Fall!

Winter 1994 Vol. 26, No.2 & 3

3Introduction.

Marine Biotechnology, the Possibilities 6

8DNA Analysis, A Powerful Molecular Tool.

Genetic A9alyses of Billfishes 11

Release That Fish-But Tag It First! 13

On the Trail of MSX 15

TheFray 18

Journeying Toward the Future 19

End Notes. 22

arine biotech-nology is anuncertain but

of exploration. Oceanic lifefonDS can be pronouncedly differ-ent than terrestrial ones. It is thisdifference that interests research-ers. The variety of organisms,and their chemical uniquenessmay be a storehouse of infonna-tion for the future.

The extent of marine biotechresearch can only be hinted uponin the following pages, but itranges from using the neurologi-cal systems of cuttlefish and octo-pus as a research basis for fifth-

generation computer develop-ment, to finding a way to convertan alga-which only uses sun-light as an energy source-to theproduction of unlimited amountsof hydrogen for a source of

power.*The intent of this issue of the

Bulletin is to provide readerswith a sampler of marine biotechpossibilities, and to highlight afew Virginia projects.

surprising in its results: a product

from the marine environment is

used as it naturally occurs, or is

changed into another form for

human use. Examples: the dis-

covery of oil-degrading microor-

ganisms in the marine world has

led some researchers to believethat a new means has been foundfor environmental remediation.Crab shells-an environmentalheadache for processors--can bealtered into a number of forms,ranging from beads which absorb

*Specifically, a Japanese research group is seeking to transform a spe-

cies of thermophilic (an organism that thrives at high temperatures) alga,

Synechococcus elongatus, by introducing the genes from the bacterial spe-

cies Clostridium that code for enzymes producing hydrogen gas.

Marine Biotechnology

...What Exactly Is it?The definition of marine

biotechnology appears to be asgeneral or specific as an individ-ual author's personal bias. Evenso, one way of explaining whatmarine biotechnology can encom-pass is to divide it into threebroad and oftentimes overlap-

ping categories.The first category is some-

what obvious, though at times

4

low value. The supply of shellsis seasonal and uncertain. Aplant to reprocess shells for me-dicinal or other purposes is toofar removed from local blue crab

processors. As it currentlystands, it is more feasible to proc-ess crab shells into meal or for itto be taken to a landfill. This,however, could change. + +

M arine biotechnology is notnecessarily new. Innova-

tive people have always lookedto the resources around them fornew products. A particularly in-ventive soul thought to weavebyssal threads from a bivalveinto what has been called cloth-of-gold, an exceptionally sheerfabric with a metallic sheen. Abyssal thread is a fine thread thata bivalve uses to attach itself to astationary object. The source ofthe byssal threads for cloth-of-gold was the pen shell Pinna no-hilis, a Mediterranean bivalvewhich can grow to a length oftwo feet and has a tuft of byssal

heavy metals, to artificial skinfor bum victims.

The second division is anatural outcome of the last. In-stead of using a specific marineproduct, scientists enlist the infor-mation they have obtained fromthe natural world and utilize itfor a human application. To de-velop drugs which treat inflam-matory diseases such as arthritis,California Sea Grant researchershave been examining marine or-ganisms, searching for a modern-

day elixir-pain inhibitingcompounds which could be thebasis for synthetic versions.

The third category entails us-ing biology as a tool toward anobjective, one which can rangefrom genetic manipulation tostock identification. Growth en-hancement of fish has been an ac-tive field, with scientists workingboth with growth hormones andwith transgenics, the transferal ofgenetic traits from one species toanother. Another example of bi-ology as a tool would be theDNA "fingerprinting" of a stockto determine if the species repre-sents one, two or more genetic

groups.Even though marine biotech-

nology is a promising frontier, itis an uncertain one. The degreeto which any area is developed issubject to the availability of fund-ing and the practicality of appli-cations. Case in point: crabprocessors in Virginia will prob-ably continue to process crabshells into meal for livestock.The crab shells are now consid-ered waste, with an inherently

threads, gold-bronze in color. Atuft of threads (as opposed to asingular thread for each bivalve)made collection easier. Even so,the amount of threads needed forthree ounces of fabric is size-able-in the neighborhood of a

pound.Cloth-of-gold is believed by

some historians to be the GoldenFleece of Greek mythology.Even if that were not the case, ithas been used for the raiment ofroyalty right up to fairly modemtimes. Even Queen Victoria wassaid to have worn this fabricfrom the sea.

5

Molecular Genetics

.Certain Antarctic fish pro-duce a natural antifreeze,called glycoproteins, whichinhibit ice crystal growth intheir tissues; use of molecu-lar techniques may permittheir large-scale productionfor research and practical ap-plication. It may be possibleto transfer the genes codingfor this natural antifreeze toother species in order to im-prove their growth and sur-vival in cold environments.

.DNA-fingerprinting tech-niques demonstrated that tra-ditional stock assessments ofAtlantic salmon were errone-ous, resulting in a large shareof valuable United Statesstocks being given over toEuropean fishing interests.

stricting their growth. Bryo-zoans and tunicates have alsoyielded novel compoundswith highly specific antitu-mor activity; some are nowundergoing clinical trials.

.Halenquinone, isolated froma sponge, is a powerful newantibiotic, while didemnin,from a tunicate, exhibits an-tiviral and anticancer activity.

SeaGrant College Pro-gram sponsors re-search in support of

marine biotechnology. The SeaGrant role is expected to increaseand may be given a substantialboost through an act which ispending congressional considera-tion, the Marine BiotechnologyInvestment Act.

With adequate funding, re-search could potentially opennew avenues for monitoringhealth and treating disease; pro-vide innovative techniques torestore and protect aquatic eco-systems; increase the food supplythrough aquaculture; enhance sea-food quality and safety; developnew types and sources of indus-trial materials and processes; andexpand knowledge of biologicaland geochemical processes in theworld ocean.

In the following list are afew examples of recent marinebiotech developments. The cate-gories are general groupingswhich the National Sea GrantCollege Program has identifiedas areas worth pursuing. The pro-jects cited are not necessarily theproduct of Sea Grant research,but illustrate areas of interest.

Immunobiology and

Pathology.Gene probes have been de-

veloped for several viral dis-

eases of shrimp. These have

made possible the estab-

lishment of disease-free

brood stock for United States

shrimp hatcheries and will

help prevent the loss of

stocks.

Bio-Organic Chemistryand Pharmacology

.Manoalide, an anti-inflamma-

tory and analgesic agent iso-

lated from a Pacific sponge,

is now in clinical trials. Its

action differs from that of

standard drugs and it appears

free of the side-effects of

steroids..A substance isolated from

shark cartilage inhibits blood

supply to tumors, thus re-

6

Endocrinology and

Developmental and

Reproductive Biology.Cloning of growth honnone

and growth promoting factorgenes of some commerciallyimportant fish has led to pro-duction of rapidly-growingstocks. Injections of growthhonnone ~iosynthesized bybacteria containing this geneincrease the growth rate oftrout, while transgenic carpand catfish containing copiesof this gene grow up to 50%faster than controls.

.Identification of factors con-trolling spawning and settle-ment of abalone and oystershas allowed synchronizedspawning in captivity, lead-ing to the development ofcommercial hatcheries forthese valuable shellfish.

important fornls such as phy-toplankton and zooplankton.This work is currently ex-tremely labor intensive, andcan be a major bottleneck inoceanographic research.

.Investigation of harmful ef-fects of the ozone "hole" inthe Antarctic are being car-ried out by examination ofDNA of marine organismsexposed to increased UVradiation. UV -tolerate fornlSare also being examined forpotential natural sunscreens.

Jellyfish

Aquaculture.Development of a triploid

oyster that makes possibleyear-round rather than sea-sonal oyster production hascontributed greatly to the re-vival of the West Coast oys-ter industry.

.Development of vaccinesagainst two major diseasesof salmon, IPN and IHN vi-

.rus sets the stage for com-mercial development ofimproved vaccines to in-crease survival in culturedtrout and salmon.

Environmental and

Evolutionary Biology.Use of gene probes to rap-

idly identify and enumeratemarine organisms, particu-larly small but ecologically

Environmenltal

Remediation

.Oceanic bacteriahave been dis-covered that di-rectly oxidizeand precipitlte

iron, mangalleSe,cobalt, nickf~land other valu-

.~

.'0' ~"- l,,' .'"'0

-'.':::;:...':::~;~)

-

Sponge .-J

;;7"

7

able and strategic metals.The genes and enzymes ofthese bacteria may be thekey to separating these met-als from low"grade ores, by-passing more expensive and

environmentally -damagingindustrial processes now

employed..A test plant employing natu-

rally occurring bacteriawhich degrade phenols hasdemonstrated a 99% drop inthe concentration of chlorin-ated phenols, from 100 to 1part per million in a bioreac-tor system. These procedureswere tested successfully bythe Environmental Protec-tion Agency at a Superfundsite, and two fIrms have con-tracted to use these proce-dures in commercial

he article in theApril 1953 issue ofNature was little

more than a page long, inbetween a confluence of sci-entific, anthropologic andagricultural studies. Yet, the"Molecular Structure ofNucleic Acids," in which

standing how the DNA mole-cule encodes and transmitsgenetic information.

Scientists Watson andCrick no doubt realized theimportance of what theywere describing, but theymay not have anticipatedhow widespread and even

NUCLEUS

DNA"""

5'

DNA is made up of two strands encoded in a four letter alphabet, the nucleotidesthymine (T), adenine (A), cytosine (C), and guanine (G). A's are always opposite T'sand G's are opposite C's. Illustration courtesy of Scientific American Inc., Georgev: Kelvin@.

authors James D. Watson pragmatic the eventualand Francis Crick postulated applications of DNA analy-the structure of deoxyribonu- sis would be. DNA analysiscleic acid (DNA) was one of as a molecular tool is beingthe most influential scientific utilized in many scientificpapers of the century; the disciplines-includingdouble helix structure was a marine science.substantial clue to under-

DNA is the blueprint forlife. Both in animals andplants, the majority of DNAis found in the chromosomesof a cell's nucleus. The in-formation is encoded inDNA's four letter alphabet,its nucleotides- thymine (t),adenine (a), cytosine (c), and

guanine(g). A

specificsequenceof these

"letters,"is calleda gene,and haspart ofthe infor-mationneces-sary forcon-

structingcompo-nents of

I :m organ-

Ism.

~

~

~

Although 99.9% of DNA isfound in an animal cell's nu-cleus, the remaining is lo-cated in the mitochondrion, aspecialized subcellular struc-ture, the "powerhouse" ofthe cell. The number of nu-cleotides in this location isfar less daunting; in a fishthe mitochondrial DNA com-

prises approximately 16,000-20,000 nucleotides, asopposed to information inthe nucleus which cannumber a staggering threebillion units.

Another factor which ad-vanced DNA-aided researchwas the discovery of restric-tion endonucleases, enzymeswhich cut strands of DNA atspecific points- molecularscissors of a sort. With theaid of these enzyme tools,scientists could more easilyfocus on a specific DNA se-quence. Researchers coulduse the restriction endonu-cleases for genetic engineer-ing (see article on page 18),or to develop a "probe," amolecular tool used to iden-tify a genetic sequence in theDNA chain.

At VIMS, a number ofDNA projects are being con-ducted, two of which are fea-tured on the following pages.

+ + +

~ Tosay that

the information within achromosome's DNA is mas-sive, is only to hint at themagnitude of information;the DNA chain can containmillions or billions of nucleo-tides.

For some DNA research,it proved easier to work withmitochondrial DNA.

Molecular MappingThe oceanic world can pose seemingly intractable challenges for fishery managers and

scientists. Prior to DNA testing, it was difficult to know if a worldwide species like blue marlinwas made up of one stock or more. Traditional tools like tagg,ing could ascertain movements,but they could not determine if groups were genetically homogeneous or if they were distinctfrom one another.

The management ramifications of DNAtesting may not be immediately obvious tothose beyond the realm of marine science.A number offish stocks were assumedto be worldwide or specific to, say,an ocean basin. Historically,the stocks were man-aged as if theywere large

units,

1"""'#r~/

~

10

evolving piece of genetic mate-rial known as mitochondrialDNA (mtDNA). Researchers pu-rify mtDNA from heart tissueand analyze the molecule with re-striction endonucleases-en-zymes that recognize specificDNA sequences and break themolecule at that site, generatingfragments. Differences in theDNA sequence between individu-als can result in different frag-ment patterns. By employing adozen or so enzymes to analyzethe DNA, researchers produce acomposite genotype or "geneticfingerprint" for each individual.If the distribution of genetic fin-gerprints is different among fishfrom different areas, then onecan infer that very little mixing isoccurring. On the other hand, ifthe distributions are similar, onecan assume that there is at leastsufficient exchange (on the orderof 10 individuals per generation)to prevent the accumulation ofgenetic differences.

ByJohn Graves and Jan McDowell

Virginia Institute of Marine Science

Why Genetic Studies?

and conservation ofthe billfishes will re-

derstanding of each species'

population genetic (stock) struc-

ture. Without such information it

is not possible to infer the ranges

and composition of management

units (do all Pacific striped mar-

lin comprise a single stock or are

several genetically distinct stocks

present?), or to determine if

unique genetic variation is re-

stricted to specific regions (will a

fishery collapse in one area re-

move genetic variation from the

species?). Unfortunately, little is

known of the population genetics

of billfishes, even though several

recent scientific and management

panels have identified the deline-

ation of billfish stock structure as

a major management need. To ad-

dress this issue, researchers at the

Virginia Institute of Marine Sci-

ence (VIMS) have been applying

new advances in biotechnology

to investigate the population ge-

netic structure of several species

of billfish.

What Has Been Learned?For most species of tuna,

little genetic differences exist be-tween samples from within anocean or even between oceans.For example, yellowfin tunafrom five samples across the Pa-cific Ocean and one from the At-lantic Ocean had virtually thesame distribution of mtDNA fin-gerprints. Each sample containedseveral different types of mtDNAfingerprints, but they occurred in

Biotechnology and

BillfishesTo investigate the popula-

tion genetics of billfishes, scien-tists study a small, rapidly

similar frequencies across allsamples. These results suggestthat there is some movement(and subsequent mating) of yel-lowfin tuna within and betweenoceans. Genetically they repre-sent a single stock.

It was generally assumedthat billfishes, like tunas, wouldalso show little genetic diver-gence among fish from distantareas. Surprisingly, this is not thecase. Striped marlin from foursites within the Pacific Oceanwere genetically distinct. The dis-tribution of mtDNA fingerprintsof each sample was unique, indi-cating that there is very limitedexchange between areas. Theseresults indicate that PacificOcean striped marlin do not com-prise a single genetic stock andthat significant within-oceanpopulation structure exists. Toconserve the unique genetic vari-ation, current management mod-els need to be revised to accountfor the population structure.

Blue marlin, like striped mar-lin, show stock structure withinocean basins as well as betweenoceans. VIMS' results indicatethat the Atlantic and Pacific bluemarlin are the same species. Sev-eral Atlantic blue marlin have thesame or similar genetic finger-prints as all Pacific blue marlin,representing a family of DNA fin-gerprints scientists call "ubiqui-tous." However, about 45% ofAtlantic blue marlin havemtDNA fingerprints not found in

Pacific fish (see Figure 1). Thisdifference has provided a meansto positively identify Atlanticblue marlin, and consequently toenforce the U.S. Fishery Manage-ment Plan for Billfishes whichallows the sale of Pacific bluemarlin but not Atlantic blue mar-lin.

comprehensive, globalanalysis of sailfish popu-lation structure.

Added BenefitsScientists at VIMS have

now analyzed more than

750 billfish. While this

data set has allowed re-

searchers to determine

the stock structure of sev-

eral billfish species, it

also provides an ex-

tremely useful resource

for identifying unknown

specimens. Last fall

VIMS researchers deter-

mined that a 1,635 lb.

marlin, tentatively identi-fied as a black marlin,

was actually a blue mar-

lin.

Figure 1. Blue Marlin Population Structure. Aunique family of DNA fingerprints occurs in al-most one-half of blue marlin from the Atlantic

Closer to home, re- Ocean, while the other half of the Atlantic fishsearchers also identify have genetic fingerprints similar to their Pa-marlin tissues from sea- cific relatives.food markets. Last yearVIMS researchers were asked toanalyze fish marketed in Virginiaas "striped marlin from Ecua-dor." Fortunately, VIMS just hap-pened to have a reference sampleof 42 striped marlin from Ecua-dor. While researchers couldhave identified the geographic lo-cation of the striped marlin sam-ples, they dido'{ need to. The fishfillets were Atlantic sailfish, aproduct that cannot legally besold in the U.S. ..Busted. ..

Within the Atlantic Ocean,blue marlin appear to have popu-lation structure. Several blue mar-lin collected in Jamaica during1991 and 1992 had a genetic fin-gerprint not found outside theCaribbean. Although prelimi-nary, these results suggest lim-ited mixing of Caribbean fish.

In addition to blue marlinand striped marlin, the VIMSlaboratory is studying white mar-lin and sailfish. Samples of whitemarlin from the u.S. Atlanticcoast in 1992 and 1993 are quitesimilar to one another, as aresamples of white marlin fromBrazil in 1992 and 1993. How-ever, some differences are appar-ent between U.S. and Brazilsamples. To determine the signifi-cance of these differences moreCaribbean samples will need tobe examined.

Sailfish show the most popu-lation structuring of any billfish,but like the blue marlin, they rep-resent a single, circumtropicalspecies. About 15% of Atlanticsailfish have mtDNA fingerprintsidentical to those found in Pa-cific fish, but the remaining 85%are unique. To find out what hap-pens throughout the rest of theworld's oceans VIMS research-ers are collecting samples for a

management and conservation.VIMS researchers typically com-municate their results throughpeer-reviewed articles in interna-tional fishery journals. Whilethey have published several pa-pers on billfish population struc-ture, management agencies arenot always current with the scien-tific literature. So, VIMS re-searchers take their results tothem.

In addition, VIMS scientistspresented their results at a spe-cial International Commissionfor the Conservation of AtlanticTunas (ICCAT) billfish work-shop in 1992. + +

Management and

ConservationA major goal of VIMS re-

search is to produce good science

that will result in better fishery

12

eas. In contrast to the historical

perspective provided by geneticanalysis, tagging studies providea "snapshot" of the movements afish makes during part of its life.Tagging data quickly and effec-tively demonstrate interactionsbetween fish from different geo-graphic areas, as well as fishtaken by differenttypes of fisheries(for example, fishtaken by recrea-tional and com-mercial fisheries).Tagging data alsoprovide critical in-formation on

studies go hand-in-hand. Neither analy-sis alone provides

infonnation on both short-tenDand long-tenD movements offish, data that-is essential for ef-fective management. Geneticstudies give an evolutionaryoverview of the movement offishes between areas (migrationand spawning). They are effec-tive in delineating stock structureand revealing the spatial distribu-tion of genetic variation. Why ge-netic studies fail to do is provideshort-tenD infonnation on the in-teraction of fish from different ar-

growth rates and longevity ofbillfishes.

So while geneticists greatlyappreciate the tissue sample af-forded by boated fish, importantscientific information can begained from those that aretagged and released.

Daysfree

Area

returned

Season

returnedArea and

SeaSOl11 tagged

Caribbean Sea

Summer

Summer

Summer

Fall

Fall

Fall

Fall

Winter

1,453427

187

153

55

636

426

2,906

Caribbean

Bahamas

W. Africa

W. Africa

Mid-Atlantic

Caribbean

Caribbean

Caribbean

Summer

Fall

Winter

Winter

Fall

Summer

Fall

Winter

.'

~ Gulf of Mexico

Summer

Sumrner

Fall

Winter

Gulf of Mexico

Bahamas

Bahamas

Gulf of Mexico

492

208

134

63

Fall

Winter

Winter

Spring~.' .c ~ US~ IslBnds

Bahamas

Summer

Summer

Summer

Winter

315

357

130

911

Caribbean

Bahamas

Bahamas

Gulf of Mexico

SpringSummer

Winter

Summer

.~o~

U.S. East CoastSummer

Summer

Fall

Summer

Fall

Summer

1,042412

263

Bahamas

Caribbean

U.S. East Coast

Summary of Atlantic blue marlin tag/recapture data by geo-graphic location and season (calendar), 1954-88. (From"Blue Marlin, Makaira nigricans, Movements in the WesternNorth Atlantic: Results of a Cooperative Game Fish Tag-ging Program, 1954-88," by W. N. Witzell and E. L Scott.)

Although tag returns indicate that blue marlin are capableof trans-Atlantic and even transoceanic movement, mostanimals are recaptured close to the site of tagging even af-ter long periods at liberty.

13

rdinarily, a spe- of Marine Science (VIMS) and

Rutgers University have reacheda milestone in MSX work, onewhich could facilitate elucidatingMSX's life cycle. The researchersdeveloped a DNA probe-a se-quence of DNA material specificto MSX-and a primer pair-aDNA tool to detect even minuteamounts of the disease. The re-searchers who made these impor-tant findings are Nancy Stokesand Gene Burreson at VIMS, andDunne Fong and Susan Ford at

Rutgers.

same gene from a parasite relatedto MSX. By comparing the two,VIMS researchers were able tolocate two regions of the MSXDNA sequence which wereunique to MSX, and not charac-teristic of related forms of theparasite. One of these regionswas used as a DNA probe andtested against Dermo, and againstother parasites similar to MSX.Results indicated that the probewas, in fact, very sensitive andvery specific for MSX.

How the probe operates maybe routine for DNA researchers,but may constitute modem-dayalchemy to outsiders. Very sim-ply, a very thin slice of tissue isplaced on a slide, and treatedwith an enzyme to poke minus-cule holes in the tissue (to allowthe probe entry into the cells).The tissue is heated, causing the

(Continued on page 17)

DNA ProbeAt Rutgers' Haskin Shellfish

Research Laboratory, scientistssequenced a gene of MSX, andpinpointed a portion of that se-quence which may be specific tothe pathogen. VIMS researchersindependently confmned this se-quence, and then sequenced the

bay, with only in-dividuals-not a large group-falling prey to a pathogen. Forany number of reasons, occasion-ally a disease garners an advan-tage. These reasons can rangefrom favorable environmentalconditions, to what must be seren-dipity for a pathogen: introduc-tion into a new system withplentiful hosts that have weak-ened or undeveloped defenses.The latter may be part of theplight of Crassostrea virginica,the Virginia oyster, in the

Chesapeake Bay.The collapse of the Chesa-

peake Bay oyster fishery is cur-rently believed to be the com-bined result of overfishing, envi-ronmental stresses and two per-sistent diseases, Haplosporidiumnelsoni (MSX) and Perkinsus ma-rinus (Dermo), pathogens whichharm oysters- not humans.Even if overfishing and environ-mental stresses could be kept incontrol, MSX has been especiallytroublesome since its life cycle isunknown. Containing a disease,or at least minimizing its impact,is improbable if the life cycle ofthe pathogen and its mecha-nism(s) for transmission are notunderstood. On a human level, itwould be like trying to controlmalaria without knowing it istransmitted by mosquitoes.

In a collaborative effort, sci-entists from the Virginia Institute

MSX.

Opposite page: Researchers Nancy Stokes

and Gene Burreson.15

Basics of the MSX probe.

From top to bottom: .

1) The DNA double helix separates

and the probe attaches to the

complementary section of DNA.

Any unbound probe is washed away.

2) An antibody with an enzyme binds

to the label attached to the end of the

probe.

3) Free substrates are processed by

the enzyme, resulting in precipitated

substrates. The color change, caused

when the enzyme processed the sub-

strates, indicates to researchers that

MSX is present.

16

DNA double helix to separate(see illustration on adjacentpage). The probe is added, and ifMSX DNA is present in a tissue,the probe will attach to the com-plementary section of DNA inthe host tissue. Any unattachedprobe is washed away, and an an-tibody with an enzyme is added.If any probe has attached, the an-tibody will bind to it. Then a sub-strate (a sub~tance with which anenzyme reacts) is added. The sub-strate will change color as it isprocessed by the enzyme. Detec-tion of the colored product meansMSX is present.

DNA to be copied. The two re-gions of the MSX DNA that theVIMS researchers found to beunique for the parasite work asvery specific and sensitive prim-ers for PCR.

Very basically, this is whatthe PCR process entails: cells arebroken open and the DNA is iso-lated. Some of this DNA isplaced in a small tube containingthe primers, an enzyme (DNA po-lymerase) and nucleotides.When heated, the DNA doublehelix will separate, exposing thenucleotides (T ,A,C,G), the basesencoded with genetic informa-tion. The temperature is then de-creased and the primers attach tothe complementary sequence ofthe DNA. DNA polymerasecauses the "free" nucleotides-ones that are not part of the twostrands-to attach to the twostrands in predictable DNA fash-ion-A's opposite T's, and G'sopposite C's. Two new, com-plete strands have been formed,each containing the informationin the original DNA strand. Theprocess of separation, primer at-tachment, and making new DNAis repeated 30 to 40 times, a pro-cedure which takes about fourhours. If no MSX DNA is pre-sent, the primers will not attachand no copies will be made. Ifonly one copy of the MSX geneis present, billions of copies canbe made in four hours.

probe and PCR primers. Re-searchers are keenly interested indetermining if MSX utilizes anintermediate host before it infectsoysters. This scenario seemsplausible since, to date, scientistshave not been able to infect oys-ters by exposing them to MSX inthe lab. In contrast, Dermo isreadily transmitted from oyster tooyster. Another reason that scien-tists suspect an intermediate hostis that despite the current, lownumber of oysters in the lowerChesapeake Bay, MSX is still

pervasive.Currently, researchers do not

know the structure and form ofMSX at every point in its life his-tory. An organism mayor maynot appear the same in its differ-ent life stages, or for that matter,in different hosts. This lack ofknowledge has an obvious disad-vantage; how would a scientistrecognize MSX in a differentform? The MSX probe and prim-ers nicely solve this human prob-lem, since on a molecular levelthe two DNA tools always recog-nize MSX, with a rapidity andcertitude of which Homo sapiensis incapable-at least not withoutthese subcellular components.No matter what the form of theorganism, its DNA is always the

same.

Polymerase Chain

Reaction PrimersThe polymerase chain reac-

tion (PCR) is little more than adecade old, but has been responsi-ble for monumental advances inDNA work. PCR can be utilizedfor many applications, including,in this case, the detection of m-inute amounts of MSX.

The DNA double helix islong and extremely complex.Plus, every cell contains all thegenetic information to constructthe entire organism. Extracting aspecific genetic sequence from aDNA strand would be like locat-ing a single volume in a large butuncatalogued library. Instead ofthe nearly impossible task of sort-ing through massive amounts ofDNA, a scientist could have ac-cess to an unlimited number of asequence or a gene through PCR.To use PCR a scientist needs twoprimers, short segments of DNAwhich flank the portion of the

Early portions of this researchwere funded by the Virginia SeaGrant Consortium and the NewJersey Sea Grant program. Re-cently, the research has beenfunded by the National Oceanicand Atmospheric Administration.

Interim HostsDetennining if an oyster is

infected with MSX is not themain intent behind the MSX

17

while the furor began to dimin-ish. Established were guidelinesfor working with recombinant

DNA, record-keeping mecha-nisms, and laws governing the re-lease of genetically engineeredorganisms. Risk assessment ex-periments indicated that the prob-ability of wreaking havoc withrecombinant DNA were slightfor a number of reasons. At thispoint, the laws governing theseareas will obviously evolve, asthey do in any realm.

There is some irony in the re-combinant DNA debate. Whilethis new type of genetic manipu-lation was creating a tempest,few people seemed cognizant ofa hazard that has been the unin-tended result of traditional ge-netic manipulation. Manyimportant world food crops aremonocultures, meaning that onestrain is cultivated instead ofmany varieties. In a worst casescenario, a disease could deci-mate an important crop like rice.Some researchers believe that the

human and economic cost couldbe devastating.*

Beyond the furor about re-combinant DNA are more subtlequestions about biotechnology.Patents on substances from lessdeveloped countries might enrichthe country that has the patentand create an inequity for the lessdeveloped country. For example,if an important drug were foundin the rainforest-a substance de-veloped by the indigenous peo-ple- and if another countryre-engineered and then patentedthe substance, which groupshould profit and to what extent?Some researchers would questionthe ethics of any patent (or Intel-lectual Property Rights) of thissort.

Space limitations preclude alengthy discussion about all as-pects of the debate about biotech-nology. As with any revolution,and it is fair to place biotechnol-ogy in that ca1[egory, it will taketime before both the benefits anddrawbacks art: obvious and fullyunderstood.';" .;..

iotechnology whichemploys genetic ma-nipulation is a power-ful tool. And, as the

case with many potent tools, ithas both positive and negative ca-pacities. When scientists beganunraveling the mechanisms ofDNA, they discovered that theDNA strand can be severed andnew material inserted, creating al-tered or new genetic material.The possibilities of recombinantDNA seemed endless. For some,the potential for creating havocwas beyond imagination; forthem, it was the biologicalequivalent of opening Pandora'sbox. For others, the commotionabout recombinant DNA was un-

founded; genetic manipulationemploying classical meth-ods-crossbreeding, for in-stance-has been in use forthousands of years.

A great debate about recom-binant DNA ensued, world con-ferences were conducted, and thebasis for countless sci-fi moviescripts was established. Mter a

*To protect biodiversity, "Gene Banks" have been established allover theworld. Germ plasm is collected, evaluated and preserved. In the face of a mas-sive crop failure caused by disease, researchers would- ideally-be able to findgenetic material resistant to the disease. This would not avert the immediate im..pact of the crop failure.

18

segment of scien-tific inquiry is re-ally an adventure

tion of cosmetics and phanna-ceuticals.

Delaware Sea Grant was atthe forefront of U.S. chitin re-search. More than a decade ago,researchers developed a methodto extract chitin, and contributedto the development of several ma-jor applications. Fourteen chitin-related patents were issued toDelaware Sea Grant researchersand licensed by companies in theUnited States and abroad. In theUnited States, DuPont and Con-Agra created a subsidiary calledYanson, a company which is re-portedly seeing success in sev-eral chitin applications.

which researchers are workingwith concepts that are futuristic.This is not to say that the ideasare improbable. Rather, all that islacking is a catalyst in whateverform it might appear-a politicalevent (oftentimes a war), an eco-nomic need, an environmentalconcern, a societal problem or op-

portunity.Chitin-a principal compo-

nent of crustacean shells and in-sect exoskeletons-is a resourcewhose time has not quite arrivedin the U.S. Articles appear aboutthe substance's promise and asmall chitin/chitosan* operationoccasionally appears and some-times disappears. Still, chitinresearch continues for the sub-stance has a number of desirablequalities, including its ability toattract, adsorb, or absorb water;its biocompactability and biode-gradability; and its capacity to .absorb some heavy metals andeven proteins. Ion exchangersmade from chitosan have beenused in food and ph!lflnaceuticalprocesses, medical and agricul-tural drugs, and in wastewatertreatment.

Light- Weight BiologicalArmor?

When the Gulf War was atits peak, a few people in the mili-tary were reportedly looking forfabric made of chitin. The think-ing was this: since chitin in someforms is highly absorbent, andcan serve as an environmentalsink for heavy metal ions as wellas pesticides, it might provideprotection against toxic agents.

Fabric from crab shellsmight sound far-fetched, but notif one thinks of the naturalsources of fabric: silk (a worm);wool (an animal); cotton (aplant). Even if a fabric were notthe intended end result, the wayin which the chitin resource canbe altered for human use is far-

reaching.It can be made into sutures,

gauze bandages, and into artifi-cial skin for burn victims. It canbe used for paper coating, anti-static fabric finishing, water clari-fication and even topsoil preser-vation when added to water usedfor irrigation, by preventing therun-off of organic matter. Chi-tosan is also useful in removinghazardous metal contaminantsfrom wastewater and proteinsfrom processing effluents.

Since chitin is non-toxic andbiologically degradable, muchresearch with chitosan has alsofocused on its use in edible coat-ings for food, and for the prepara-

Crab Waste-

Bane or Bounty?Chitin is found abundantly

in crustaceans, notably crabs andshrimp, and the amount of shellwaste from crab processing op-erations is sizable. In Virginiathe approximate landings of hardblue crabs average 40 millionpounds yearly. That translatesinto only four million pounds ofcrab meat-the rest is consideredwaste. ..36 million pounds. Yetthat waste can be broken downinto (on a dry basis) approxi-mately 50% calcium of one formor another; 35% of protein; and15% chitin.

Until recently, the easiestway to deal with crab waste wasto simply bury it. In recent times

*Chitin is a component of crustaceanshells and insect exoskeletons. Chitincan be regenerated into a fiber formor into chitosan, a powderous sub-stance soluble in dilute acetic acid. (Continued on page 21)

10

From top left, clockwise:1) Callinectes sapidus, the blue crab, is an ample source of chitin, a substancewhich in certain forms has significant human applications, from artificial skin tobeads which absorb heavy metal ions.

2) Fabric (non-woven) made from chitin.

3) Hydrogel beads from chitosan taken through an optical microscope, in aque-

ous medium. The beads are useful for ion exchange of heavy metal ions. They

are formed by adding a chitosan solution in dilute acetic acid into weak alkali.

4&5) Photomicrographs of the surfaces and tom edges of 100% chitosan fibridpaper, and 100% cellulose paper.

20

diving submersible, enabling sci-entists access to an estimated 98percent of the world's oceanfloor. In addition to a differentworld view, the Japan~se appearmore likely to invest in researchwhich will not show an immedi-ate financial return, but may havea long term benefit.

landfill regulations havechanged; in Virginia, the wastemust be low in moisture. So,many processors convert theshells into dried crab meal, a feed

supplement.

call a "wonder macromolecule."These researchers may be aheadof their time-at least in the U.S.

While the U.S. utilizes rela-tively little of the chitin resource,Japan is reportedly producing inthe neighborhood of 700-1,000tons a year.* Japan is now consid-ered the world leader in chitin re-search and is actively pursuingapplications. Why the differ-ence? Undoubtedly, many rea-sons exist, but two seem readilyapparent. Bounded by the seaand with a land mass that ismostly mountainous (74%) andnot suitable for agriculture, Japan

Chitin Brewing?An obstacle to more U.S.

chitin use, as mentioned before,could partially be the seasonalsupply of, say crab shells. Yetthere are other sources of chitin.In an extensive book on chitin,Riccardo Muzzarelli, with theUniversity of Ancona, Italy,

describes an intriguingsource: "... largequantities of fungicurrently grown in fer-

mentation sys-tems producingorganic acids, an-tibiotics and en-

zymes, constitute apotential source of

chitin." Some fungal speciescontain high quantities of

chitin.Beyond the means for extrac-

tion and the accessibility of alarge source of raw material isthe biggest obstacle to U.S. chi-tin production: a market. Tenyears from now a substantialU.S. market might exist, or chi-tin/chitosan might still be on theverge. ..of being utilized.

+ + +

Chitosan beads

views the oceanic world differ-

ently than a good portion of the

U.S. population. Case in point:

Japan is the world's largest fish-

ing nation, the world's largest

consumer of fish products, and is

the world leader in aquaculture.

Japan owns the world's deepest-

Seconds Instead of HoursAt the Virginia Polytechnic

Institute (VPI) in Blacksburg, re-searcher Wolfgang Glasser be-lieves he has a means to readilyisolate chitin, it process which tra-ditionally uses large amounts ofacid to demineralize the shells.With research associates BobWright, Willer de Oliveira, andseveral graduate students, Glas-ser examined the use of steam ex-plosion to isolate chitin inhundreds of seconds as com-pared to hours usingconventional methods.Steam explosion isakin to pressurecooking; the materi-als are subjected tohigh pressure steam fora period of time. Withsteam explosion, the re-lease of steam causes thematter to explode into a fibrousmass. This work was performedat the VPI demonstration steamexplosion facility, the only oneof its kind in North America, afacility which has the capacity totreat up to one ton of organicwaste per hour.

Glasser and his associatesare keenly interested in this newmeans for extracting chitin, see-ing it as a major step toward util-izing a plentiful and very muchrenewable resource, envisioningthe extensive use of what some

*Sources varied in their estimates. The amount of chitin/chit6san might be more.One source indicated that the University of Tokyo reported 1,270 tons in 1986;most of that amount was chitosan.

21

Biotechnology. ..Continued

There are simply too manybiotechnology projects to cover,and it seems a shame not to beable to devote more space tothem. In the future issues of theBulletin, a column will be de-voted to bringing readers morenews about work in this field.

tional Weather Serviceof the National Oceanicand Atmospheric Ad-ministration, STORMwill operate through anetwork of up to 300volunteers in majorcoastal towns fromMaine to Florida.

Stephen Leatherman,director of the Labora-tory for Coastal Re-search and STORM,currently is recruitingvolunteers.

"We need peoplewho live or work onthe beach, who walk itevery day, who knowtheir beach," saidLeatherman. Althoughformal training in science is notnecessary, volunteers should"have a genuine interest in learn-ing about beaches and the impactthat storms have on them," hesaid. "STORM will study the re-

lationship between a storm'scharacteristics, such as centralpressure, wind speed and waveheight, and its impact in terms ofcoastal flooding, beach and duneerosion and overall propertydamage," said Leatherman. Peo-ple who are interested in becom-ing volunteers should call theSTORM Center at the Univer-sity of Maryland at (301) 405-4074.

New STORM Observation

Center EstablishedThis year when a powerful

hurricane or northeaster hits theEast Coast, information aboutthe condition of beaches andbeach property will be availablealmost immediately through theStorm Tracking and Observa-tional Reports to Media(STORM) Center.

Recently established by theUniversity of Maryland's Labora-tory for Coastal Researchthrough a grant from the Na-

New Publications

To order the following pub-lications, write Virginia SeaGrant, Marine Advisory Pro-gram, Virginia Institute of Ma-rine Science, Gloucester Point,VA 23062. Please make checksout to VIMS.

22

The Economic Impact of

Marine Aquaculture on

Virginia's Eastern Shore

Author: Sarra Thacker

In The Economic Impact ofMarine Aquaculture on Vir-ginia's Eastern Shore, BusinessSpecialist Sayra Thacker exam-ines the current status of marineaquaculture on the EasternShore, and the potential impactof increased production. Specifi-cally, the author surveys bluecrab shedding, and culturing ef-forts with clams, oyster, andother bivalves.

The fIrst copy of this advi-sory is free to Virginia residents,and additional copies cost $2.00.The cost of the publication forout-of-state residents is $2.00

The Crab Industry in

Venezuela, Ecuador,and Mexico.

Implications for the Chesapeake

Bay Blue Crab Industry

A quaculture is the fastestgrowing agricultural indus-

try in the United States. Duringthe 1980s, United States

aquaculture pr.oduction quadru-pled. By 1990, the estimatedU.S. production was 860 millionpounds with a farm gate (dock-side) value of 760 million dol-lars. For coastal regions wheretraditional fisheries and relatedemployment may be in decline,development of marine aquacul-ture provides employment oppor-tunities that maintain links totraditional lifestyles. Marineaquaculture may also provide abasis for rejuvenating the localseafood industry.

Virginia's Eastem Shore rep-resents an area with potential formarine aquaculture developmentprimarily because of the abun-dance of its coastal areas and be-cause the Eastern Shore lacksmany of the man-made influ-ences of more developed coastalregions. Designated as a UnitedNations Biosphere Preserve, theEastern Shore's Atlantic coast-line has one of the longest chainsof barrier islands on the EastCoast.

Authors: Michael J. Oesterlingand Charles Petrocci

I n recent years, ChesapeakeBay crabmeat processors have

been besieged by imports intotheir traditional domestic market-places. This comes at a time ofrising production costs and localresource fluctuations. Asian andLatin American countries havebeen significantly contributing tothis influx of picked crabmeat.Without some grasp of foreigncrab production (present and po-

tential), marketing strategies, andbiological parameters of the ex-

ploited species, Chesapeake Baycrabmeat producers will continueto lose their share of the domes-tic crabmeat market.

With the completion of a1992 overview of a portion ofthe Asian crab industry (TheWarmwater Crab Fishery inAsia, Petrocci and Lipton), thefIrst step has been taken in pro-viding the Chesapeake Bay crab-meat industry with a betterunderstanding of their globalcompetition. However, morethan any other region, LatinAmerica has the longest historyof exporting crabmeat to theUnited States, and yet very littleis known about the crab indus-tries in those areas.

In The Crab Industry inVenezuela, Ecuador, and Mex-ico, the authors characterize ex-isting crab fisheries/productionfacilities, and identify potentialareas of expansion. The authorsexamine the industry's harvest-ing and marketing strategies andits production capabilities. Addi-tionally, the authors give reasonswhy successful crabmeat indus-tries have developed in some ar-eas but not in others.

The Crab Industry in Vene-zuela, Ecuador, and Mexico is ajoint Virginia/Maryland SeaGrant project The cost of thepublication is $3.00.

23

On the cover: Fertuizedfish embryos

"'-

Sea Graat Communications

AddrE;ss correction requested