Molecular-grade preservation

There are many ways to preserve millipedes for subsequent molecular work like DNA sequencing and genomics.

* The trick for molecular-grade preservation of tissues is to dry out the specimen quickly and thoroughly.

Two common ways of doing this is with 100% ethanol or silica gel. These two preservatives draw out all the water rather quickly (the way to really do this is with a dewar of liquid nitrogen but liquid N is often not accessible).

Alternatively, live specimens can be placed in a -20 C to -80 C freezer (the colder the better, but preferably not a frost-free freezer* like we have in our kitchens).

If you find a cool millipede for me, I will gladly supply some tubes of 100% ethanol or (particularly, if you’re in another country) some tubes of silica gel.

Alternatively, one can extract DNA directly from live individuals. If you found a millipede and want to keep it alive, tell me about your specimen  (it might be useful for scientific research on the biodiversity and evolution of millipedes) and consider shipping it to me at the University of Arizona. I put together a primer on how to safely ship live specimens.

[*Note about frost-free freezers (FFFs) - Most folks commonly have a FFF in their kitchen that automatically cycles through a warm-up stage to prevent frost from accumulating. FFFs are generally avoided for DNA-grade specimen preservation because there are many temperature shifts, especially re-freezes. Re-freezing really damages the DNA causing it to degrade and become less useful.]

100% ethanol method

1. •Plop the whole live millipede into a vial of 100% ethanol
   

2. •The greater volume of ethanol, the better (at least 10 volumes of EtOH per 1 volume of millipede)
   

3. •The colder the ethanol, the better (for both short term and long term storage)
   

4. •The tighter the seal, the better (I use Sarstedt plastic vials found here through Fisher or here through Sarstedt, Nalgene bottles are good too)
   

5. •If the millipede is big and juicy, you should replace the EtOH enough times that EtOH cloudiness/discoloration disappears
   

Silica gel method (more complicated but excellent for shipping internationally)

1. •(Silica gel is the stuff in the little bag that comes with a new electrical gadget. “Silica Gel - Do Not Eat” is typically printed on the bag. Some fancy cat litter is also composed of silica gel)
   

2. •Prepare a vial or centrifuge tube (fill about half way with silica gel and then stuff a thin wad of clean cotton, or tissue on top)
   

3. •While the millipede is alive, remove 5 - 10 legs, depending on the size of the individual (for this apheloriine millipede pictured here I’d remove 4 legs)*
   

4. •Place the legs on top of the cotton, which is on top of the silica crystals
   

5. •Cap the vial tightly
   

6. •The silica gel will quickly draw out the water from the legs thus desiccating them
   

[*Note: to prevent contamination with non-millipede DNA (even your own DNA) keep everything as clean and sterile as possible.]

Importance of a “voucher” specimen

I will extract DNA from the legs using a method in which I grind up and lyse the tissue. As a result nothing, except good clean DNA (and/or RNA), will remain. How will I know what species the legs came from? That’s why I would like the millipede from which the legs were removed too. This is called a “voucher specimen”. By having a voucher specimen, I’ll be able to identify the species and thereby associate the DNA that I extracted with its unique species name (after all it’s rather difficult to identify a millipede based just on legs).

Ultimately, I will give the voucher specimen a unique code (on a tiny slip of paper with the millipede in its vial). The voucher specimen’s DNA will get the same code (written on the tube containing the DNA).

The voucher specimen can be similarly preserved in 100% ethanol (best case scenario) or dried out (and drying works best with small millipedes that aren’t juicy - juicy ones tend to rot before they dry out).