AMER. ZOOL., 22:635-646 (1982)
The Role of Dissolved Organic Material in the Nutrition of
Pelagic Larvae: Amino Acid Uptake by Bivalve Veligers1
DONAL T. MANAHAN 2 AND DENNIS J. CRISP
N.E.R.C. Unit of Invertebrate Biology, Marine Science Laboratories,
Menai Bridge, North Wales, United Kingdom
SYNOPSIS. Studies on the role of dissolved organic material in the nutrition of marine
invertebrates have largely been confined to adults. However larval forms, with a higher
surface area to volume ratio, have a greater weight specific capacity for absorbing dissolved
organic material than adults. Autoradiographic, biochemical, and kinetic experiments with
bivalve larvae all indicate that amino acid uptake and translocation mechanisms can operate efficiently at naturally occurring substrate concentrations. The mechanisms operate
throughout the life-span of the animal, from fertilized egg to adult. Experimental evidence
is presented to show that the kinetics of uptake by larvae allow them to compete with
bacteria for dissolved organic material in sea water. In larvae, supplementary sources of
energy may be more important than in adults since larvae are provided with minimal
food reserves by the parent and must pass through periods when paniculate feeding
cannot occur.
INTRODUCTION
Dissolved organic material (DOM) in sea
water is generally expressed as dissolved
organic carbon. In surface layers of the sea,
dissolved organic carbon ranges in concentration from 0.5 to 3.0 x 10~3 g C/liter
(Williams, 1975). In contrast, particulate
organic carbon occurs at concentrations
approximately one tenth the concentration
of dissolved organic carbon, varying between 0.02 and 0.2 x 10"3 g C/liter (Parsons, 1975). Although dissolved amino
acids represent only 1-2% of the total
DOM, the organic matter of dissolved amino acids is in the same range as that available from phytoplankton (J0rgensen, 1966)
Undoubtedly, DOM in marine ecosystems
represents a very large pool of potential
energy for organisms adaptated to the uptake of these compounds from dilute solution.
Unfortunately, measurements of total
dissolved organic carbon vary considerably
with the methods used (Williams, 1975)
probably concealing spatial variations. For
1
From the Symposium on The Role of Uptake of Organic Solutes in Nutrition of Marine Organisms presented
at the Annual Meeting of the American Society of
Zoologists, 27-30 December 1980, at Seattle, Washington.
2
Donal iVtanahan's present address is: Department
of Developmental and Cell Biology, University of
California, Irvine, California 92717.
amino acid concentrations however, new
sensitive fluorometric techniques (North,
1975; Stephens, 1975), especially when
coupled with High Performance Liquid
Chromatography (Lindroth and Mopper,
1979), are much more precise and reveal
clearer relationships with the origin of the
sea water. Thus for offshore waters, measurements of dissolved amino acids range
from 0.2-0.5 x 10"6 M (Garrasi et al.,
1979), for inshore and estuarine waters
0.2-2.0 x 10-fi M (North, 1975; Lee and
Bada, 1977), while for interstitial water
from coastal sediments Henrichs and Farrington (1979) report values of 1.4—
1.7 x 10~3 g/liter, i.e.,: approximately
14-17 x 10"fi M. We know of no good data
for bottom water close to sediments; however these values must lie between those
for surface inshore waters and interstitial
values. Since larvae are predominantly
found in coastal and often near interstitial
waters, the higher recorded values for
amino acids in solution in sea water are
probably the more relevant. More information on dissolved organic nutrient concentrations in the larval environment is
needed.
The transepidermal uptake of DOM directly from sea water by marine invertebrates has received considerable attention
during this century (see reviews by J0rgensen, 1976; Stephens, 1981). No one now
doubts that radioactively labelled nutrients
635
636
D. T. MANAHAN AND D. J. CRISP
pass through the epidermis and become
incorporated into animal tissue, but
whether this represents a net influx has
been a controversial issue (e.g., Johannes
etal., 1969). However, net uptake of amino
acids in solution in natural sea water has
been established for the adult mussel, Mytilus edulis, using High Performance Liquid
Chromatography (HPLC) and fluorometric detection (Manahan et al., 1982).
The examination of the nutritional role
of DOM has been largely confined to adult
invertebrates. Since the ratio of surface area
to body volume is of obvious importance
in any surface absorption process, larval
forms might well have a much greater
weight-specific capacity for taking up DOM
than adults. Moreover the majority of larval forms (other than arthropod larvae)
have ciliated epithelia covered with microvilli surfaces normally associated with nutrient absorption. Uptake of DOM would
be advantageous to larvae especially as the
energy reserve provided by the parent is
minimal (Crisp, 1974). In the early developmental stages of all larvae, the gut is absent or pooly developed. Indeed some larvae are entirely non-feeding during their
life span. Also the reorganisation of tissues
necessary to transform a larva into an adult
may stop particulate feeding for a period
of several days {e.g., Hickman and Gruffydd, 1971). In all these circumstances a
supplementary role of DOM may be important.
There are some indications in the literature that DOM can be utilized by larval
forms. Fontaine and Chia (1968) using autoradiography noted that, relative to adults,
embryos of the ophiuroid Amphipholis
squamata had a greater capacity for assimilating dissolved glycine and glucose into
their tissues. Bass et al. (1969), experimenting with the polychaete Nereis virens,
compared amino acid uptake rates by larvae with those found in adults; a larva took
up 200 times more leucine per gram wet
weight than did the adult worm. In an
abstract Crane et al. (1957) reported that
larvae of the bivalve Mactra incorporated
[14C]glucose. Tolisand Monroy (1963) stated that radioactive amino acids are incorporated into the eggs of the calm SpLsula
solidissima. More recently, Fankboner and
deBurgh (1977) showed that Pacific oyster
larvae, Crassostrea gigas, could take up a
[14C]labelled algal exudate from sea water.
Rice et al. (1980) demonstrated net uptake
of amino acids from sea water at concentrations exceeding 9 x 10~(i M by larvae of
the European oyster, Ostrea edulis.
Because of their commercial importance, the nutrition and growth of bivalve
larvae have been more exhaustively studied
(Loosanoff and Davis, 1963; Bayne, 1976)
than those of any other marine invertebrate
larva. Walne (1965) who measured feeding
rates, assimilation efficiency, and growth
rates of O. edulis larvae commented that
the concentration of algal cells used in his
cultures was greatly in excess of those normally thought to exist in the oyster's environment. The most rapid growth rate of
O. edulis larvae was obtained when 50-100
cells//u,l of the nanoplankton species Isochrysis galbana were provided. Furthermore, energy-budget calculations based on
filtration rates of O. edulis larvae showed
that the larvae obtained insufficient food
when the algal concentration fell below
50-100 cells/Ail (White, 1976). Yet in determinations of the abundance of nanoplankton in waters over oyster beds counts
of over 10 cells//i.l are uncommon (Waugh,
1957; Driwkwaard, 1961). Since growth
rates of larvae in nature and in the laboratory are thought to be similar (Walne,
1963), an explanation for this inconsistency could be that larvae in nature are using
additional sources of nutrition to supplement the apparently inadequate algal diet.
Other likely sources include particulate detritus, bacteria, and dissolved organic nutrients. Davis (1953) failed to demonstrate
that oyster and clam larvae could utilize
particulate organic detritus to support
growth. Davis also found that none of the
13 species of marine bacteria fed to C. gigas larvae increased their growth. Similar
claims rejecting any contribution from bacteria were made by Walne (1963) and by
Millar and Scott (1967). However, none of
these early investigations convincingly
eliminate detritus and bacteria as important food sources in the nutrition of bivalve larvae. Although the evidence for
DISSOLVED ORGANICS AND LARVAL NUTRITION
DOM uptake cited above is much stronger,
nevertheless whether the uptake of DOM
makes a significant contribution to the energ)' requirements of larvae is still a question.
In this review we shall first describe the
autoradiographic evidence for amino acid
uptake sites by larvae and subsequent
translocation of labelled material to subepithelial tissues. Next to be considered will
be the stage of embryonic development at
which uptake mechanisms are activated.
The biochemical fate of [14C]label after absorption by larvae will then be followed.
The kinetics of uptake will be considered
with particular reference to competition for
dissolved organic nutrients between larvae
and bacteria. Lastly, the role of DOM in
the nutrition of bivalve larvae will be discussed, emphasising the potential that the
availability of axenic larvae offers to this
research.
637
S
B
T H E SITE OF ABSORPTION OF
DISSOLVED AMINO ACIDS
Pequignat (1973) demonstrated by autoradiography that in adult mussels, Mytilus edulis, the gill was the major organ responsible for the uptake of small organic
molecules from sea water. This was an important observation since bivalves maintain high rates of water transport over their
gills and are thus ideally suited to absorb
DOM. In bivalve larvae, the gill has not yet
developed; instead the larva possesses a
velum (Fig. 1A). This large organ consists
of two semi-circular lobes bearing cilia
along their margins and is used for swimming and filtering paniculate foods.
In experiments with larval oysters, C. gigas, 0. edulis, and larval mussels, M. edulis,
which had been exposed for various time
intervals to 1 X 10~B M [3H]glycine (Manahan, 1982), isotope activity first appeared
in the velum (e.g., Fig. IB, C). At 1 min
exposure silver grains were seen over sections of larvae fixed in glutaraldehyde
which binds free amino acids present in
the tissues (Peters and Ashley, 1969). However, Carnoy-fixed larvae showed no radioactivity at this stage, indicating that there
was no incorporation of the label into macromolecules. Within 10-100 min, activity
FIG. 1. A. Diagram of the setting stage (pediveliger)
of C. gigas. ad, adductor muscle, d, digestive diverticula. e, oesophagus, f, foot, g, gill rudiment, m,
mantle, s, stomach, v, velum. B. Phase contrast of
longitudinal section of C. gigas pediveliger. Organs
annotated as in A above. Scale bar is 25 /u.m. C. Same
as B above as seen under dark field illumination
showing sites of uptake in velum and border of foot.
The larva was exposed for 1 min to 1 x 10"" M
[3H]glycine in sea water, fixed in 2.5% glutaraldehyde, sectioned in Spurrs resin.
began to appear in the deeper tissues and
Carnoy-fixed material indicated progressive incorporation. Eventually all tissues
and organs became labelled, as was observed at 16.6 hr with O. edulis veligers.
Experiments with larvae at various stages
638
D. T. MANAHAN AND D. J. CRISP
of development provided similar evidence
of direct epidermal uptake and tissue incorporation of soluble nutrients at comparative time scales. Uptake via the digestive tract was negligible. Scanning electron
micrographs of 0. edulis larvae showed that
there were no adherent bacteria on the
surface of the velum that might have acted
as an intermediate step in transport. The
velum of bivalve larvae must therefore be
considered to function as an organ both
for the transport of paniculate food to the
mouth and for the uptake of dissolved nutrients. It presents an extensive surface area
for absorption and since the velum is extended for swimming, it will continuously
encounter a renewed source of DOM.
After the pediveliger has settled, the juvenile bivalve ("spat") is unable to filter
particulate foods during a period of several days while the velum is being resorbed
and its functional replacement, the gill, is
not fully developed (Hickman and Gruffydd, 1971). The young spat has previously been assumed to rely solely on its
stored energy reserves during this metamorphosis period (Holland and Spencer,
1973). Autoradiographic studies with 24hr-old, 1-2-day, and 3^-day spat of C. gigas, and the 5-6-day-old spat of the scallop, Pecten maximus, have shown that all
these stages are capable of absorbing
[3H]glycine from a 1 X 10"6 M solution
mainly via the developing gill buds (Manahan, 1982). These studies with setting larvae have linked the phenomenon of larval
uptake with the extensive literature on the
uptake of dissolved nutrients by adult bivalves (e.g., C. gigas, Bamford and Gingles,
1974; M. edulis, Wright and Stephens,
1978).
ACTIVATION OF AMINO ACID
TRANSPORT MECHANISM
Since veligers, pediveligers, spat, and
adults can absorb dissolved amino acids,
the question arises as to when transport
mechanisms in bivalves become activated.
Experiments with unfertilized eggs of C.
gigas showed that they did not accumulate
dissolved glycine from sea water even after
three hours exposure (Manahan, 1982).
Figure 2 illustrates activation of glycine
3500
3000
2500
2000-
1500
1000-
500
Sperm
0
30
60
90 120
Time (mil)
150 180
FIG. 2. The uptake of [14C]glycine at 5.7 x 10~7 M
(25°C, 25%c salinity) by C. gigas eggs following fertilization. "F" = addition of sperm to unfertilized eggs.
Eggs and embryos were separated intact from sea
water medium containing isotope by centrifugation
through a sucrose cushion. Similar procedure using
medium with sperm, but without eggs, have the results shown as "Sperm."
transport following fertilization. At 60 min
after the addition of sperm (F), the embryos begin to absorb glycine from sea
water. Activation coincides approximately
with the first cell cleavage of the fertilized
gg
The activation of uptake mechanisms in
embryos is not unique to bivalves. Epel
(1972) demonstrated that mechanisms for
the transport of amino acids in eggs of the
sea urchin, Strongylocentrotus
purpuratus,
were activated 10 min after the addition of
sperm. Adult echinoids are also capable of
absorbing dissolved amino acids (deBurgh
et al., 1977). However, we know of no data
for uptake by setting stages of echinoids.
Thus only for bivalves has it been shown
that uptake mechanisms operate throughout the life span of the animal from fertilized egg to adult.
639
DISSOLVED ORGANICS AND LARVAL NUTRITION
2001
TABLE 1.
Metabolism of [l4C]glycine* by C. gigas pedi-
veligers.
180
Metabolic fraction
Time
160-
10 min
140-
120
100 min
2
- ioa
~
80
60
(10—g
[»C]gl>/larva)
14
CO
2
(% of total)
Total uptake
Lipid
Proteinb
CO 2
TCA soluble
42.3
0.9
16.5
8.4
16.5
2.1
39.0
19.9
39.0
Total uptake
Lipid
Protein"
CO 2
TCA soluble
283.0
4.2
133.6
109.0
36.2
1.5
47.2
38.5
12.8
a
Concentration of [14C]glycine = 5.9 x 10"7 M.
Temp. = 25°C.
b
Denned as insoluble in 5% cold trichloroacetic acid
(TCA).
40-
the oxidation of 1.02 X 10"12 g [14C]glycine
larva"1 min"1. It is important to measure
14
CO2 produced, since in this experiment
for example, the uptake would have been
20
40
60
80
100
underestimated by 38% without such a
Time ( m i n )
correction. Therefore the total uptake of
14
FIG. 3. Time-course of [ C]glycine uptake and ox[14C]glycine from a solution of 5.9 x 10~7
idation by C. gigas pediveligers. [14C]glycine concenM was 2.7 x 10~12 g larva"1 min"1.
tration = 5.9 x 10"7 M. Temperature = 25°C. Salin14
A more complete picture of the fate of
ity = 25%o. Open circles represent C recovered in
larvae (b ± SE = 1.68 ± 0.15). Closed circles repre[14C]glycine after absorption by a pedive14
sent CO2 produced by larvae (b ± SE = 1.02 ± 0.07).
liger is given in Table 1. After 100 min
Data points are x ± SE.
exposure, 47% of the glycine has been synthesized into protein and 38% oxidized to
CO2. Only 2% was used in lipid synthesis,
RATES OF UPTAKE AND METABOLISM
the remainder still being present as low
We have seen from the autoradiograph- molecular weight compounds soluble in 5%
ic studies on veligers and pediveligers that cold trichloroacetic acid. These data may
incorporation into larval tissue takes place be compared with the total energy metabvery rapidly; the time scale of 10-100 min olism of the O. edulis pediveliger as decovers the main period of translocation and scribed by Holland and Spencer (1973).
incorporation. Quantitative measurements Their data showed that 41% of the respiof metabolism of the [14C]glycine were ratory loss in unfed larvae is derived from
therefore performed over this time-course protein (see Crisp,141975), a figure close to
(Manahan, 1982). Figure 3 shows glycine that of 38% for [ C]glycine oxidation by
uptake and oxidation to 14CO2 by C. gigas C. gigas pediveligers. It thus appears that
pediveligers. T h e total uptake of glycine taken up transepidermally is not
[14C]glycine by the animal is the sum of 14C treated differently by the animal from the
in the larva and the I4CO2 produced by the rest of the amino acid pool.
larva. Measurements of radioactivity in the
Bamford and McCrea (1975) and Stewlarva gave the uptake rate as 1.68 x 10~12 art and Bamford (1975) snowed that in the
14
1
1 I4
g [ C]glycine larva"" min" ; CO2 released adult cockle, Cerastoderma edule, and the
by the larva into sterile filtered sea water clam, Mya arenaria, 95% of the labelled
in a parallel experiment corresponded to amino acid taken up remained unmetab20
640
D. T. MANAHAN AND D. J. CRISP
TABLE 2. Kinetic constants1' for amino acid transport by oyster (C. gigas,) and mussel (M. edulis,) larvae.
Species
C. gigas
M. edulis
25°C
15°C
Salinity
Substrate
25%c
32%c
glycine
L-alanine
K, ±SE
3.7 /xM ± 1.0
3.5 fiM ± 0.5
1.8 x 10"9 g glycine larva"1 hr"
0.9 x 10"9 g alanine larva"'hr"
a
Values were obtained by exposing larvae for 5 min periods to substrate concentrations ranging from 10~7
to 10~5 M. K, and Vmax constants were calculated from Hofstee plots (Manahan, 1982).
olized after 1 hr exposure and M. arenaria
required 24 hr to metabolize 50% of the
label in its tissues into macromolecules
(Stewart, 1977). In larvae the time scale is
much shorter, for example by 100 min exposure (Table 1) at least 87% of the absorbed [14C]glycine has been metabolized
either by incorporation into protein and
lipid, or by oxidation to 14CO2. Note in Table 1 that as the exposure time was increased from 10 to 100 min, the amount
of 14C-label recovered in lipid, protein, and
14
CO2 increased correspondingly by about
an order of magnitude. Hence, both the
rate of uptake and metabolism appear to
be linear with time.
KINETICS OF UPTAKE
The relationship of uptake rates by marine invertebrates to ambient substrate
concentration is well described by Michaelis-Menten kinetics. The transport constant K, is the substrate concentration at
which uptake is experimentally determined to be half maximal (i^Jmax)- There
is an extensive literature on the kinetics of
amino acid transport by adult bivalves (see
reviews by Stewart, 1979; Wright, 1982).
Values for Kt, when allowance is made for
unstirred boundary layers, generally range
from 1-5 X 10"fi M. The kinetic data for
oyster pediveligers and mussel veligers
(Manahan, 1982) given in Table 2 show a
K, of 3-4 x 10~6 M for glycine and alanine
uptake. These values accord well with the
values for adult mussels (e.g., Wright and
Stephens, 1978) indicating that the transport mechanisms in the velum of the larva
and the gill of the adult function efficiently
at the micromolar concentrations that occur in natural sea water.
Table 3 summarizes the present data on
bivalve lateral uptake kinetics and compares the data with those of Wright and Stephens (1978) for an adult Mytilus californianus. We have not included data for O.
edulis larvae by Rice et al. (1980) because
the authors express skepticism about their
kinetic data. Their values for K, are an order of magnitude greater than those presented in Table 3. This may have resulted
from mutual interference in the dense larval suspensions (400 larvae/ml) used in their
experiments. On a weight specific basis, it
can be seen in Table 3 that mussel larvae
remove eight times as much amino acid
from sea water as the adults, although the
temperature for the larvae was 5°C lower.
Presumably at equal temperatures weight
specific larval uptake would exceed adult
FABLE 3. Comparison of dissolved amino acid (DAA) uptake rates per unit biomass between larval and adult bivalves.
Animal
(stage; °C>
Crassostrea gigas"
(pediveliger; 25°C)
Mytilus edulisa
(veliger; 15°C)
Mytilus californianus'"
(adult; 20°C)
Organic dry wt.
g D A .A animal
hr
1.84
X
10"
1.8 x 1 0 -
0 .18
X
10 "
0.9 x 10
1.0
9
6.0 x 10 <
" Manahan. 19H2.
h
Wright and Stephens, 1978 (dry wt. estimated as 20c/c wet tiesh wt.).
K,
Molar
Weight specifii Jm
g DAA (g dry
wt.) ' hr '
3.7 x 1 0 -
1.0 x 10 »
3.5 x 1 0 "
5.0 x 10 -1
3.0 x 10 "
0.6 x 10
:1
641
DISSOLVED ORGANICS AND LARVAL NUTRITION
TABLE 4. Comparison of kinetic constants for amino acid
transport by bivalve larvae and bacteria.
Organism
Bacteria"
Larvae"
K,
0.03
3.0
Jmax
1 x 10"12 mol ml-' hr"1
10 x 10"12 mol larva"1 hr"'
a
The biomass of suspended bacteria in 1 ml of sea
water was estimated from bacterial counts of 0.5 x 106
bacteria ml"1 (Hobbie, 1979) and an assumed bacterial volume of 0.5 (im1 with 20% weight of organic
matter, giving 50 X 10~8 g bacteria ml"1.
b
The main organ of uptake, the velum (see Fig.
1A), is assumed to be xh of the total tissue of the larva.
The dry organic weight of the larva is 0.18 x lO"6 g
(Crisp, 1976), giving the velum approximately equal
dry organic weight with that of bacteria present in 1
ml sea water.
uptake by an order of magnitude. Whereas the K, value, indicating affinity for substrate, in the pediveliger of C. gigas is similar to that of larval and adult mussels, the
weight specific uptake is low. In the pediveliger stage the velum is being reduced,
while the gill rudiments are enlarging (Fig.
1A). Since the velum is the major organ
absorbing dissolved amino acids, a drop in
weight specific rates is not surprising. The
limited evidence available suggests that,
weight for weight, larvae can remove much
greater amounts of dissolved amino acids
than adults. This probably results from a
greater surface to volume ratio. A more
fundamental comparison of relative transport efficiencies would be on the basis of
uptake per unit surface area; unfortunately accurate measurements of surface
area of living organisms are beyond presently available techniques.
2
- 100-
0.15
0.45
0.75
1.10
1 .30
Substrate concentration
(micro-moles/1)
FIG. 4. Predicted uptake of glycine by a larva and
its equivalent biomass of bacteria. Uptake rates were
calculated from the Michaelis-Menten equation using
the kinetic constants presented in Table 4.
dons than are invertebrates with a K, of
around 10"H-10~5 M. However, when
comparing the relative transport capacities
of two organisms, it is important to view
the entire kinetic response to substrate
concentration and not just the calculated
Kt. Table 4 shows a comparison of the kinetic constants K, and Jmax for amino acid
THE ABILITY OF LARVAE TO COMPETE WITH uptake by M. edulis larvae and bacteria. The
BACTERIA FOR AMINO ACIDS
bacterial constants are representative valMarine biologists generally consider het- ues for amino acid transport taken from
erotrophic bacteria to be the main users of Sepers (1977). The larval values are from
DOM in sea water (see reviews by Williams, data on alanine uptake by M. edulis larvae
1975; Sepers, 1977; and Newell, 1979, p. (Table 2) but assumed to apply to glycine
344). This conclusion is based largely on a transport also. The bacterial and larval kicomparison of the K, for dissolved nu- netics presented in Table 4 have been comtrient transport by bacteria and inverte- pared on the basis of equal biomass; this is
brates. For instance, Sepers (1977) noted biased in favor of higher Jmax values for
that since bacteria have a K, for amino acids bacteria. If, on the other hand, Jmax values
in the 10~8-10~7 M range that they are had been expressed in terms of equal surmuch better adapted to utilize dissolved face areas, then the corresponding value
substrates at sub-micromolar concentra- for bacteria would have been lower. Know-
642
D. T. MANAHAN AND D. J. CRISP
ing the Kt and Jmax for both a larva and
the equivalent biomass of bacteria, a prediction of their respective kinetic curves can
be made by calculating the rate of solute
uptake at a given substrate concentration
(S) using the Michaelis-Menten equation:
Uptake = (Jmax'S)/(Kt + S). Figure 4 shows
the predicted kinetic response of a larva
and bacteria to concentrations of dissolved
glycine, ranging from 10~8 to 10~6 M. From
this model, it appears that the larva is
out-competing its equivalent mass of bacteria at substrate concentrations above
0.3 x 10~6 M, while below this concentration bacterial uptake exceeds larval uptake. However, even below 0.3 x 10"6 M,
the transport mechanisms in the larva's
velum can still compete with those of the
bacteria for available amino acid. The apparent first order response (linear) of glycine uptake by larvae over a substrate concentration range of 10~8—10~6 M is
consistent with a K, of 3 x 10"6 M for larvae. Conversely, the near zero order
(curved) response of bacterial uptake over
the same concentration range is to be predicted from their low K, of 0.03 x 10~6 M.
Likewise, the consistently greater uptake
by larvae as substrate concentration increases is also expected given the 10-fold
larger maximal transport rate (Jmax) of larvae compared to bacteria (Table 4).
To test the validity of the model proposed in Figure 4, Manahan (1982) incubated M. edulis larvae with natural bacteria
in sea water containing [I4C]glycine at concentrations ranging from 10~8 to 10~B M.
The result is shown in Figure 5. Clearly,
larvae are continuing to absorb [wC]glycine
despite the presence of bacteria. The possibility that [14C]glycine was first taken up
by bacteria on which the larvae then fed
was eliminated by direct counts of acridine
orange stained bacteria. Neither bacterial
growth nor removal of bacteria by larvae
was detected.
The experimental data fit the predicted
kinetic model quite well. Hence, on both
theoretical and experimental grounds larvae with a K, value 100 times that of bacteria can still remove amino acids from sea
water. Inspection of the Michaelis-Menten
equation (see above) shows that high val-
0
0.15
0.75
1.10
1 -30
C-glyclne concentration
(micro-mole s/l)
FIG. 5. Uptake of [l4C]glycine by M. edulis larvae
and natural bacteria (0.4 x 10" ml*1) at 7°C. Larvae
and bacteria were separated by differential filtration
prior to determining their respective radioactivity.
Data points are x ± SE.
ues of Jmax can compensate for high values
of K, at concentrations lower than Kt, while
above Kt, Jmax is the main determinant of
uptake rates. Effective larval uptake relies
both on the high values of J max (Table 4)
and on the fact that certain amino acids
are maintained in sea water above
0.1 x 10 fi M, which prevents bacteria exhausting the supply. It is therefore invalid
to assume that one transport system cannot compete with another solely on the basis of a lower K,.
SIGNIFICANCE OF DISSOLVED AMINO ACIDS
IN NUTRITION
Whether DOM provides an appreciable
contribution to the nutrition of marine invertebrates is still a controversial issue. To
resolve the issue, two criteria can be employed. First, one can measure the proportion of total energy, or of some speci-
DISSOLVED ORGANICS AND LARVAL NUTRITION
fied biochemical component such as
protein, which is derived from DOM. Secondly, the importance of this contribution
to the animal might be tested directly in
terms of growth and survival. The first criterion can usually be met by short term
experiments using labelled amino acids
(assuming net flux). Semi-sterile conditions are sufficient for such experiments as
they terminate in a few hours at most.
Growth and survival studies however, require much longer time scales. Since in the
presence of growing bacteria it would not
be clear whether uptake was direct or indirect via bacteria, this second important
approach has not as yet been pursued.
However, the availability of axenic animal
material would open up this field of research.
We shall now consider bivalve larvae
from both these viewpoints.
Energy calculations
The uptake of dissolved amino acids has
usually been compared with oxygen consumption on the basis that approximately
1 g combined amino acids requires 1 liter
of oxygen for combustion. This oxygen
demand comparison approximates to an
energy demand comparison because major
biochemical constituents of food have almost equal oxycalorific equivalents of 4.8
Kcal/liter O2. The comparison will therefore be on a total energy basis but will be
valid only if all food intake in oxidized aerobically and none is conserved for growth
or reproduction. For larvae, about 70% of
food intake is allocated to growth (Holland, 1978) hence respired oxygen does not
represent total energy uptake. Uptake of
[l4C]glycine by C. gigas pediveligers at 0.6
and 6 x 10~(i M was compared with total
energy uptake of oyster larvae based on
biochemical composition and growth data
from Holland and Spencer (1973) and respiration rates from Manahan (1982). For
this single amino acid, the contribution was
0.6 to 2.8% respectively. Glycine has a low
caloric content, hence its contribution to
growth may be more important than to energy. Assuming 50% of [14CJglycine uptake
is incorporated into protein (Table 1), the
corresponding contribution to protein syn-
643
thesis would be 2 to 9.5%. It should be
remembered that these figures, although
small, refer to glycine which is only one of
many small molecular weight organic compounds in solution in sea water (Williams,
1975). Many of these are independently
transported by marine invertebrates (Stewart, 1979). The combined contribution
from the uptake and metabolism of other
organic compounds could, therefore, be
substantial.
Growth studies using axenic larvae
The availability of axenic microorganisms and phytoplankton allowed major advances to be made in understanding the
nutritional requirements of these organisms. Conversely, the lack of axenic material has severely hampered attempts at
providing chemically defined diets for most
marine invertebrates. Provasoli and coworkers pioneered the use of chemically
defined media to establish the nutritional
requirements of axenic crustaceans, notably Artemia (Provasoli et al., 1959, 1969,
1970). However, since no arthropod has
been clearly shown to take up DOM from
sea water, they are not suitable for such
studies.
Langdon (1980) succeeded in obtaining
axenic cultures of C. gigas. Using axenic C.
gigas larvae, Manahan (1982) carried out
preliminary experiments to determine
whether larvae could grow when presented with a single substrate diet. Larvae
presented with the addition of either dissolved glycine, alanine, or glucose each at
micromolar concentrations, failed to show
any significant increase in shell length over
starved controls, but their tissues appeared
less wasted. Obviously, the dietary requirements of this larva could not be met by a
single substrate. The data in Table 5, taken
from Langdon (1980), clearly demonstrate this point. In order to grow axenic
larvae on dissolved nutrients, they were
presented with a complex Tissue Culture
Medium (199) at a high concentration (1
g/liter) which consisted of 21 amino acids,
13 water soluble and 4 fat soluble vitamins,
8 purines/pyrimidines, and 8 other organic
compounds, one of which was glucose.
Even so, growth was limited. Unfortu-
644
D. T. MANAHAN AND D. J. CRISP
TABLE 5. Beneficial effects on the growth of axenic C.
gigas larvae by the addition of dissolved organic nutrients.'
Mean shell
length (fJ.m)
Diet
Starved larvae
1 g/liter Tissue Culture Medium (199)
"Complex I" particles'1 alone
"Complex I" particlesd with
1 g/liter Tissue Culture Medium (199)
80.0"
88.0b
86.2C
92.7°
a
(Data from Langdon, 1980.)
' Means are significantly different at the 99.9%
level.
d
Egg albumin/starch particle at 100 particles//il
culture.
bc
nately, no tests were carried out with Tissue Culture Medium alone at lower concentrations. Langdon's attempts to grow
larvae on an artificial particulate diet
("Complex I") were equally unsuccessful,
although growth was improved by the addition of the above Tissue Culture Medium. Evidently the artificial diets tested did
not meet the nutritional requirements of
the larvae, even though the concentrations
of nutrients tested in both the dissolved
and particulate fractions of the diets were
considerably higher than those found in
natural sea water. Only by the further use
of axenic cultures will it be possible to define their specific nutritional requirements
and to determine the nature and significance of DOM in the growth and development of bivalve larvae.
CONCLUSIONS
Practically all the evidence presented on
larval uptake of DOM refers specifically to
bivalve larvae. However, bivalve larvae are
probably not atypical since some evidence
exists that echinoderms and polychaete
larvae also possess this capacity. All larvae
have high surface-volume ratios and in the
veliger, uptake is mainly located at the velum surface. Autoradiographic, biochemical, and kinetic evidence all indicate that
amino acid uptake and translocation
mechanisms can operate efficiently at naturally occurring substrate concentrations.
The mechanisms are initiated shortly after
fertilization and continue to operate
throughout larval development and adult
life. There is now experimental evidence
to show that the kinetics of uptake by larvae allow them to compete with bacteria
for DOM in sea water.
Over the range 0.6-6 x 10~(i M glycine,
the rate of uptake contributes only
0.6-2.8% of total energy needs, but 2-9.5%
of the protein synthesis of growing oyster
larvae. As in adults uptake of DOM must
be regarded from the viewpoint of total
needs as supplementary to particulate
feeding, but our ignorance of dietary requirements makes the evaluation of the
importance of micronutrient uptake impossible at present. In larvae supplementary sources of energy are probably more
important than in adults since larvae are
often provided with minimal food reserves
by the parent and must pass through transitional periods when particulate feeding
cannot occur. Clearly, the role of DOM in
the nutrition of invertebrate larvae deserves more attention than it has received.
Further advances in understanding the
role of DOM uptake to larval growth and
development will rely heavily on recently
introduced axenic techniques. These
methods have so far been used only for
oyster larvae, but should be extended to
other species and phyla in the pursuit of
long term nutritional studies. A combination of axenic cultures with recently developed detection techniques, such as High
Performance Liquid Chromatography, now
makes possible the unambiguous evaluation of the role of DOM in the growth and
survival of marine invertebrates at concentrations which are similar to those occurring naturally.
ACKNOWLEDGMENTS
One of us (D.M.) would like to thank
Drs. K. E. Arnold, C. J. Langdon, G. C.
Stephens, and S. H. Wright for many useful discussions and comments on earlier
drafts of this manuscript.
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