Friday, May 11, 2007

Mollusc Genomes Update

After whining last week about my tribulations with molluscan cells, my advisor, Herr Doktor Professor T. Ryan Gregory sent me two emails. One basically said "stop kicking ideas around and do something, you slacker!" and the other included contact details for a mollusc-oriented mailing list, the "molluscalist".

So I sent off my questions to the molluscalist, and the wonderful world of malacology replied with many excellent suggestions, and links (or PDFs) to relevant papers that my earlier literature searching had not found. The upshot of most of this is that menthol crystals, crushed and sprinkled on the water's surface, act as a good "narcotizing agent" for freshwater invertebrates, especially snails. The basic protocol is to sprinkle a small amount (less than one gram) of menthol crystals on the water that contains snails (e.g. in a beaker), and wait roughly 24 hours for the snails to "relax" so they can be removed from their shells without damaging the soft tissue or the taxonomically-important shell.

I tried menthol this week. I ordered some from Sigma-Aldrich* on Wednesday, and, amazingly, the menthol arrived yesterday. So I took some locally-collected 'pond snails'** from the 'fridge where I'd been slowing them down, and dropped 1g of menthol on them. Today I dissected the snails and smeared some of their cells onto glass slides. The menthol worked very well - I think I actually overdid it, given the relatively small volume (perhaps 50 mL) of water the snails were in. They were apparently dead - no response at all to mechanical stimulation (I poked them with an insect pin). A pin bent to a hook was a very useful tool for 'winkling' snails out of their shells - it worked much better than I expected.

The downside of the dissections was (as is so often the case) the smell. Dead freshwater molluscs smell unpleasant, but these had a minty-fresh overtone from the menthol that interacted to provide a truly awful stink. I may have trouble brushing my teeth this evening when I smell the toothpaste.

I also used the menthol treatment on a caddisfly larva*** I had also collected. I think it was much more susceptible to the menthol (have I mentioned how chemically tough snails are?), as it looked like it had died sometime quickly after the menthol went in, and was very floppy and difficult to dissect. I don't know how floppy they are when they're alive, though.

The dissections went fairly well, to the point I could sort-of identify some internal organs of the snails, a big surprise to me given that when the shell has to be crushed to remove a snail, the resulting blob of tissue is a homogeneous mess. I'm pretty happy with this method; one major advantage of menthol relative to other potential narcotizing agents is its total harmlessness to humans - it's a food additive, and the LD50 value for rats is so high that it suggests the toxic effects are a result of osmotic balance disruption, rather than any particular cell-physiology impact, besides being a mild anaesthetic.

* Edit June 23 2011. I received an email from a representative of Sigma-Aldrich Corporation, requesting I change my link from "Sigma" to "Sigma-Aldrich" as that more properly represents the company's name and brands. I have made the change, and I'd like to apologize to Sigma-Aldritch for the oversight. And I'd like to thank them for taking the time to read my little corner of the internet!

** My inter-library-loaned copy of Burch, 1980 and Clarke 1981 helped me to fairly-confidently identify the snails as Pseudosuccinea columella, Fossaria parva, and Stagnicola elodes.

*** I used Pennak 1978 to identify (after three tries - can you tell easily how many sclerotized plates cover the mesonotum?) the caddisfly larva as genus Heterophylax.


Necator said...

Minty molluscs? Sounds like a song title to me. No ut seriously, after working with mice for three years, dissection starts to suck. I can only imagine that working with something so anatomically different freom vertabrates, identifying organs becomes tough. It brings back flash backs of my zoology course where we cut up a different invert every two weeks and we were stuck thinking how different the location of everythings was in, the gonads near the dorsal surface, above the stomach(s), the crazy number of limbs and the derived mouth parts.

The use of menthol on snails reminds me of the intoxicating properties of sour milk on the aliens in Alien Nation.

Also, SDS can and does dissolve nuclear membranes (1% is more than enough). That's why it's a common detergent in lysis buffers. If you're trying to lyse tissues, you will have to homogenize them first since connective tissue can be rather tough.

TheBrummell said...

Thanks for the note about SDS. I wasn't seriously considering it, but it's one of those readily-available lab chemicals and I ran into a problem a while ago with surface tension - I wanted to freeze some Drosophila melanogaster in various solutions, and they just froze solid sitting on top of the liquid. Sodium citrate at about 0.1% seems to work, and is also used in Flow Cytometry so we know it won't trash the nuclei.

Dissecting snails is really difficult. Getting them out of the shell is merely the first step. Then one is faced with a blob of rapidly-dehydrating and self-lysing tissue that is an infinite variation on the theme of "brown". Some bits are yellowish, some bits are whitish, and some bits are greyish, but overall, it's a blob of brown goo. A de-shelled snail looks remarkably like bird droppings, with the white part removed.

Then I read all these papers about taxonomy, identifying species and working out phylogenies, where they discuss the finer anatomical points of the reproductive organs. The pineal sheath? Would that be this deflated brown sac or that one?

The shells are pretty, though.

Laelaps said...

Thanks for sharing this; should I ever need to kill/dissect an invert, I'll know to make sure they're "minty fresh" first...

"The downside of the dissections was (as is so often the case) the smell. Dead freshwater molluscs smell unpleasant, but these had a minty-fresh overtone from the menthol that interacted to provide a truly awful stink. I may have trouble brushing my teeth this evening when I smell the toothpaste."

This reminds me of my various experiences with sprays that are supposed to eliminate/cover-up odors; in the end you just end up with a sickly-sweet combination of the two.

Goestaf said...

I did my experiment on use of "common anesthetics" for my pearl oysters. Menthol liquid was in the list with two different concentrations. Since timing is important for pearl operation, menthol liquid (unfortunately) its not the one I recommend. It took long time to induce and more deaths I recorded than other anesthetics. The paper is being submitted.

TheBrummell said...

Thanks for the info, Goestaf. I haven't encountered liquid menthol anywhere, but the solid crystals are still working well, at least for my purposes. I haven't tried to tightly control the time-to-relaxation, since I'm not too concerned about how thoroughly dead the animal is.