Mark Roberts
Anaerobic metabolism in snails
ABSTRACT
The existence of an anaerobic metabolic pathway which accumulates
alanine and succinate was tested for in two snails which inhabit the
intertidal region of the central Californian coast. Anaerobic con¬
ditions were induced by maintaining experimental animals in nitrogen
flushed vials for 64 hours after which the whole soft tissue was
assayed for alanine, succinate, and lactate. These assays were com¬
pared against the same for a set of controls. Results did not de¬
monstrate an accumulation of more alanine, succinate, or lactate in
the snails under anaerobic conditions than in the aerobic controls.
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Mark Roberts
Anaerobic metabolism in snails
INTRODUCTION
Nst of the animals which inhabit the midtide forest are adapted
for metabolizing carbohydrates via the oxygen requiring respiratory
pathway common to most members of the animal kingdom. Being ma-
rine, they are primarily adapted for respiration in an aquatic me¬
dium, though many are quite capable of aerial respiration when the
tide is out. (12,14).
However the capacity for aerial respiration is functional on¬
ly so long as the gill surface of the animal remains relatively
wet.(12,14). When evaporative water loss becomes severe,aerial
respiration must be halted to prevent the possibility of death due
to dessication. (12,14).
Numerous well known strategies and combinations of strategies
are employed by the intertidal invertebrates for coping with this
problem. These include: the use of oxygen stored in body fluids
via respiratory pignents such as in the worm Urechis.(12), the use
of oxygen trapped in mantle cavities as for example in certain.
Littorine snails (12), and the use of some form of metabolic slow
down akin to hibernation, such as the lowering of heart rate which
occurs in the bivalve Mytilus Edulis (12).
A final strategy, and the one of particular relevance to this
2 -
Mark Roberts
Anaerobic metabolism in snails
paper is the transfer from aerobic to anaerobic metabolism which
often occurs after the potential of the other methods has been
exhausted(7,12,14,20).
In most species, the switch to anaerobic metabolism is a
switch to the lactate producing pathway of glycolysis (8). But not
all invertebrates accumulate lactate during glycolysis (11,21). A¬
nother glycolytic pathway that employs the linkage of amino acid
with carbohydrate metabolism has been shown to exist in a number
of invertebrates (1,7,8,18) including the parasitic round worm
Ascaris (15), and the intertidal bivalves Rangia, Crassostrea,
and Volsella (2,6,18,19). The pathway is shown in figure 1. Some
features to note in it are: 1). 8 moles of AIP are produced as
oppossed to the 2 moles made during lactate-producing glycolysis
and so it conveys an adaptive advantage to any organism capable of
using it. 2). it accumulates succinate, alanine, and sometimes
propionate instead of lactate.
The existence of this pathway was investigated in two of the
gastropods which inhabit the intertidal zone of central California 's
rocky coast - Nucella Emarginata, and Acanthina Puntulata. These
were selected for the following reasons: One of them, the Nucella
belongs to a genus in which some other species have been shown to
possess quantities of the enzymes malic dehydrogenase, lactate dehy¬
drogenase, pyruvate kinase, and pyruvate-carboxykinase in proportions
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Mark Roberts
Anaerobic metabolism in snails.
that are characteristic of the animals which are known to have this
pathway (2,16,17,18). In the lab, both species demonstrated an
ability to survive for ar least 30 hours in a nitrogen flushed en¬
vironment - in the case of Nucella, the length of survival exc eded
5 days for 75% of the specimens. Both species are left dry for
reasonably long periods during low tide, and are therefore  suscep¬
table to the type of dessication stress that leads to anaerobiosis.
Both have shells into which they may retreat during dry periods,
thus closing off the organism from the oxygen containing medium
which surrounds it.
The build up of the end products alanine, and succinate can
be used as a means to detect the existence of the pathway. The
hypothesis upon which this paper is based is this - If this path¬
way is utilized in these snails, then those which have been placed
under conditions which would induce anaerobiosis should possess a
greater amount of these end products in their tissue then thos
left to respire aerobicly. Furtheremore, there should be no sig¬
nificant difference in the lactate levels between the two. An
assay for lactate was therefore also added.
MATERTALS AND METHODS
Two snails each were placed in 30 ml vials containing 0.5 ml
of seawater, all flushed with 99.9% nitrogen gas for 60 seconds.
The vials were capped, and taped closed, and the snails left to
Mark Roberts
Anaerobic metabolism in snails
respire for 64 hours in a bath of 17° C water. An equal number of
controls were placed in open vials and kept under the same condi¬
tions except that there was free exchange of oxygen between the
vial and the air.
After the test period, all animals were frozen in liquid
nitrogen, and homogenized in perchloric acid according to the pro¬
cedure of Williamson (24) with only one modification; the whole
snail was dropped into the liquid nitrogen instead of being frozen
between freeze clamps. The extracts were then assayed for the
end products according to Williamson (23) for alanine ; Williamson
(24) for succinate with a modification in the concentration of AlP
by a 10 fold increase; and according to Gutman (5) for lactate.
All assays were spectrophotometric measurements of the change
in absorbtion resulting from a change in the concentration of NADH
according to the equations shown in figure 2. Figure 2 also gives
a flow chart of the major steps in the experiment.
RESULITS
Based on an average consumption rate of 124 ul/g/hr of oxygen
for a group of similar gastropods (4,12), and on an average animal
weight of 2 grams per vial for the Nucella, and 5.3 grams'per vial
for Acanthina, theloxygen in these vials should have been exhausted
in less than 23 hours in the case of Nucella, and in less than 9
5 -
Mark Roberts
Anaerobic metabolism in snails
for the Acanthina - even if the vials were not flushed with any
nitrogen. Nitrogen flushing served to minimize both the possibilty
of aquatic and aerial respiration, and also it minimized the possi¬
bility of death due to dessication because seawater could be left
in the vial.
A preliminary check on the sensitivity of the assays showed
the alanine and lactate assays to be about 10 times less sensitive
than expected. The succinate assay did not work at all, presum¬
ably because of instability in the enzyme succinic thiokinase which
initiates the whole reaction. The succinate assay was therefore
eliminated. The results (in change of absorbance) of the remaining
two assays are shown in figure 3.

CUSSIOV
The high number of negative values for both the aerobic and
anaerobic samples are difficult to explain. Both normal change in
absorbance in blanks, and when NADH concentration is changed cause
only positive changes in absorbance. Negative readings may have
resulted from localized heating in the cuvettes, producing convec¬
tion currents which alter the absorbance of the solution. Another
possibility is that there are some other compounds present in the
mixture whose absorbance in UV light also decreases upon addition
of the final reagent.
The other explanation of negative results is that these numbers
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Mark Roberts
Anaerobic metabolism in snails
represent random changes in the overall absorbance of the solu¬
tion. If this were the case, all positive results within the same
range of absolute values would also have to be considered random.
Indeed, statistical tests indicated that the whole of the values
obtained for both the aerobic, and anaerobic extracts were not
significantly different than zero. This of course means that no
difference between the experimental and control animals could be
determined.
Zero readings lend themselves to two interpretations. 1).
there is no measurable increase in the alanine or lactate concen¬
trations when snails are kept under anaerobic conditions over when
they are not, and so the animals do not possess the pathway in
question, or they utilize a similar pathway which may accumulate
succinate for exanple, but does not accumulate alanine or lactate.
2). The assay itself is not sensitive enough to measure the small
concentrations of these products which for alanine should be near
.78 mg/gram fresh tissue - assuming a value near the average for
other gastropods (3).
In light of the statistical finding that the numbers are not
different than zero, the later explanation is most probable.
In fact, when the concentrations inplied by these results were
conpared to the minimum resolving ability of the assays according
to the original authors it was found that the highest reading cor¬
responded to a concentration that was lower than the confident
Anaerobic metabolism in snails
Mark Roberts
level of resolution by a factor of three.
However if some justifiable reason for elimination of the
negative readings could be found, some results fall out of this
data. Certainly negative readings are impossible to interpret as
they directly correspond to negative concentrations - which are
impossible. If any of this data had a remote chance of being cor¬
rect, it would have to be the positive readings.
Knowingly taking the experimental bias of considering all ne-
gative values as zero readings, and all positive values as real,
the results in figure 4 can be obtained. In the Nucella, the ave¬
rage accumulation of alanine was greater in the anaerobic animals
than in the aerobic ones. The ratio of anaerobic to aerobic con¬
centration is an indication of how much greater. For Nucella this
ratio is greater than 2.5. Also, the accumulation of lactate in
the anaerobic tissue is only slightly greater than the same in the
aerobic tissue. For the Acanthina, both the accumulation of ala¬
nine and lactate are nearly the same for both extracts.
If these results were acceptable, they would indicate an af¬
firmitive conclusion regarding the existence of the pathway in fi¬
gure one in Nucella, and would imply that either the Acanthina
does not have it, or that conditions were not sufficiently vigorous
to force the snail into anaerobic metabolism.
Anaerobic metabolism in snails
Mark Roberts
ONCLUSION
Only by allowing for extreme experimental bias may it be said that
any of this data provides evidence for or against the existence of
succinate-producing glycolysis in these two snails. In light of
the prior conclusion that the assays employed were not sensitve e-
nough to detect any end products, the ultimate conclusion of this
paper must be that the existence of this interesting biochemical
pathway still awaits further investigation in these species.
Mark Roberts
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Anaerobic metabolism in snails
OS
FIGURE CAPT
Essentials of the succinate producing anaero¬
bic metabolism found in several invertebrates.
Note the evolution of 8 moles of ATP and the
accumulation of alanine, succinate, and propi¬
onate.
Flow of steps in this experiment. Final assay
for end productswere based on change in concen¬
tration of NAD according to these equations.
Results of assays for alanine and lactate.
The units are absorbtion, and represent the
change in absorbtion which occurs in the
assay mixture after addition of the enzymes.
Change in absorbtion is directly related to
concentration of end product.
Relativeanounts of end products accumulated
in anaerobic and aerobic tissue of Nucella
and Acanthina. Results are based upon extreme
expiremental bias as described in text.
G3P
2 Moles ATP
PEP
ASPARTATE

OXOX
MLATE MATE
PRVATE
FUMRATE

—
2 Moles ATP
—
E

—
FIGURE 1
GLUTAMTE
KGA

GLUTAMTE
KGA
— SUCCI-OA
PETHAL-COA
PROPIOENL-CA
PRPIOWATI
—) 2 Moles ATP
2 Moles ATP
SAILS
SMAILS
ONTROL
DPERIFENTAL

e ES-
EPEEE
(Liquid Nitrogen)
HODGEVIZE
(Perchloric Acid)
& EXTRACT
CETTRIGUGE
supernatant
(Spectrophotometer)
ASSAV
ALANINE + NAD + H,O ADH PYRUVATE + NHA + NADH
Increase in Abs.
LACTATE + NAD + H,O — LDH PYRUVATE + Hr + NADH
Increase in Abs.
SUCCINATE + ATP + COA STK
SUCCINYL-COA + P + ADP
ADP + PEP PK
ATP + PYRUVATE
PYRUVATE + NADH + H LDH
LACTATE + NAD
Decrease in Abs.
ALANINE
Absorb.

AR
-.233
.103
.300
.470
.050
.060
.140
.070
-.920
-.890
-.069
—.043
-.088
-.044
.006
.097
-.047
265
.020
-.853
-.024
.171
020 070
ACANTHINA
PUNCIULATA
NUCELLA
EMARGINATA
JURI
LACTATE
Absorb.
—
AR
-.056
-.066
430
.104
-.161
.042
.090
.320
-.028
-.055
-.388
-.394
.160
300
.085
.086
010
.064
2
Anaerobic
Extract
Rerobic
Extract
RATIO ANAEROBIC
AEROBIC
2.66
1.7
.75
1.11
Nucella
Emarginata
ALANINE
LACTATE
Acanthina
Puntulata
FIGURE 4
Mark Roberts
Anaerobic metabolism in snails
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Anaerobic Metabolism in Snails
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