The Effect of Thermal Acclimation On the Heat Tolerance of the Intertidal Prosobranchs Littorina Littorea (L.) and Monodonta Lineata (Da Costa)

The influence of temperature on the survival of sublittoral and intertidal organisms has received considerable attention in the past. Huntsman & Sparks (1924) and Henderson (1929), for example, showed that the upper limit of thermal tolerance of a variety of marine organisms could be correlated with the temperatures experienced under natural conditions. Both latitudinal differences in thermal tolerance and variations with shore level were apparent in the species they studied. Again, Gowanloch & Hayes (1926) showed that in Littorina littorea and L. saxatilis upper-shore individuals had a higher limit of thermal tolerance than lower-shore individuals. Broekhuysen (1940) showed that the sequence of thermal death points in a series of six South African gastropods corresponded in general with their zonational sequence on the shore, and similar results have been obtained on trochids from the British Isles by Micallef (1966). Much the same correspondence between environmental temperature and thermal tolerance has been demonstrated in many other intertidal organisms by Evans (1948) and in Uca by Edney (1961).

The situation is complicated in many organisms, however, by seasonal acclimation to thermal stress. In Uca, for example, the upper lethal temperature may be raised by 2°C in summer compared with winter (Edney, 1961). Similar acclimation effects have also been noted in the upper limit of thermal tolerance of other species of Uca (Vernberg & Tashian, 1959) and in the gill cilia of the mussel Modiolus demissus and the oyster Crassostrea virginica (Vernberg, Schlieper & Schneider, 1963). Another complicating factor is that much of the earlier data on the upper lethal temperature was obtained by slowly raising the temperature of a water bath in which the animals were contained, at a rate of approximately 1°C in 5 min (Huntsman & Sparks, 1924; Gowanloch & Hayes, 1926; Henderson, 1929; Broekhuysen, 1940). This same procedure was adopted by Evans (1948) and by Southward (1958) to facilitate comparison with earlier work, much of which is summarized by Heilbrunn (1952) and by Gunter (1957). Other work has shown that the duration of exposure to each temperature is also of importance. Rees (1941), for example, showed that the upper lethal temperature of the flatworm Monocelis fusca was 37°C when heated slowly at 1°C per day, but that the lethal temperature was as high as 44°C when the heating rate was 1°C per 30 min. Fry, Hart & Walker (1946) and Fry (1947, 1957) showed that exposure time was also of importance in freshwater fishes and much the same has been found to be true of a variety of marine organisms by Orr (1955), Fraenkel (1960) and by Micallef (1966). None of these last three authors concerned themselves with the influence of thermal acclimation on the heat tolerance of the organisms they studied, although Micallef (1966) acclimated all his trochids to 10°C before making interspecific comparisons. We therefore investigated the effect of both exposure time and thermal acclimation on the heat tolerance of two common intertidal gastropods: the winkle Littorina littorea (L.) and the topshell Monodonta lineata (da Costa). The results are not only in general agreement with those obtained by Southward (1958) and Fraenkel (1960) on Littorina littorea and Micallef (1966) on Monodonta lineata, but also demonstrate the influence of thermal acclimation on the heat tolerance of these animals.

Approximately 1000 similar-sized specimens of Monodonta lineata were collected from Wembury Reef, Devon, on 20 July 1970 and stored in Polythene buckets of sea water overnight. On the following day groups of 200 animals were placed in each of five large shallow vessels of aerated sea water held at 5, 10·5, 16, 21 and 25°C. The temperature was controlled in each vessel by means of a glass coil through which water was pumped from a refrigerated water bath (Grant, model L.B.I X) and aeration was provided by two aerator cubes in each vessel connected to a rotary pump. The vessels were filled nearly to the brim with sea water and a lid was placed over each to prevent the animals from climbing out. The sea water was replaced at intervals of 48 h and any dead or unhealthy specimens were removed daily. It was found that despite the plentiful aeration there was a heavy mortality at 25°C after 4 days whilst after 8 days the mortality had reached 80%. The remaining animals were therefore rejected and subsequent measurements were made only on those specimens surviving at the remaining four acclimation temperatures. There was a mortality of less than 10% at the other acclimation temperatures, except for animals in the vessel at 21°C where a mortality of 21% had occurred after 14 days. A similar procedure was adopted with the winkle Littorina littorea. Approximately 1000 similar-sized animals were collected from mid-tide level on 21 July 1970 at Whitstable, Kent, and groups of 200 were placed to acclimate at 5, 10·5, 16, 21 and 25°C as described above for Monodonta. The water was changed each 48 h as before and the mortality was not greater than 5% even after 14 days at 25°C.

Animals from each acclimation temperature were removed after 14 days from the vessels of sea water and equal numbers were placed in containers of sea water held at temperatures between 30 and 40°C. Only healthy and active animals were used for the experiments and the results described below thus apply only to those individuals of the population which were capable of survival at each acclimation temperature. Preliminary trials had shown that if either Littorina or Monodonta was removed from the experimental vessels and failed to retract when the operculum was touched with a seeker, then they did not subsequently recover when returned to the original acclimation vessels. We therefore adopted failure to retract as a criterion of irreversible heat damage, and the dead or moribund animals were removed at regular intervals. The percentage mortality could then be expressed as a function of time of exposure to each temperature and the time taken for 50% mortality could be read off from the cumulative curves.

The percentage mortality of Littorina littorea acclimated for 14 days to 5, 10·5, 16, 21 and 25°C and tested at 2°C intervals between 30 and 40°C is plotted against exposure time in Fig. 1. From this it is apparent that at any one test temperature the animals acclimated to 5 and 10·5°C survived for a shorter time than those acclimated to 16 and 21°C. But animals acclimated to 25°C in general did not survive any longer than those which had been stored at somewhat lower temperatures. Inspection of Fig. 1 also shows that at any one experimental temperature the cumulative mortality curves for animals acclimated to low temperatures are shallower in shape than those for animals acclimated to high temperatures. That is, most of the group of warm-acclimated animals had a similar heat-lethal temperature. This implies that the increase in survival which occurs after warm-acclimation is accomplished by raising the heat tolerance of the less resistant animals more than that of the other members of the population. In winter, when the heat-lethal temperature has little if any significance in survival, a considerable variation in heat-lethal temperatures would occur In summer, however, when the upper limit of thermal tolerance may acquire an ecological significance, the population of Littorina littorea would have a high and relatively uniform thermal tolerance. It is also apparent from Fig. 1 that at 40°C most of the winkles survived for a similar period despite the different acclimation temperatures to which they had been exposed. At these high experimental temperatures, therefore, thermal acclimation has little effect in increasing the thermal tolerance of L. littorea.

The influence of experimental temperature on survival time in each group of winkles is shown in Fig. 2, in which the time in hours to 50% mortality for animals acclimated to 5, 10·5, 16, 21 and 25°C has been read from Fig. 1 and plotted against experimental temperature. The most obvious features are that survival time is reduced at high experimental temperatures and that acclimation to temperatures above 21°C results in little further enhancement of survival at each experimental temperature.

The precise effect of acclimation temperature on mortality at various experimental temperatures is shown in Fig. 3 in which the time to 50% mortality at each experimental temperature has been read from the thermal resistance lines illustrated in Fig. 2. It is evident that thermal acclimation has its most marked effects in enhancing survival at 30°C and that as experimental temperatures are raised to 40°C, mortality is nearly as rapid in groups of animals acclimated to 25°C as in those acclimated to only 5°C. Further, as is also apparent from the thermal resistance lines shown in Fig. 2, acclimation to temperatures up to 16°C has a more important influence in increasing thermal tolerance than acclimation to temperatures between 16 and 25°C. Seasonal variations in the temperature of the sea may therefore play a more important part in increasing the thermal tolerance during the summer months than periodic exposure to high temperatures when the winkles are uncovered by the tide.

Littorina littorea is not normally uncovered for more than 8 h per tidal cycle at Whitstable, Kent, and occurs mainly on parts of the shore which are uncovered for approximately 6 h per tidal cycle. The thermal tolerance of such winkles is thus the temperature required for 50% mortality after an exposure time of not more than 6–10 h. Graphs relating the thermal tolerance after 6, 8 and 10 h to acclimation temperature are shown in Fig. 4. The winkles are likely to be acclimated to sea temperatures of approximately 15°C in the summer, thus temperatures which would kill more than 50% of the animals within one tidal cycle are 37·5°C for high-level individuals (uncovered for 8 h) and 39°C for animals of the mid-shore. If the upper-shore animals were uncovered for more than one tidal cycle during the neap tide period, then the limit of thermal tolerance may be lower than the values cited above. However, it is unlikely that temperatures greater than 36°C would occur on the shore for as much as 10 h per day so that a thermal tolerance of 37·5−39°C would allow survival of winkles from all shore levels even if they were exposed in direct sunlight.

The thermal resistance lines for Monodonta lineata are shown in Fig. 5, which was constructed from cumulative mortality curves similar to those shown in Fig. 1 and which shows that survival at 30°C is longer than at 40°C for groups of animals accli-mated to 5, 10·5, 16 and 21°C respectively. Further, acclimation to 10·5°C resulted in a marked increase in survival time compared with specimens acclimated to 5°C, but acclimation to higher temperatures than 10·5°C resulted in little further extension of survival. This result is similar to that described above for L. littorea.

The influence of thermal acclimation on survival time at various experimental temperatures is shown in figure 6 from which it is evident that, as in L. littorea, survival is most influenced by thermal acclimation at experimental temperatures between 30 and 34°C. At experimental temperatures above 36°C thermal acclimation has progressively less effect in enhancing survival.

Finally, it is of interest to estimate the thermal tolerance of M. lineata after exposure times which might occur under natural conditions much as has been shown in Fig. 4 for L. littorea. The results are illustrated in Fig. 7, which also shows the influence of thermal acclimation on the heat tolerance of Monodonta. This animal normally experiences a maximum of 6−8 h exposure to high temperatures during the summer when the tide has ebbed. Assuming that the animals are acclimated to approximately 15°C at this time, it is clear that the lethal temperature lies between 36 and 39°C, which is very similar to the lethal limits for L. littorea.

The data presented above agrees in general with those obtained by Southward (1958) and Fraenkel (1960) for L. littorea and by Micallef (1966) for M. lineata and shows that the thermal tolerance of these two species in water is similar. We have also shown that the heat-lethal temperature is profoundly influenced by thermal acclimation which is accomplished by raising the heat tolerance of the less resistant animals more than that of the other members of the population. It is of interest that acclimation to temperatures up to 16°C has the most pronounced effects in enhancing survival at high temperatures. This implies that even a modest increase in the sea temperature with the onset of summer would be sufficient to increase the thermal tolerance, so that unusually high temperatures could be tolerated when the animals were uncovered by the tide. It will have been noticed, however, that the thermal tolerance of both L. littorea and M. lineata considerably exceeds the maximum temperatures likely to be experienced by the organisms, especially when the cooling effect of evaporation of water from the body is taken into account. This has led several workers, including Broekhuysen (1940), Evans (1948) and Southward (1958), to suggest that lethal temperatures may not directly control the vertical zonational sequence of intertidal organisms.

More recently, Micallef (1966; see also Sandison, 1967) has shown that heat coma occurs before heat death and that under these conditions Monodonta and several other trochids may fall or be passively washed to lower levels on the shore where thermal stress is less severe. Indeed, Janssen (1960) found that Littorina littoralis becomes positively geotactic and thus crawls to lower shore levels at temperatures below 3°C or above 15°C. Micallef (1966, 1968; see also Newell, 1970) found that re-establishment of zonation patterns after a period of extreme temperatures was accomplished in trochids by negative geotaxis coupled with the selection of characteristic temperatures, and that high-level species had higher thermal optima than low-level species. Thus the data of Micallef (1966) on trochids and Janssen (1960) and Sanderson (1967; see also Lewis, 1964) on littorinids suggests that behavioural responses to temperature, coupled with evasion of extreme temperatures, may be of prime importance in controlling the characteristic zonational sequence of prosobranchs in the intertidal zone. The acclimation effects we have described would allow a seasonal adjustment of the upper limit of thermal tolerance and may acquire an ecological significance when unusually high temperatures prevail on the shore.