Download Aspects of Lipid Metabolism in Crustaceans Department of

AM.
ZOOLOGIST,
8:529-539 (1968).
Aspects of Lipid Metabolism in Crustaceans
JOHN
D. O'CONNOR AND
LAWRENCE
I.
GILBERT
Department of Biological Sciences) Northwestern University)
Evanston) Illinois 60201
SYNOPSIS. Lipid is the predominant organic reserve of many crustaceans and is
important in the metabolism of many of these animals. Ingested lipid is digested by
gastric lipase and apparently absorbed into depot-lipid as .a-monoglycerides. The
variation in the content and composition of the depot-lipid is a function of both the
external environment and internal control systems. Evidence suggests that lipids from
marine organisms contain more long-chain polyunsaturated fatty acids than does the
lipid of fresh water organisms which in turn have a high proportion of ~6 and C18
fatty acids. The fatty-acid composition of the sub-tropical land crab, Gecarcinus
lateralis, resembles that of the fresh-water crustaceans. In addition, our studies indicate
that aspects of lipid metabolism filay be under endocrine control. The induction of
premolt by destalking markedly increases the synthesis of lipid from metabolic
precursors and its subsequent incorporation into the depot-lipid of the hepatopancrease In the late premolt stages there is a decrease in the lipid content of the
hepatopancreas. This occurs as the lipid is mobilized from the hepatopancreas to meet
the energy demands of all those processes resulting in ecdysis. This sinusoidal variation
in the lipid metabolism of the hepatopancreas is influenced by an eyestalk
factor (s),
Until recently, studies regarding the
function and metabolism of lipids in invertebrates have been restricted for the
most part to insects (cf. Gilbert, 1967).
This is partly a consequence of the current in teres t in mechanisms of synthesis,
catabolism, and transport of insect hormones and pheromones, the majority of
which appear to be lipoidal. In addition,
there is little doubt that extraordinary
precautions must be taken in the analysis
of lipids, which unlike proteins, carbohydrates; and nucleic acids, are extremely
pron,e to auto-oxidation and rarely yield a
crystalline product when purified. These
properties have for many years. discouraged the physiologist and biochemist from
studying lipids. Particularly with the advent of thin-layer and gas-liquid chromatography, a renascence in lipid bioThis work was supported by Grants AM-02818
from the National Institutes of Health, and GB6366 from the National Science Foundation.
The senior author was a Pre-doctoral Fellow
(NIH Training Grant GM-903).
We would like to thank members of the staff of
the Bermuda Biological Station for Research for
their help in collecting G. lateralis and providing
facilities for some of the experiments conducted
there.
chemistry has taken place, and the role of
lipid in the life of an organism can now
be studied with routine techniques.
The importance of lipids to the life of
the organism is without question. Not
only do they play a metabolic role and
provide energy for almost all endergonic
processes, but they are of utmost importance in maintaining the structural and
physiological integrity of cellular and
sub-cellular membranes. Their role in
transport of substrates uia the circulatory
system in both vertebrates and invertebrates is a vital one.
Since it now appears that the hormones
controlling metamorphosis (Costlow, 1968)
and molting (Gilbert, 1963) in crustaceans
may be similar to those controlling analogous phenomena in insects, we have turned
our attention to crustacean lipid metabolism to lay the groundwork for studies of
crustacean endocrinology at the biochemical
level. A byproduct of this research has been
a comparison of the lipid composition
and metabolism of terrestrial and fresh
water crustaceans in order to determine
whether, indeed, there has been adaptation
a t this level. The purposes of this
pa per are to generally review the little that
529
530
JOHN
TABLE
D.
O'CONNOR AND LAWRENCE
1. Mean corn-position of hepatopancreas
(Cancer pagurus),
Substrate
% Fresh weight % Dry weight
Protein
Glycogen
Lipid
6.22
0.80
8.23
Modified from Vonk
Renaud (1949).
23.81
2.34
29.02
(1960) after data from
is known of lipid content and metabolism in
crustaceans, present some of our unpublished data on the subject, and point out
the many gaps in our knowledge in this
most important area of research.
LIPID CONTENT
Although lipid was shown to be an organic reserve in the crustacean digestive
gland 75 years ago (Cuenot, 1893), only a
few studies on the regulation of lipid
metabolism have since been conducted,
(e.g.) Scheer, 1957; Vonk, 1960). The im~
portance of lipid to the crustacean is suggested by its percentage contribution to
the fresh weight of the organism and particularly to the hepatopancreas. Although
the average lipid content of Cancer pa ..
gurus is 2.96% of the fresh weight, lipid
constitutes almost 30% of the dry weight of
the hepatopancreas (Table 1). This is
about double the relative quantity found
in the liver of several species of mammals
(Altman and Dittmer, 1964). Although
measurements of total lipid content point
to its importance as a metabolic reserve,
they give little definitive data. Perhaps a
qualitative analysis of the fatty acid composition of these lipids will suggest a role
in terrestrial adaptation.
COMPOSITION OF FATTY ACIDS
Fatty acids are most commonly composed of an even number of carbons in a
straight chain, and when unsaturated,
usually have a double bond between C,
and C 1 .O • Of all the known fatty acids, oleic
acid is the most abundant in nature.
Typically, oil extracted from marine organisms contains a relatively high proportion of long chain, polyunsaturated fatty
I.
GILBERT
acids, whereas the lipid of fresh water
forms characteristically yields a relatively
larger quantity of saturated C I 6 and CIS
fatty acids. Table 2 reveals that the marine crustaceans, P. borealis and M. norvegica, do indeed contain significant
quantities of highly unsaturated (polyenic) fatty acids. The saturated fatty acids
account for 21.5% of the total content of
fatty acids of P. borealis and 22.7% in M.
noroegica, while the monoenic and polyenic fatty acids constitute 45.8% and 32.6%,
respectively, of the total fatty acids in P.
borealis and 52.3% and 24.7% in M. norvegica (Ackman and Eaton, 1967).
The terrestrial form, Gecarcinus latera lis, possesses a pattern of fatty acids more
closely related to that of the fresh water
species than of the marine forms. That is,
the hepatopancreatic lipid of Gecarcinus
contains a relatively high percentage of
C I S : 1 and C 18 : 2 fatty acids. In addition)
whereas the fatty acid composition of
phospholipids and glycerides is dissimilar
in marine crustaceans (Ackman and
Eaton, 1967), they are remarkably similar
in Gecarcinus.
Unfortunately, for the various fatty acids we know little about the adaptive significance of their structural features
(Rhodes, 1964). The more unsaturated a
fatty acid is, the more it tends to remain
in the liquid state at low temperatures.
Differences between the fatty-acid composition of marine and fresh-water organisms may not be due to the salinity of the
environments, but to the size of the body
of water (Farkas and Herodek, 1964).
One consequence of smaller, fresh-water
ecosystems is that they show a greater rise
in temperature in the summer than does
the ocean and thus inhabitants of such
an environment would profit from utilizing a more saturated fatty acid as depot
and structural lipid.
Fresh water forms depend to a greater
degree on food originating on land and
thus have "more in common" with terrestrial forms than do marine organisms. Gecarcinus itself is a semi-tropical terrestrial organism not subjected to very low
Ackman and
Eaton, 1967
4.8
2.0
15.3
4.9
8.9
Ackman and
Eaton, 1967
0.2
4.8
0.6
15.2
11.8
0.7
1.3
15.3
1.3
Trace
Trace
10.5
1.1
9.3
13.1
8.1
0.3
2.9
0.5
14.9
14.1
0.3
2.6
20.2
0.8
1.0
......
Total lipid
Zandee, 1967
1.6
1.4
17.7
10.3
5.8
3.9
29.9
0.1
Trace
Trace
10.7
Trace
Hepatopancreatic lipid
M. noroegica H. gammarus
Total lipid
P. borealis
Zandee, 1966a
Wolfe, et al., 1965
O'Connor and Gilbert
O. virilis
Hepatopancreatic lipid
NL
PL
O'Connor and Gilbert
G. lateralis
Hepatopancreatic
lipid
NL
PL'
Terrestrial
=II:
NL == neutral lipid; PL == phospholipid.
Values are those of winter animals.
t Carcass lipid without exoskeleton.
t Number preceding the colon represents the number of carbons in fatty acid. The number following the colon represents the number of unsaturated
carbon-to-carbon bonds.
The hepatopancreas of o. virilis and G. lateralis was extracted with chloroform-methanol (2: 1). The extracted lipid was fractionated by column chromatography using activated silicic acid. The neutral lipids were eluted with chloroform and the phospholipids with 100% methanol. The methyl esters
of the fatty acids were obtained by methanolysis and were analyzed with an' F & M gas chromatograph equipped with a flame ionization detector. The
samples were injected at a port temperature of 200°C onto a 6% DEGS column at 170°C. The carrier gas was argon delivered at 30 ml /rnin from an
outlet pressure of 40 psi.
Reference:
upto 14:0t
14:0
15:0
16:0
16:1
17:0
18:0
18: 1
18:2
18:3
20:0
20:1
20:4
20:5
22:1
22:6
Fatty acid
(number of
carbons)
2. Percent fatty acids in lipids from crustaceans living in different environments.
Marine
TABLE
"'""""
~
~
en
>
Z
~
~
en
C
~
o
Z
a::
C
en
o
~txl
t%1
~
~
o
~
~
~
532
JOHN
D.
O'CONNOR AND LAWRENCE
temperatures and thus would be expected
to have less polyenic fatty acids if the
above hypothesis is correct. We wish to
stress that this is speculation and in fact
there is good evidence that the environmental temperature has no effect on the
character of lipid synthesized (ct. Van
Handel, 1966).
There have been suggestions that the
fatty-acid composition of crustaceans
varies with the stage of the molt cycle.
For example, a greater relative quantity
of C I S : I was found in Astacus lipid in December than in April (Zandee, 1966a).
Of course this may be a consequence of a
change in the composition of the diet
rather than biosynthetic mechanisms, but
our data do. indicate a rise in CIS:1 of the
phospholipid fraction of the hepatopancreas from destalked Gecarcinus compared to controls. This suggests the possibility of endocrine control. The fattyacid composition of an animal is a conseqllence of both biosynthesis and diet.
What is known about digestion and absorption of lipids in the Crustacea?
DIGESTION AND ABSORPTION OF LIPIDS
I.
GILBERT
been demonstrated in both the gastric
juice and the hepatopancreas of Astacus
astacus and Cambarus affinis (Kleine,
1967). Although four esterase fractions
have been electrophoretically separated
from Astacus hepatopancreatic tissue,
only one of these fractions appears to be
present in the gastric juice, and' its exact
function is unknown. The pH optimum
of the crustacean gastric lipase falls in the
acidic range in all cases studied· (Agrawal,
1964; Kleine, 1967; Vonk, 1960): In the
case of hepatopancreatic lipase.Q.owever,
there is a shift in optimal pH to the alkaline range (pH 8 to 9) (Kleine~ 1967).
As in the case of insects, there are few
data available regarding absarption of
lipid in crustaceans. Van Weel (1955)
suggested that lipid droplets observed in
the L cells of the hepatopancreas were
not phagocytized but rather resynthesized
from the fatty acid and glycerol products
of gastric digestion. Recently, an elegant
experiment by Brockerhoff and Hoyle
(1967) demonstrated that the digester'
lipid is finally absorbed into the depotIipid in the form of p-monoglycerides and
fatty acids. In this experiment the triglyceride containing oleic-D, 10-3 H acid in
the fi position and 0Ieic-I- I4C acid in the a
position was fed to lobsters and the distribution of label in the depot-lipid then
analyzed. The original ratio o£3H/14C at
the f3 position was to a large extent retained while the ratio at the. a position
was randomized, indicating retention of
the original p-rn;onoglyceride structure
during triglyceride digestion and absorption. Once lipid is stored, it is then available for catabolism, and the energy released can be captured in the form of
high energy compounds such as ATP.
Digestion of dietary lipids occurs in the
pyloric region of the crustacean stomach.
The major digestive enzyme acting upon
lipids in the gastric juice appears to be a
Iipase hydrolyzing the esteric bonds of
triglycerides. Release of fatty acids from
olive oil by the digestive juices of Astacus
was demonstrated almost 100 years ago
by Hoppe-Seyler (Vonk, 1960). Thereafter, crustacean enzymes capable of splitting the esteric bonds of lipids were
studied in some detail (Yonge, 1924;
Hasler, 1935, 1937).
Mechanisms of lipid digestion In
METABOLISM OF LIPIDS
crustaceans appear to be the same as
those known in mammals and insects
The organ of central importance in a
(Brockerhofl, et al., 1967; Gilbert, 1967). discussion of crustacean metabolism is the
For example, the gastric lipase of the lobhepatopancreas, which is analogous to
ster attacks the primary esteric bond of the vertebrate liver and insect fat body.
the triglyceride so that the hydrolytic pro- . The early work of Smith (1915) and Paul
ducts are first a,. ,a-diglycerides followed by and Sharpe (1919) demonstrated the
p-monoglycerides. Esterase activity has sinusoidal variation in hepatopancreatic
LIPID METABOLISM IN CRUSTACEANS
533
tivity of both the neutral lipid and the
phospholipid fractions following destalking. Sixteen days .after removal of the
400
eyestalks, the specific activity of the neuPhospholipid
Neutral Lipid
tral lipid fraction is close to that of the
controls,
whereas the specific activity of
~300
:.J
the phospholipid fraction remains at an
E
increased level in the destalked animals.
"f> 200
I t should be noted that the specific activity of the neutral and phospholipids from
destalked animals decreases dramatically
100
from day I to day 16 after the operation,
while that of the control group' is more
constant. Along with the decrease in spe16
7
7
16
I
cific
activity from day I to day 7 is an
Days After Destalking
increase
in the quantity of hepatopanFIG. 1. The specific activity of neutral lipid and
creatic
lipid
in the destalked land crab.
phospholipid fractions of hepatopancreatic lipid
From day 7 to day 16, however, there is
from normal and destalked G. lateralis. Animals
were sacrificed 4 hr after the injection of 10 P.c of
no appreciable increase in hepatopanHe-I-acetate into the hemocoel through the arthrocreatic lipid, and the decrease in specific
dial membrane. Solid bars, destalked animals. Open
activity
represents either a decrease in net
bars, control animals.
synthesis of lipid or possibly an increased
lipid during the 'molting' cycle. These ear- rate of lipid catabolism. These data sugly observations were extended by Renaud g-est that a factor (s) in the eyestalks influ(1949) who demonstrated a rise in hepa- ences lipid metabolism.
To test this hypothesis, a saline extract
topancreatic lipid content until midpremolt, followed by a decrease after of eyestalks was administered to crabs and
ecdysis. This variation in the hepatopan- the incorporation of labeled acetate folcreatic lipids of Cancer differs from the lowed in the hepatopancreatic lipids
data of Neiland and Scheer (1953) work- (Fig. 2). The total incorporation of 14C_
ing with Hemigrapsus nudus. They found acetate into lipid is ag-ain appreciably
no decrease in the lipid content of inter- higher in animals destalked for 10 days
than in normal animals. Administering
molt animals after 23 days of fasting.
However, removing the sinus gland led to eyestalk extract partially reverses the
a marked decrease in the content of lip- effect of destalking while administering- a
id. The histochemical data of Travis saline extract of gills to controls is with(1955) on the lobster, Panulirus argus, out effect, This supports the postulate
confirmed the stage-dependent variation that an eyestalk factor (s) is involved in
the regulation of lipid metabolism in Geof hepatopancreatic lipid.
The effect of destalking upon the meta- carcinus.
bolism of lipids has been investigated in
Additional studies on incorporation of
our laboratory using several crustacean substrate have been performed using- 14C_
species. The results of a typical experiI-palmitate and 14C-glycerol as lipid prement performed with G. lateralis are il- cursors. The results of these experiments
lustrated in Figure I. Four hours follow- support our earlier results in that de,ing the injection of 14C-I-acetate, the ani- stalked animals incorporate these labeled
mals were sacrificed, the total lipid ex- substrates into lipid at a much greater
tracted from the hepatopancreas, chroma- rate than controls. Experiments in vitro
tographed into neutral and phospholipid utilizing the isolated hepatopancreas of
components, and radioassayed. The re- both G. latera lis and o. v irilis also
suI ts reveal an increase in the specific ac- demonstrate a dramatic increase in the
500
01
~
JOHN
140
~
120
l-
D.
O'CONNOR AND LAWRENCE
....
'~2 100
if(J
....
-
If)
f-
....
-
80
~
60
40
-
~
f-
I 2 3 4
r.
234
A
B
FIG. 2. Total counts per minute (CPM) incorporated into the hepatopancreatic lipid of male G.
lateralis following the injection of 10 p'c of HCl-acetate. Group I, normal animals. Group 2, animals destalked 10 days prior to injection of isotope.
Croup . 3, destalked animals injected with three
eyestalk equivalents in saline on alternate days
during a 6-day period. Group 4, destalked animals
injected with a saline extract of gill tissue following
the same protocol used for the eyestalk injection. Experiment A: animals sacrificed 2 hr after
injection of acetate. Experiment B: animals sacrificed 8 hr after injection.
capacity of the hepatopancreas of destalked animals to synthesize lipid (Fig.
3). The rise in the specific activity of the
hepatopancreatic lipid of destalked G.
lateralis from day 20 to day 40 is due, at
Ieast in part, to a decrease in lipid content. When similar in vitro studies are
performed with o. virilis hepatopancreas
using differentially labeled glucose as substrate, the results indicate that the rate of
incorporation into hepatopancreatic lipid
decreases after destalking (Table 3).
Similar results have been reported by
Zandee (1966 b) for Astacus.
In addition, it has been reported that
the induction of premolt activity by removing eyestalks leads to a reduction in
the activity of the pentose phosphate pathway with a concomitant increased rate
of glycolysis (McWhinnie and Chua,
1964). The data in Table 3 support this
thesis by demonstrating a decrease in the
ratio of 14C0 2 originating from 14C-
I.
GILBERT
1-glucos'ej14C-6-glucose from 1.68 in inter..
molt to 1.50 in premolt. However, such
an increase in the glycolytic activity of the
hepatopancreas should not a priori suggest a subsequent increase in the incorporation of label from glucose into hepatopancreatic lipid. It has been shown that
less than 1% of the label administered as
14C-glucose can be recovered in intermediates of the Krebs cycle (Huggins, 1966).
Most of the 14C recovered from 14C-glucose
appears in alanine and to a lesser extent
in lactic acid. These data suggest that the
degradation of glucose via the glycolytic
pathway terminates principally in the
transamination of pyruvate to alanine or
in its reduction to lactic acid rather than
by oxidative decarboxylation of pyruvate
to acetate.
The observation that the lipid of the
hepatopancreas is labeled when incubated
in the presence of radioactive glucose
indicates either that some small percentage of the glucose is oxidized to acetate
and then synthesized into fatty acids, or
that the label from the glucose finds its
way into lipid via a-glycerophosphate.
The former suggestion is supported by
our data demonstrating the evolution of
.-----------------~
I
4.5
I
I
I
4.0
I
3.5
2.5
-I
01
1.0
0.5
01
E
40 :2
....
.9..J
I
2.0
~
a. 1.5
0
50i
at
I
I
.~
s
i
I
I
I
I
I
I
'0
- 3.0
:'S!
T
6Ol.
I
I
I
If)
70
.~
300
e
I
I
I
I
20
•
o----~------------------~
i
J
10 ~
0
0
10
20
30
40
DAYS
FIG. 3. The in vitro incorporation of HC-I-acetate
into the hepatopancreatic lipid of G. lateralis. The
solid lines refer to the left ordinate which
expresses the data as specific activity. The dashed
lines refer to the right ordinate which expresses
the incorporation as a function of fresh weight.
Open circles (0), normal animals; solid circles (.),
animals destalked at zero time.
535
LIPID lVIETABOLISM IN CRUSTACEANS
TABLE
3. The metabolism of
and 1~C-6-glucose by the hepatopancreas of the crayfish,
'0. virilis, in vitro.
l~C-'l-glucose
.
14C02 CPM!
mg fresh wt.
EC-I ~gl ucose 14C-6-g1 ucose
Stage
CS-4 (Intermolt)
D0-3 (Premolt)
131 (6)
51 (12)
CPM! mg lipid
HC-I-gI ucose 14C-6-g1ucose
78 (8)
34 (11)
7590 (8)
2501 (16)
9908 (8)
3149(16)
CPM in totalli pid/
mg fresh wt.
HC-I-gl ucose 14C-6-g1ucose
183 (6)
144 (12)
215 (6)
190 (11)
The excised tissues were incubated for one hour at 30°C in the presence of 1.5 P.c of the labeled substrate. - Both types of labeled glucose molecules possessed equal specific activities. In every experiment
the hepatopancreas from each animal was divided equally. Identical halves from each animal were then
incubated in the presence of either 14C-l-glucose or 14C-6-glucose. The numbers in parentheses indicate
the number of samples.
14C0 2 from glucose labeled in the 6 position since it can only be oxidized by way
of acetate and the Krebs cycle. The second suggestion is supported by the high
rate of incorporation of 14C-glycerol into
hepatopancreatic lipid. Decrease in the
rate of incorporation of label from glucose in to lipid as the animals pass from
intermolt to premolt could then be the
effect of dilution of isotope resulting from
an increased glycolytic rate.
The oxidation of lipid sU'bstrateshas
also been investigated in the shore crab,
Carcinus maenas, where the oxidation of
14C-Iabeled fatty acids has been demonstrated (Munday and MUl\n, 1962), and
the presence of fatty acids has been shown
to cause an increase in Q02 (Munday
and Munn, 1965). No quantitative differences in the rate of oxidation of fatty
acids have been demonstrated during different stages of the molt cycle although
such variations have been implied (Bliss,
1953).
In order for lipid to be utilized at different body sites it must be transported to
them from the storage depot. How is this
accomplished in crustaceans?
-and Scott. {I966) reported that' almost
65% of the total1ipid present in thehernolymph of rhe lobster is phospholipid and
the remainder is almost equally divided
between triglycerides and sterols, with
only a small amount .(2.4%) of free fatty
acids. We have extended these findings to
Cardiosoma guanhumi and Gecarcinus
lateralis. The virtual lack of partial glycerides (mono- or diglycerides) in the hemolymph of these crust.aceans contrasts
sharply with the lipid composition of insect hemolymph, in which there is an
abundance of diglycerides (Chino and
Gilbert, 1965).
Our 'experiments with G. lateralis have
demonstrated an increase in the specific
activity of lipid in hemolymph following
destalking (Fig. 4). This occurs despite
the decrease in the total concentration of
lipid in the hemolymph of animals destalked 10 days previousiy (O'Connor and
500
5OOs:;
0..
~
"0
E
:!
~
l"""-
400~
:.J
1
I--
TRANSPORT OF LIPID
Any hypothesis involving the mechanism of lipid transport must be based on
knowledge of the lipids present in the
hemolymph. The paucity of· information
available on lipids found in the hemolymph
of crustaceans did not even permit a
discussion of the subject in one review
(Florkin, 1960). Recently, however, Bligh
100
300
J
~
Q.
o
~
FIG. 4. The specific activity of hemolymph lipid of
male G. lateralis following the injection of. 10
P.c of He-I-acetate. Animals were des talked 10 days
prior to the injection of isotope. The right ordinate
expresses the radioactivity present per unit volume
of hemolymph. Solid bars, destalked animals, Open
bars, control animals.
536
JOHN
D.
I.
O'CONNOR AND LAWRENCE
P"-4~
~
]bS:a
a
0./'0 •
0...,
°
~c:c:~
~ ....
P"-4
Cl:S
""O~C:
..., Cl:S
GILBERT
V,)
~.0-4
~~~~
..... O"'l:f4CC')
t-o:~
"'l:f4CC')
~ c..~""
n
A
It')lt)CO
tO~lL"i
ocuQj]
~~ M =
~
o
o .5
n
-
B
}I'IG. 5. Ratio of the specific activity of the hepato-
pancreatic lipid to the specific activity of the hemolymph lipid in normal and destalked G. lateralis.
.Animals were injected with 10 p'c of He-I-acetate and
sacrificed at 2 (A) and 4 (B) hours. Solid bars,
destalked animals. Open bars, control animals.
oa~
..., =0 V,)
=.0-4 • Cl:S
....."t:S
..., cu
"Ocua:1-I
~
Cl:S
S..d
I
V,)
v.g~g
1-1 ~
00 co
e-.i"";lt')(O
...
~g~
~
..........
C'fCC')
lC)"'¢l
~"l:f'Io)
oooc.o
CC')CC')(I(')
~..oS~
~=I-Icu
U U cu...c::
.5 ~
• "0
Gilbert, unpublished) . This decrease
coupled with an increase in the specific
activity of the lipid results in similar cpm
values for equal volumes of hemolymph
from normal and destalked animals. The
ratio of the specific activity of hepatopancreatic lipid to the specific activity of lipid in hemolymph is a measure of the
relative rate of release of the 14C-lipid
from the hepatopancreas into the incubation-medium. The fact that this ratio is
higher in destalked animals indicates
that although the hepatopancreas of destalked animals synthesizes lipid at a
greater rate than controls, it releases this
newly synthesized lipid at a relatively
lower rate (Fig. 5). Therefore, the increase in synthesis of lipids in the early
stages of premolt is probably not compensated by a more rapid release of lipid into
the hemolymph.
Preliminary in vitro experiments with
prelabeled hepatopancreas support the
above, since the release of lipid from the
hepatopancreas into the medium is not
increased in destalked animals (Table 4).
In fact, the amount of lipid released
from the hepatopancreas of destalked animals is actually less than from controls
when adjustments are made for the
amount of labeled precursor incorporated
into lipid. The rate of release of lipid
from the hepatopancreas increases in the
later stages of premolt (stage D 3 ) . This
~
Cl:S
cuC:~c..
.
B·a~
a:.::..d
].~ ~
=Cl:S~
.0-4
cu bO
~tJs
00 00"l:f'l 00
CNlr)~O
..... O')OO~
..... ~C(')~
0C'f C'f(o~
00 CC') lr)ooCN
~oo
~ei
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LIPID METABOLISM IN CRUSTACEANS
537
til very late in stage D, just prior to
ecdysis (Adelung, 1967).
Our data demonstrate an increase in
synthesis of lipid immediately after removing the eyestalks. This increase
reaches a plateau in mid-premolt. In late
premolt stages there is a decrease in synthesis of lipid coupled with an increase in
the rate of their release from the hepatopancreas. Studies in which we have attempted to stimulate an increase in synthesis of lipids by the addition of crustecdysone both in vivo and in vitro have
so far been unsuccessful. Thus; the possibility remains that removing the eyestalks
alters metabolic events by removing the
sources of molecules that normally regulate or inhibit specific metabolic steps. It
is possible, therefore, that removing the
eyestalks resul ts in the dramatic increase
in synthesis of lipids that is characteristic
of the early premolt stage, while an increasing titer of molting hormone during
the later premolt stages stimulates a
marked release of lipid from the hepatopancreas and general catabolism of lipid.
Past literature in crustacean endocrinology favors the interpretation that removing
CONCLUSIONS AND SUMMARY
the eyestalks stimulates the v-organs,
which
in turn secrete molting hormone
There is a factor (s) in the eyestalks of
several decapod crustaceans that appears and thus bring about various metabolic
to restrain or regulate the biosynthesis of alterations that culminate in ecdysis. Yet
lipids. When this factor is absent due to the possibility of a direct effect of an
extirpation of eyestalks, there is a resul- eyestalk factor (s) upon the hepatopan..
ting increase in the synthesis of lipid from creas and perhaps upon other organs as
precursor molecules. Extirpation of eye- well cannot be ruled out.
stalks results in numerous other metaIf the polyhydroxy steroid, crustbolic alterations as well. These effects may ecdysone (possibly the molting hormone
be due to the absence of an eyes-talk of crustaceans), can be termed a lipid,
hormone (s) or to an increase in the ac- then the study of the biosynthesis of molttivity of the Y-organs. Present data do not ing hormone is a problem in the bioallow us to distinguish between these al- chemistry of lipids. In addition, the
ternatives. Although molting is inhibited crustacean eyestalk is a rich source of maby removing both the eyestalks and Y-or- terial with the activity of juvenile horgans (Passano, 1960), there is no evidence mone when assayed on insects (Schneithat such surgical procedures inhibit the derman and Gilbert, 1958). The juvenile
induction of metabolic events characteris- hormone in silkmoths has been shown to
be a terpene derivative (Roeller, et al,
tic of the early premolt stages. On the
other hand, there is some indication that 1967). Thus, we can predict exciting dethe titer of molting hormone in Carcinus velopments in the study of the biochemistry of lipids in crustaceans.
maenas does not increase appreciably un-
increase correlates with the decreased rate
of incorporation of 14C-palmitate into hepatopancreatic lipid. Thus, immediately
following destalking and in the early
stages of premolt there is an increase in
synthesis of lipid in the hepatopancreas
that is not coupled with an increased rate
of release. The result is an accumulation
of lipid. In the late premolt stages there
is a decrease in the rate at which lipid is
synthesized and an increase in the rate at
which .it is released, resulting in a net
decline in the content of lipid in the hep,atop'ancreas. This decline continues until the enforced starvation-period of the
molt cycle ends in stage C (Renaud,
1949).
Numerous questions remain unanswered concerning the release and transport of lipids in crustaceans. What is the
chemical nature of the released lipid? Is
the transported lipid bound to a specific
protein? Are transport and release of lipid hormonally mediated? These questions
are currently being studied in this laboratory.
538
JOHN
D.
O'CONNOR AND LAWRENCE
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