The Invasive Mussel Project (IMP)

Using A Simple PCR Test to Distinguish between Native and Non-Native Mussels in Puget Sound

Click here for web version of Powerpoint  presentation

Click here for downloadable Powerpoint presentation 

        The introduction of non-native (exotic) species can present serious problems for ecosystems, often displacing native species. The predominant native species of mussel in the Puget Sound region is Mytilus trossulus, sometimes called blue mussels.  These small (usually less than 6 cm) mussels can be found on rocks and pilings throughout the region.  However, a non-native mussel, Mytilus galloprovincialis, also can be found in the area, although its native habitat is the Mediteranean Sea and eastern Atlantic Ocean.  Mytilus galloprovincialis were probably introduced to our area both by ships' ballast water and by aquaculture.  The exotic mussels are known as "Penn Cove" mussels, so-called because one of the areas where they have been farmed is Penn Cove on Whidbey Island.   Several seafood companies raise these more desirable faster-growing mussels in Puget Sound, forming a multi-million dollar industry.  However, little is known about the extent to which these exotic mussels are spreading throughout the region or impacting the native marine ecosystem.  Although M. galloprovincialis tend to grow larger, it is difficult to distinguish them from M. trossulus using just morphology.  It is known that the two species can easily interbreed and produce hybrids.  The two species and their hybrids can be detected by the molecular technique described below.  The goal of our project is to involve students in gathering data on the incidence of the mussels and their hybrids in the Puget Sound region.  Contact Dr. Peter Wimberger (wimbo@ups.edu ) at the University of Puget Sound for more detailed information.

        The Invasive Mussel Project takes advantage of a significant difference between the M. trossulus and M. galloprovincialis in a gene that codes for a foot protein (adhesive protein) used for making the byssal threads, which mussels use to attach themselves to a substrate.  In M. galloprovincialis, there is a deletion in the adhesive protein gene, resulting in a shorter fragment when amplified with primers spanning the region.  When using the primers Me15 and Me16, M. trossulus yields a fragment that is 168 bp long, whereas M. galloprovincialis will produce one that is only 126 nucleotides long.  This difference is clearly distinguishable on a 2.5% agarose gel.  Also, hybrids show up easily with both of the above bands present (see photo below):

2.5% gel of mussels in Totten Inlet near Olympia, WA.

Lanes 3, 6, 7, 8 are all M. trossulus.

Lanes 2 and 5 are M. galloprovincialis.

Lanes 1 and 4 are hybrids of the above species.

 

Collecting Methods

           Before setting out on a collecting trip, obtain a shellfish license (for info see sources section below).  At this point, one has the opportunity to talk about the importance of sampling and contrast different types of collecting methods and the different conclusions that can be drawn from different methods.  For example, if one collects only the largest mussels one will gain the best information about presence/absence of M. Galloprovincialis.   This "size" method might be a good first collecting method, since this will provide some idea of whether the Gallo's are even present in the area.  If one wants to determine the relative frequency of M. Galloprovincialis and hybrids, then one needs to design a random sampling scheme.  In order to collect mussels of a variety of sizes from a site, you might collect, say, 50 mussels, and then sort them back in the classroom into size groups, such as less than 30 mm in length, 30 - 50 mm, and greater than 50 mm.   Measure the length of each mussel, and, if possible, the width and height (see photos below- note whitish blotches where barnacles were stuck; you may need to scrape these off).  Then, decide how many you want to process, and try to do the same number for each size range.  (Under "method" in the table, this would be the "range" method.  If you collected only the largest mussels, you would write "size" under method).  Set up an Excel spreadsheet with categories such as those shown below.  Possible substrates might be rocks or pilings.  If collected from a piling, was it near the top, middle, or bottom?  If collected from an area exposed to the rise and fall of the tide, then Tide zone = intertidal.  If the mussels were collected from a dock, where they are always submerged, then tide zone = subtidal.   Species can be filled out only after PCR identification (T = M. trossulus, G = M. Galloprovincialis, T/G = hybrid).

ID # Location Date Method Substrate/loc. Tide Zone Length (mm) Height (mm) Width (mm) Weight    (g) Species
FI-1 Fox Isl. 6/18/02  range piling/top sub 54 28 23 32 T    
FI-2 Fox Isl. 6/18/02  range piling/ bottom sub 74 37 33 45.6 T/G  
FI-3 Fox Isl.  6/18/02 range  piling/middle sub 68 41 28 42.3 G

 

Length (64.5 mm for this example):

 

 

Width (26 mm):

 

 

Height (32 mm):

 

Obtaining Tissue for DNA preparation:

        Total DNA can be prepared from almost any tissue of the mussel, but for consistency, the mantle tissue just inside the rim of the shell is recommended (see photo).

        Mantle tissue (under the metal spatula):

 

Snip off about 3 mm of the mantle tissue, rinse it with a little distilled water, and put in 1.7 ml eppendorf tube.  

            The DNA extraction protocol below is written for use with a kit from Qiagen Corp.  See sources section below for possible free kit.   The purpose of each step is indicated in italics.  Essentially, the tissue is digested to release the DNA, which is then bound to the material in a minicolumn.  The minicolumn is washed a couple of times with a high-salt buffer to remove unwanted material, then with a low-salt buffer which removes the DNA.

Protocol for DNA Extraction using Qiagen kit

1)  Important: write an ID number for that particular mussel on the tube (and in your notebook).

2)  Add 180 ul ATL buffer to each tube containing rinsed tissue.  (Check ATL for undissolved white crystals- if found, warm in hands for a couple minutes).  The ATL buffer contains a detergent that causes the cell and nuclear membranes of cells to break open, releasing the DNA and other cellular material.

3)  Add 20 ul of Proteinase K stock solution, mix by flicking the tube (or use vortex if available), and incubate at 50 - 55oC for 3 hours to overnight until the tissue disappears completely or is mostly dissolved (lysed).  Mix occasionally during incubation to disperse the sample, or place in a shaking water bath.  Proteinase K is an enzyme that digests all protein in the cell into amino acids, which will later be washed away.

4)  Add 200 ul of Buffer AL to the sample, mix thoroughly and incubate at 70 o C for 10 minutes (rinse hands afterwards- AL is a little caustic).  The AL buffer and hot temperature result in the destruction of the proteinase K, so that it will not interfere in later steps.

5)  Add 200 ul of ethanol (95 100%) to the sample, and mix thoroughly.  This causes the DNA to begin to precipitate and stick to the material in the column.

6)  Label the cap of a QIAamp spin column with mussel ID number. (Note: Collection tube that column is in need not be labeled.  Query: Why not?).  Carefully pour the mixture from step 5 (including any precipitate if there is any) to the spin column, close the cap and centrifuge at at least 10,000 rpm (8,000 x g) for 1 minute.  During this step, the DNA will stick to the white material in the column, and most of the rest of the cellular material will pass through into the collection tube.

7)  Take the spin column out of the collection tube, pour the liquid out of the collection tube into the sink, and put the spin column back in the tube.  This gets rid of unwanted cellular junk.

8)  Carefully open the spin column and add 500 ul of Buffer AW1.  Centrifuge at 10,000 rpm for 1 minute.  Again, carefully remove the spin column from the collection tube, pour the liquid out of the collection tube, and place the spin column back in the now empty collection tube.  In this step, you are washing away any unwanted cellular material that may have stuck to the column.

9)  Carefully open the spin column and add 500 ul of Buffer AW2.  Centrifuge at full speed (14,000 rpm) for 2 minutes (to dry the column material and make sure that residual ethanol does not does not contaminate the product in the final step).   This gets rid of any traces of unwanted cellular material, and prepares the DNA for elution. 

10)  Place the spin column in a clean eppendorf (labeled with mussel ID number) and discard the collection tube containing the filtrate.  It is the column that has the DNA stuck to it, so you keep that, and collect your DNA in an eppendorf tube in the next step..

11)  Carefully open the spin column.  Elute the DNA into your labeled eppendorf tube, by adding 200 ul of Buffer AE to the spin column.  Let tube sit at room temperature for 1 minute then centrifuge at 8,000 rpm for 1 minute.  In this step, we switch to a low-salt buffer, which causes the DNA to become unbound from the column material, so that it can be collected in the eppendorf tube.

The liquid you have collected contains the DNA.  DO NOT THROW THIS TUBE AWAY even after you have finished the whole lab project.  Your teacher may want to perform further analyses on the DNA.

Agarose Gel:   Visualize your total DNA on a 1% agarose gel (1 g agarose in 100 ml TBE or TAE buffer).  Run 10 ul of your sample (with 1 ul loading dye).  Also, remember to run a size standard (also known as ladder), such as a 1 kb ladder.  Depending on which ladder you use you will probably load 2 - 3 ul of the ladder with 1 ul of the loading dye.  If you are using a loading dye that has two blue stains (methylene blue and cyanol) you should run the gel until the two blue bands divide the gel into thirds (usually about 30 min. at 100V).  After running the gel, it will need to be stained to see the bands.  Depending on the stain used, gels are stained anywhere from 30 minutes (for ethidium bromide) to overnight, and are then washed before visualizing on a light box.  

 

Polymerase Chain Reaction

        For a description of how PCR works, click here.  An excellent article by the inventor of PCR, Kary Mullis, can be found in the April, 1990 issue of Scientific American (see ref.'s: Mullis, 1990).

The primers that can be used to distinguish between M. trossulus and M. galloprovinciallis as described above are Me15 and Me16 (see Inoue, 1995).  The sequences of these primers are:  (5' to 3')

Me15:    cca gta tac aaa cct gtg aag a

Me16:    tgt tgt ctt aat agg ttt gta aga

Primers are relatively inexpensive.  For ordering information, see Sources section below.  

Note: The primers will arrive dry in eppendorf-like tubes.  You will need to add distilled water to redissolve them.  The amount of primer present in the tube will be printed on the tube's label.  The amount will usually be around 50 nmol.  You will need to make stocks of 10 pmol/ul for use in PCR.  Start by making a 100 pmol/ul stock by dissolving primer in 10X as much water (in ul) as you have nmol.  So, if you have 50 nmol, dissolve in 500 ul of water and you will have 100 pmol /ul.  (Remember, 50 nmol = 50,000 pmol.  So, 50,000 pm/500 ul = 100 pm/ul, right?).  Then you can dilute 1:10 to get your 10 pmol/ul working stock.    

For those who have done Project GROWS before, the protocol shown below is exactly the same, except for the use of different primers (and only one round of 30 cycle PCR).

Protocol for using liquid Taq polymerase: 

 

1)   Number thin-walled 0.2 ml PCR tubes on top and side using thin permanent marker.  

2)  Make a "cocktail" that includes 10X PCR buffer, nucleotides, magnesium choloride, primers, Taq polymerase, and water (but not the DNA). Calculate what you would need to add to the cocktail using the following recipe.  Let's say you are making enough cocktail for the class, which is doing 18 samples (just to make the math easy).     You will want enough cocktail for these 18 samples plus 1 negative control (for the class).  To ensure that you have enough cocktail, calculate enough for one extra tube.  Note that you will be adding 2 ul DNA separately to each tube, so you want to have 23 ul of the cocktail for each tube (for a final per-tube volume of 25 ul).

                                                        Per tube                                         x 20                                                                                                                      (18 samples, plus
                                                                                    1 negative control, 1 extra)
 
10X PCR buffer                                   2.5 ul                                             50 ml
5 mM dNTPs                                        1 ml                                               20 ml
25 mM MgCl2                                      1 ml                                               20 ml
5 Primer (Me15) (10 pmol/ml)           1 ml                                               20 ml
3 Primer (Me16) (10 pmol/ml)          1 ml                                               20 ml
Taq polymerase                               0.15 ml                                                3 ml                
ddH2                                          16.35 ml                                            327 ml
Total:                                              23 ul                                       Total in cocktail: 460 ml

  (Note: If each lab group is making their own cocktail, just multiply the "per tube" amounts by one more than the number of samples your group is doing; e.g. if your group is doing 4 samples, make enough cocktail for 5 samples, so you have some extra).

DNA:                    2 ml (not in cocktail!)

 

3)  Add 23 ml of the cocktail to each small PCR tube. 

4)   Add 2 ml of your DNA to the PCR tube, so the final volume is 25 ul. 

 

5)   Mix the contents of the tube by gently pipetting in and out.

 

6)   Place the samples in the thermal cycler and start the cycler as instructed by your teacher.   The thermal cycler will be programmed with the following general profile (the optimal cycling profile for a given thermal cycler will vary and must be empirically determined): 

    

      1 cycle at 95O C for 2 minutes, followed by 30 cycles of 95 O C for 15 s,  54 O C for 40 s, 72 O C for 1 min. and 20 s;  1 final extension of 72 O C for 5 minutes.  This program usually takes about 2 hours;  the cycler can be programmed to hold at  4O C after the cycling is complete (if desired not necessary).  Store samples in fridge (room temp. is O.K.).

 

 

Gel Electrophoresis of PCR Product

 

              Since the PCR product is very small and you are trying to distinguish between fragments that are 168 bp (M. trossulus) and 126 bp (M. galloprovinciallis), you will need to visualize the DNA on a high percentage gel (2.5% agarose = 2.5 g agarose dissolved in 100 ml of running buffer (TAE or TBE).  Run 5 ul of PCR product (mixed with 1 ul load dye).  Use a 100 bp ladder (available from New England Biolabs- see supply section below).

 

               See sample PCR gel at top of page to learn how to identify the species of mussel.  Record the data in your Excel data table, and please email as an attachment to Dr. Peter Wimberger at:    wimbo@ups.edu     The data will be integrated into an ongoing study.  Thank you for your contribution!

     

 

Supplies and Equipment

 

     Click here for required supplies, sources, and equipment (Note: same as for GROWS salmon project, except you do not need the restriction enzyme). 

 

 

Sources

 

To obtain a shellfish collecting permit, go to: http://www.wa.gov/wdfw/lic/vendors/vendors.htm  and you can do a search for vendors in your area who will sell you a permit.  For more detailed information about the regulations, go to: http://www.wa.gov/wdfw/fish/regs/fishregs.htm and download the relevant sections of the "2002 Fishing in Washington Regulation Pamphlet" (highlighted on the left side of the webpage).  

 

To request a free DNA extraction kit, as well as Taq polymerase kit (ask about dNTPs), call Sonya Dobias at Qiagen Corp. at:  1-800-426-8157  Ext. 23544. 

 

To request free DNA ladder (or restriction enzymes, dNTPs, and other biotech supplies), call Sherry Leavitt at New England Biolabs at 1-800-632-5227  Ext 275.   For free NEB catalogs, dial Kate at Ext. 378.  We recommend the 100 bp ladder (Cat. #N3231) for the mussel project.  Dilute the ladder 1:5 in TE buffer and then add 10X load dye as needed.

 

To order primers, go to http://www.fisheroligos.com.  There you will find out how to order either by phone, fax, email, or online (easiest) using a credit card or PO number.  First call their customer service number to get a teacher discount and account number.  When ordering online, use  the default settings for amount of primer (0.05 umol), and purification (desalt).  Then simply type in the sequence in the 5' to 3' direction in the box indicated.

 

To order PCR tubes, go to http://www.islandsci.com/bisland.htm where you can buy 0.2 ml rainbow colored tubes (Cat. # IS-430R) for $39.66/bag of 1000.

 

To borrow equipment (and you have taken their workshop), contact the Science Education Partnership Program (SEP) at Fred Hutchinson Cancer Research Center (http//chroma.mbt.washington.edu/outreach/sep.html).  Once you have taken their workshop for teachers you may borrow equipment from them.  

 

      You may borrow thermal cyclers from:

N. end:  Joe Day, Lynwood H.S., 425-670-7520, DayJ@edmonds.wednet.edu

Bellevue area:  Christy Shiers, Newport H.S., 425-456-7400, Shiers_Christy/53@belnet.bellevue.k12.wa.us

South Sound:  Dr. Peter Wimberger, University of Puget Sound,  253-879-2712, wimbo@ups.edu.

      You can also contact Washington State University's equipment loan program (http//www.sci.wsu.edu/bio/equiploa.html).

 

For staining DNA, you can use either ethidium bromide (EtBr), SYBR Green, or Biosafe.  EtBr is highly carcinogenic and should not be handled by students- only by teachers in a prep room. You can buy a 10 mg/ml solution from Sigma (www.sigma-aldrich.com , 800-325-3010, catalogue # E1510, $34.55).  Dilute to 5 ug/ml with water or running buffer and stain for 30 minutes.  The stain can be poured back into a brown bottle and reused many times.  Stained gels and used ethidium can be disposed of by soaking overnight in bleach, or as hazardous waste material.

      SYBR green nucleic acid stain is much safer, but is much more expensive when added to staining buffer.  A good alternative is to add it directly to your 10X load dye, so that after running your gel it is ready to go on the UV light box.  However, you must first dilute the stock solution by 1:10,000 or else it will distort the movement of the DNA in the gel.  SYBR green may also be obtained from Sigma (cat. # S9430, 0.5ml = $155.10).  More information can be obtained at the manufacturer's website at http://www.molecularprobes.com

      Bio-Safe is a nice non-toxic alternative DNA stain available from BioRad, which has the advantage that the bands are visible in normal light, so no UV box or camera is required.  It's also very cheap.  I recommend using it at the 1:200 dilution for 1-3 hours and then destaining overnight.  You can then dry the gel down on a special membrane that BioRad sells (kind of pricey), or take a picture of the gel with a regular camera.  You can download the information about Bio-Safe at the BioRad website; go to Google and search for BioRad, then search their website for Biosafe.  Bio-Safe is not as sensitive as ethidium or SYBR Green, but it should work fine for visualizing PCR product.

 

 

To borrow a thermal cycler, contact Peter Wimberger at wimbo@ups.edu.

 

 

References

 

Inoue, K., Waite, J.H., Matsuoka, M., Odo, S., and Harayama, S.  1995.  Interspecific variations in adhesive protein sequences of Mytilus edulis, M. galloprovincialis, and M. trossulusBiological Bulletin 189: 370-375.

 

Mullis, K.  The unusual origin of the polymerase chain reaction.  Scientific American April 1990: 56-65.

 

Suchanek, T.H., Geller, J.B., Kreiser, B.R., and Mitton, J.B.  1997.  Zoogeographic distributions of the sibling species Mytilus galloprovincialis and M. trossulus (Bivalvia: Mytilidae) and their hybrids in the north Pacific.  Biological Bulletin 193: 187-194.