WEB EXCLUSIVE

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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Squid
Squid
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.
Squid-Map
Squid-Map

The region's main squid fisheries are at Santa Rosalía and Guaymas. Mexicanfisher men 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.

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

After packing his truck at Stanford's Hopkins Marine Station, in Pacific Grove,California, Gilly drove 900 miles without sleeping to Santa Rosalía, in Baja California, Mexico. Down the long peninsula, where the cacti troop to the indigo sea, past the lava fields and boulders and "pale, burned mountains" that fascinated John Steinbeck. In 1940 the novelist sailed with a marine biology expedition to the Sea of Cortés, or the Gulf of California. It is October 2001, and Gilly is leading a team of seven researchers on an expedition there. He turned 51 on Sept. 11, a birthday well left behind. At Santa Rosalía the heat has slipped below 100 degrees, and the fishy air of the harbor is tickled with marijuana smoke, for the strangers have attracted a small crowd. The Mexicans are curious to see the scientists who, according to the posters they had put up in town, will pay fishermen $50 for each Humboldt squid that is turned in with a yellow tag in its mantle.

Gilly is an electrophysiologist, an expert on the wiring of the nervous system.Ordinarily the test subject for his research is the 8-inch-long squid Loligo opalescens, which seafood lovers know as the market squid. At Hopkins he studies the function of the giant axon, a thick, 5-inch-long nerve fiber that triggers the squid's escape mechanism. The axon sends a signal to the muscle cells of the mantle,where a powerful contraction expels water from the squid's internal siphon, jetting the animal forward or backward. During the last 25years in the lab, Gilly has penetrated ever smaller domains of the squid neuron, into the cell and down to the conductive protein molecules on its membrane, and from there to the genes responsible for producing those proteins. Now, in an abrupt shift of scale that also represents an abrupt career turn, he finds himself launching an uncontrolled experiment in the open ocean on an invertebrate creature that can reach six feet in length and weigh 60 pounds — the Humboldt squid.

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

The Humboldt, or jumbo flying 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 up through the food chain rapidly, so that at two years of age, which is as old as they get, only the biggest fish and the toothed whales can prey on them. But before then most have become meals in the cafeteria of the eastern Pacific. Squid may have become even more important to the food chain in recent years, Gilly notes, since aggressive fisheries have depleted the marine species with backbones. There are indications that squid populations worldwide are increasing, as they move into the niches opened by the harvests of finfish.

Squid are brainy mollusks. They once had shells and were relatively immobile, but in the grand battle of evolution, says Gilly, "squid had to give up their heavy armor and become free-swimming predators with vertebratelike eyes and 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 system to help me escape from certain people and situations — but also for jetting over and getting closer to others."

From spring to fall the port of Santa Rosalía hosts a small-scale but intense fishery. Men race their fiberglass skiffs out onto the flat sea at night. They turn on battery-powered lightbulbs in an effort to lure schools of squid. With jigs — cylindrical lures ringed with hooks — 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 of Marine Life, an ambitious multi-project attempt to record the diversity and 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 on large Pacific fauna like tuna, sharks, whales, elephant seals, sea turtles and now squid. The lightweight devices track the animals intheir migrations across the ocean. Some tags communicate directly with satellites; others store their data until the animal is caught again or comes ashore.

From the marriage of microelectronics and telemetry a new line of marine studies has been born. Gilly has hired two fishermen and a panga, as the 20-foot skiffs are known. While the fishermen catch the squid, Gilly and his young assistants will tag and release them. This is a preliminary experiment, in that the costly electronic tags won't be used. Gilly wants to learn how manymarked animals will be turned in if the fishermen who catch them are offered 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 in Mexico), along with a reward message. A fisherman turning in a tag isasked to report when and where the squid was caught.

The Sea of Cortés is dark. Mars is aloft, glowering red like the jumbo squidbelow, and a sweet breeze is blowing from the desert. Less than five minutes after the panga's anchor is set, the first squid is hooked. In their New York Yankee baseball caps and yellow rain pants, the Mexicans haul the lines from opposite ends of the boat. A plywood board 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. Then they raise one end of the board, and the squid slides back into the sea. The entire operation, after a little practice, takes 30 seconds, tops.

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

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

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

A diver on the expedition has made a tape of two animals flashing under the surface. At first the pulses are out of phase, then they synchronize as the two squid come together. "What does this signaling mean?" 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 a couple of the squid, too ripped up to release, are held for analysis ashore. 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 the squid out of its misery.

"Before then I never felt small — by that I mean diminished — in sacrificing such animals for scientific work," he recalls. "Maybe it was the size of the eye — it made it seem more living."

Gilly
Gilly

William Gilly found himself marveling at Humboldt squid in the wild. He has written that "looking into the eye of a living, breathing, swimming 60-pound, 6-foot squid from an arm's length  away in the open ocean stuns you."

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

 The Hopkins MarineStation is a complex of buildings sitting among the cypress trees a few yards from Monterey Bay. Although the Cannery Row waterfront and the Monterey Bay Aquarium are nearby, busy with tourists, the Gilly lab is serene, a sort of science sanctum. Wearing his regular outfit of khaki shorts, a black T-shirt beneath a short-sleeved sport shirt, and rubber-soled walking shoes, Gilly shows a visitor around. He needs glasses for distance vision and carries reading lenses as well, but instead of alternating them he simply puts one pair on top of the other.

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

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

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

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

Four or five graduate and postdoctoral students wander about, each engrossed in a research task. The clock on the wall seems superfluous. The best way to tell time in the Gilly labis by the low music coming from the CD player: Classical during the day, 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 to explore new angles and find more questions. It becomes more and more exciting and consuming. Not necessarily Faustian, but probably close. I need to keep from self-destructing, as with any addiction." Divorced,with a son away at college, Gilly lives alone, and it's not certain whether the lab is his primary or secondary residence.

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

The poster is meant to be ironic, yet it's placed like an altar. Gilly says that in his boyhood he was "a card-carrying Lutheran with seven years of medals for perfect attendance in Sunday school." One day when being examined by the pastor, he could not accept that the bread and the wine were literally the body and blood of Christ. "I loved the church before that moment," 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 scientist who tweaks animals with electrodes and toxins, heartless to their suffering. Indeed, he has spent the bulk of his afternoon exposing Loligo squid to domoic acid, a marine poison that is the stuff of red tides."They [the squid] will recover immediately from this," he says. "They aren't distressed. In fact, it's virtually impossible to say if an invertebrate animal feels pain."

Having served on a Stanford committee that reviewed the protocols for handling vertebrate animals,Gilly is not in favor of extending such standards to squid. "Already there 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? It turns out he's written an account of the incident. The Humboldt is about the size of a human being, he observes. No microscopes or telescopes lie between the scientist and his subject. "Why should the size of an organism affect the way we perceive its qualities?" he writes. "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 are glistening. "Don't use this to promote squid rights!" he says gruffly.

In the late 1980s Bill Gilly took the renowned four-week training course at the Cold Spring Harbor Laboratory on Long Island in New York.The Cold Spring lab is the cradle of molecular biology. It represents the triumph of reductionism, whereby living systems are broken down to their 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 got seriously interested in the molecular aspects of voltage-gated ion channels," Gilly recalls. "This was after the successful cloning of some channel genes was reported."

Voltage-gated ion channels are complex proteins in the membranes of nerve and muscle cells. They are structured to function like pores; they permit electrically charged atoms, known as ions, to pass in and out. Cells have separate channels for ions of sodium, potassium, calcium and chloride. The sodium channel, which became Gilly's target of interest, transports the positively charged sodium ions into the negatively charged interior of the squid's giant axon.

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

Human nerves basically fire by the same mechanism. If the ion channels don't function correctly because of a genetic disorder, say, a neuromuscular disease may result, such as myotonia, a stiffness of the muscles. In multiple sclerosis, disruption of the sodium channels may cause some symptoms of the disease.

Hence Gilly's thinking: Biomedical science would benefit if more were known about the genes that direct the formation and placement of the ion channels. Researchers can't dissect the nervous systems of living people, but the superthick and superlong nerve fibers of the squid make for a fine substitute. Gilly already knew how the channels perform at the gross level of the organism. Below that, at the genetic level — well, that's why he was at Cold Spring Harbor, 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 the keys to the entire machine.

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

But having learned the steps that would be needed to isolate and clone (make unlimited copies of) the squid channel genes, he brought aboard a Ph.D. student, technically more adept than he, to do the heavy lifting. In 1992, the Gilly lab won a major grant from the National Institutes of Health. It was not a blank check, but the researchers were given a lot of leeway to explore the genetic bases of sodium and potassium channels in squid.

Eight years later, the work ended in disappointment. Gilly and his students did manage to create a working model of one of the squid channels. Using frog eggs to take up the squid genes and make the channels, they were able to measure action potentials and nerve activity. They published papers on their findings in journals, but an overall understanding of the system eluded them.

"We were banging our heads against the wall," Gilly explains ruefully. "We got sidetracked into some questions that just did not give easy answers, and it was felt by the [National Institutes ofHealth] review panel — as well as myself in my heart — that it was time to move on to something new."

Funding for the project was cutoff. Gilly was out $320,000 a year. As every scientist knows, the dead ends are far more frequent than the breakthroughs, but that's no consolation when your lab's very survival depends on a grant.

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

Then a Stanford colleague offered Gilly an assignment with the Census of Marine Life, at the opposite end of the research spectrum. He'd never done formal field biology before, but, at a crossroads, he agreed to lead the squid-tagging project to Baja, which he knew from his fishing jaunts.

This experiment succeeded. The team put tags on nearly 1,000 squid and got back about 80. Gilly was pleased with not only the rate of return but also the locations of the catches, which sketches the seasonal migration of the groups across the Sea of Cortés. "Thereis a two-week gap when there were no returns," he says. "Before that they were from the Santa Rosalía area [on the west side] and afterward from 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 all interrelate: 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 the warm wind, their mission accomplished. He's a little nervous because this time the tags cost $250 apiece. Somewhere offshore there are 95 Humboldt squid carrying quarter-size, battery-powered devices, which periodically record the water temperature and depth and so will give a picture of the squid's vertical movements. Each tagged animal is worth$100 to whoever recovers it, whenever that will be.

The high point of the trip was swimming with the squid at night. With snorkel and mask Gilly tested the reputation of the "red devils." "You'd see these 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 feeding mode, they were delicate and retiring.

"As I was swimming, I had my hand stretched out. It was pretty moving. It was as close as you could 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 awareness was still there. We didn't take any more squid [as bait for other squid] than we would need. I insisted that their heads be cut off — to stop thought quick in those animals. They don't think the way we do, but still, suffocating, expiring, they do not enjoy it."

As for the tagging results, instead of an 8 percent return, this time he had just one: A single tag was turned in by a Mexican fisherman a month later. It was all he needed to show that the project was feasible."Stayed up practically all night plotting out the data," he wrote later in an email. "It is beautiful. The most regular phenomenon is a rapid rise [of the squid] right at sunset. ... Got to go now to do some electrophysiological recordings. Back to the day job, you know."

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