Adaptation to Anaerobiosis in Tigriopus californicus
INTRODUCTION
Tigriopus californicus (Baker, 1912), a harpacticoid copepod lives
mainly in the high tide pools along California's coast. The abundance
of high tide pools; the large density of indigenous animals; and their
ease of collection and maintenance all support the use of Tigriopus for
respiration studies.
Prosser (Prosser, 1961) divides animals into four categories based
on their utilization of oxygen: obligate anaerobes; infrequent aerobes;
aerobes which can withstand multi-hour periods of oxygen deprivation
(ie. frogs, aquatic snails); and, aerobic animals which depend on oxygen
but briefly accumulate an oxygen debt upon exposure to anoxia (ie. ceph¬
alopod molluscs). From studies done on the respiratory rate of harpacti¬
coid copepods (Pallares, 1974) there is little information that would
indicate how Tigriopus would fit in Prosser's classification.
The oxygen debt is recognized as an increase to an above normal con¬
sumption rate of oxygen following exposure of the animal to an anoxic
medjum. At exactly what level of oxygen saturation a medium becomes an¬
oxic varies for the animal in question. The biochemical pathways for an¬
aerobic respiration in invertebrates have been concisely reviewed by Ho¬
chachka (Hochachka, 1973, 1972). The debt appears to -come from the an¬
jmal's oxidation rate of substrates accumulated during anoxia being added
onto its normal consumption rate when the anoxic animal is re-introduced
into a non-anoxic medium. The variety of sustrates which build-up varies
with the animal (Stokes, 1968; Saz, 1971 ; Hochachka, 1975 ). Enzymatic
and fluorometric methods are known (Bergmeyer, 1965; Williamson, 1969)
allowing the changing concentrations of: glycerophosphate, succinate,
alanine, malate, lactate, etc., to be determined. These substrates form
Page 1
Page 2
Adaptation to Anaerobiosis in Tigriopus californicus
so that the catabolic system achieves redox balance while partially operating
without oxygen. Thus the possession of an oxygen debt shows catabolism sup¬
plies energy needs during the anoxic period imposed.
Anoxic survival without accumulating an oxygen debt, by employing other
biochemical or physiological adaptations, is documented (Prosser, 1961).
Some animals accumulate the substrates and excrete them as waste. Activity
has been seen to diminish in ascarid worms as oxygen in the medium drops,
with hyperactivity noticeable upon return to aerobic conditions. Therefore
the lack of debt does not imply a dearth of energy for an organism during
anoxic conditions.
MATERIALS AND METHODS
Tigriopus californicus collected from high tide pools ranging from
Cabrillo Point south to Carmel Beach acted as the experimental stock. All
animals were aquarium acclimated at ambient temperature (16-24 C) for at
least twelve hours and fed Tetranin" fish food on odd numbered days.
A Yellow Springs Instrument Model 53 Biological Oxygen Monitor attach¬
ed to a self-contained stirring probe registered the amount of oxygen
present in the chamber on a 0—100% of saturation scale. The read-out was
continuously recorded on a twelve inch Beckman Chart Recorder. Membrane
condition was followed daily using the Voltage Plateau Test and replaced
as necessary. For the test chamber a one-half liter Erlenmeyer flask pro¬
vided the appropriate aperture for the probe, while concurrently limiting
the sites where environmental oxygen diffusion could occur. Similar
methods have been used previously (Bayne, 1971; Sassaman, 1973 and 1972).
Placement of artificial lights; use of the Hopkin's seawater system
as a reseryoir; and, submerging the chamber in a regulated bath maintained
Page 3
Adaptation to Anaerobiosis in Tigriopus californicus
light, salinity and temperature, respectively, as constants. The variables
of the set-up were the amount of Tigriopus sequestered for each run and the
lenth of anoxia each group experienced. Anoxic periods had been chosen short
enough to avoid signifigant mortality.
Tigriopus to be used for standard oxygen depletion runs were strained
from the aquarium stock and placed in one liter of ambient temperature sea¬
water. Within ten minutes these animals passed through a strainer, to be
caught and subsequently rinsed into the experimental chamber with saturated
water. The oxygen probe had to be immediately inserted without trapping any
bubbles. Any trapped air was expelled upon seating of the rubber seal about
the probe.
As oxygen disappeared the percent of saturation fell resulting in a
smooth depletion curve being generated. Upon reaching 0% saturation the
Tigriopus remained anoxic for varied lengths of time while the water circu¬
lated in the chamber. Time spent at 0% saturation is considered as the
anoxic time. Re-introduction of the animals to saturated water terminated
anoxia and immediately post-anoxic depletion curves were generated.
Calibration of the oxygen electrode monitored over similar time courses
saw a total drift for the 100% and 0% line of only 2%. The absolute amount
of oxygen in the water was found by extrapolating out from literature data
(Harvey, 1955) for seawater at a salinity of 36 parts per thousand. Tigrio¬
pus dry weights could be determined after the post anoxic curve was record¬
ed. The animals were dried at 90 C in a petri dish, then dislodged, collec¬
ted and re-dried. The balance used was a Mettler accurate to 0.0001 grams.
Oxygen consumption rates were calculated by placing the best straight
line through linear or near linear sections of the consumption curves and
then determining the slope, which is the average rate of the period looked
10/t-t.
K.
at. Q's could be found by applying the formula: 910
K.
Adaptation to Anaerobiosis in Tigriopus californicus
Where K, is the consumption rate at the temperature T,
RESULTS
The plot of percent saturation vs time as the Tigriopus respired was
a smooth curve. These were transformed into smooth curves of cc 0,/gram of
Tigriopus vs time for the three temperatures: 30, 25, 22 C (Figures 1, 2, and
3). Since three standard depletion runs exist for each temperature a mean
rate for various time periods can be calculated (Table 1). Rates as stated
earlier, came from slopes of linear of near linear sections of the consump¬
tion curves. The minimal consumption rate is also included in the table.
It represents the lowest determinable rate before the recorded rate reached
zero.
Each one of the standard depletion runs has its respective post-anoxic
run (Figures 4-12). The differing periods of anoxia per run are noted.
Table 2 tabulates the determined anoxic rates per time period in contrast
to the respective mean rate and the length of anoxia undergone. Graphically
these appear for 30 and 22 in Figures 13 and 14.
From the temperatures and rates listed in Table 2, a 0 was calculata¬
ble. The results are found in Table 3.
DISCUSSION
Using the minimal rates calculated from the standard depletion curves,
one may ascertain the minimum debt which could be accumulated by Tigriopus
during the periods of anoxia. Since the post-anoxic rates generally are
less than the mean rates for the first three time periods, Tigriopus is not
repaying an oxygen debt. Calculations show that if the minimum debt were
Page 4
Adaptation to Anaerobiosis in Tigriopus californicus
repayed at any point in the first ten minutes, the rate of consumption would
be from one-half to three times that which is observed.
It is important to note that Tigriopus become torporous prior to reach¬
ing anoxia. Using the rate comparisons in Table 2, one sees a decreasing
consumption rate post-anoxia for correspondingly longer periods spent anoxic.
Apparently the Tigriopus stategy adapts it to reducing oxygen concentration
by first reducing the animal's metabolic rate. Secondly, torpor occurs at
some low oxygen concentration with further metabolic suppression occurring
for longer periods of exposure to anoxia, as evinced by the lower post anoxic
rates.
Activity of Tigriopus during oxygen depletion changes drastically. Once
the percent of saturation falls below 4-9%, one finds Tigriopus immobile,
on the chamber's bottom. Observationally, this state appears to be one of
low oxygen induced torpor. Since Tigriopus depletes oxygen in the chamber
completely, torpor occurs prior to the total disappearance of oxygen. When
depletion is allowed to occur without turbulence the torporous state occurs
similarily, showing the state to be oxygen dependant. Return to saturated
water causes total revival to occur generally within 1-3 minutes at the
temperatures used.
The Qo's listed in Table 3 range from 1.4—1.9. This falls within
the acceptable range for marine invertebrates and does not warrant more of
a discussion.
Page 5
Adaptation to Anaerobiosis in Tigriopus californicus
SUMMARY
Tigriopus californicus activity varies from normal at 100% of oxygen
saturation to that of a torpid condition at 0%.
2. Gross physiological torpor occurs around 4-9% of oxygen saturation.
3. Post-anoxic oxygen consumption rates show no repayment of an oxygen
debt.
Increasing periods of anoxia show subsequently depressed rates of
4.
oxygen consumption.
5. Q0 values ranged from 1.4—1.9.
Page 6
Adaptaptation to Anaerobiosis in Tigriopus californicus
REFERENCES
Bayne, B.L.
1965 Oxygen Consumption by Three Species of Lamellibranch Mollusc in
Declining Ambient Oxygen Tension. Comp. Biochem. Physiol.
40A: 955-970.
Bergmeyer, H. U.
1965 Methods in Enzymatic Analysis. Academic Press, (London).
Harvey, H. W,
1955 Chemistry and Fertility of Sea-Waters. Cambridge University Press,
(Cambridge).
Hochachka, P. W., et. al.
1975 Metabolic Sources of Power For Mantle Muscle of a Fast Swimming
Squid. Comp. Biochem. Physiol. 52B: 151-158.
Hochachka, P. W., Jeremy Fields, and Tarig Mustafa
1973 Animal Life Without Oxygen: Basic Biochemical Mechanisms. Amer.
Zool. 13: 543-555.
Hochachka, P. W. and T. Mustafa
2 Invertebrate Faculative Anaerobiosis. Science. 178: 1056-1060.
Pallares, Rosa E. and Carlos A. Duville
1974 Measurement of the Respiratory Rate in Harpacticoid Copepods. (Eng.
summary). Physis Secc. A Oceanos Org. 32 (85): 441-466.
Prosser, C. L. and F. A. Brown, Jr.
1961 Comparative Animal Physiology. Second Edition. W. B. Saunders Co.,
(Philadelphia).
Saz, H. J.
1971 Faculative Anaerobiosis in the Invertebrates. Amer. Zool. 11: 125-135.
Sassaman, C. and C. P. Mangum
1973 Relationship Between Aerobic and Anaerobic Metabolism in Estuarine
Anemones. Comp. Biochem. Physiol. 44A: 1313-1319.
Sassaman, C. and C. P. Mangum
1972 Adaptations To Environmental Oxygen Levels in Infaunal and Epifaunal
Sea Anemones. Biol. Bull. 143: 657-678.
Page 7
Adaptation to Anaerobiosis in Tigriopus californicus
REFERENC
Stokes, T. M. and J. Awapara
1968 Alanine and Succinate as End -Products of Glucose Degradation in
Some Invertegrates. Comp. Biochem. Physiol. 25: 883-892.
Williamson, J. R. and B. E. Corkey
1969 Assays of Intermediates of the Citric Acid Cycle and Related Compounds
by Rluorometric Enzyme Methods. Methods in Enzymology. 13: 434-512.
ACKNOWLEDGME
I wish to thank the faculty and staff of Hopkins Marine Station
for all of their help in making this research possible. Special notice
goes to my advisor, Dr. Fred Fuhrman, for all of his help. He smoothed
out the rough spots.
Page 8
The following Standard Depletion Curves were made at
the constant temperatures noted. The weight of animals
used can be calculated from the Y-intercept in all these
cases if you know that 4.37, 4.78, and 5.03 cc 0,/liter
are the saturation concentrations of 0, at 30, 25 and 22
degrees centigrade respectively; and that, all experiments
were run in a one-half liter chamber. Salinity is a
constant at 36 parts per thousand. The different symbols
represent different weighted runs, identical in conditions.
c02
GRAM
10
0
30
FIGURE 1- 30°C STANDARD DEF
MINUTES
ZTION RUNS
120
CO.
GRAM
25
20
P
30
FIGURE 2- 25 C STANDARD DEPLETION RUNS
04

120
MINU
150
CO
GRAM
D
30
FIGURE 3-22 C STANDARD DEPLETION RUN:
MINUTES

120
STANDARD DEPLETION RATE
TABLE 1
Temp. Of Amount of anoxia
Run
laterincurred
TIME PERIODS WITH CONSUMPTION RATES FORO
0-10 10-20 20-30 30-40 50-60
50-70 MINIMUM
30 c
32 min.
17.4 12.6 10.4 8.7
0.64
6.0
30
105
16.5 12.4 9.9 8.8
7.9
1.56
30
240
22.4 15.3 13.7 11.3
9.2
MEAN RATES
18.8 13.4 11.3 9.6
7.7
25°C
48 min.
13.2 12.0 9.6 7.0
0.52
5.3
25
104
14.7 9.6 9.0 7.3
6.0
2.2
25
201
14.1 11.4 10.1 8.7
6.4
1.6
MEAN RATES
14.0 11.0 9.6 7.7
5.9
22°0
44 min.
13.7 9.6 7.8 6.3
1.2
0.33
22
133
11.5 8.7 7.8 7.2 5.5
0.3
22
233
12.8 9.6 7.2 5.4 2.5
0.18
MEAN RATES
12.7 9.3 7.6 6.3
1.1
Table 1. The above rates were calculated from the curves in
Figures 4-12 as the slopes through the time periods noted.
The rates listed are in the units of co 02 /gram/Hour
and
are for animals with no anoxic history. Individual curves
are identified by the amount of anoxia they were later sub-
jected to. The time periods are in minutes from 100% satur-
ation.
Figures 4 through 12 represent different post-anoxic curves
graphed against their initial depletion curves. Open circles
trace out the post-anoxic curve, while crosses show for the
respective standard depletion curve. 0, utilization rates
for various time periods are arrived at by taking the slopes
of these curves through the times desired.
ccO,
GRAN
13
XO
FIGURE 4- 22 C: 44 Minutes of Anoxia
MINUTES
90
cc O2
GRA
13

30
FIGURE 5-22 C: 133 Minutes of Anoxia
MINUTES
90
120
c02
GRAM
30
FIGURE 6-22 C: 233 Minutes of Anoxia
MINUTES
0
120
CCO
GRAM
13
XO
30
FIGURE 7- 25 C: 48 Minutes of Anoxia
* 0
c
MINUTES

120
CO
GRAN
O
FIGURE 8- 25 C: 104 Minutes of Anoxia
MINUTES
30
120
c02
GRAN
13
XO
FIGURE 9-25 C: 201 Minutes of Anoxia
*
O
*
(
MINU
30
90
120
cc O,
GRAM
XO
XO
FIGURE 10- 30 C: 32 Minutes of Anoxia
0
X (
O
MINUTES
30
cc 0.
GRAM
13
FIGURE 11- 30 C: 105 Minutes of Anoxia
MINUTES
30
120
c02
GRAM
13
10
FIGURE 12- 30 C: 240 Minutes of Anoxia
MINUTES
120
TABLE 2 —POST-ANOXIC RATES OF RESPIRATION
Length of Anoxia
Temp. of
TIME PERIODS (in minutes,
Incurred
Run
0-10
10-20 20-30
50-70
30-40 50-60
MEAN 18.8
13.4
9.6
11.3
32 min.
30°C
13.4 11.2 11.2 9.9
5.1
30
105
14.7 11.6 10.8 8.7
5.5
30
240
8.4
10.6 9.2 8.5 9.1
25 C
MEAN 14.0 11.0 9.6 7.7
5.9
25
48 min.
11.4 9.0 8.7 7.2 5.8
25
104
11.2 11.3 9.0 8.3 5.4
25
10.5 10.8 9.3 8.7 8.0
201
22°0
MEAN 12.7 9.3 7.6 6.3 4.1
44 min.
9.4 8.7 7.6 7.5 4.5
133
8.4 8.1 7.6 7.1
6.4
233
4.2
7.9 6.3 6.6
5.7
Table 2. Summarized above are the anoxic rates from the anoxic
curves in Figures 4—12. Notice the anoxic rates are in gen¬
eral lower at all points in comparison to the calculated mean
rates for the listed time periods. Inconsistencies in this
table are most likely due to the fact that post-anoxia the
animals can maintain higher intermediate rates since oxygen
depletion in the chamber was not as severe during the first
ten minutes. Thus the numbers overlap with time.
Figures 13 and 14 are g
aphical representations of the
data in Table 2 for the temperatures 30° and 22°0.
CCO.
GRAe
1
FIGURE 13
0-10 —
10-
20-30 30-40 40-50
TIME PERIODS
14
cc O,
GRAN
A
e
FIGURE 14
0-19—
10-20
20-30 30-49 40-50
TIME PERIODS
TABLE 3 - 9,
VALUES
TIME PERIODS
T1.
0-10 min.
10-20 min
200
910 For:
1.6
30, 25
1.9
25, 22
1.4
1.7
Table 3. Above are the 0's as calculated according to
the steps previously outlined. They are within the range
for marine invertebrates.