Online exhibits : Field notes : Stephanie Bush

Squid science: Field notes from Stephanie Bush

By UCMP grad student Stephanie Bush, September 11, 2007–January 10, 2008

Stephanie is a graduate student at UCMP currently doing research at the Monterey Bay Aquarium Research Institute (MBARI). She wants to figure out why deep sea squid release ink even though there's very little light in the deep sea. Check out an overview of Stephanie's research and previous updates, or read on to find out what she's been working on recently …

September 11, 2007
Stephanie Bush
Stephanie cruising for squid on the deck of the research vessel, the Western Flyer.
I'm building an "ink ejector" to test reactions of deep-sea animals to deep-sea squid ink. The ejector will attach to the arm of an ROV (a remotely operated, underwater vehicle) so that squid ink can be expelled toward an organism living in the wild. We'll be able to catch the animal's reaction on video. Of course I am interested in knowing how squid predators react to ink, but the only squid predators we'll be able to see using an ROV are medium to small fish (which prey only on juvenile squid) and other squid. Although we probably won't see the main squid predators such as elephant seals and toothed whales because they avoid ROVs, I can at least begin to answer some questions about ink functionality. How does a squid react to ink from the same species? How does a squid react to ink from other squid species? How do midwater fish react to ink?

The first step in building the ink ejector was to find a suitable ink storage device that can be taken deep underwater. The best option I found was an "IV" bag because it is flexible and compressible and therefore does not have to be built to withstand great pressure. Next, I needed a pump to pull the ink from the storage bag and expel it into the water. The submersible pumps commonly used on oceanographers' measuring equipment are just right for this job. The last step was simply attaching the ink storage bag to the pump with aquarium hoses.

Ink ejector
Stephanie designed this ink ejector to see how deep-sea animals in the wild react to squid ink.

We'll use different squids' ink in different trials. But I'm worried that animals might react to just seeing the cloud, and I really want to know if they are reacting to particular chemicals in the ink. To help me figure out whether animals are just reacting to visual cues or if they are actually sensing something in the ink, I'm going to use what's known as an experimental control. Sometimes, instead of expelling squid ink from the injector, I'll expel a chemically inactive dye called fluorescein. And to rule out the possibility that animals are responding to the rush of liquid from the ejector, I'll also run tests where I expel plain sea water at the animals. By comparing the reactions of animals to actual squid ink, to fluorescein, and to sea water, I'll be able to figure out what effect the visual/physical cues alone have and can see if chemicals in the ink cause animals to react differently.

September 17, 2007
Today's dive was the first in which I used the ink ejector. I thawed some squid ink and filled the IV bag the night before the research cruise. The ROV pilots helped me put an additional arm on the ROV to hold the ink ejector and we attached each part carefully so no loose hoses would be snagged.

The ink ejector worked well and produced convincing looking ink clouds. The most difficult part was aiming the ink. It's difficult to tell an animal's distance from the ROV in midwater! We had to simultaneously look at different camera views to triangulate the animal's position. Much ink was accidentally released nowhere near the animal and went to waste. After we successfully tested two squids, Galiteuthis phyllura and Gonatus onyx, we were out of ink. I decided I better come up with a larger storage bag if I wanted to test more than two animals during future ROV dives.

Histioteuthis squid in an ink cloud The ink ejector expels fluorescein dye
Left: Histioteuthis heteropsis in an ink cloud expelled by the ink ejector. Right: The ink ejector expels fluorescein dye at a Gonatus onyx squid.

September 18, 2007
The second dive on which I used the ink ejector. I didn't have as much luck this day as the previous one. The beginning of the dive consisted of our monthly quantitative video transects. The ROV runs in a straight track for 10 minutes at 50 m, 10 minutes at 100 m, 10 minutes at 200 m, etc. to 1000 m depth. Later, we count the animals that were seen. Since we know the ROV velocity, we can calculate the volume of water encountered and the number of animals per volume of water. The process of taking the transects generally takes three hours. By the time we were done and were ready to expel ink around an animal, there was no ink in the bag. I guess the ink was sucked out of the hose while we flew through the water column. I'll have to incorporate a check valve to prevent this from happening again.

October 10, 2007
Octopoteuthis deletron bioluminescing
Octopoteuthis deletron showing off its bioluminescence.
I've added a hydraulic actuator as a check valve for the ink ejector. This device is placed on the open end of the hose, preventing the ink from flowing out when closed. Hydraulic fluid, which controls the arms and sampling devices on the ROV, opens and closes the actuator so we can use the ink ejector when we want to. I also found a larger ink storage bag that more than triples the amount of ink we can store. That means I'll be able to test more organisms on a single dive!

December 18, 2007
I'm working with MBARI engineers to construct a device that mimics the bioluminescence of the squid Octopoteuthis deletron. I'm working on describing some of the bioluminescent patterns of this squid — but we still don't know what function the bioluminescence serves. It might be used to lure potential prey, distract or startle predators, or attract mates. The "Pseudosquid" that we're building will be used in experiments that I'm hoping will help me figure out how squid react to the bioluminescence and what its function might be.

The PseudoSquid will be made of blue LED lights that put out the same light wavelength as the squids' bioluminescence, all controlled by a circuit board. We'll be able to control each LED light and program them for different patterns of flashing, from simultaneous to random flashing. The PseudoSquid will be partially waterproof so that we can put the LED lights inside an aquarium with a squid and observe how the squid reacts.

Left: PseudoSquid's simultaneous flashing pattern. Right: PseudoSquid's random flashing pattern.

January 10, 2008
The PseudoSquid was finished a few days ago. After the software programming was complete and the circuit board made, I had my first soldering lessons and decided to make the LED part of the PseudoSquid myself. It took much longer than expected. I think I'll leave the building to the professionals next time.

Testing the PseudoSquid revealed a problem though — the device was not long enough. While the LEDs are immersed in the aquarium, the other end is attached to a laptop for inputting the commands. With the current length, the circuit board was dangling in midair between the aquarium and computer. Despite my best efforts to keep it dry, a few drops of water splashed the circuit board, shorting the whole device out. Back at the drawing board, we designed a new version that was much longer and less sensitive to water. We put the whole circuit-board in a small, waterproof box.

A scientist's luck is kind of like Murphy's Law. Throughout the process of designing, building, testing, and coming up with a functional PseudoSquid, we have not seen a single Octopoteuthis deletron that I could collect and test it on …

Ink ejector and both Pseudosquid animations © 2008 Stephanie Bush; Histioteuthis heteropsis and Gonatus onyx © 2007 MBARI; Octopoteuthis deletron © 2004 MBARI.