Table Of ContentReference: Biol. Bull. 196: 177-186. (April
Protein Metabolism in Lecithotrophic Larvae
(Gastropoda: Haliotis rufescens)
JAY VAVRA AND DONAL T. MANAHAN*
Department ofBiological Sciences, University ofSouthern California,
Los Angeles, California 90089-0371
Abstract. Rates ofprotein depletion, synthesis, and turn- theless. our understanding of metabolic processes during
over were measured in larvae ofthe abalone Haliotis rufe- lecithotrophic larval development is very limited compared
scens as an approach to understanding macromolecular me- to what is known about metabolism and the dynamics of
tabolismduring lecithotrophic development. Proteincontent biosynthetic processes in species with planktotrophic devel-
decreased linearly during development to metamorphic opment (e.g., protein synthesis in sea urchin embryos:
competence, with 34% ofthe initial protein in eggs depleted Goustin and Wilt, 1981; Bedard and Brandhorst. 1983).
during the 8-day larval life span. Fractional rates ofprotein Most studies ofthe biochemistry and physiology oflecitho-
synthesis (percentage of total body-protein synthesized per trophic larvae have focused on the composition of energy
day) decreased during development, from 40% (1-day-old reserves in the early stages of development (Turner and
trochophore larva) to 14% (7-day-old veliger larva). Sepa- Rutherford. 1976; Jaeckle and Manahan, 1989a; Anger,
ration of proteins by one-dimensional gel electrophoresis 1996). The importance of maternally endowed energy sup-
showed that protein pools in larvae are dominated by two ply to larval life span has also been studied (Shilling and
high-molecular-weight protein classes (88 and 121 kDa). Manahan, 1994; Ben-David-Zaslow and Benayahu. 1998),
When the proteins of 1- and 3-day-old larvae were labeled as have respiration rates and amino acid transport (Jaeckle
with a mixture of 3SS-methionine and cysteine. the pattern and Manahan. 1989a. b; Shilling et ai, 1996; Hoegh-
on two-dimensional gels showed that the turnover process Guldberg and Emlet, 1997).
(protein synthesis and degradation) involved hundreds of The aim of this study is to quantify the energetics of
different proteins. The energy gained from loss of protein protein degradation and synthesis (turnover) in a lecithotro-
could account for 20% of the protein turnover rates for phic larval form. Protein metabolism was chosen as the
trochophore larvae and 79% ofthe lower turnover costs for focus of this study because protein, not lipid. has been
late-stage veligers. Lecithotrophic larvae of H. rufescens reported (Jaeckle and Manahan, 1989a) to be the major
maintained high biosynthetic activities, with up to 40% of endogenous energy reserve utilized by larvae of Haliotis
their whole-body protein being turned over each day. Such rufescens (red abalone), a species with lecithotrophic devel-
dynamic processes during development of nonfeeding lar- opment.
vae would contribute significantly to maintenance metabo-
lism.
Materials and Methods
Introduction Larval culture
Nonfeeding (lecithotrophic) larval forms are widely rep- Adult abalone (Haliotis rufescens) were spawned and
resented in the life cycles ofmany marine invertebrate taxa fertilized at acommercial hatchery (Ab Lab, Port Hueneme,
(Thorson. 1950; Strathmann, 1978; Pearse, 1994). Never- California). All cultures were started using gametes from 1
male and 2-3 females (different adults were used for each
culture). Zygotes and larvae were maintained at 14-15C in
Received 28 May 1998: accepted 6 January 1999.
*To whom correspondence should he addressed. E-mail: manahan@ unstirred, UV-irradiated seawater that had been passed
usc.edu through a 5-jum (pore size) filter; the larvae were reared on
177
178 J. VAVRA AND D. T. MANAHAN
80-/u,m mesh screens. Under these culturing conditions, it
took6-8 days forlarvae toreach metamorphic competence,
as determined by ability to undergo metamorphosis when Tau
induced with 7-aminobutyric acid (GABA). 1
Change in totalprotein
M
Throughout development, samples (Culture 1. n = 4-5 03
=
independent samples; Culture 2, H 3 independent sam-
ples) containing 50-75 individuals were taken formeasure-
ment ofprotein content (Bradford assay, 1976; as modified H R
by Jaeckle and Manahan, 1989a). The rate of protein loss
during development was calculated from the linear regres-
sion of total protein per individual with time, during the 10 20 30
period from egg to metamorphic competence. Protein con- Elution time (minutes)
tent of eggs (day zero) was determined only for Culture I.
B
Rates ofamino acid transport
20
-i
To determine absolute rates ofprotein synthesis, rates of "oo
umino acid transport by larvae were measured during the 5.~
14C-labeling experiments (see Manahan, 1983, for meth- S 15 -
ods). All physiological rates were measured at 15 0.1C. o 2
Each transport assay used 5000 larvae in 10 ml of filtered E
(pore size0.2 /nm) seawaterto which was added 1 /zCi ml ' if 10 -
M
of C-(U)-glycine (New England Nuclear; 110 juCi 3= CD
/IJLa\msoamrp1l).eTehveerryat2e-3ofmiisno)todpueritnrganaspo1r6t-1w8a-smimneaesxuproesdur(e50o0f 'Cco "SP5 5-02468
larvae to 9 ^M glycine in seawater. _O>,
Rules ofprotein synthesis
Age (days)
A modified radioisotope-labeling protocol, based on that
of Fry and Gross ( 1970) for sea urchin embryos, was used rufFeisgceunrse. 1(.A)GlCyhcrionmeatinogtrheamfresehoawmiinngotahceidseppoaorlatoifonlaorvfaeamoifnHoalaicoitdiss
to measure absolute rates of protein synthesis in abalone extractedfrom3-day-oldveligerlarvaebyreverse-phasehigh-performance
larvae. Our modifications included trace labeling over a liquid chromatography: D, aspartic acid; E, glutamic acid; N, asparagine;
short time interval (<18 min) to minimize possible effects S. serine; H.histidine;G.glycine; R.arginine;Tau,taurine;A.alanine; Y.
of perturbations, caused by high transport rates of exoge- tyrosine; M, methionine; F, phenylalanine; I, isoleucine; L, leucine; K,
nshoourst 1e4Cx-ploasbuerleedalasmoinaovoaicidde,dofthteheifnrteeercaomnivnerosaicoindopfool1.4CA- lisneynstifnreee(1uanmSkiEnnooofwnatchpeiedmaekpsoaonalrs(enutn=hlrao6buefgloherd)el.aarcv(haBl)stCdahgeeva)en.lgToephmeiennettqh.ueaatEimroornournftobraotrfhseglrlyeicpnireneae-r
glycine, the tracer used in our measurements, into other regressionispmolglycinelarva"1 = 1.51x + 5.16;where.v = ageindays
amino acids (confirmed by analysis with high-performance (r = 0.50; n = 48).
liquid chromatgraphy, HPLC). Short experiments also min-
imized any effects from protein degradation (i.e., reutiliza- Rates ofprotein synthesis were determined by measuring
tion of 14C-labeled amino acids due to protein breakdown). the rate ofincorporation of l4C-glycine intoprotein [defined
Glycine was used as the tracer because it is transported at a as the 5% trichloroacetic acid (TCA(-insoluble fraction of
high rate and is a measurable fraction ofthe free amino acid larval homogenates, shell included]. For measurements
pool in larvae ofH. ntfescens (Fig. 1 ). These characteristics from the free amino acid pools, the intracellular contents of
allowed for accurate HPLC measurement of the change, larvae were extracted overnight into 707r ethanol. The spe-
with time, in the specific activity of glycine in the free cific activity of glycine was then determined with HPLC
amino acid pools of larvae (Fig. 2A). Also, glycine in (Welborn and Manahan, 1995) foreach larval stage studied.
acid-hydrolyzed, whole-body protein extracts of H. rufe- After detection of fluorescence (i.e., moles of glycine: Fig.
scens larvae (Table I) could be measured with HPLC; this 1A, B), post-column samples ofeluent were collected every
value is required for calculation ofabsolute rates ofprotein 30 s (LKB fraction collector) and mixed with scintillation
synthesis (see below). cocktail to determine the amount of radioactivity in the
PROTEIN METABOLISM IN ABALONE LARVAE 179
5- glycine peak. All measurements of radioactivity (as counts
per min. CPM) were corrected forquenching and converted
tototal disintegrations permin (DPM). By correcting forthe
change in the specific activity of glycine in the free amino
acid pool (Fig. 2A), the incorporation rate of '4C-glycine
cOo I 3' (Fig. 2B) could be converted to the total amount ofglycine
incorporated into protein (both I2C- and 14C-glycine). The
<<JD O3. value for the incorporation oftotal glycine into protein was
Q.
co a. then converted to an absolute rate ofprotein synthesis (Fig.
2C) by determining the mole-percent of glycine in larval
0.5J protein and the mole-percent-corrected molecular weight of
12 16 all amino acids in larval protein of H. rufescens (Table I).
The components required forthe calculation ofthe absolute
rate of protein synthesis are
MW
_
MW
where Jt5 is the rate of protein synthesis, /t is the mole-
percent-corrected molecular weight ofamino acids in larval
protein, S,,, is the mole-fraction of glycine in protein. Sf, is
the amount of radioactivity in protein, and S/M, is the spe-
cific activity of glycine in the free amino acid pool, which
changed during the time course ofexposure to isotope (Fig.
2A). The above equation was solved foreach sampling time
interval during which the incorporation of C-glycine into
protein was measured (Fig. 2B|.
>
lOOOi
Patterns ofprotein synthesis
For the analysis ofthe electrophoretic patterns ofprotein
w
.Ic I synthesis, each labeling experiment used 10.000-15,000
cw c^ lmaertvhaieoniinne1/4cymslteionfef(i1lt1er0e0d-1se2a0w0atCeirmwmiot!h"'2.00Ne/uw.CiEnofgl3a5nSd-
600
Nuclear). Non-radiolabeled methionine was added to sea-
O
'm water (final concentration of 500 nM) to increase substrate
concentration and, hence, transport rates. After a 3-h incu-
400
OQ). 4 8 12 16 bcaetsisoinv,eusneianwcaotreprorwaatesdheissotafotpeerwtahse rlearmvoaveedwewriethpetlhlreeteedsuci-n
Time (minutes) 15-ml conical tubes with a hand centrifuge. Protein samples
for electrophoresis were prepared by ultrasonicating em-
Figure 2. Determination of the absolute rate of protein synthesis in bryos orlarvae in 50 mMTris-HCl at pH 7.2. Samples were
7-day-oId veliger larvae of fialiotis rufescens. (A) Specific activity of
intracellular glycine in the free amino acid pool following transport of kept on ice during this process to prevent any rise in
'4C-glycine. Each point represents the specific activity determined by temperature. Homogenates were centrifuged to remove lar-
high-performance liquid chromatography using fluorescence detection val shell (30 min at 15.000 X g, 4C) priorto electrophore-
(moles of glycine) and measuremenl by liquid scintillation counting of sis. The amount of ?5S-methionine/cysteine incorporated
radioactivity in the glycine peak collected from the chromatographic into protein was determined by TCA (5%) precipitation of
eluent. (B) Radioactivity in the irichloroacetic acid (TCA)-insoluble frac- =
tion (protein). (C) Rate of protein synthesis after correcting the TCA- 10-/J.1 aliquots (n 2) ofthe homogenate. Tissue solubilizer
msoluble traction for the change in intracellular specific activity of L4C- (0.5 ml. Scintigest. Fisher Co.) was added to these precip-
glycine. the mole percent of glycine in protein, and the average mole- itates before the radioactivity was counted. These data were
percent corrected molecular weight of amino acids in larval protein required for loading of equal amounts of radioactivity on
(Table I). each gel to permit comparisons of different larval stages
(see Results). The amount ofprotein loaded on each gel was
also determined.
180 J. VAVRA AND D. T. MANAHAN
Table I
Atninit ticiilcomposition (mole-percent) ojproteins in larvalstages of Haliotis rutescens
Age (days after fertilization)
Amino acid Mean SEM)
(
Glutamic acid &
PROTEIN MF.TABOLISM IN ABALONE LARVAH 181
Age (days)
Figure 3. Total protein content during development ofHaliotis rufe-
scens. Datafromtwoculturesareshown: Culture I (n = 4-5 independent
samples perdata point: O) and Culture2 (n = 3 independent samples per
datapoint; ). Proteincontentofeggs wasdeterminedonly forCulture 1.
Rates ofprotein loss were not statistically different between cultures (see
text),andalldatawerecombinedtocalculatetherateofproteinlossduring
development: ng protein individual"1 = 21.2.v + 498.6. where.v = age
in days (SE ofslope = 3.52. n = 68). Errorbars are 1 SEM.
4). Combining all glycine transport data for both cultures
revealed that transport rates were significantly higher at
metamorphic competence (based on larvae before and after
day 6: /|24| = 2.81. P < 0.005).
Table II
Protein synthesis andfractional rates ofprotein turnoverduring lamil
development ofHaliotis rufescens
182 J. VAVRA AND D. T. MANAHAN
O glycine in the proteins of 2- to 8-day-old larvae was 8.0%
(0.2 SEM), and this value was used for all calculations of
protein synthesis rates. The mole-percent corrected molec-
I ular weight of amino acids in larval protein ranged from
1.1 133.7 to 136.8 g amino acid mol~', with a mean value of
lO-g<U 100H 135.5 (0.3. SEM) used for all calculations (Table I).
O
i 02468 Rates ofprotein synthesis
tr o
a
O) Rates ofprotein synthesis decreased during development,
from 193 to 49 ng protein synthesized larva" ' day"1 (cal-
culated from analysis given in Table II ofdatashown in Fig.
Age(days) 5). The decrease was nonlinear and was best described by a
menFtiguofreHi5i.lintAibxsroiliufteseccrnasl.esEaocfhprdoatteainposiynntthreespirsesdeunrtisngalraartvealofdepvreoltoepi-n speercolnadr-voard=er3.p7o6lyxn2omia5l4.(0n9an.vog+ra2m4s3.o5f2.prwohteeirnes,yvn=theasgiezeidn
synthesisdetermined from theslopeofaseparate time-courseexperiment, days). The fractional rate ofprotein synthesis, expressed as
bwiatsheddoenvemluolptimpelnetsawmapslefsit(teads itnoFaig.se2c).onTdh-eorddeecrrepaosleynionmpiraolt:einngsynptrhoteesiins the percentage of total whole-body protein synthesized per
synthesizedlarva"1 day"' = 3.76.x2 54.09.v + 243.52, where v = age day. also showed a steady decline through larval develop-
in days. ment and decreased from 40% (1-day-old) to 14% (7-day-
old) (Table II: comparison oftotal protein content per larva
and absolute rate of protein synthesis).
Age (days) Age (days)
12357
Std. kDa 1 2 3 5 7
H8SBR
r
21
14
A. Coomassie-stained gel B. Autoradiogram showing
showing protein classes 35S incoporation into protein
Figure 6. One-dimensional sodium dodecyl sulfate polyacrylamidc gel electrophoretic analysis of total
suluhk i .>, in larvae of Huliolis rufescens. (A) Coomassie-stained protein pools. (B) Corresponding
autorudhr.i.ii, -I proteins labeled with 15S-methionine/cysteine (i.e., newly synthesized proteins). Each lane
contains adilt.rein larval stage (day 1. 2, 3. 5, and 7) andequal radioactivity (1 X 10"DPM loadedperlane).
Bunds marked P, and P, are high-molecular-weight proteins present at each stage of larval development as
shown in the Coomassie-stained gel (A) and are not radiolabeled in the autoradiogram (B). Molecular-weight
standards arc represented with markers from 97 to 14 kDa.
PROTEIN METABOLISM IN ABALONE LARVAE 183
kDa
97
66
45
31
21
.
14
7.0 4.0
Pi
Figure 7. Autoradiogram of proteins in 3-day-old veliger larvae ofHaliotis rufescens labeled with 35S-
methionine/cysteine and separated by two-dimensional gel electrophoresis. Areabounded by the dashed box is
displayedinexpandedforminFigure8B. Molecular-weightstandardscorrespondtoCoomassie-stainedgel(not
shown) with markersfrom97 to 14 kDa. Isoelectric points(pi)fall withinalinearpH gradient from 7.0to4.0.
Patterns ofprotein synthesis The main point illustrated in Figures 7 and 8 is that protein
synthesis in these larval forms is not limited to any single
At the analytical resolution of one-dimensional gel elec- group (MW or pi) of proteins, but involves many proteins
trophoresis, similar patterns of Coomassie-stained proteins and complex patterns.
were evident during development (1- to 7-day-old larvae).
Two classes of high-molecular-weight protein (Fig. 6A: Discussion
labeled as P, and P2 at 121 and 88 kDa, respectively)
dominated the Coomassie-stained proteinpool ateach larval Larval stages of Haliotis rufescens lost protein reserves
stage analyzed. The patterns ofthe existing pool ofproteins continuously during development (Fig. 3). By day 8, velig-
(Fig. 6A: Coomassie-stained proteins) were different than ers that were competent to metamorphose contained 34%
those of the newly synthesized proteins (Fig. 6B: autora- less protein than the egg. The average daily loss of protein
diogram oflabeled proteins), with nodetectable synthesisof was 21.2 ng protein larva"1 day"1 (average rate of both
the two high-molecular-weight protein classes at any larval cultures. Fig. 3). equivalent to 509 juJ day"1 (24.0 kJ g~'
stage (note lack of signal in Fig. 6B autoradiogram for P, protein; Gnaiger, 1983). During this period of protein loss,
and P2). the absolute rates of protein synthesis decreased fourfold,
The patterns of synthesis of individual proteins were from 193 to 49 ng protein synthesized larva"' day"' for
compared, using two-dimensional gel electrophoresis (Figs. 1-day-old trochophores and 7-day-old veliger larvae, re-
7. 8). for the trochophore stage (1-day-old) and a veliger spectively (Fig. 5; Table II). Note that the rate of protein
stage (3-day-old). The two-dimensional separation resolved loss was linear with time (Fig. 3). whereas the decrease in
over300proteinsthat were being synthesized in larvae (Fig. the rate of protein synthesis was nonlinear (Fig. 5). The
7). The results of a qualitative analysis (visual absence or relationshipofthese ontogenetic changes in protein loss and
presence) are given in Figure 8 to show some of the differ- synthesis was analyzed further by calculating the fractional
ent proteins being synthesized by 1- and 3-day-old larvae. rates of protein synthesis during development. When
kDa
* + TT,T I /* * A* 445c
R~l f '
* R2
f T
A
* i * .
\ A
A A A A
1 31
R3
4*
21
5.8 4.8
Pi
B
kDa
*
Rl
A T <^
RP92
?
31
' ; '
_:
R3 A *' R4
4 21
5.8 4.8
Pi
Figure 8, Autoradiograms of two-dimensional separation of "S-labeled proteins in (A) I-day-old tro-
chophuiv larvae ;md(B)3-day-oldveligerlarvaeofHaliotisrufescens(seeFig.7fortheentiretwo-dimensional
pattern ofpro;.-in synthesisforthe veligerstage). Eachgel was loadedwithequal radioactivity (3 x I0h DPM)
andexposed u> X-ray film for8days. Fourdifferentproteinsthat were labeled ineachdevelopmental stage are
markedas reference points(R1-R4)forcomparison ofproteins ineach gel. Arrows show proteinssynthesized
at one stage but not the other (by visual comparison ofoverlaid autoradiograms oftrochophore and veliger).
Orientationofarrows is not intended to indicate greaterorlesseramountsofprotein,just presence orabsence.
Molecular-weightstandardscorrespondtoCoomassie-stainedgel (notshown)withmarkersfrom45 to2\ kDa.
Isoelectric points (pi) fall within a linear pH gradient from 5.8 to 4.8.
184
PROTKIN METABOLISM IN ABALONE L.ARVAE 185
expressed as the percentage of a larva's total (whole-body) II). The energy gained from the loss ofprotein (24.0 kJ g ')
protein content that was synthesized per day. the fractional during development ofH. rufescens was constant at 509 p.}
rates ranged from 40% (1-day-old trochophore) to 14% day . Assuming complete oxidation of the protein de-
(7-day-old veliger) (Table II). These rates of synthesis rep- pleted, this input of energy from the loss of protein could
resent the turnover rates ofwhole-body protein in larvae of account for only 20% (509/2548) of the cost of protein
H. rufescens. When there is no net deposition (growth) of turnover in 1-day-old larvae. Obviously, during early devel-
total protein, as in the lecithotrophic larvae ofH. rufescens. opment most of the costs of protein turnover have to be
the rate of protein synthesis is the measure of the rate of supplied from sources other than protein degradation. How-
protein turnover (Waterlow et /., 1978). Protein turnover, ever, later in development (near metamorphosis) when rates
the continual breakdown and replacement of cellular pro- of protein turnover decrease, the energy made available
teins, is asignificant componentofmaintenance metabolism from protein reserves could account for a large percentage
in adult marine invertebrates (Hawkins. 1991). Few data are (79%) of the costs of turnover.
available in the literature forrates ofprotein turnoverduring Protein turnover plays a major role in establishing the
development of marine invertebrates, making comparisons metabolic rate and physiological state ofanimals (Waterlow
with our own data difficult. However. Berg and Merles et til.. 1978: Reeds et al.. 1985; Hawkins, 1991). For leci-
(1970) measured a rate of protein turnover in sea urchin thotrophic larval forms, little is known about rates of mac-
embryos (Lvtechinux ananiesits at 19C) of 23% day"1, a romolecular synthesis during nonfeeding development. Our
value that falls within the range we determined forlarvae of findings with larvae of H. rufescens show that these pro-
H. rufescens (149r-40% at 15C, Table II). These synthesis cesses proceed at a high rate and undergo dynamic ontoge-
rates in abalone larvae are not limited to a few specific netic changes, with up to 40% of a larva's whole-body
proteins. The electrophoretic patterns of protein synthesis protein being turned over per day compared to depletion
showed widespread incorporation of 35S-label, indicating a rates ofonly 4%-6%. Ifabalone larvae are typical ofother
high synthesis rate of multiple proteins (Figs. 6B. 7. 8). as lecithotrophs. at least in theirbiochemical andphysiological
has been found in developing stages of other species of activities, then biosynthetic rates in nonfeeding larval forms
marine invertebrates (e.g., sea urchin embryos: Bedard and may be much higher than expected.
Brandhorst, 1983). These complex patterns of synthesis
show that many proteins are involved with the synthesis
side of the turnover process (synthesis and degradation). Acknowledgments
The specific proteins that are degraded to provide the pre-
cursors for new synthesis have not been identified in this We thank John McMullen and Mike Machuzak ofthe Ab
study, but the lack of synthesis of two high-molecular- Lab (Port Hueneme, California) for supplying animals and
weight proteins (Fig. 6B: PI and P2 at 88 and 121 kDa. supporting this project. Our thanks also to Nicholas Appel-
respectively) suggests that these proteins might be degraded mans for his help in rearing the larvae. This research was
to support the new synthesis ofother proteins. Amino acids supported by a grant from NOAA, Office of Sea Grant
dissolved in seawater are unlikely to have been a source of (U.S.C. Sea Grant) and by a grant from the Office ofNaval
precursors for synthesis in our experiments, because the Research (NOOO14-90-J-1740).
very high numbers oflarvae (500 ml"') that we used would
have quickly depleted any substrates present at low concen-
tration in natural seawater. Literature Cited
=
larTvhaeeoefnHe.rgryufecsocsetnsofcanprboetecianlctuulrantoevdefror(compsyanrtihseosnisw)itihn Angedre,velKo.pm1e9n96t.anPdhyesairollyogjiucvaelnilaendgrbioowcthhemiincatlhechnaonrtgheesrndusrtionngelcarravbal,
the energy made available from the degradation of protein Lithodesmaja (Decapoda: Anomura). Mar. Biol. 126: 283-296.
(Fig. 3). The costofprotein synthesis appears tobe constant Bedard. P. A.,and B. P. Brandhorst. 1983. Patternsofproteinsynthe-
in different animals and stages ofdevelopment [e.g.. Reeds sis and metabolism during sea urchin embryogenesis. Dev. Bid. 96:
et til.. 1985: mammal = 11.52 1.12 J (mg protein syn- 74-83.
thesize=d)"1: Hawkins et ai. 1989: juvenile marine bi- Ben-gDeavivtiyd-iZnasplloatnutl,aeR.,ofansedveVr.alBesonfatycaohrual.s.19M9a8r.. ECcoolm.pePtreong.ceSearn.d1l6o3n:-
valve 1 1.38 8.88 J (mg protein synthesized)"1; Vavra 235-243.
ft id., unpubl.: veliger larvae of the bivalve Crassostrea Berg, VV. E., and D. H. Merles. 1970. Rates ofsynthesis and degrada-
=
gigas 13.2 4.2 J (mg protein synthesized)"1]. Using tion ofprotein in the seaurchin embryo. .v/>. CellRes. 60: 218-224.
our value for molluscan larvae, the protein turnover rate Bradford, M. 1976. A rapidandsensitive methodforthequantitationof
measured for trochophore larvae of H. rufescens of 193 ng microgram quantities of protein utilizing the principle ofprotein-dye
protein would equate to a requirement of 2548 ju.J larva Fry,biBn.diJn.,g.anAdnalP..RB.ioGcrhoesms.. 7129:702.48P-a2t5t4e.rnsandratesofproteinsynthesis
day"1, decreasing in late-stage veliger larvae (7-day-old) to in sea urchin embryos: I. Uptake and incorporation of amino acids
647 fiJ larva"1 day"1 (turnoverrate of49 ng protein; Table during the first cleavage cycle. Dev. Biol. 21: 105-124.
186 J. VAVRA AND D. T. MANAHAN
Gnaiger, E. 1983. Calculation ofenergetic and biochemical equivalents sea Benthos, K. J. Ecklebarger and C. M. Young, eds. Columbia
of respiratory oxygen consumption. Pp. 337-345 in Polarographic University Press. New York.
Oxygen Sensors, E. Gnaiger and H. Forstner. eds. Springer-Verlag, Reeds, P.J.,M. F. Fuller,and B.A. Nicholson. 1985. Metabolic basis
Berlin. ofenergyexpenditurewithparticularreferencetoprotein.Pp.46-57in
(.(piisiin. A.S.,and F. H. Wilt. 1981. Protein synthesis, polyribosomes, SubstrateandEnergyMetabolism, J. S. Garrow and D. Halliday.eds.
and peptide elongation in early development of Strongylocentrotus John Lihbey. London.
/>t<ri>tii\iiii.\. Dcr. Biol. 82: 32-40. Shilling, F. M., and D. T. Manahan. 1994. Energy metabolism and
llnukins. A.J. S. 1991. Protein turnover: afunctional appraisal. Fund. amino acid transport during early development of antarctic and tem-
Ecnl. 5: 222-233. perate echinoderms. Biol. Bull. 187: 398-407.
Hawkins,A.J.S.,J. Widdows,andB. I,. Bayne. 1989. The relevance Shilling, F. M., O. Hoegh-Guldberg, and D. T. Manahan. 1996.
of whole-body protein metabolism to measured costs of maintenance Sources ofenergy for increased metabolic demand during metamor-
and growth in Myrilus eilulis. Physiol. Zool. 62: 745-763. phosis ofthe abalone Haliotis riifexcens (Mollusca). Biol. Bull. 191:
Hoegh-Guldberg, O., and R. B. Emlet. 1997. Energy use during the 402-412.
development of a lecithotrophic and a planktotrophic echinoid. Biol. Strathmann,R. R. 1978. Theevolutionandlossoffeedinglarval stages
Bull. 192: 27-40. ofmarine invertebrates. Evolution 32: 894-906.
Jaeckle, W. B., and D. T. Manahan. I989a. Growth and energy im- Thorson, G. 1950. Reproductive and larval ecology of marine bottom
balance during the development of a lecithotrophic molluscan larva invertebrates. Biol. Rev. 25: 1-45.
(Haliotix rufescenx). Biol. Bull. 177: 237-246. Turner, R. L., and J. C. Rutherford. 1976. Organic, inorganic, and
Jaeckle,W.B.,andD.T.Manahun. 1989b. Feedingbya"nonfeeding" caloriccompositionofeggs,pentactulae,andadultsofthebroodingsea
larva: uptakeofdissolveduminoacidsfrom seawaterby lecithotrophic cucumberCuiumariacnralaCowles (Echinodermata: Holothuroidea).
larvae ofthe gastropod Halioii\ nifescens. Mar. Biol. 103: 87-94. J. Ev/>. Mm: Biol. Etal. 24: 49-60.
Laemmli,U. K. 1970. Cleavageofstructural proteinsduring the assem- Waterlow, J. C., P. J. Garlick, and D. J. Millard. 1978. Protein
bly ofthe head ofbacteriophage T4. Nutiiiv 227: 680-685. Turnover in Mammalian Tissues and in the Whole Body. North-Hol-
Manahan, D. T. 1983. The uptake and metabolism ofdissolved amino land. Amsterdam.
acids by bivalve larvae. Biol. Bull. 164: 236-250. Welborn, J. R., and D. T. Manahan. 1995. Taurine metabolism in
Pearse,J.S. 1994. Cold-waterechinodermsbreak"Thorson'sRule."Pp. larvae of marine molluscs (Bivalvia. Gastropoda). J. .v/>. Bid. 198:
26-43 in Reproduction, LarvalBi(>tt>^\: andRecruitmentin theDeep- 1791-1799.
