Biological Activity of Biosynthetic Rainbow Trout Growth Hormone in the Eastern Oyster, Crassostrea virginica

4 Pages·1991·1.7 MB·English

by  K T Paynter

Reference: Biol. Bull 181:459-462. (December, 1991)
Biological Activity of Biosynthetic Rainbow Trout
Growth Hormone in the Eastern Oyster,
Crassostrea
virginica
KENNEDY T. PAYNTER1* AND THOMAS T. CHEN2
^Department ofZoology, University ofMaryland, College Park, Maryland 20742 and-Cenler of
Marine Biotechnology, University ofMaryland, 600 E. LombardSt., Baltimore, Maryland21202
Abstract. Juvenile oysters werMe exposed to seawater that mollusks possesscomplexendocrine systems(Joosse
containing 1CT9, 1CT8, and 1(T7 biosynthetic rainbow and Geraerts, 1983), and several recent findings suggest
troutgrowth hormone (rtGH); thetreatment wasapplied that bivalve growth may be regulated at least in part by
for one five-hour period per week for five weeks. At the an endocrine system.
end ofthe five weeks, the animals treated with the two In general, comparative endocrinological studies of
highest concentrations of hormone were significantly mollusks have been limited to gastropods and cephalo-
longer and had dry tissue weights 50% greater than did pods, and in many ofthose studies, endocrine control of
the lowest treatment group or the control group. Con- reproduction hasbeenamajorfocus. InthesnailLymnaea
tinuing in vitro experiments on isolated oyster tissue stagnalis. however, an endogenous growth-promoting
showedthatthehormonetreatmentsignificantlyincreased hormone hasbeen identified and well characterized. This
oxygen consumption. Boiled hormone had no effect. In hormone, calledthe molluscan insulin-like peptide(MIP),
both sets ofexperiments (whole animals and isolated tis- hasbeen isolated from Lymnaeaneuroendocrine cellsand
sues), the% drywt(drywt/wetwt)wassignificantly higher reported to affect various aspects ofgrowth, metabolism,
in all animals and tissues that responded to rtGH. The andshell formation in thesnail(Geraerts, 1976;Dogterom
results demonstrate that rtGH has biological activity in elal., 1979; Dogterom andJentjens, 1980;Dogteromand
oystertissues, and thisactivity may bedirectly associated Robles, 1980; Smit el al., 1988).
with growth regulation in the whole animal. The results While much is known about the physiology ofthe bi-
further show that bivalve growth is not directly limited valves, little attention hasbeen paid totheendocrine reg-
by environmental parameters. ulation oftheir metabolism and growth. In early, rather
crude experiments, forexample, thegrowth ofoysterswas
Introduction arrested after their cerebral ganglia had been extirpated
(Galtsoff, 1964), [and similar results were obtained with
The growth ofmany marine and estuarine pelecypods the gastropod, Crepidula fornicata, by Lubet (1971)].
haslongbeenthoughttobedetermined by environmental Morerecently, Toullec et al. (1988) reported thatextracts
conditions. Many studies have revealed specific correla- ofcerebral ganglia prepared from Mytilus edulis stimu-
tionsbetweenbivalvegrowth rateandsuch environmental latedtheincorporation ofleucineintothemacromolecular
factors as water flow, salinity, temperature, oxygen, and fraction ofmantlecellsthat had been isolated from several
food availability (Dame, 1975; Hall, 1984; MacDonald bivalvespecies. Thesestudiessuggestthat bivalvegrowth,
and Thompson, 1985a,b; Brown, 1988; Brown and Har- likethat ofsnails, iscontrolled byendocrine mechanisms
twick, 1988a,b). Yet endogenous control of growth by that are not species specific.
neural orhormonal systemshasbeen investigated in only Conservation ofsequence and function among verte-
a few species. A significant body ofknowledge indicates brate growth hormone polypeptides is also well estab-
lished. Furthermore, bovine growth hormone (as well as
Received 14January 1991;accepted 20 September 1991. insulin) has been shown to accelerate the growth ofaba-
* Towhom correspondence shouldbeaddressed. lone larvae (Morse, 1981, 1984), and a growth hormone
459
460 K. T. PAYNTER AND T. T. CHEN
like polypeptide has been identified in the abalone (Mo- Mean values offinal height, total weight, shell weight,
riyama et a/., 1989). Therefore, we have begun to inves- wet tissue weight, and dry tissue weight were calculated
tigate the effects of biosynthetic rainbow trout growth for each treatment group. Values were statistically com-
hormone (rtGH) on the growth and metabolism of the pared using a t-test.
eastern oyster, Crassostrea virginica. Here we report that
exogenously applied rtGH stimulates the growth ofju- Oxygen consumption ofisolatedtissues
venile oysters and increases oxygen consumption in iso- Respirometry was conducted according to standard
lated oyster tissues. manometric procedures. Oyster gills (two gill pairs per
individual) were dissected and split into gill pairs. One
Materials and Methods pair from an individual was used as a control, the other
wastreated with boiled or native rtGH. Therefore, oxygen
froOmysttheerVilragrivnaiea(ICnrsatsitsuotsetroefaMavriirgnieniSccai)enwceer.eThoebtlaairnveade tchoenssuammpetiaonnimwaals, cboumtptahraetdweinregiltlrsetahtaetdwdeirffeerteanktelyn.fTrhoem
wAlelrecormapiesetdenatccloarrdvaiengwetroesitnadnudcaredd thoatucnhdeerrygoteccuhlnticqhuleess.s gill pairs were immediately placed in manometric flasks
containing 2 ml filtered seawater inoculated with buffer,
wmietthameopirnpehpohsriisne(n(o1t0a6ttMa)chiendtthoessuebaswtartaetre.)Tbhyetrreesautlmteinntg ibnosiulleidnrtaGnHd,boorvnianteivegrrotwGtHh (h1o0r6moA/n)e. N(aBtSiTv)e awnedreboiallesdo
juvenile oysters were raised in downwelling chambers in tested. The flaskswereaffixedtoadifferential respirometer
the hatchery at theChesapeake Bay Institute (CBI) ofthe
(Gilson), immersed in thewaterbath(25C),andallowed
Johns Hopkins University.
o(AfgBreitlolGsoHynnt(ehAtegate/i.l.clor1n9t8Ga8nH).dwTCahhseepnrc,eopm1ap9rl8e6ed)maewsnaptsraecrvlyioonDuesNdlyAadne(dscciDrniNtbrAeo)d- rtnooommeeeqttueielrrissbrywasettreeemforwreac3so0rcdlmeoidsn.e.dAatAfntd1e5ri-nmeitqiiunailliirnbetraeadrtivianolgnss,tothnhereaelarlfemtsepari-,-
manometer readings were recorded. Incubations typically
duced intGoHE. coli for large scale production ofthe poly- lasted for 2 h. Oxygen consumption was usually linear
peptide. inclusion bodies were isolated from E. coli
cells, dissolved inaG5HMguanidine hydrochloride solution, alfitneerarthdeecfriersatse1s5-inmionxyrgeeandicngo.ncAenntyrastaimopnlewserneotdisschaorwdiend.g
and thedenatured polypeptidewasre-natured bydif-
ferential dialysisagainst 50 mMammonium bicarbonate wAfetreerrtehmeorveesdpifrroommettrhyehfaladsksb,eewneicgohmepdl,edtreyde,dtihneati6ss0ueCs
binuff1e0rm(pMH 1a0m.0m).oTnhieulmyobpihiclairzbeodnaptreotebiunffwearsprHesu10s.p0enadteda oven for 72 h, and reweighed.
concentration of 10 mg/ml and used in appropriate Because oxygen consumption rates in gill pairs from a
amounts to inoculate the experimental seawater. single individual were being compared, the oxygen con-
sumption data were analyzed by means ofa paired t-test
to minimize the effects ofbetween individual variation.
Oystergrowth
Results
In September and October 1989, during the natural
growingseason oftheoysterintheChesapeakeBay region, After three weeks of treatment (Table I), the oysters
an experimental hormone treatment was begun. Four treated withMthe highest concentrations ofrtGH for five
groups of25 juvenile oysters were constructed such that weeks(10~7 and 10~8 A/)were significantly largerwith
the initial mean heights ofthe groups were not different. respect to shell height and dry tissue weight than either
One group (control group) was incubated in seawater in- thecontrolgrouporthelowesttreatmentMgroup(10~9A/).
oculatedwithbuffer,theotherthreegroupswereincubated But only the oysters treated with 10~7 hormone had
in seawatercontaining 10 7 A/, 10~8 M. or 10~9 A/rtGH, shell weights that were significantly larger than those of
respectively. Each group was incubated for 5 h, once per thecontrol group. Increases in total weight andwet tissue
week, in Petri dishes containing 50 ml of 1 ^-filtered am- weightwerenotsignificantlydifferent fromcontrols; vari-
bient seawater(=s8%o)at 23C. Initially, and beforeeach ation was high in these categories due to the large con-
treatment thereafter, the shell height ofeach oyster was tribution of water to the weights. Tissue hydration, as
measured from the umbotothe ventral shell margin. The measured by the dry wt/wet wt ratio, was significantly
animals were maintained in an upwelling system with a higher in the highest treatment groups when compared
continuousflowof100^ filteredseawaterduringtheentire to the control group. There was no mortality in any of
experiment. After five weeks the oysters were sacrificed, the groups.
GH
and the final height, total weight, shell weight, wet tissue Trout induced significantly higher rates ofoxygen
weight, and dry tissue weight were determined for each consumption in isolated gill tissue (Fig. 1). The control
individual. Whole oyster volume was calculated as the oxygen consumption values for both boiled rtGH and
difference between total animal weight and shell weight. native rtGH experiments were not different and were
EFFECT OF GROWTH HORMONE ON OYSTERS 461
Table I
EffectofexogenouslyappliedIroulgrowth hormoneon oystergrowth
Treatment
462 K. T. PAYNTER AND T. T. CHEN
although sufficient nutrients must havebeen available for oyster, Crassoslreagigas I. Absolute and allometric growth. Aqua-
theenhanced growth tooccur, thecontrol oystersdid not culture1Q: 231-251.
use them. We conclude that endogenous metabolic con- Brown, J. R., and E. B. Hartwick. 1988b. Influences oftemperature,
salinity and available food upon suspended culture ofthe Pacific
trolsregulategrowth rateswith respect to, but notdirectly oyster,CrassostreagigasII.Conditionindexandsurvival.Aquacullure
limitedby,theenvironment, andthatexogenouslyapplied 70: 253-267.
growth hormone causes a shift in that regulation. Ifthe Dame, R. F. 1975. Day degree growth models for intertidal oysters.
concentration offood in the upwellers was not limiting, Conlrih. Mar. Sci. 19: 107-1 12.
thetreatedoystersmayhave fedatagreaterratetosupport Dogterom, A. A., and B. R. Robles. 1980. Stimulation ofornithine
theirfastergrowth. On the otherhand, iffood levelswere dwietcharmboolxlyluassceanacgtrioviwttyhihnoLrymmonnea.eaGesnt.agCnaolmips.afEtnedroacrsiinnogll.e i4n0j:ec2t3i8o-n
limiting,ornearlyso, thetreatedoystersmayhavebecome 240.
more efficient and may have been able to assimilate and Dogterom, A. A.,andT.Jentjens. 1980. Theeffectofthegrowthhor-
use a higher percentage ofration. These possibilities will mone of the pond snail Lymnaea stagnalis on periostracum for-
bedirectly tested by performing feeding, assimilation, and Dogtmeartioomn,.AC.oAm.,p.H.BiVoacnheLmo.enPhhoyusti,ola.ndAR6.6:Va68n7d-e6r90S.chors. 1979. The
utilizationexperimentsandotherscope-for-growth studies effect ofthegrowth hormone ofLymnaea stagnalison shell calcifi-
on treated and control oysters. cation. Gen. Comp. Endocrinol. 39: 63-68.
Our results also indicate that the effect ofrtGH in oys- Galtsoff, P. S. 1964. The American oyster. Crassostrea virginica
ters, likethatofitsevolutionary relative, prolactin, in fish Gmelin. U. S Fish li'ildl. Sen:, Fish. Bull 64: 480pp.
(see Prosser, 1974), may be involved in osmoregulation, Geraerts,W.P.M.1976. Controlofgrowthbyneurosecretoryhormone
because it influenced tissue, and possibly cellular, hydra- oGfentheColimghpt gErnedeoncrcienlolls.in29t:h6e1-f7re1s.hwater snail Lymnaea stagnalis.
tionlevels. Thus, theeffectongrowthcouldbeeven more Hall,S. 1984. Amultipleregressionmodelofoystergrowth.Fish.Res.
indirect. Oystersgrow fasterat highersalinities wheretis- 2: 167-175.
sue hydration is normally slightly lower (Paynter and Hawkins, A.J. S., P. N. Salkeld, B. L. Bayne, E. Gnaiger, and D. M.
Mallonee, 1991; Paynter, pers. obs.). Changes in tissue Lowe. 1985. FeedingandresourceallocationinthemusselMy/i/iis
hydration could affect numerousphysiological functions, eSdeur/is2:0:e2v7id3e-n2c8e7.for time-averaged optimization. Mar. Ecol. Prog.
including feeding and digestion rates, which would ulti- Joosse,J.,andW.P.M.Garaerts. 1983. Endocrinology. Pp.317-406
mately lead to more rapid growth. Studies are currently in TheMol/itscu. t'ol4, Physiology. Part I. A. S. M. Saleuddin and
underway to examine the in vitro effects ofrtGH on var- K. M. Wilbur, eds. Academic Press, London.
ious aspects ofbivalve metabolism and growth. Lubet, P. 1971. Influence desganglions cerebroides sur la croissance
deCrepidulafornicalaPhil.(mollusquemesogasteropode).C R.Acad.
Sci. 273:2309-2311.
Acknowledgments
MacDonald, B. A., and R.J. Thompson. 1985a. Influenceoftemper-
The authors would like to thank Y. L. Tang for puri- ature and food availability on the ecological energetics ofthegiant
fying the trout growth hormone and M. E. Mallonee for scallopPlacopeclenmage/lnniciis. I.Growthratesofshellandsomatic
tissue. Mar. Ecol. Prog. Ser 25: 279-244.
hisassistance in conductingthe growth experiments. The MacDonald, B. A., and R.J.Thompson. 1985b. Influenceoftemper-
authors are deeply indebted to M. Castagna and K. Kir- ature and food availability on the ecological energetics ofthe giant
kowski, VIMS, forprovidingtheoysterlarvae. The bovine scallopPlacopeclenmagel/anictis. II. Reproductiveoutputandtotal
somatotropin was a generous gift ofDr. E. Popper, Lilly production. Mar. Ecol. Prog. Ser. 25: 295-303.
Research Lab, Greenfield, Indiana. Moriyama, S., S. Atsuta, M. Kobayashi, and H. Kawauchi. 1989.
Growth hormone-likesubstanceofAbalone. Haliotisdiscushamai.
XIlh International Symposium on Comparative Endocrinology.
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