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Glowwith the

FlowLight-prod u c i n g plankton r e v e a l details of

"Living light" time-lapse photograph of plankton excited by a kayaker in Bioluminescent Bay in Puerto Rico.

(inset) Researcher Michael Latz swirls bioluminescent dinoflagellates into action.

S C R I P P S I N S T I T U T I O N O F O C E A N O G R A P H Y

B Y P A I G E J E N N I N G S

c e l lular dynam i c s

ANY PEOPLE have experiencedthe roll of a boat on a rough

body of water—along with a queasystomach and uneasy legs. The pitch ofthe boat and the distasteful physiologi-cal effects can be blamed on fluidmotion. Luckily, people can escape anuncomfortable boat ride by eventuallyreturning to port. But for organismsthat live in the ocean there is noescape. They exist continuously in adynamic fluid environment, which sci-entists do not yet fully understand.

Scripps marine biologist MichaelLatz and his graduate students arestudying the effects of fluid motion onsingle-celled algae known as dinofla-gellates.

In the ocean, tur-bulence is created bywind, waves, tides, and cur-rents. This turbulent motion notonly carries around plankton, includingdinoflagellates, but also directly affectstheir biology.

“We know essentially nothing aboutthe physiological effects of flow onmarine cells,” explains Latz. “But we doknow that dinoflagellates are among themost flow-sensitive cells, far more sothan mammalian, plant, or insect cells.”

Dinoflagellates commonly occurthroughout the world’s oceans and haveseveral interesting characteristics.

Certain species form red tides,which occur when populations congre-gate and reproduce so densely that theydiscolor the water red or brown. Somedinoflagellate “blooms” can degradewater quality and some produce toxinsthat are harmful to other marine organ-isms, such as seals or whales. The sametoxins also affect humans through para-lytic shellfish poisoning.

Many dinoflagellate species are bio-luminescent, emitting brightemitting bright flashes oflight at night in response to aginight in response to agitation.In larlargege accumulations, theyaccumulations, they produce“phosphorescent seas”“phosphorescent seas” in win which crestsof waves, surf , and of waves, surf , and waters aroundboats and swimming oboats and swimming organisms glowelectric blue. In San Diego, a commondinoflagellate named Lingulodiniumpolyedrum is responsible for these lightdisplays, which are most prominent duringred tides.

M

Phot

o co

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rank

Bor

ges

Llosa

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Although luminescent dino-flagellates have been studied atScripps by Latz for several yearsand by others since the 1950s,some of the cellular and environ-mental factors affecting the biolu-minescent response are still notunderstood.

STIMULATING A RESPONSE

There are theories, but no concreteexplanations as to how dinoflagel-lates sense their fluid environment,such as the turbulence caused bywind and breaking waves. Scientistsalso have not identified the internalpathways or mechanisms in theorganisms that trigger physiologicalresponses to flow, such as biolumi-nescence and changes in growth rate,nutrient uptake, and structure.

Flows capable of stimulating abioluminescent response in dinofla-

gellates must be quite strong.Beneath the ocean surface on awindy day the turbulence usual-ly isn’t strong enough to stimu-late bioluminescence, but it doesaffect the cells in other ways, caus-ing them to reproduce more slowlyand even to change shape.

Dinoflagellates and othermicroscopic plankton experiencetheir environment much differentlythan do larger animals. Because oftheir small size, dinoflagellates feelturbulence as laminar shear, a dif-ference in flow velocity across thecell diameter.

“If you are on a ship in windyconditions, you feel lots of acceler-ation,” explained Latz. “For plank-ton, acceleration isn’t as important;they are so small that they live in aviscous world dominated by shear.”

According to Latz, although

Clockwise: Graduate

student Andrew Juhl

monitors the health of

natural populations of

dinoflagellates from

Scripps Pier. Research

vessel monitoring red

tide in San Sebastian,

Spain. Red tide on

Tanabe Bay, Japan.

the velocity difference across adinoflagellate is extremely small,there is sufficient shear for them tosense and respond to.

Latz is using dinoflagellate bio-luminescence as a way of reportinghow cells are affected by flow.Agitating water containing dinofla-gellates results in flashes of lightfrom the cells. The flashes arebright and nearly instantaneous,allowing Latz to observe exactlywhere, when, and to what types offlow the dinoflagellates respond.

In the lab, Latz and graduatestudents Andrew Juhl and Peter vonDassow create carefully definedexperimental flow conditions to testdinoflagellate flow sensitivity.Simple fluid shear is created usingspecial flow chambers consisting oftwo clear, concentric cylinders. Thespace between the two cylinders-

filled with water containing dinoflagel-lates, and the outer cylinder is rotated atdifferent speeds while the inner cylinderis held stationary. This causes a lineargradient of velocity in the gap betweenthe two cylinders, resulting in a constantshear. This type of flow is calledCouette flow and isused by the scientists totest the response of dinofla-gellates to an exactly definedshear.

At one time it was thought thatdinoflagellates responded only to therapidly changing, chaotic nature of tur-bulent flow. Using Couette flow, Latzhas been able to show that smoothunchanging laminar flows can stimulatebioluminescence too.

Latz and his collaborator Jim Rohr, aphysicist at the Space and Naval WarfareSystems Center in San Diego, have con-ducted other studies combining experi-mental fluid mechanics with more com-plex flows. For example, in some studiesthey send water filled with dinoflagel-lates through a clear pipe. By controllingthe flow rate and thus the characteristicsof the flow, they test how the biolu-minescent response isaffected by the

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Above, Many marine

organisms, such as this

large species of brittle

star, are bioluminescent.

Left, Latz and graduate

student Peter von Dassow

observe the glow of

dinoflagellates surging

through a flow chamber.

ON THE WEB - See video of dolphins swimming through bioluminescent waters and brittle starslighting up for science at SIO’s homepage at www.scripps.ucsd.edu.

cont inued on page 10

shear stress (the shearing force of thefluid flow) compared to other flow char-acteristics, such as flow acceleration orchanges in the laminar or turbulentnature of the flow.

IMPLICATIONS FOR ALL CELLS

The results of these studies have power-ful implications for understanding howall cells might be affected by fluid flows.But scientists still know very little aboutthe internal pathways by which dinofla-gellates detect their fluid environment.How do flows trigger physiologicalresponses such as bioluminescence inorganisms, or changes in growth rate,nutrient uptake, and cell structure?

“Not all cells are bioluminescent,”explains von Dassow, a third-year

graduate student with Latz.“But probably the trigger

that controls thatresponse in dinofla-

gellates has gener-al similarities to

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cean pollution is a seriousenvironmental concern, espe-

cially in developed coastal areaswhere shoreline and bay sedimentsmay become reservoirs of urban andindustrial pollutants, including heavymetals. As a consequence, organisms livingin or on the sediment, or those that preyupon bottom-living organisms, face heavy-metalexposure.

Marine organisms living in direct contact with contaminatedcoastal sediments may serve as biological indicators to help sci-entists determine the locations and severity of pollution. DimitriDeheyn, a postdoctoral fellow from Belgium, is working inMichael Latz’s lab at Scripps to develop new ways of using com-mon bioluminescent brittle stars for this purpose.

“Brittle stars are cousins of starfish, and their name refers totheir tendency to release an arm when stressed,” explainsDeheyn. “They live in contact with the sediment on which theyfeed and are sensitive to environmental quality, being easily killedby exposure to high enough levels of certain heavy metals.”

Commonly, bioluminescent bacteria are used to determinesublethal effects of toxicity, but Deheyn thinks they are not accu-rate indicators of how heavy-metal toxicity will affect larger mul-ticellular organisms with nervous systems.

Deheyn explains,“Bacteria lack the complex organization oftissues and organs found in higher organisms, including humans.The human nervous system can be very sensitive to pollutants,while other tissues in our bodies are less sensitive or even helpremove pollutants. “This appears to be the case for brittle stars.

In luminescent brittle stars, light production is under thecontrol of the nervous system and originates in photocells in thefive arms. By comparing bioluminescence from individual photo-cells to that from the arms, Deheyn’s experiments at Scripps willdetermine whether the effect of heavy metals on biolumines-cence is due to nervous system toxicity or a more general phys-iological impairment.

One of Deheyn’s main field projects is taking place this sum-mer in San Diego Bay at six sites exposed to varying levels of

heavy metals. He will transplant brittle stars collected fromuncontaminated areas outside of the bay into mesh cages buriedin the sediment at each site.The locations range from the rela-tively clean waters at the mouth of the bay, to the back of thebay, where the sediments support high levels of contaminationdue to heavy industry and a slow rate of water recirculation.

During the course of one month,Deheyn will remove samples from eachsite for evaluation in the lab. He willdetermine the types and quantities ofmetals in the tissues and will measurethe light production of animals fromeach site. He will then compare the

physiological toxicity to the amount ofheavy metals in the tissue as measured

by mass spectrometry.“If light production decreases, pollution

might limit the fitness of any individual brittlestar.The bay brittle star can be considered a model

for other organisms in which such a causal link between anthro-pogenic pollutants and individual fitness may not be so obvious,”says Deheyn.

Deheyn’s field work is being conducted in collaborationwith the Marine Environmental Quality Branch of the Spaceand Naval Warfare Systems Center in San Diego. He alsoreceives research support from NATO, the Belgian-AmericanEducational Foundation, and the UC Toxic Substances Researchand Training Program.

O

Top left and middle, In the backwater of San Diego Bay, Dimitri

Deheyn prepares to dive to one of his research sites. Above, Collecting

during the dive. Next page, With a keen eye and gentle grasp, Deheyn

searches for tiny brittle stars living within a locally collected kelp holdfast.

Indicators of Pollution

S T U D I E S I N S A N D I E G O B AY

“My goal,”

explains Deheyn,

“is to use the

light production

as a physiologi-

cal indicator of

pollution, with

the understand-

ing that the

amount of light

produced will

vary with the

amount of heavy

metals that an

animal encoun-

ters and absorbs

into its tis-

sues.”

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Left, Deheyn's

research requires a

large, healthy popula-

tion of brittle stars.

In Scripps's experimen-

tal aquarium he enlists

the help of Latz and

UCSD undergraduate

Laura Brams to tend the

specimens maintained

for his experiments.

Facing page, UCSD

undergraduate Lisa

Schile handles sterile

cultures of luminescent

dinoflagellates.

Science Foundation, von Dassow,Latz, and John Frangos in theUCSD Department of Bioengi-neering are studying how the fluidforces acting on the cell are trans-lated into a biochemical signal thattells the cell to produce light. VonDassow is conducting tests todetermine if shear causes calciumions from seawater to enter thedinoflagellate cell, triggering biolu-minescence. Shear is known toresult in calcium entry into mam-malian endothelial cells (cells thatform the lining of blood vessels). Ifhis hypothesis is correct, it would

indicate that shear affects biolumi-nescent dinoflagellates—which,unlike endothelial cells, are notattached to anything and must movewith the fluid—in a similar way. Inthe future, Latz plans to testwhether other biochemical eventsin the cells are triggered by fluidmotion.

The relatively large shearforces that stimulate dinoflagellatebioluminescence are higher thantypical levels of oceanic turbu-lence. But dinoflagellates are alsoaffected by lower levels of fluidmotion, such as those present near

those controlling other responses inother cells. A big lesson of the lastfew decades of cell biology is thatall cells use the same basic buildingblocks for a large number ofprocesses. We just need to figureout which fundamental elementsare being used by the biolumines-cent cells, in order to know how ourstudies of dinoflagellate biolumi-nescence might be relevant toother cells in flowing fluid.”

T R A C I N G B I O C H E M I C A L

PAT H WAYS

In a project funded by the National

S C R I P P S I N S T I T U T I O N O F O C E A N O G R A P H Y

the surface on a windy day.“Because dinoflagellates swim to

surface layers during the day,” explainsLatz, “they are exposed to stronger lev-els of turbulence than exist in deeperlayers.”

Generally, dinoflagellate red tidesoccur during calm conditions. In con-trast, other planktonic algae, such asdiatoms, thrive in more turbulent condi-tions, which stir up nutrients from deep-er layers. Juhl, who is just completing hisdissertation, has been studying whetherthe dinoflagellates’ preference for calmconditions results from their extraordi-nary flow sensitivity. It is possible thatred tides don’t occur during turbulent con-ditions because the turbulence preventsdinoflagellate populations from growing.

“The idea that flow affects cellphysiology is well developed in otherfields of biology, but it is a novel idea foroceanography,” says Juhl. “Typicallypeople only think of oceanic flow interms of its ability to move things fromhere to there, not in terms of what it isactually doing directly to the cell. I’mlooking at whether the growth ofdinoflagellates is sensitive to oceaniclevels of flow.”

I N T H E L A B O R ATO R Y

Juhl has designed special Couette flowchambers that allow him to grow dinofla-gellates in the gap between the twocylinders. He studies and records thisgrowth by periodically removing a sam-ple of water and counting the cells itcontains.

He exposes dinoflagellates to verylow levels of shear for an hour or moreeach day. Other Couette flow chambersremain stationary to serve as experimen-tal controls. Juhl finds that the populationgrowth rate, the rate at which the dinofla-gellates are increasing in number, is muchreduced in the sheared chambers

“Because dinoflagellates

swim to surface layers duryers dur--

ing the day they aree

exposedexposed to stronger levels of

turbulence than exist in

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lates to shear, their toxin levels goway up. So there might be fewercells because of their decreasedgrowth rate, but they might be moretoxic.” This may be importantbecause cell counts of toxic speciesmight not represent the potentialtoxicity of the seawater and shell-fish during turbulent conditions.

E X P LO R I N G P R AC T I C A L

AP P L ICATIO N S

In addition to using dinoflagellatesas models for understanding howcells are affected by flows, Latz also

At night, when they can’t beseen by predatory fish, many

zooplankton swim to the upperlayers of the ocean to feed onalgae, including dinoflagellates.

However, when a zooplanktonattacks bioluminescent dinofla-gellates, the dinoflagellates arestimulated to flash, making the

zooplankton vulnerable to beingeaten by a nearby fish alerted by

the light. According to MichaelLatz, the bioluminescence acts asan alarm when the dinoflagellateis being attacked by a zooplank-

ton and results in fewer dinoflagellates being eaten.So, if dinoflagellates use bio-

luminescence for protection,why is the light response also

triggered by waves or the flowaround swimming organisms?

Just as a car alarm can be inadvertently triggered by an

accidental bump or a heavy rainstorm, dinoflagellate biolumines-

cence is stimulated by flow conditions that have sufficient

force to set off the “alarm.”

has been exploring practical appli-cations for dinoflagellate biolumi-nescence. Fluid physicists cannotyet measure flows directly at thevery small scales of individualplankton. Latz’s studies of the rela-tionship between fluid shear andbioluminescence suggest thatdinoflagellates can be used asmicroscopic flow sensors to studythe complexities of fluid flow.

In a dramatic example of thispossibility, Latz and Rohr observedbioluminescence generated arounddolphins as they swam through

compared with thosethat remain still.Juhl has studied the local

red tide dinoflagellate speciesLingulodinium polyedrum extensivelyand is now working with Alexandriumfundyense, a toxic dinoflagellate thatcauses paralytic shellfish poisoningin other areas of the world, includ-ing the East Coast and the PacificNorthwest.

“This species causes huge eco-nomic losses to fisheries and canmake people sick,” Juhl explains.“When you expose the dinoflagel-

bioreactor and observe the areas that light up.Those are areas of high shear.”

This same technique might also be used todevelop artificial hearts that are safer and moreeffective. The shear caused by blood pumpingthrough an artificial heart must not be too highor blood cells will be damaged, but the shearmust also not be too low or clots might devel-op. Latz believes it might be possible to usebioluminescent dinoflagellates to determinewhether the shear is too high, too low, orjust right.

As work in the lab continues, Latzplans to pursue new collaborations with-

in marine biology and inother fields where his

innovative techniquescan help physicists

and engineers learnmore about intri-cacies of fluidflow.

Above, Deheyn removes the

arms of a large brittle star for

use in his experiments. Brittle

stars can detach their arms

for self-defense and then

regenerate them. Middle,

Michael Latz cultures dinofla-

gellates in a lab incubator.

water containing dinoflagellates.By comparing their results to math-ematical solutions and to flows cre-ated around objects in the laborato-ry, they identified different regionsof flow around the dolphins’streamlined bodies.

The success with swimmingdolphins demonstrates the poten-tial usefulness of the approach.According to Latz, “We are nowinterested in applying this knowl-edge to other flow conditions, suchas in bioreactors, the laboratorychambers used to grow culturedcells from which important com-pounds can be harvested for medi-cines or biomedical research.”

Cells grown in this manner

include plant,yeast, insect, and evenmammalian cells. These cells can-not swim, so when grown in biore-actors, they must be supplied withnutrients and dissolved gasesthrough constant mixing. Thisrequirement can cause moreharm than good—mixing toovigorously will damage or killthe cells.

“The ideal bioreactor mixeswell but with low shear. Usingbioluminescence is a way to ver-ify the claims of a bioreactormanufacturer that their productis more gentle than the compe-tition,” Latz explains. “You canput the dinoflagellates in a

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S C R I P P S I N S T I T U T I O N O F O C E A N O G R A P H Y

Visit the Latz laboratory website at http://siobiolum.ucsd.edu