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Squid Sensitivity

The more electrophysiologist William Gilly learns about these mysterious denizens of the deep, the more they seem like an alien intelligence

By Jeff Wheelwright, Grant Delin|Tuesday, April 01, 2003
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This Humboldt squid is a preserved
specimen five feet in length. William
Gilly can study small squid in captivity,
but the Humboldt must be
studied in the field or preserved;
they don't live long in a lab.

Late in the afternoon, WilliamF. Gilly, a professor of biology at Stanford University, lugs hisequipment across the dirt-topped wharf. A stubble of beard pushesthrough the sunburn on his cheeks. He is a happy man. Why shouldn't hebe? Free of the introspection of the laboratory, he's off to tag thewild Humboldt squid on the Sea of Cortés.

After packing histruck at Stanford's Hopkins Marine Station, in Pacific Grove,California, Gilly drove 900 miles without sleeping to Santa Rosalía, inBaja California, Mexico. Down the long peninsula, where the cacti troopto the indigo sea, past the lava fields and boulders and "pale, burnedmountains" that fascinated John Steinbeck. In 1940 the novelist sailedwith a marine biology expedition to the Sea of Cortés, or the Gulf ofCalifornia. It is October 2001, and Gilly is leading a team of sevenresearchers on an expedition there. He turned 51 on September 11, abirthday well left behind. At Santa Rosalía the heat has slipped below100 degrees, and the fishy air of the harbor is tickled with marijuanasmoke, for the strangers have attracted a small crowd. The Mexicans arecurious to see the scientists who, according to the posters they hadput up in town, will pay fishermen $50 for each Humboldt squid that isturned in with a yellow tag in its mantle.

Gilly is anelectrophysiologist, an expert on the wiring of the nervous system.Ordinarily the test subject for his research is the eight-inch-longsquid Loligo opalescens, which seafood lovers know as themarket squid. At Hopkins he studies the function of the giant axon, athick, five-inch-long nerve fiber that triggers the squid's escapemechanism. The axon sends a signal to the muscle cells of the mantle,where a powerful contraction expels water from the squid's internalsiphon, jetting the animal forward or backward. During the last 25years in the lab, Gilly has penetrated ever smaller domains of thesquid neuron, into the cell and down to the conductive proteinmolecules on its membrane, and from there to the genes responsible forproducing those proteins. Now, in an abrupt shift of scale that alsorepresents an abrupt career turn, he finds himself launching anuncontrolled experiment in the open ocean on an invertebrate creaturethat can reach six feet in length and weigh 60 pounds—the Humboldtsquid.

The Sea of Cortés is the gash formed when BajaCalifornia split away from the Mexican mainland. For a narrow body ofwater it is extremely deep. Within the vertical volume of the sea swarmmarlin, sailfish, red snapper, grouper, yellowfin tuna, sweet-tastingmahimahi, and groups of Humboldt squid.

The Humboldt, or jumboflying squid, is one of the largest squid that anyone has seen alive.(So-called giant squid are much larger but are known only from rare,dead specimens.) Fast growing and short-lived, the jumbos move upthrough the food chain rapidly, so that at two years of age, which isas old as they get, only the biggest fish and the toothed whales canprey on them. But before then most have become meals in the cafeteriaof the eastern Pacific. Squid may have become even more important tothe food chain in recent years, Gilly notes, since aggressive fisherieshave depleted the marine species with backbones. There are indicationsthat squid populations worldwide are increasing, as they move into theniches opened by the harvests of finfish.

Squid are brainymollusks. They once had shells and were relatively immobile, but in thegrand battle of evolution, says Gilly, "squid had to give up theirheavy armor and become free-swimming predators with vertebratelike eyesand big brains. They had to outsmart and outperform fish, which aremore agile and explosive and maneuverable. The giant axon system is onesolution for escape response and, probably, prey capture." He pausesand arches his eyebrows. "I often wish that I had a giant axon systemto help me escape from certain people and situations—but also forjetting over and getting closer to others."


Theregion's main squid fisheries
are at Santa Rosalía and Guaymas.
Mexicanfishermen haul in
110,000 tons of Humboldt
squid each year.

Image provided by ORBIMAGE,
© Orbital Imaging Corporation,
and processing by NASA
Goddard Space Flight Center.

From spring to fall the port of Santa Rosalía hosts a small-scalebut intense fishery. Men race their fiberglass skiffs out onto the flatsea at night. They turn on battery-powered lightbulbs in an effort tolure schools of squid. With jigs—cylindrical lures ringed withhooks—they snag the reddish animals 500 feet down and pull them up handover hand. The meat of the mantle is processed onshore and exported,most of it to Asia.

Gilly is here on behalf of the Census ofMarine Life, an ambitious multi-project attempt to record the diversityand distribution of as many of the world's ocean creatures as possible.The goal of this part of the census is to mount electronic tags onlarge Pacific fauna like tuna, sharks, whales, elephant seals, seaturtles, and now squid. The lightweight devices track the animals intheir migrations across the ocean. Some tags communicate directly withsatellites; others store their data until the animal is caught again orcomes ashore.

From the marriage of microelectronics andtelemetry a new line of marine studies has been born. Gilly has hiredtwo fishermen and a panga, as the 20-foot skiffs are known.While the fishermen catch the squid, Gilly and his young assistantswill tag and release them. This is a preliminary experiment, in thatthe costly electronic tags won't be used. Gilly wants to learn how manymarked animals will be turned in if the fishermen who catch them areoffered a $50 reward. If enough tags come back, the data-logging"archival tags" will be deployed next time. Thus the marker is a ringof yellow plastic with a phone number printed on it (an 800 number inMexico), along with a reward message. A fisherman turning in a tag isasked to report when and where the squid was caught.

The Seaof Cortés is dark. Mars is aloft, glowering red like the jumbo squidbelow, and a sweet breeze is blowing from the desert. Less than fiveminutes after the panga's anchor is set, the first squid ishooked. In their New York Yankee baseball caps and yellow rain pants,the Mexicans haul the lines from opposite ends of the boat. A plywoodboard lies across the center thwarts. When a squid is flopped onto theoperating table, glistening like a newborn calf, Gilly threads the taginto its mantle, and a graduate student snips the plastic tie. Thenthey raise one end of the board, and the squid slides back into thesea. The entire operation, after a little practice, takes 30 seconds,tops.

Each squid, as it's captured, frantically tries to freeits tentacles from the hooks by emitting great jets of water. Normally,the hydraulic force through its siphon tube would propel the animalquickly away. Held at the side of the boat, however, the squid is likean errant fire hose, soaking anyone in range. Gilly has a snorkelingmask over his glasses, but the water gets in anyway, and he flings themask off.

The smaller ones are often in bad shape, some intatters, because of attacks by other squid during their helplessascent. The creatures are cannibalistic, terroristic, rushing on theirprey with a forward thrust of the tail. Gilly has heard rumors abouthow dangerous Humboldts are—how the "red devils" have dragged downcapsized fishermen. So far the one casualty of the evening is a bite onhis finger. The squids' blood where the tag pricks them is blue, andthe water in the bottom of the panga is black with their ink.

Surprisingly, the squid grow still once they are on the table. Theylower a flap over black eyes the size of eight balls and show theirdistress only by the rapid blushing or blanching of their coloration.Humboldts can be maroon or ivory or any tone in between, according tothe action of their chromatophores, or pigment cells. The color theydisplay may depend on the depth of water; biologists don't really know.

A diver on the expedition has made a tape of two animals flashingunder the surface. At first the pulses are out of phase, then theysynchronize as the two squid come together. "What does this signalingmean?" Gilly asks. "I'm not the only one who believes they communicate."

Close to 90 Humboldts are tagged in the first night of work. But acouple of the squid, too ripped up to release, are held for analysisashore. One, lying in the inky bilge, seems to have set its eyes onGilly. The creature is dying, its color fading, its electricity ebbing.Air rasps through its siphon. Overtaken by what he later describes as a"profound sadness" that "must have been lying latent for 25 years,"Gilly pulls out his knife, bends between the thwarts, and puts thesquid out of its misery.

"Before then I never felt small—bythat I mean diminished—in sacrificing such animals for scientificwork," he recalls. "Maybe it was the size of the eye—it made it seemmore living."

+++


WilliamGilly found himself marveling at Humboldt
squid in the wild. He haswritten
that "looking into the eye
of a living, breathing, swimming 60-pound,
six-foot squid from an arm's length
away in the open oceanstuns you."

It is April,six months after Gilly's epiphany—not that he'd use that term. He willsay he had a bout of "squid sensitivity" in Baja, but it has notdeterred him from continuing to experiment on squid or on the nervoussystems of small captive invertebrates.

The Hopkins MarineStation is a complex of buildings sitting among the cypress trees a fewyards from Monterey Bay. Although the Cannery Row waterfront and theMonterey Bay Aquarium are nearby, busy with tourists, the Gilly lab isserene, a sort of science sanctum. Wearing his regular outfit of khakishorts, a black T-shirt beneath a short-sleeved sport shirt, andrubber-soled walking shoes, Gilly shows a visitor around. He needsglasses for distance vision and carries reading lenses as well, butinstead of alternating them he simply puts one pair on top of theother.

The main corridor houses the research equipment. Someof the technology is obsolete, but Gilly is loath to throw away thingsthat work, and besides, the devices are links to his past. There areoscilloscopes, amplifiers, and voltmeters. There are aquariums andpiping and pumps. There are micromanipulators, which, with the aid of amicroscope, allow the scientist to press a tiny electrode into livingtissue and measure the drop in electric potential as the membrane isbreached.

The animals occupy a side room. Cone snails, whichsting their prey with nerve poison, are kept in small tanks on a shelf.The cuttlefish (ovoid bottom-dwelling cousins of squid) are in themedium-size tanks on benches. The very large, round fiberglassstructures are the tanks for the Loligo squid, one tank for themales and the other for the females. Huddled, the two dozen male squidback away as far as they can from the humans leaning over thechest-high wall. A few sidle forward, then gently propel themselvesback.

"We keep them separate because they mate like crazy,"Gilly says. "The males are aggressive and do unspeakable things to thefemales. The male gets underneath and wraps its tentacles around andbasically fillets the skin of the female."

On the bottom ofthe females' tank is a white, spiky formation: squid eggs in theircases. The mating took place before they were caught. After mating,females store sperm in pockets around their mouths. Responding to somemysterious cue, they all squeeze eggs out through their siphons andfertilize them. "You'll come in the morning and there'll be a huge pileof eggs," Gilly says.

Four or five graduate and postdoctoralstudents wander about, each engrossed in a research task. The clock onthe wall seems superfluous. The best way to tell time in the Gilly labis by the low music coming from the CD player: Classical during theday, folk as evening arrives, then rhythm and blues after dark.

"There's an addictive and an escapist component to science," Gillyallows. "The more you do and see and answer, the more you want toexplore new angles and find more questions. It becomes more and moreexciting and consuming. Not necessarily Faustian, but probably close. Ineed to keep from self-destructing, as with any addiction." Divorced,with a son away at college, Gilly lives alone, and it's not certainwhether the lab is his primary or secondary residence.

At thevery center of the lab is a poster that Gilly has owned since he was anundergraduate at Princeton University, in the late 1960s. It's aportrait, Norman Rockwell-style, of a Catholic Boy Scout. A smallfigure of a priest stands behind the earnest visage of the Boy Scout.The caption asks, "Is God Calling Me?"

The poster is meant tobe ironic, yet it's placed like an altar. Gilly says that in hisboyhood he was "a card-carrying Lutheran with seven years of medals forperfect attendance in Sunday school." One day when being examined bythe pastor, he could not accept that the bread and the wine wereliterally the body and blood of Christ. "I loved the church before thatmoment," he says wistfully.

In his small, chock-full office down the hall, while a computer monitor displays Got Squid?in undulating type behind his head, Gilly considers whether or not asquid feels pain. He's well aware of the stereotype of the scientistwho tweaks animals with electrodes and toxins, heartless to theirsuffering. Indeed, he has spent the bulk of his afternoon exposing Loligosquid to domoic acid, a marine poison that is the stuff of red tides."They [the squid] will recover immediately from this," he says. "Theyaren't distressed. In fact, it's virtually impossible to say if aninvertebrate animal feels pain."

Having served on a Stanfordcommittee that reviewed the protocols for handling vertebrate animals,Gilly is not in favor of extending such standards to squid. "Alreadythere are too many restrictions on what you can do with fish and frogs.Where do you stop? Where do you draw the line? At bacteria?"

Still, how does he explain his response to the dying squid in Baja? Itturns out he's written an account of the incident. The Humboldt isabout the size of a human being, he observes. No microscopes ortelescopes lie between the scientist and his subject. "Why should thesize of an organism affect the way we perceive its qualities?" hewrites. "Perhaps animals that are of a size comparable to our own arethose that lead us most easily to a conscious respect for life.

As Gilly hands over the piece of paper with his essay, his eyes areglistening. "Don't use this to promote squid rights!" he says gruffly.

In the late 1980s Bill Gilly took the renowned four-week trainingcourse at the Cold Spring Harbor Laboratory on Long Island in New York.The Cold Spring lab is the cradle of molecular biology. It representsthe triumph of reductionism, whereby living systems are broken down totheir smallest and, presumably, simplest parts. Like many biologists,Gilly was excited to learn how to manipulate DNA and the genetic code,which were newly sprung from the nucleus of the cell.

"I gotseriously interested in the molecular aspects of voltage-gated ionchannels," Gilly recalls. "This was after the successful cloning ofsome channel genes was reported."

Voltage-gated ion channelsare complex proteins in the membranes of nerve and muscle cells. Theyare structured to function like pores; they permit electrically chargedatoms, known as ions, to pass in and out. Cells have separate channelsfor ions of sodium, potassium, calcium, and chloride. The sodiumchannel, which became Gilly's target of interest, transports thepositively charged sodium ions into the negatively charged interior ofthe squid's giant axon.

The axon conducts a nerve signal byharnessing the ion channels studded along its length. As the "gates" ofthe channels open and close in rapid succession, an "actionpotential"—a sharp reversal of electrical charge—ripples from the hubof the axon to its tip. It happens with the speed of a sprintercovering 30 yards in a single second.

Human nerves basicallyfire by the same mechanism. If the ion channels don't functioncorrectly because of a genetic disorder, say, a neuromuscular diseasemay result, such as myotonia, a stiffness of the muscles. In multiplesclerosis, disruption of the sodium channels may cause some symptoms ofthe disease.

Hence Gilly's thinking: Biomedical science wouldbenefit if more were known about the genes that direct the formationand placement of the ion channels. Researchers can't dissect thenervous systems of living people, but the superthick and superlongnerve fibers of the squid make for a fine substitute. Gilly alreadyknew how the channels perform at the gross level of the organism. Belowthat, at the genetic level—well, that's why he was at Cold SpringHarbor, poring over a textbook as a centrifuge spun out strands of DNA.If he could master the channel genes of the squid, he might gain thekeys to the entire machine.

The course left him a bitfrustrated. "I soon realized I wasn't cut out for the benchwork," hesays. "All of the lab procedures were superficially identical,regardless of what you were trying to do. Most distressingly, theregenerally was nothing to see or touch except a tiny pellet of DNA."

But having learned the steps that would be needed to isolate andclone (make unlimited copies of) the squid channel genes, he broughtaboard a Ph.D. student, technically more adept than he, to do the heavylifting. In 1992 the Gilly lab won a major grant from the NationalInstitutes of Health. It was not a blank check, but the researcherswere given a lot of leeway to explore the genetic bases of sodium andpotassium channels in squid.

Eight years later, the workended in disappointment. Gilly and his students did manage to create aworking model of one of the squid channels. Using frog eggs to take upthe squid genes and make the channels, they were able to measure actionpotentials and nerve activity. They published papers on their findingsin journals, but an overall understanding of the system eluded them.

"We were banging our heads against the wall," Gilly explainsruefully. "We got sidetracked into some questions that just did notgive easy answers, and it was felt by the [National Institutes ofHealth] review panel—as well as myself in my heart—that it was time tomove on to something new."

Funding for the project was cutoff. Gilly was out $320,000 a year. As every scientist knows, the deadends are far more frequent than the breakthroughs, but that's noconsolation when your lab's very survival depends on a grant.

Taking a 25 percent pay cut for himself, he scrambled for new projectsthat would maintain his academic standing and also keep his graduatestudents busy. "For the past year," he said in 2001, "I've been tryingto get funds here and there to keep people at the lab."

Thena Stanford colleague offered Gilly an assignment with the Census ofMarine Life, at the opposite end of the research spectrum. He'd neverdone formal field biology before, but, at a crossroads, he agreed tolead the squid-tagging project to Baja, which he knew from his fishingjaunts.

This experiment succeeded. The team put tags on nearly1,000 squid and got back about 80. Gilly was pleased with not only therate of return but also the locations of the catches, which sketchesthe seasonal migration of the groups across the Sea of Cortés. "Thereis a two-week gap when there were no returns," he says. "Before thatthey were from the Santa Rosalía area [on the west side] and afterwardfrom Guaymas [on the mainland to the east]. No overlap. Pretty neat."

Baja, moreover, seems to have overturned his research philosophy:"I'm combining biophysics with ecology now. Ideally, the scales allinterrelate: the muscle work, the chromatophores, the genetic studies,the tagging . . . the squid spawning, the toxicology, populations,behavior. I do find myself thinking more holistically."

September 2002. The sky over the Sea of Cortés is pink and purple.Gilly and his helpers are camped on the beach, eating lobster in thewarm wind, their mission accomplished. He's a little nervous becausethis time the tags cost $250 apiece. Somewhere offshore there are 95Humboldt squid carrying quarter-size, battery-powered devices, whichperiodically record the water temperature and depth and so will give apicture of the squid's vertical movements. Each tagged animal is worth$100 to whoever recovers it, whenever that will be.

The highpoint of the trip was swimming with the squid at night. With snorkeland mask Gilly tested the reputation of the "red devils." "You'd seethese pulsating white blobs coming out of the blackness," he says."They were definitely curious. A squid would come up at you, feel you,and touch your hand. Even when four and five at a time were in feedingmode, they were delicate and retiring.

"As I was swimming, Ihad my hand stretched out. It was pretty moving. It was as close as youcould imagine to meeting an alien intelligence." Like a scene from E.T., perhaps. Or, in keeping with the poster in his lab, like the touch of the fingertips on the ceiling of the Sistine Chapel.
"So the awarenesswas still there. We didn't take any more squid [as bait for othersquid] than we would need. I insisted that their heads be cut off—tostop thought quick in those animals. They don't think the way we do,but still, suffocating, expiring, they do not enjoy it."

As forthe tagging results, instead of an 8 percent return, this time he hadjust one: A single tag was turned in by a Mexican fisherman a monthlater. It was all he needed to show that the project was feasible."Stayed up practically all night plotting out the data," he wrote laterin an e-mail. "It is beautiful. The most regular phenomenon is a rapidrise [of the squid] right at sunset. . . . Got to go now to do someelectrophysiological recordings. Back to the day job, you know."

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