Yes, Tina Turner, you had a question?

"What’s methyl farnesoate got to do, got to do with it?

What’s methyl farnesoate, but a sesquiterpenoid?

What’s methyl farnesoate got to do, got to do with it?

Who needs a heart when you’ve got mandibular organs?"

Wow, that's a good question Tina (or three, really). I'm surprised you're so familiar with crustacean hormones, but I guess I should never underestimate Ms. Turner.

mm-hmm!

Well, now that I've been set straight, let me answer your big question: what does methyl farnesoate (MF) have to do with crab love? That is the big question in my graduate thesis, and I'm afraid I don't have all the answers... YET. But here's what I do know:

You'll remember that MF is synthesized in the mandibular organs (MO):

methyl farnesoate leaving the mandibular organs and heading for the gonads

When the MF leaves the MOs (ah, science and acronyms), it binds to specific receptor sites that can change the physiological activity of the target (such as the gonads). What results is increased gonad development, so far recorded in these crustaceans:

monsoon river prawn Macrobrachium malcolmsonii (pictured is a congener)

and red swamp crayfish Procambarus clarkii

green crab Carcinus maenas,

Indian field crab Oziotelphusa senex senex (another congener picture),

and spider crab Libinia emarginata

In the red swamp crayfish, green crab, spider crab, and Tanner crab (you remember Chionoecetes bairdi), higher MF also correlates with more reproductive behavior. And, since MF stimulates secretion of ecdysteroids by the Y-organs (which in turn stimulates the production of a new shell and shedding of the old one), mating and molting are hormonally linked!

MF stimulating the secretion of 20-hydroxyecdysone (20-HE) from the Y-organs

My research will look at the mating/molting relationship in snow crabs: will MF be lower in post-molt males? Will it correlate with lower gonads (GSI)? I can't wait to find out!

So you see, Tina, MF has a lot to do, lot to do with it!

Ask A Grad Student: Miranda Westphal

Miranda is my crab lab mate -- she's co-advised by Dr. Sherry Tamone (my advisor) and Dr. Ginny Eckert (my committee member) -- and my office mate in the Lena building, as well as my ELISA accomplice!

Age: 34

Degree: Candidate for Master’s of Science, Fisheries

Current City: Juneau, Alaska

1. Describe your project, in 4 sentences or less.

I study the growth physiology of red king crab (Paralithodes camtschaticus), specifically comparing wild caught red king crab (from Southeast Alaska) and hatchery-raised red king crab (from the Alutiiq Pride Shellfish Hatchery in Seward, Alaska). There are two aspects to my project: 1. is to determine how often the crabs molt (molt interval) and how much they grow with each molt (growth increment) 2. look at the fluctuations in circulating growth ecdysteroids (20-hydroxyecdysone) in the hemolymph throughout the molt cycle. Additionally, I conduct monthly beach surveys (during the low low monthly tides in Auke Bay) to measure the wild juvenile red king crab that live in the intertidal in order to help determine if there are any differences in growth that may result from rearing crabs in a laboratory over time. By understanding the early life history of king crabs, specifically how they grow and behave during their most vulnerable time (first few years of life), managers will be empowered to manage the fishery in a more informed and predictive manner; moreover, this research may be used to better classify (or “age”) young crabs that are encountered during surveys or other field research opportunities, giving managers and researchers a deeper understanding of how movement and behavior of differing age classes of juvenile king crabs in the field will ultimately affect the fishery into the future. (I know that last sentence is kind of long and a run-on but I only had four sentences!)

a red king crab's growth over subsequent molts (all photos scaled the same)

2. You were living in Florida before. What drew you to Alaska?

I loved working in the warm waters off of Florida but couldn't pass up the opportunity to work on an amazing crab fishery in the last frontier. It really was the project and my advisors that drew me to Alaska. The bonus is that Southeast Alaska is an amazing place to live and work!

at work on ADF&G's R/V Medeia with a blue king crab

3. What has been the most challenging aspect of your project? The most fun?

The most challenging part has been learning time management. It's a real challenge to fit in all of your research as well as classes, meetings, lab work and all of the other little things that tend to come up! The most fun is, without a doubt, working with live animals. Even though it can be challenging and time consuming, it brings me a lot of joy watching their behavior and watching them grow. I often feel more like I have thousands of little pets rather than research animals!

first stage juvenile red king crab

4. Will you continue to work with crabs after you graduate?

I sure hope so! I love working with juvenile shellfish and I really do prefer working with decapod crustaceans (e.g. crabs, shrimp, lobster, etc.). I am considering continuing on with my education and pursuing a PhD but I haven't made any decisions yet.

5. What is your favorite piece of crab paraphernalia?

When working with a specific organism or class of organisms, you really do tend to accumulate a lot of paraphernalia! Without a doubt, my favorite is my crab beach ball that I got for my birthday from my sister-in-law. I also really love the crab business card holder that I got for Christmas.

the greatest beach ball EVER!

a card holder with crabby bling

Thanks Miranda!

ELISA: not just a pretty name

We’ve talked about methyl farnesoate, the reproductive hormone, but what about the hormone that makes crustaceans molt? Ecdysteroids!

from Kumar et al., 2004

These bad boys stimulate the production of a new cuticle, or soft shell, under the old hard one, as well as stimulate the production of enzymes to separate the new and old shells.

production of a new cuticle before and after the molting process

(Thanks to Miranda Westphal for the artistic idea!)

You can tell when a crab is getting ready to molt because the ecdysteroids increase significantly:

ecdysteroid profile for Dungeness crabs

How do we measure ecdysteroids? With a competitive enzyme-linked immunosorbant assay (ELISA), of course! An ELISA works by taking advantage of the specific nature of antibodies. Rabbits are injected with crab hemolymph (blood) to make a primary rabbit antibody (1° Ab), and then goats are injected with rabbit blood to make a secondary goat anti-rabbit antibody (2° Ab). This way the 1° Ab will only bind to the 2° Ab, and ecdysteroids from crab hemolymph will only bind to the 1° Ab.

The competition comes in with the crustacean ecdysteroids: the ecdysteroids will bind to the rabbit antibodies, but in order to measure how many ecdysteroids are in the hemolymph we add marked ecdysteroids to the mix.

ELISAs are run in tiny wells. Within the wells, the 2° Ab first binds to the well wall. Next we put both the crab hemolymph and the marked ecdysteroids (“horseradish peroxidase-conjugated ecdysone”) into the wells. They can’t bind to anything yet, but we finally add the 1° Ab. This is when things go nuts! The 1° Ab is busy binding to the 2° Ab, while the two types of ecdysteroids are competing for spots on the 1° Ab. After we let the wells sit for a bit, anything that hasn’t bound to an antibody gets dumped.

binding!

The horseradish peroxidase-conjugated ecdysone turns blue during the development process. When you see a well with bright blue, it means that sample of hemolymph did not have a lot of ecdysteroids in it, which allowed for more of our marked ecdysteroids to bind to the rabbit antibodies.

an ELISA plate after development

Wells that appear mostly clear represent crabs that had a lot of ecdysteroids in their hemolymph. These crabs are the ones that are ready to molt!