THE LOSS OF HEAT RESISTANCE
AND
THE RECOVERY FROM HEAT TORPOR IN
SRIOPUS CALIFORICUS
Reed F. Kratka, Spring 1977
The harpacticoid copepod, Tigriopus Californicus, an inhabitant of the high
intertidal splash pools of the California coast, displays a profound ability to
withstand large fluxuations in the physical parameters of its environment. Water
temperatures ranging over a 30'Cspan have been observed, along with rapid
fluxuations in temperature. The heat resistance of T. Californicus and the role
of thermal acclimation in this process have been demonstrated. (Kontogiannis 1973)
But the nature and mechanism of resistance, while explored by Matutani (Matutani
1961), is still very much a mystery. The present study was initiated in an attempt
to better understand this process. The initial approach was to examine the time
course of the loss of heat resistance. In contrasting the temporal nature of the
acquisition and loss of heat resistance information about the process itself can
be obtained. A close corollary to this approach is the examination of the time
course of recovery from heat induced torpor. This was also pursued.
Materials and Methods
The T. Californicus used in these experiments were obtained from 2 large deep pools
at the south end of the Carmel City Beach, Carmel, California, located at about
the plus 8 foot tidal level. Animals were collected from the center of the pools
by straining water through a fine netting and then washing the captured animals into
a holding container. Throughout the course of experimentation approximately
100,000 animals were collected in this manner. The specimens were maintained in
the laboratory in three 10 gallon aquaria at 16 C - 24°C with a density of
approximately 1,500 animals per gallon. They were fed one half a teaspoon of
Tetramin (fish food) every odd day. The animals remained in these conditions for
2 - 4 days before the initiation of an experiment.
The procedure for determining the rate of loss of heat resistance is
straight forward, yet it can be divided into 4 sections: pretreatment, acclimation
to high temperature, loss of resistance to high temperature, and heat stress and
(2)
recovery. Initially, 96 groups of approximately 160 animals per group were placed
in 16X150 mm test tubes containing 20 ml of fresh sea water and the entire unit was
placed in a constant temperature bath at 20 C. The animals were maintained for 2
days in this condition before initiation of acclimation. To begin acclimation half
of the tubes in the 20 C bath were transferred to a 30 C constant temperature bath.
After 12 hours of acclimation, these animals were transferred back into the 20 C bath
and then tested at intervals of 0, 12, and 48 hours for loss of resistance to heat
acquired while at 30 C. Testing consisted of administering various thermal "dosages
to the animals and assaying for survival rate. These "dosages" consisted of exposure
to a 37 C water bath for times ranging from 18 to 40 minutes. One tube was used to
monitor each dosage, i.e., 12 in all. The populations in each tube were split into
2 groups before testing. Dosages were controlled by removing tubes at two minute
intervals from the 37 C bath and pouring the contents into a petri dish filled with
fresh seawater at 13 C. From the percent effect of these dosages an LT was calculated
first by the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949) and
then more exactly by the use of linear regression analysis after conversion of percents
to probits. This was accomplished with the use of two H-P 65 programs. The LTg was
used to compare different groups in terms of there heat resistance. The animals were
assayed for survival 5 - 7 hours after this heat stress. Survival was defined as
responsiveness to physical stimulation with a pipet. The assay time of between 5 and
7 hours was determined by making repetitive measurements for survival at 2,4, and
10 hours after heat stress. From graph il we can see that the 4 to 10 hour period
appears to be a plateau, i.e., no further recovery is taking place. A more detailed
analysis of recovery from heat torpor was done later with 0,7,11,16,24 and 48 hour
periods being assayed. In this case the time course of recovery from torpor was
(3)
monitored in animals subjected to several different thermal "dosages",i.e., different
times spent at 37 C. The criteria used to distinguish between torpor and non-torpor
wereidentical to the survival criteria. The recovery conditions were also identical
to the heat resistance recovery conditions.
Results
In graph 2 it is appearent that the first 12 hours spent in 20 C after acclimation
at 30 C are most important interms of the loss of heat resistance. It also suggests
that this 12 hour time period should be examined more carefully. To do this another
experiment was conducted utilizing 0,3,6,12 and 48 hour deacclimation periods. All
other procedural aspects were similar to the first experiment. The results are
shown in graph 13. From this we see that the loss of heat resistance occurs rather
rapidly, a significant portion of it within the first 3 hours. Intuitively, the
graph depicts a very rapid decay of the ability to survive high temperature stress.
The results of the torpor recovery examination can be seen in graph #4. It illustrates
the recovery from torpor seen in the first 7 hours and then the death of individuals
for the rest of the time period assayed. This result seems to contradict the model
of recovery from torpor suggested by graph #1. In one case it appears that recovery
from torpor has plateaued and yet, in the same general interval in graph #4 it
appears to peak. Consider the fact that graph l involves sampling on either side of
the peak in graph 14 and the contradiction is resolved. In analyzing the data from
graph#4 we can arrive at two conclusions: torpor and death involve two different
mechanisms, and the torpor response is much more sensitive to temperature than the
death response. The first of these ideas is supported by the general shape of the
curved produced. Animals are recovering from torpor and then dying. If death was
simply an extention of the physiologic modifications which induce torpor, this result
would not be expected. Animals would simply go into torpor and die or achieve total
recovery. This not the result. Secondly, if one plots the thermal dosages vs.
percent "survival",i.e,, percent responsive to tactile stimulation, after half an
hour of recovery time and then again at 24 hours of recovery time, we see very
different responses to temperature stress. This can be seen in graph #5. Response
I is the torpor response and 2 the death response. Analternate way to illustrate
this is shown in graph 6. The mathmatical combination of these two curves yields
graph 14.
Discussion
The loss of heat resistance occurs rapidly. Considering the thermal regime experienced
by Tigriopus Californicus the retention of heat resistance at lower temperatures
would be at best inefficient and at worst maladaptive depending on the nature of the
change which induces resistance. But more interestingly, when one considers the work
of Kontogiannis (Kontogiannis 1973) the time course of the loss of heat resistance
appears to be symetrical to its acquisition. This has two implications. First, the loss
of resistance probably involves the remodification of a structure or process by the
same mechanism by which it was initially change. Speculatively, the modification is
a simple one, at least in terms of stretural reorganization, if not biochemical.
Hochachaka and Somera (Hochachaka and Somera 1973) have proposed that immediate
acclimation to temperature involves the modification of enzme affinities for
substrate or co-factors in energy pathway enzymes. With the modification of Km
values in these enzymes, the general restructuring of biochemical pathways caused
by increases in temperature (Johnson 1954) could be avoided. In this way heat death
may be circumvented. If not offering any concrete support for this point of view,
my results are at least consistant with it. Affinity changes can be affected simply
by modifications in the 3' and 4' structure of enzymes. Temperature is capable of
inducing such changes. The fact that modification and remodification appear symetric
is also easily contained within this model.
In dealing with recovery from torpor it is most interesting to examine the
nature of the two dosage response curves produced by torpor and death respectively.
The torpor response is more dramatic and temperature sensitive than the heat death
response. For torpor a change in dosage of two minutes produces an approximate 707
change in animals scored as "surviving", while that same change in dosage produces
an approximate 302 change in the heat death response. The truncated nature of the
torpor response seems to implicate either very high "toxicity" of temperature or
alternatively, that a behavioral rather than a physiologic mechanism is being tapped
here. Considering the smooth signoid nature of the death response curve it seems more
logical to conceptualize torpor as a sensory response rather than an environmentally
imposed phsiologic response. This is consistant with the view of torpor as an
adaptive response to stress conditions. In this sense Tigriopus Califoricus chooses
to go into heat torpor while death is imposed upon it.
Summary
1) The loss of heat resistance in T. Californicus occurs rapidly within the
first 3 hours after removal from a high temperature condition.
2) In light of studies by Kontogiannis 1973 the acquisition and loss of heat
resistance appears symetrical in relation to time course.
3) The mechanisms involved in torpor and thermal death are different.
References Cited
Hochachka, P.W., and Somero, G.N., Strategies of Biochemical Adaptation
W.B. Standers Company, Philadelphia-London-Toronto, 1973, pp. 179-271
Johnson, F.H., The Kinetic Basis of Molecular Biology, John Wiley and Sons,
Inc., New York, 1954, pp. 187-205
Kontogiannis, j, Acquisition and Loss of Heat Resistance In Adult Tide
Pool Copepod Tigriopus Californicus, Physiol. Zoology 46: 50-54 1973
Litchfield, J.T., and Wilcoxon, F, A Simplified Method of Evaluating Dose
Effect Experiments, Pharmacol. Exptl. Therap. 96: 99-113 1949
Matutani,K, Studies on the Heat Resistance of Tigriopus Japonicus,
Publ. Seto Mar Biol Lab 9 (2) 1961
Acknowledgements
I wish to express appreciation to the students and staff of Hopkins Marine
Station, Pacific Grove, California, with special thanks going to my
advisor Dr. F.A. Fuhrman, and to Dr. Robin Burnett for his help in
statistical analysis.
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