue in rockfish management is the problematic identification of larvae. Many stock assessment methods are based on larval abundance, requiring the accurate species identification of larvae. Indeed, many of the methods we will use in the lab have been primarily developed for larval identification (e.g. Li et al. 2006).

We will use three different molecular techniques for species identification: RFLP (Restriction Fragment Length Polymorphism) of the mtDNA (mitochondrial DNA) ND 3/4 genes, sequencing of the mtDNA cytochrome b gene (see chart) and allozyme electrophoresis. All techniques rely on the comparison between unknown mystery fillets and reference species.

Project Report

The results of the project will be written up in a short (10 pages double spaced) project report, which should contain all the relevant background, methods, results and discussion. The report should be presented in the format of a scientific paper - here are some guidelines of how to do that. The report should only cover mtDNA RFLP and mtDNA sequencing. We will do the allozymes in week 8, which is too late for the report.

For the RFLP, we will run a class gel so that we have a single photograph with all known controls and the mystery fillets. Use the positive controls to work out the species of the mystery fillets. If none of the controls match, try to work out from the data in Li et al. (2006) which other species our mystery fillets may be.

You should also use the mtDNA cytochrome b sequence data we collected. The approach is similar to the DNA barcode of life approach, which aims to identify all species by DNA sequences (see also Smith et al. 2008). We will analyze these data in week 7, and will post the relevant information here.

Consider both markers when drawing your conclusions about the identity of the mystery fillets.

During the quarter, we will use each lab to talk about a specific section of the project report. Take heed of these pointers, as they may help you to write your final project report:

Instructions for the preparation of the project report

Updated species key

Comments of Parentage Result section

Comments and key for the Material and Methods section. The last part of this document lists aspects you should definitely have in your final report.

Comments on Results Draft - this is mainly a list of stuff you should include in your results.

Grading Key from 2008: This key may be slightly modified this year, but it should give you a good idea what we are looking for.

Links

These links may be useful for your project report, but will not be on the exam

For more information on rockfish, check out the webpage of the Lab of Milton Love. There are also lots of rockfish pictures.

Rockfish Game by the Alaska Fisheries Science Center - test if you know your rockfish. They also produced a very nice guide to rockfishes

If you are interested in the issue of accurate labeling (or lack thereof), have a look at the FDA's guide to acceptable market names for seafood.

References

Li ZZ, Gray AK, Love MS, Goto A, Asahida T, Gharrett AJ (2006) A key to selected rockfishes (Sebastes spp.) based on mitochondrial DNA restriction fragment analysis. Fishery Bulletin 104, 182-196.

Seeb LW (1986) Biochemical systematics and evolution of the Scorpaenid genus Sebastes. Ph.D. Dissertation. University of Washington, Seattle, Washington

Smith PJ, McVeagh SM, Steinke D (2008) DNA barcoding for the identification of smoked fish products. Journal of Fish Biology 72, 464-471.

Redbanded rockfish (Sebastes babcocki)

Shortspine Thornyhead, Sebastolobus alascanus (aka Kinki)

Rockfish (and others) for Sale!

Not a happy fish!

But a happy Prof!

Our mystery fillets

Flow chart of our lab project. We will use three methods to identify the mystery fillets, but only two methods (mtDNA RFLP and mtDNA sequencing) will be considered in the project report.

Schematic of the mtDNA with the two regions we will analyze (white boxes). The longer region are the ND3 and ND4 genes, which we will screen by RFLP. The shorter cytochrome b region will be sequenced.