AN ABSTRACT OF THE THESIS OF
Bruce Leon Boese
for th
degree of
in _____Department of Zoology__ presented on
Title:
Doctor of Philosophy
March 15, 1979
Heart Rate, Survivorship, Oxygen Consumption and Metabolic End
Product Accumulations in the Intertidal Limpets Collisella
pelta and Collisella digitalis in Relation to Desiccation and
xia
Redacted for Privacy
Abstract approved:
Austin W. Pritchard
The heart rate, oxygen consumption, survivorship, and 1-lactate
accumulation were determined in the intertidal limpets Collisella pelta
and Collisella digitalis in relation to laboratory desiccation at 15°C
or 30°C.
Water loss was computed as a percentage of total initial body
water (wet weight - dry weight).
Although water was lost faster in C.
digitalis than in C. pelta, C. digitalis tolerated more water loss and
survived longer than C. pelta at both experimental temperatures.
During
the first few hours of desiccation at 15°C the heart rate of C. digLta1is
was constant while in C. pelta the heart rate declined rapidly.
During
desiccation at 30°C the heart rates of both species declined rapidly.
No change was noted in control heart rates, determined under moist
aerial conditions, in either species during a 40 hour exposure period.
Oxygen consumption of C. digitalis declined gradually during desiccation
at 15°C to about 60% of the undesiccated aerial control rate after 46
hours of exposure.
During desiccation at 30°C the decline in oxygen
consumption was much more pronounced.
In C. pelta during desiccation
at 15°C oxygen consumption progressively declined with increasing
length of exposure to approximately 50% of control rates for the first
16 hours of exposure at which time rates became constant.
At 30°C a
more pronounced reduction in oxygen consumption occurred.
Whole-body
lactate accumulated in both species during desiccation at 15°C and 30°C
in comparison with controls.
At 15°C accumulation in C. pelta began
after 15 hours of desiccation while at 30°C accumulations were evident
after 4 hours.
In contrast, C. digitalis gradually accumulated lactate
during the entire length of exposure to desiccation conditions at 15°C.
Upon exposure to moist nitrogen atmosphere both species exhibited
a rapid bradycardia.
Heart rates recovered rapidly after exposure and
overshot pre-exposure rates indicating oxygen debt repayment.
Of the three anaerobic end products examined (alanine, succinate
and lactate) only lactate accumulated to any degree in either species
during anoxia.
C. pelta.
During anoxia C. digitalis accumulated more lactate than
C. pelta reached a maximum accumulation after 4 hours of
anoxia while C. digitalis continued to accumulate lactate over the
entire length of exposure to moist nitrogen.
The results suggest differences between C. digitalis and C. pelta
in metabolic adaptations which may be related to intertidal zonational
differences.
Heart Rate, Survivorship, Oxygen Consumption,
and Metabolic End Product Accumulations
in the Intertidal Limpets Collisella pelta and
Collisella digitalis in Relation to
Desiccation and Hypoxia
by
Bruce Leon Boese
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed March 15, 1979
Commencement June 1979
APPROVED:
Redacted for Privacy
Professor of Zoology in charge of major
Redacted for Privacy
Chairman of Departmentof
Redacted for Privacy
Dean of Graduate School
Date thesis is presented
March 15, 1979
Typed by Kathryn Miller for
Bruce Leon Boese
TABLE OF CONTENTS
I.
Heart Rate, Oxygen Consumption, Lactate Accumulation, and
Survivorship in the Intertidal Limpets Collisella pelta
(Rathke, 1833) and Collisella digitalis (Rathke, 1833) in
Relation to Desiccation
Introduction .........................
2
Materials and Methods ....................
3
Results........................... 7
II.
Discussion ..........................
10
Figures ...........................
16
Literature Cited .......................
25
Survivorship and Accumulation of Metabolic End Products in
Collisella pelta (Rathke, 1833) and Collisella digitalis
(Rathke, 1833) Under Conditions of Low Oxygen Exposure
Introduction .........................
29
Materials and Methods ....................
30
Results ...........................
31
Discussion ..........................
32
Figures ...........................
37
Literature Cited .......................
42
LIST OF FIGURES
I.
Heart Rate, Oxygen Consumption, Lactate Accumulation, and
Survivorship in the Intertidal Limpets Collisella pelta
(Rathke, 1833) and Collisella digitalis (Rathke, 1833) in
Relation to Desiccation
Percent of water loss in Collisella digitalis exposed
to dry air at 15°C
16
2.
Percent of water loss in Collisella pelta
17
3.
Survival of Collisella pelta and Collisella digitalis
in dry air at 15°C and 30°C
18
Aerial heart rate of Collisella digitalis when exposed
to moist air (controls) at 15°C, dry air at 15°C, and
dry air at 30°C
19
5.
Aerial heart rate of Collisella pelta
20
6.
Aerial oxygen consumption of Collisella digitalis when
exposed to moist air (controls) at 15°C, dry air at
15°C, and dry air at 30°C
21
7.
Aerial oxygen consumption of Collisella pelta
22
8.
Lactate Accumulation in Collisella digitalis when exposed to moist air (controls) at 15°C, dry air at 15°C,
and dry air at 30°C
23
Lactate accumulations in Collisella pelta
24
1.
4.
9.
II.
Survivorship and Accumulation of Metabolic End Products in
Collisella pelta (Rathke, 1833) and Collisella digitalis
(Rathke, 1833) Under Conditions of Low Oxygen Exposure
1.
2.
3.
4.
Aerial heart rate of four individual Collisella pelta
before, during, and after exposure to moist nitrogen
at 15°C
37
Aerial heart rate of three individual Collisella digitalis
before, during, and after exposure to moist nitrogen at
15°C
38
Survival of Collisella pelta compared to Collisella
digitalis during exposure to moist nitrogen at 15°C
39
Lactate accumulation in Collisella digitalis and
Collisella pelta during exposure to moist nitrogen at
15°C
40
LIST OF TA3LES
I.
Heart Rate, Oxygen Consumption, Lactate Accumulation, and
Survivorship in the Intertidal Limpets Collisella pelta
(Rathke, 1833) and Collisella digitalis (Rathke, 1833) in
Relation to Desiccation
1.
II.
Mean survival time and water loss tolerated by Collisella
pelta and Collisella digitalis when subjected to desiccation stress at two temperatures
11
Survivorship and Accumulation of Metabolic End Products in
Collisella pelta (Rathke, 1833) and Collisella digitalis
(Rathke, 1833) Under Conditions of Low Oxygen Exposure
1.
2.
Mean values of three anaerobic end products present in
Collisella pelta after 12 hours exposure to moist nitrogen at 15°C
Mean values of three anaerobic end products present in
Collisellla digitalis after 12 hours exposure to moist
nitrogen at 15°C
32
33
Heart Rate, Oxygen Consumption, Lactate Accumulation,
and Survivorship in the intertidal Limpets
Collisella pelta (Rathke, 1833) and
Collisella digitalis (Rathke, 1833)
in Relation to Desiccation
Bruce L. Boese
Department of Zoology
Oregon State University
Corvallis, Oregon
and
Austin W. Pritchard
Department of Z.ology
Oregon State University
Corvallis, Oregon
and
Oregon State University Marine Science Center
Newport, Oregon
2
Introduction
Numerous attempts have been made to relate the tidal zonation of
marine gastropods to differences in temperature tolerance (Southward,
1958; Hardin, 1968; and Sandison, 1968), osmotic tolerance, and desiccation resistance (Wolcott, 1973).
Enhanced tolerances in upper inter-
tidal limpets of the family Acmaeidae have been attributed to increased
water-holding capacity (Shotwell, 1950; Segal, l956a; Segal and Dehnel,
1962), lower desiccation rates, and cellular adaptations for the toleration of high electrolyte concentrations (Wolcott, 1973).
One of the problems associated with these zonational studies has
been the use of mortality as a measure of physiological adaptation.
Although differences in tolerance occur under laboratory conditions, the
extremes in temperature and exposure time necessary for mortality are
usually not approached in the field (Wolcott, 1973).
Marine gastropods
generally live in an exposure zone where temperature and the duration of
desiccation are well below the tolerance limits for the species (Newell,
1970).
Sublethal effects on energy utilization and behavior may more
realistically reflect the effects of the physical environment on intertidal distributions.
Zonational differences in aerial oxygen consumption and heart rate
have been observed in numerous marine gastropods (Segal, 1956b; Sandison,
1966; Micallef, 1967; Russell-Hunter and McMahon, 1974; and McMahon and
Russell-Hunter, 1977).
Data on the responses of these "metabolic
indices" to desiccation stress are lacking from the literature, although
several authors have suggested that some gastropod species may resort to
anaerobic energy pathways when exposed to prolonged desiccation (Emerson
3
and Duerr, 1967; Newell, 1973; and McManus and James, 1975).
Although
the role of anaerobic metabolism is well established in bivalves
(DeZwaan and Wijsman, 1976), less has been done on gastropods (Von Brand
etal., 1950; Oudejans and Van Der Horst, 1974; McManus and James, 1975;
and Storey, 1977).
It is possible that upper intertidal gastropods may be able to
regulate metabolic processes during aerial desiccation to a greater
extent than species in the lower intertidal zone.
This may, for example,
allow the upper species more time during tidal exposure to exploit
resources.
To test this hypothesis changes in heart rate, oxygen con-
sumption, and lactic acid accumulation were compared in zonationally
separate upper intertidal limpets during aerial desiccation.
The upper
intertidal species, Collisella digitalis (Rathke, 1833), is often
exposed for more than one tidal cycle in its upper zonational limit and
can tolerate a large amount of water loss (Wolcott, 1973; Roland and
Ring, 1977).
C. pelta (Rathke, 1833), whose upper distribution overlaps
that of C. digitalis on the Oregon coast, is submerged on every tidal
cycle.
Materials and Methods
Collisella digitalis (15-25 mm in length) and C. pelta (25-40 nmi in
length) were collected at low tides from Yaquina Head and Boiler Bay,
Oregon.
C. pelta were collected from 0.4 m to 1.5 m above MLLW while
C. digitalis were collected from 1.0 m to 2.5 m above MLLW.
Before
experimentation the limpets were kept in a circulating sea water system
(30 °/) at 12-15° C under constant light, for 3-5 days.
Two experimental
temperatures (15°C and 30°C) were used in this study.
Fifteen degrees
C is near the maximum sea surface temperature recorded on the Oregon
Coast (Gonor, 1970), while internal temperatures of 30°C have been
observed in C. digitalis (Wolcott, 1973).
Aerial heart rate at 15°C and 30°C was determined by impedance
recording.
Two leads from a length of bathytherinograph wire were inser-
ted under the shell through a small hole drilled to expose the mantle
tissue over the heart.
The leads were sealed in place with beeswax and
cyanoacrylate glue and attached to an impedance pneumograph and chart
recorder.
The limpet was allowed to attach to a raised platform sur-
rounded by nylon screening, which was within a 1 liter plexigalss
aquarium filled with sea water at 15°C.
After 3 hours of submergence
the sea water was drawn off and silica gel desiccant placed around the
outside of the screening.
The aquarium was then placed in a constant
temperature room maintained at one of the experimental temperatures,
and aerial heart rate recorded.
The experiment was terminated when
there was no discernable heart beat.
Data were reduced to number of beats per minute by counting and
averaging
three 1 mm
than 15 beats per mm
intervals every 30 mm.
a 10 mm
sample was taken.
irregular records the entire 30 mm
For heart beats of less
For extremely slow and
interval was counted and averaged.
Survivorship and total water loss with desiccation was determined
at the two experimental temperatures.
Limpets were weighed after blot-
ting with Kimwipes to remove excess water, placed ventral side down in
individual plastic disposable beakers (100 ml), and sealed in a desiccator containing silica gel.
The desiccator was kept in a constant
5
temperature room.
No more than three limpet test groups (20 limpets per
group) were kept in the desiccator during any experimental run.
Desiccation times in each experimental run were staggered so that at
least 5 hours elapsed between the removal of one experimental group and
the next.
This was done to ensure that exposure to moist constant
temperature room air was kept at a practical minimum, and that sufficient
time was allowed between desiccator openings to allow maximum moisture
extraction by the desiccant.
The above experimental procedure was
repeated several times in order to obtain data values separated by 2 to
5 hours during the duration of the survivorship curve.
After removal
from the desiccator the limpets were reweighed and submerged ventral
side down on a plastic tray in running sea water.
The criterion for
mortality was failure to reattach to the substrate and the absence of
discernible foot movement after 24 hours.
All limpets were then dried
to a constant weight at 70°C and the weight of the shell and body determined.
For experiments where lactate determinations were made, animals
were weighed and desiccated as above.
A rough estimate of percent
water loss was determined, based on the amount of weight loss during
desiccation.
If this estimate exceeded the maximum percent later loss
tolerated in the survivorship experiment or if the limpet displayed
obvious signs of mortality (blackened, dry, head and foot), lactate
values were not determined for that individual.
Control limpets were
treated as in the survivorship experiment except that moist paper towelling was substituted for desiccant.
In order to obtain enough tissue
for extraction of lactate it was necessary to pool limpets (3 to 5 for
Collisella digitalis, 1 to 3 for C. pelta).
Eight to ten pooled samples
for each desiccation or control exposure time were extracted separately.
After removal from the desiccator, the litnpets were weighed, the viscera
separated from the shell and quickly frozen and powdered in a mortar and
pestle filled with liquid nitrogen.
The frozen powder for each separate
extraction was weighed and homogenized with 3.5 vol
of perchlorlc acid
(6%) in a chilled tissue homogenizer (Potter, Elvehjem, 15 ml).
centrifuging (25,000 g, 10 mm
After
at 4°C), the pellet was reextracted with
2.5 vol of perchloric (6%), recentrifuged, and the supernatants combined.
Supernatants were filtered (0.45 urn Millipore), and lactate values
determined on 0.1 ml of the extract by spectrophotometric measurement
of NAD/NADH conversion using kits provided by Sigma Chemical Co., with
internal standards.
Final concentrations were corrected to a fresh
undesiccated shelless weight value.
Aerial oxygen consumption (ui
g dry
wt1
h1) was determined
at 15°C and 30°C in a Gilson differential respirometer.
Twenty to 40
limpets were handled and desiccated as above at either 15°C or 30°C.
After a predetermined desiccationtime all the limpets were removed from
the desiccator and weighed.
Maximum desiccation time was based upon
survivorship data for each species at the given desiccation temperature.
If estimates of percent water loss exceeded the maximum tolerance
observed in the survivorship experiments or limpets showed obvious signs
of mortality (see above) the limpet was discarded.
Control limpets
(15°C) were removed from the flowing sea water system, blotted dry, and
weighed.
Three to six desiccated animals were placed in a dry 100 ml
respirometer chamber (Collisella pelta) or 15 nil respirometer chamber
7
(C. digitalis) and allowed to equilibrate for 2 hours at their exposure
Control limpets were
temperature, with 10% KOH as a CO2 absorbent.
handled as above except that a minimal volume of sea water was added to
moisten the floor of the chamber.
For desiccated limpets, half hour
For control limpets half
readings were taken for 3 hours and averaged.
hour oxygen consumption measurements were taken.
determinations
After oxygen uptake
all the limpets were removed from the respiration cham-
bers, reweighed, and dried to constant weight.
If the weight had
changed by more than 5 mg (approximately 0.5%) during the course of the
experiment, the data were discarded.
Results
Figures 1 and 2 depict the percent of water loss during desiccation
of Collisella digitalis and C. pelta at 15°C.
Each value is the mean
water loss of all surviving organisms at that particular exposure time.
The figures show that there is a more rapid initial water loss with
exposure in C. digitalis than C. pelta.
Both species show a rapid water
loss for the first 10 hours of exposure with a more gradual rate of loss
after this time.
Figure 3 compares survivorship of Collisella digitalis and C. pelta
during aerial desiccation at 15°C and 30°C.
Although C. digitalis loses
water at a greater initial rate, Figure 3 shows that C. digitalis is
more tolerant of desiccation than C. pelta at both 15°C and 30°C and
survives a longer exposure.
Table 1 summarizes these data and shows
that mean survival time and mean water loss tolerated (LD50) was considerably greater for C. dIgitalis than for C. peita at both experimental
temperatures.
rI
Figure 4 represents the averaged aerial heart rate data from individual Collisella digitalis during the period of exposure to dry air
(15°C and 30°C) and moist air (15°C).
The maximum aerial heart rate at
both 15°C and 30°C occurred approximately 90 mm
after the sea water
was drained from the chamber and the animals exposed to aerial conditions.
The maximal heart rate recorded at 30°C was approximately 1.5
times higher than the maximal rates (moist and desiccated) at 15°C.
The moist aerial control rate at 15°C was nearly constant for 30 hours,
but exhibited a gradual decline to one half the maximal rate from 60 to
80 hours after the start of desiccation (not shown).
were measured beyond 80 hours of exposure.
No control rates
At 15°C heart rate remained
constant and near the maximum aerial rate for 6 to 10 hours after the
start of desiccation.
After that period the rates dropped rapidly with
loss of a recordable heart rate beyond 17 hours of exposure.
At 30°C
heart rates dropped rapidly after 2 to 3 hours of exposure with loss of
a recordable heart rate 5 to 7 hours after the onset of exposure (Figure
4).
The heart rate responses of Collisella pelta to aerial conditions
are shown in Figure 5.
In contrast to C. digitalis, maximum aerial
heart rate occurred immediately after exposure to aerial conditions.
Moist aerial control rates were nearly constant for 30 hours with a
gradual decline in heart rates to approximately one half of the aerial
maximal occurring from 40 to 60 hours after exposure to aerial conditions
(not shown).
those at 15°C.
Maximum aerial rates at 30°C were apprOximately 1.5 times
C. pelta in contrast to C. digitalis, exhibited a pro-
gressive reduction in heart rate at 15CC, with loss of a recordable
trace occurring between 33 and 27 hcurs after the start of desiccation.
At 30°C the reduction of heart rate was much more rapid, with loss of a
recordable heart rate occurring 12 to 18 hours after the start of
desiccation (Figure 5).
Figure 6 depicts the results of desiccation on the oxygen consumption of Collisella digitalis at 15°C and 30°C, in comparison to moist
aerial controls (15°C).
At 30°C oxygen consumption decreased throughout
the length of exposure.
At 15°C the rate of oxygen consumption is near-
ly constant with only a gradual decline to about 60% of the undesiccated
aerial control rate (15°C) after A6 hours of desiccation.
Moist aerial
control rates remained constant throughout the exposure period.
Oxygen
consumption experiments were terminated at both temperatures when excessive mortalities occurred after prolonged desiccation.
Figure 7 depicts the effect of desiccation on the oxygen consumption on Collisella pelta at 15°C and 30°C, in comparison to moist aerial
controls (15°C).
Oxygen consumption declined with increasing length of
desiccation at both experimental temperatures.
After about 16 hours of
exposure to 15°C desiccating conditions, the oxygen consumption rate
remained constant at approximately half of the undesiccated control rate.
A more pronounced reduction in oxygen consumption occurred at 30°C compared to 15°C.
Moist aerial controls exhibited a constant oxygen uptake
during the exposure period.
Figure 8 summarizes the accumulation of whole-body lactate in
Collisella digitalis during desiccation (15°C and 30°C) in comparison to
moist aerial controls (15°C).
At both 15°C and 30°C under desiccation
conditions, lactate accumulates gradually over moist aerial control
10
amounts.
Control lactate values remained constant throughout the length
No lactate values were determined at 30°C beyond 12 hours
of exposure.
of exposure due to excessive mortalities.
Figure 9 summarizes the accumulation of whole-body lactate produced
in Collisella pelta during desiccation (15°C and 30°C), in comparison
with the amount present in moist aerial controls (15°C).
At 15°C in dry
air lactate accumulation begins after about 15 hours of desiccation.
30°C lactate accumulation begins at 4 to 7 hours of desicciation.
At
Total
amount of lactate accumulated in C. pelta is similar to that of C.
digitalis.
Control lactate values did not change significantly during
No data were taken beyond 12 hours of
exposure to moist air at 15°C.
desiccation at 30°C due to excessive mortalities.
Discussion
The observation that Collisella digitalis tolerates more water loss
than C. pelta is not surprising.
Upper intertidal gastropods are con-
sidered by several authors to have relatively larger amounts of extravisceral water than lower intertidal forms (Shotwell, 1950; Segal, 1956a;
Segal and Dehnel, 1962).
The usefulness of this water in delaying appre-
ciable loss of tissue water has been questioned seriously by Wolcott
(1973), who suggested that the greater tolerance of desiccation in upper
intertidal Acmaeidae is due to a greater tolerance of internal osmotic
concentration.
Although mean survival times at 30°C were considerably shorter than
at 15°C in both species, similarities in LD50 values for water loss at
both temperatures (Table 1) indicate that mortality was caused by water
11
Table 1.
Mean survival time and water loss tolerated by Collisella
pelta and Collisella digitalis when desiccated at two temperatures.
Mean Survival Time
(hours)
15°C
30°C
Mean Water Loss Tolerated (%)*
(LD5O estimate)
15°C
30°C
Collisella pelta
50
8
31.8 ± 7.1
31.5 ± 6.6
Collisella digitalis
62
13
61.1 ± 7.1
59.4 ± 8.5
*percent water loss ± 2 standard errors.
loss rather than by exceeding thermal tolerances.
were not included in this study.
Thermal tolerances
Hardin (1968), however, noted that 80%
of Collisella digitalis survived 15 hours at 29°C while 70% survived 10
hours at 31.5°C, and C. pelta have been shown to withstand 15 mm
iinmer-
sions in 34°C water (Wolcott, 1973).
The percent of water loss tolerated in this study by both species
is considerably less than reported by Wolcott (1973) and Roland and
Ring (1977).
Although zoogeographic differences cannot be discounted,
variation in technique may account for much of this difference, especially the manner in which the limpets were allowed to attach to the substrate.
Wolcott (1973) allowed limpets to attach when submerged,
while
in this study, limpets were first blotted dry and placed on a dry substrate.
This would tend to reduce the initial weight of the limpets and
therefore the percent of water lost during desiccation.
The heart rate and oxygen consumption of Collisella digitalis
(Figures 4 and 6) during the first few hours of desiccation at 15°C are
nearly constant, in contrast to C. pelta (Figures 5 and 7) which showed
12
a marked reduction in both.
This initial regulation of metabolic func-
tion during desiccation may allow C. digitalis to maintain an active
feeding stature not possible in C. pelta under similar environmental
conditions.
Although it is difficult to assess field behavior with
respect to desiccation in limpets, Craig (1968) noted that C. pelta
remains stationary while emerged, rarely moving while awash.
In con-
trast, C. digitalis commonly move while awash (Miller, 1968; Millard,
1968), and have been observed as much as 30 feet above MLLW in areas
where optimal spray conditions occur (Wolcott, 1973).
The relatively larger amount of extra-visceral water in Collisella
digitalis (Segal and Dehnel, 1962) may account for the initial regulation of heart rate and oxygen consumption during desiccation at 15°C.
Wolcott (1973) described two phases of water-loss in limpets; an initial
rapid evaporative phase where free water being lost from body surfaces
is the rate-limiting factor, and a much slower diffusion phase where
rates of water loss are determined by the rate at which water is being
supplied from the underlying tissues.
The evaporative phase of water
loss in C. digitalis is more extensive than that of C. pelta (Figures 1
and 2), possibly due to a larger relative amount of "stored water."
The
slight advantage this extra-vixceral water gives C. digitalis at 15°C
may be lost at 30°C, as no regulation can be inferred from either the
heart rate or oxygen consumption data, which decline during initial exposure (Figures 4 and 6).
Wolcott (1973) noted that upper intertidal Acmaeidae including
Collisella digitalis secrete mucous barriers that dry and retard water
loss.
The initial regulation of heart rate and oxygen consumption
13
observed in C. digitalis (Figures 4 and 6) does not appear to be due to
mucous barrier formation.
Although formation of mucous barriers was
observed in C. digitalis, this occurred only during the later stages of
desiccation, and they were not as extensive as those illustrated by
Wolcott (1973).
Surprisingly, C. digitalis shows a more rapid water
loss than C. plta, a species which does not produce a mucous barrier
(Figures 1 and 2).
Numerous authors have suggested that intertidal gastropods may
resort to anaerobic fermentation when exposed to prolonged aerial
desiccation conditions (Ghiretti, 1966; Nicallef and Banister, 1967;
Newell, 1973).
Recent research on anaerobiosis has established that
lactate, which is formed in some mammalian tissues during hypoxia, is
often only an initial end product in many bivalve molluscs, with alanine
and succinate playing a more significant role (DeZwann, IUuytmans and
Zandee, 1976).
In a companion study, Boese (1979) found that in
Collisella pelta and C. digitalis only lactate increased to any degree
during hypoxia, suggesting that pathways leading to alanine and succinate probably do not play a significant role during desiccation.
Under
desiccation stress an increase in lactate was noted in both species
suggesting some shift to anaerobic respiration, although the lactate
accumulation observed in C. pelta at 15°C occurred well beyond the time
limit for tidal exposure in that species.
Laboratory desiccation may
only roughly estimate the time course of desiccation in the environment,
but does suggest that anaerobic metabolism is not used on a routine
basis by C. pelta during periodic tidal exposures.
In contrast, C.
digitalis appears to accumulate small amounts of lactate, possibly from
14
the onset of desiccation at 15°C (Figure 8).
The literature contains relatively little information concerning
metabolic end product accumulation during desiccation in intertidal
gastropods.
Barnes, Barnes and Finlayson (1963) observed lactate
accumulations in the barnacle, Balanus balanoides, during prolonged
desiccation.
Although mean rates of accumulation in B. balanoides are
roughly comparable to rates of accumulation in Collisella digitalis and
C. pelta, B. balanoides survives desiccation for a much longer time and
can accumulate up to ten times the maximal amount of lactate observed in
either limpet species in this study.
A marine gastropod, Littorina
saxalilis rudus, which survives up to three weeks of desiccation, produces alanine, succinate, and lactate as anaerobic end products in a
2:1:1 ratio (McManus and James, 1975).
The apparent inability to undergo long term anaerobic respiration
in both limpet species in this study is compensated by an ability to
respire aerobically when severely desiccated.
Both species maintained
a high rate of oxygen consumption at 15°C when severely dried, even
after heart stoppage.
The heart is primarily associated with two respi-
ratory surfaces in Acmaeidae (the ctenidium and mantle fold), and moves
blood from these areas to the foot and viscera (Kingston, 1968).
In
severely dried Collisella pelta and C. digitalis, both of these respiratory surfaces appear dehydrated.
The mantle tissue over the visceral
hump, however, was still moist and may have served as an area of oxygen
exchange in both species but especially in C. digitalis, since little
change in oxygen consumption was noted with desiccation time at 15°C
(Figure 7).
15
Control moist aerial oxygen consumption rates of Collisella
digitalis are about one half the rates measured by Doran and McKenzie
(1972).
In their study the respiratory chambers were agitated which
may account for this difference.
In this study, no change in oxygen
consumption was noted during the length of exposure to moist air at
15°C in either limpet species (Figures 6 and 7).
Collisella digitalis has several physiological characteristics
which may enhance its ability to use the upper intertidal zone over
C. pelta during desiccating conditions.
During the first few hours of
desiccation at 15°C C. digitalis was able to maintain a relatively con-
stant heart and oxygen consumption rate, while C. pelta exposed to
identical conditions exhibited rapidly declining rates.
The initial
regulation in C. digitalis may have resulted from a relatively larger
amount of free water storage, which allows respiratory surfaces to stay
moist for a longer period than those of C. pelta.
When severaly
desiccated at 15°C the oxygen consumption of C. digitalis was greater
and had declined less from control values than in C. pelta.
C.
digitalis survives desiccation at both 15°C and 30°C for a longer period
and can tolerate more water loss than C. pelta.
Survival during exten-
sive desiccation may be enhanced in C. digitalis by the production of
mucous barriers as previously elucidated by Wolcott (1973).
Although
i-lactic acid accumulated in both C. pelta and C. digitalis during
prolonged desiccation, oxygen consumption rates of both species during
prolonged desiccation suggest that extensive anaerobic metabolism is
unlikely in either limpet species during normal tidal exposures.
16
IL
I
I
I
-
I
('p
C,)
-J
Lu
I-
-
30-
0
10
20
30
40
50
60
70
TIME (HOURS)
Figure 1.
Percent of water loss in Collisella digitalis exposed to dry
air at 15°C. Values are means of 10 to 20 individuals ±2
standard errors.
17
50
40
11
U)
U)
30
I-
20
10
0
10
20
30
40
50
60
TIt'!E (hOURS)
Figure 2.
Percent of water loss in Collisella pelta.
Figure 1.
Symbols as in
70
I-.
(I-)
(S
UJ()
Ho
UftflJRs)
Ffgu
3
15°c
Ea1is In dry air at
and
e11a
s11a
Survva1 of
and 3O°. Each value represents the Percentage of Indivjdu5i8 that sur
Vjved exposure from an Iflitial sample size of 20.
C. pelta, 15° A C.
30°c; 0 C. g115 15°c;
dIgItj8 30°c
100
T60
C')
I-
w
20
0
4
8
12
16
20
24
28
TIME (HOURS)
Figure 4.
Aerial heart rate of Collisella digitalis when exposed to moist air (controls) at 15°C (0), dry air at 15°C (Q), and dry air at 30°C (L).
Values are means of 4 to 7 individuals.
Method of estimating average
heart rate is described in text.
t,o
[:111
6O
(I)
FL-
to
40
0
4
8
12
20
16
24
TIME (FlOURs)
Figure 5.
Aerial heart rate of Collisella pelta.
Symbols as in Figure 4.
28
21
620
580
540
500
460
420
380
3;
340
00
250
220
180
140
100
4
8
12
16
20
24
28
32
36
0
44
1ME (HOUR)
Figure 6.
Aerial oxygen consumption of Collisella digitalis when
exposed to moist air (controls) at 15°C (0), dry air at
15°C ($), and dry air at 30°C (/). Values are means of
6 to 10 determinations ±2 standard errors.
22
500
460
420
340
I-
300
C4
I-
220
180
140
100
60
0
4
8
12
16
20
24
28
32
TIME (HOURS)
Figure 7.
Aerial oxygen consumption of Collisella pelta.
in Figure 6.
Symbols as
24
20
0
2
4
6
8
10
12
14
16
18
20
22
TitI[ (houRs)
Figure 8.
Lactate accumulations in Collisella digitalis when exposed to moist air
(controls) at 15°C (s), dry air at 15°C (0), and dry air at 30°C (LS).
Values are means of 8 to 10 pooled samples ±2 standard errors.
2
)
t/1
)
tz
w
I-
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
TPIE (HOURS)
Figure 9.
Lactate accumulation in Collisella pelta.
Symbols as in Figure 8.
25
Literature Cited
The seasonal
1963.
Barnes, Harold, Margaret Barnes and D. M. Finlayson.
changes in body weight, biochemical composition, and oxygen uptake
of two common boreoartic cirripedes, Balanus balanoiedes CL.) and
Balanus balanus (L.). Journ. Mar. Biol. Assoc. U. K. 43(1): 185211.
Survivorship and accumulations of metabolic end
1979.
Boese, Bruce L.
products in Collisella pelta (Rathke, 1833) and Collisella digitalis
(Submitted
(Rathke, 1833) under conditions of low oxygen exposure.
for publication)
1968.
Craig, Peter C.
pet Acmaea pelta.
The activity pattern and food habits of the lim13-19.
The Veliger 11, Supplement:
1976.
Facultative
DeZwaan, A., J. H. F. N. Kluytmans and D. I. Zandee.
Biochem.
Soc.
(Lond.),
Symposia.
41: 133anaerobiosis in molluscs.
168.
DeZwaan, A. and T. C. M. Wijsman. 1976. Anaerobic metabolism in bivalComp.
via (Mollusca): Characteristics of anaerobic metabolism.
Biochem. Physiol. 54(3B): 313-324.
Doran, Sharon Rose and Donald S. McKenzie. 1972. Aerial and aquatic
respiratory responses to temperature variations in Acmaea digitalis
The Veliger 15(1): 38-42.
and Acmaea fenestrata.
Some physiological
1967.
Emerson, David N. and Frederick G. Duerr.
effects of starvation in the intertidal prosobranch Littorina
panaxis (Philippi, 1847). Comp. Biochem. Physiol. 20(1): 45-62.
In Karl M. Wilbur
Ghiretti, F. 1966. Respiration. Vol. 2, p. 175-208.
Academic Press,
The
physiology
of
mollusca.
and C. M. Yonge led.].
New York.
Oregon coastal marine animals, their
1970.
Gonar, Jefferson John.
environmental temperatures and mans impact. Ore. State Univ.,
Tech. Rep. No. 199.
School of Oceanog.
Hardin, Dane D. 1968. A comparative study of lethal temperatures in
The Veliger 11,
the limpets Acmaea scabra and Acmaea digitalis.
Supplement: 83-88.
1968. Anatomical and Oxygen electrode studies of
Kingston, Roger S.
The Veliger 11,
respiratory surfaces and respiration in Acmaea.
Supplement: 73-78.
Temperature rela1977.
NcMahon, Robert F. and W. D. Russell-Hunter.
tions of aerial and aquatic respiration in six littoral snails in
relation to their vertical zonation. Biol. Bull. 152(1): 182-198.
26
McManus, D. P. and B. L. James. 1975. Anaerobic glucose metabolism in
the digestive gland of Littorina saxatilis rudis (Maton) and in the
daughter sporocysts of Microphallus similis (Jãg) (Digenea:
Microphillidae). Comp. Biochem. Physiol. 5(33): 293-297.
Aerial and aquatic respiration of certain trochids.
1967.
Micallef, H.
Experientia 23(1): 52.
Aerial and aquatic oxygen con1967.
Micallef, H. and W. H. Bannister.
swnption of Monodonta turbinata (Nollusca: Gastropods). Journ. of
Zool. (Lond.) 151(4): 479-482.
The clustering behavior of Acmaea
1968.
Millard, Carol Spencer.
The Veliger 11, Supplement: 45-51.
digitalis.
Orientation and movement of the limpet
Miller, Allan Charles. 1968.
Acxnaea digitalis on vertical rock surfaces. The Veliger 11,
Supplement: 30-44.
Biology of intertidal animals.
1970.
Newell, Richard C.
Elsevier Publishing Co., Inc., New York.
555 p.
American
Factors affecting the respiration of intertidal
Amer. Zool. 13(2): 513-528.
invertebrates.
1973.
Aerobic-anaerobic
1974.
Oudejans, R. C. H. M. and D. J. van der Horst.
biosynthesis of fatty acids and other lipids from glycolytic intermediates in the pulmonata land snail Cepaea nemoralis (L.). Cotnp.
Biochem. Physiol. 47(13): 139-147.
Roland, W. and Richard A. Ring. 1977. Cold, freezing, and desiccation
Cryobiology
tolerance of the limpet Acrnaea digitalis (Eschschcltz).
14(2): 228-235.
Patterns of aerial
1974.
Russell-Hunter, W. D. and Robert F. McMahon.
and aquatic respiration in relation to vertical zonation in four
littoral snails (Abstract). Blol. Bull. 147(2): 496-497.
The oxygen consumption of some intertidal
1966.
Sandison, Eyvor E.
gastropods in relation to zonation. Journ. of Zool., (Lond.) 149(2):
163-173.
Respiratory responses to temperature and temperature
1968.
tolerance of some intertidal gastropods. Journ. Exp. Mar. Mo?.
Ecol. 1(2): 271-281.
Adaptive differences in water holding capacity in
l956a.
Segal, Earl.
an intertidal gastropod. Ecology 37(1): 174-178.
Microgeographic variation as thermal acclimation in
l956b.
an intertidal mollusc. Biol. Bull. 111(1): 129-152.
27
Segal, Earl and Paul A. Dehnel.
mollusc: Acmaea limatula.
Osmotic behavior in an intertidal
1962.
Biol. Bull. 122(3): 417-430.
1950. Distribution of volume and relative linear
Shotwell, Jesse Arnold.
measurement in Acmaea, the limpet. Ecology 31(1): 51-61.
Note on the temperature tolerances of some
1950.
Southward, A. J.
intertidal animals in relation to environmental temperature and
Journ. Mar. Biol. Assoc. U. K. 37(1):
geographical distribution.
49-66.
Lactate dehydrogenase in tissue extracts of
1977.
Storey, Kenneth 3.
the land snail, Helix aspersa: Unique adaptation of LDH subunits
Comp. Biocheni. Physiol. 56(2B): 181-187.
in a facultative anaerobe.
Wolcott, Thomas G. 1973. Physiological ecology and intertidal zonation
Biol.
in limpets (Acmaea): A critical look at "limiting factors".
Bull. 145(2): 389-422.
Von Brand, Theodor, Harry D. Baernstine and Benjamin Mehiman. 1950.
Studies on anaerobic carbohydrate consumption of some fresh water
Biol. Bull. 98(3): 266-276.
snails.
28
Survivorship and Accumulation of
Metabolic End Products in
Collisella pelta (Rathke, 1833) and
Collisella digitalis (Rathke, 1833)
Under Conditions of Low Oxygen Exposure*
Bruce L. Boese
Department of Zoology
Oregon State University
Corvallis, Oregon
*Research aided by grant-in-aid of Research from Sigma Xi, the Scientific
Research Society of North America.
Introduction
It has been suggested that some intertidal gastropods resort to
anaerobic respiration during tidal exposure to dry aerial conditions
(Ghiretti, 1966; Micallef and Bannister, 1967; Newell, 1973; McNanus
and James, 1975).
Although the extent of anaerobic respiration is well
established in bivalves (Hochachka, Fields and Mustaf a, 1973; DeZwaan,
Kiutytnans and Zandee, 1976; DeZwaan and Wijsman, 1976) little has been
done on marine intertidal gastropods with respect to the nature and
amount of anaerobic end products.
For this study two species of limpets
of the family Acmaeidae were selected.
Collisella digitalis (Rathke,
1833) is an upper intertidal limpet found from California to Alaska
(Griffith, 1967).
This limpet is often exposed to aerial conditions
for more than one tidal cycle and has been observed as much as 30 feet
above MLLW in areas of optimal spray conditions (Wolcott, 1973).
Upper
intertidal representatives of C. pelta (Rathke, 1833) are distributed
from Alaska to Mexico (Griffith, 1967) and are submerged and exposed on
every tidal cycle.
On the central Oregon coast the lower distribution
of C. digitalis overlaps the upper distribution of C. plta.
Both
species have been shown to tolerate a large amount of water loss (Roland
and Ring, 1977; Wolcott, 1973), and high internal temperatures (Wolcott,
1973).
During desiccation stress, C. digitalis has been shown to secrete
mucous which drys around the shell margin and retards water loss
(Wolcott, 1973).
Extensive loss of moisture from respiratory surfaces
or "sealing off" of the respiratory surfaces during desiccation may
result in some anaerobic metabolism.
Therefore, an enhanced ability to
undergo anaerobic respiration may allow upper intertidal members of the
30
limpet family Acmaeidae to exploit areas where desiccation stress is
more likely.
This study compares the ability of these zonationally different
limpet species to survive anoxia and to accumulate certain metabolic
products (lactate, alanine and succinate) associated with anaerobic
metabolism.
Materials and Methods
Collisella pelta (25-40 mm long) and C. digitalis (15-25 mm long)
were collected from Yaquina Head, Oregon in areas where their distributions overlap.
Limpets were stored in a circulating sea water system
(30 %) under constant light, at 12-15°C for 3 to 5 days before experimentat ion.
Before exposure to anoxic conditions, limpets were first weighed
and placed in an 8 liter sealed chamber under moist aerial conditions
at 15°C for at least 3 hours.
After this equilibration period the con-
tainer was rapidly gassed with moist nitrogen for 10 mm, after which a
slight positive pressure was maintained to ensure against leakage.
Heart rate was determined prior to, during and after exposure by
impedance recording (Boese and Pritchard, 1979).
Survivorship was deter-
mined using the same criteria as Boese and Pritchard (1979).
For determination of anaerobic end products the tissues were homogenized and extracted by the same method as Boese and Pritchard (1979).
To obtain enough tissue for extraction it was necessary to pool limpets
(3 to 5 for Collisella digitalis, 1 to 3 for C. pelta).
L-lactate was
determined by the method of Sigma Chemical Co., using internal standards.
31
L-alanine was determined by the method of Williamson (1974).
Succinate
was determined by a fluorotnetric method (Williamson and Corkey, 1969).
Results
Upon exposure to moist nitrogen the heart rate of both species
(Figures 1 and 2) show an immediate bradycardia with complete bradycardia
evident in Collisella digitalis (Figure 2).
After return to normoxic
conditions heart rate recovers rapidly in both species with an initial
overshoot of the pre-exposure rate, followed by a gradual return to a
"typical" aerial heart rate.
Figure 3 depicts the survivorship of Collisella digitalis and C.
pelta when exposed to moist nitrogen at 15°C.
anoxia considerably longer (LD50
C. digitalis survives
33 hours) than C. pelta (LD50 = 19
hours).
Figure 4 is a comparison of the accumulation of 1-lactate in
Collisella digitalis and C. pelta when exposed to moist nitrogen.
Both
species accumulate 1-lactate during anoxia, with C. digitalis accumulating a larger amount than C. pelta (Figure 4).
The increase in lactate
with time of anoxia is nearly linear for C. digitalis for the entire
period of exposure, while C. pelta reaches a peak level of lactate within 4 hours (Figure 4).
Table 1 is a comparison of control levels of three end products
(alanine, succinate and lactate) in Collisella pelta with the amounts
present after 12 hours of exposure to moist nitrogen at 15°C.
Signifi-
cant increases in lactate and succinate occurred while no increase in
alanine was noted.
32
Table 1.
Mean values of three anaerobic end products present in
Collisella pelta after 12 hours exposure to moist nitrogen at
Control animals taken directly from holding tanks.
15°C.
Numbers in parentheses represent number of samples.
12 Hours
Controls
End Product
± 0.5
L-lactate
1.5
Succinate
0.05 ± 0.02 (13)
Alanine
3.1
Values are
mo1
g wet
± 0.8
wt'
( 9)
(8)
8.0
± 2.1
( 7)*
0.20 ± 0.03 (12)*
3.3
± 0.8
( 8)
± 2 standard errors.
*Significantly different at p < .01 (t-test).
Table 2 summarized similar data obtained from Collisella digitalis.
Only lactate increased significantly over controls after 12 hours of
exposure to anoxia.
Discussion
The bradycardia that occurred in both Collisella digitalis and C.
pelta upon exposure to hypoxic conditions has been observed in bivalves
(Bayne, 1971; Taylor, 1975).
Overshoots in heart rate similar to those
observed in both limpet species after termination of exposure to moist
Table 2.
Mean values of three anaerobic end products present in
Collisella digitalis after 12 hours exposure to moist nitroControl animals taken directly from holding
gen at 15°C.
Numbers in parentheses represent number of samples.
tanks.
± 1.3
L-lactate
5.2
Succinate
0.09 ± 0.03
Alanine
2.5
Values are iimol
12 Hours
Controls
End Product
± 0.8
(10)
(
6)
( 8)
g wet wt' ± 2 standard errors.
*Significantly different at p < .01 (t-test).
15.1
± 2.6
(9)*
0.12 ± 0.02 (7)
3.8
± 1.7
(6)
33
nitrogen (Figures 1 and 2) have been observed in bivalves following prolonged aerial exposure and hypoxia (Trueman, 1967; Helm and Trueman,
1967; Taylor, 1975; Bayne etal., 1976).
Post-exposure overshoots in
heart rate may be indicative of the repayment of an oxygen debt (Trueman,
1967; Helm and Trueinan, 1967) due in part to the accumulation of the
products of anaerobic metabolism.
Neither species in this study had significant amounts of alanine in
the tissues after 12 hours of anoxia.
Although succinate increased sig-
nificantly in Collisella pelta during anoxia, the maximum amount accumulated would contribute less than 4% of the ATP yield that is accounted
for by lactate accumulation.
In C. digitalis succinate did not accumu-
late significantly over control values.
In contrast, both species rapidly accumulate lactate in highly signi-
ficant amounts in a moist nitrogen atmosphere.
Collisella pelta reaches
a maximum accumulation after approximately 4 hours of anoxia while C.
digitalis continues to produce lactate over a much longer period.
This,
coupled with the enhanced survivorship of C. digitalis and the relatively
larger amount of lactate accumulated by C. digitalis over C. pelta, are
consistent with the hypothesis that upper intertidal Acmaeidae are
capable of more extensive anaerobic respiration than lower intertidal
representatives.
Although exposure to moist nitrogen does not mimic desiccation
stress, the amount of lactate present in Collisella digitalis after 12
hours of anoxia (Table 2) compares favorably to the amount present after
12 hours of exposure to dry air at 15°C and 30°C (Boese and Pritchard,
1979).
This similarity in the amount of lactate accumulated suggests
34
that anaerobic metabolism may be of importance during short term
exposures to dry air that might occur in the zonational range of C.
digitalis.
In contrast, the amount of lactate present in Collisella pelta
after 12 hours of exposure to moist nitrogen (Table 2) is considerably
greater than the amount produced after 12 hours of exposure to dry air
at 15°C.
When desiccated at 30°C, however, the amount of lactate pre-
sent is similar to the amount produced during anoxia (Boese and Pritchard,
1979).
Since C. pelta is submerged on every tidal cycle it is unlikely
that anaerobic pathways are utilized to the extent that might occur in
C. digitalis, unless it is exposed to high temperatures.
Assuming glycogen to be the primary energy source during anaerobic
respiration, an estimation of anaerobic energy production can be made
and compared to quiescent or "standard" aerobic energy production using
calculations similar to those of DeZwaan and Wijsman (1976).
The
approximate anaerobic ATP yield from lactate in both species after 12
hours of anoxia at 15°C is shown below:
ATP yield
.zmol lactate
g wet wt-
(.imo1
.
g wet wt-)
Collisella pelta
6.5
15
Collisella digitalis
9.9
10
In quiescent animals the moist aerial oxygen uptake (p1 02
wt1
hr'
g wet
± 2 SE) is 35 ± 3 for Collisella pelta and 25 ± 4 for C.
digitalis (data from Boese and Pritchard, 1979).
13.4 pmol of 02
of 02
.
g wet wt
g wet wt
This represents about
in 12 hours for C. digitalis and 18.8 pmol
in 12 hours for C. pelta.
Using 6 molecules of 02
Jii
consumed and 37 ATP's produced for each glycosyl unit utilized, the
aerobic ATP yield (imol
g wet wt) in 12 hours is 116 for C. pelta
and 82 for C. digitalis.
These values are considerably higher than the ATP yield (10-12 pmol
g wet wt) during 12 hours of exposure to moist nitrogen.
Realizing
that "standardtt metabolic rate is difficult to estimate it appears that
anaerobic energy production based on lactate accumulation is considerably
lower than quiescent aerobic production.
This is consistent with find-
ings in Mytilus edulis, where a 20-fold reduction was noted (DeZwaan and
Wijsman, 1976).
DeZwaan and Wijsman (1976) speculated that anaerobic
energy requirements may be considerably lower than "standard" aerobic
energy demands.
The results of this study together with the companion study on
responses to desiccation (Boese and Pritchard, 1979) suggest some differ-
ences in metabolic adaptations of these two intertidal limpets.
Although
tolerance limits of Collisella digitalis to desiccation (at 15°C and
30°C) and to anoxia (at 15°C) are considerably greater than those of C.
pelta, this alone would not account for the differences in tidal zonation observed in these species since environmental extremes rarely
approach tolerance limits.
However, C. digitalis does exhibit metabolic
features which may offer some advantage over C. pelta during sublethal
exposures to dry aerial conditions.
Although both species exhibited
considerable oxygen uptake during desiccation, even when severely dried,
at 15°C, C. digitalis regulated oxygen consumption and heart rate during
the first few hours of desiccation while C. pelta did not (Boese and
Pritchard, 1979).
C. digitalis shows greater capacity for anaerobic
36
metabolism than C. pelta.
Comparisons of the time course of lactate
accumulations in both species during desiccation (15°C) and while in a
moist nitrogen atmosphere suggest that anaerobic pathways may be of
importance during tidal
xposure to desiccating conditions in C.
digitalis but probably not in C. pelta.
in
I!)
I-
w
0
2
4
0
2
4
60
2
4
6
8
O
12
TLIE (houRs)
Figure 1.
Aerial heart rates of four individual Collisella Relta before, during and after
exposure to moist nitrogen at 15°C.
38
60
50
I-
z
C,,
20
10
0 1
2 3 4 5 6 0 1 2 3 4. 56 0
1
2 3 4 5 6 7 8 9101112
TIME (HOURS)
Figure 2.
Aerial heart rates of three individual Collisella
digitalis before, during and after exposure to
moist nitrogen at 15°C.
39
100
4..
O\
0
0
eoo0\o
60
\\
>
0
(I-;
40
0
'
20
0
8
16
24
32
40
48
TIME (HOURS)
Figure 3.
Survival of Collisella pelta () and C. digitalis (C) during
exposure to moist nitrogen at 15°C. Each value represents the
percentage of individuals that survived exposure from a sample
of 20.
20
ri
NJ
0
2
4
6
8
10
12
14
TIME (HOURs)
Figure 4.
Lactate accumulation in Collisella digitalis (0) and C. pelta () during
exposure to moist nitrogen at 15°C. Each value represents the mean of 8
to 10 pooled samples ±2 standard errors.
0
41
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