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The influence of temperature and salinity on heat tolerance in two grapsoid crabs, Hemigrapsus nudus… Todd, Mary-Elizabeth 1959

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THE INFLUENCE OF TEMPERATURE AND SALINITY ON HEAT TOLERANCE IN TWO GRAPSOID CRABS, HEMIGRAPSUS NUDUS AND HEMIGRAPSUS OREGONENSIS MARY-ELIZABETH TODD B.A., University of British Columbia, 1957 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Apri l , 1959 i ABSTRACT H e m i g r a p s u s nudus and H. o r e g o n e n s i s , t h e e x p e r i m e n t a l a n i m a l s , a r e s u b j e c t e d t o a wide r a n g e o f t e m p e r a t u r e and s a l i n i t y i n t h e i r n a t u r a l e n v i r o n m e n t . The i n f l u e n c e o f s e a s o n a l change, and l a b o r a t o r y a c c l i m a t i o n to v a r i o u s t e m p e r a t u r e - s a l i n i t y c o m b i n a t i o n s i n b o t h summer and w i n t e r , on h e a t t o l e r a n c e was d e t e r m i n e d . T h e r e was a s e a s o n a l change i n the 50 p e r c e n t s u r v i v a l t e m p e r a t u r e f o r 12 and 24 h o u r s when summer and w i n t e r b a s e l i n e s were compared. A d e f i n i t e s p e c i e s d i f f e r e n c e i n t h e r m a l r e s i s t a n c e was p r e s e n t , b ut b o t h s p e c i e s r e a c t e d s i m i l a r l y to a n y p a r t i c u l a r t e m p e r a t u r e - s a l i n i t y c o m b i n a t i o n . A c c l i m a t i o n t o a h i g h t e m p e r a t u r e i n t h e p h y s i o l o g i c a l t e m p e r a t u r e r a n g e g e n e r a l l y i n c r e a s e d t h e r e s i s t a n c e t o l e t h a l t e m p e r a t u r e s and a c c l i m a t i o n t o l o w s a l i n i t y . g e n e r a l l y d e c r e a s e d i t . H i g h t e m p e r a t u r e , h i g h s a l i n i t y was t h e most f a v o u r a b l e a c c l i m a t i o n c o m b i n a t i o n to r e s i s t l e t h a l t e m p e r a t u r e s . G a i n i n h e a t t o l e r a n c e a f t e r a low t e m p e r a t u r e h i s t o r y was r a p i d , l e s s t h a n one week. The low t o l e r a n c e f o u n d i n w i n t e r a n i m a l s a t the low t e m p e r a t u r e s e r i e s was n o t d e m o n s t r a t e d i n summer a n i m a l s a c c l i m a t e d t o t h e s e same c o n d i t i o n s . S m a l l a n i m a l s a p p e a r e d to be s l i g h t l y more r e s i s t a n t t h a n l a r g e r o n e s . M o u l t i n g a t t h e t e s t t o l e r a n c e t e m p e r a t u r e a d v e r s e l y a f f e c t e d t h e r e s i s t a n c e . A c e r t a i n d e n s i t y was n e c e s s a r y to p r e v e n t d e a t h f r o m o v e r c r o w d i n g a t l e t h a l t e m p e r a t u r e s . T h e r e was no d i f f e r e n c e i n t o l e r a n c e b e t w e e n t h e s e x e s . In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Mary-Elizabeth Todd Department of Zoology  The University of B r i t i s h Columbia, Vancouver Canada. Date April 17, 1959 i i TABLE OP CONTENTS INTRODUCTION MATERIAL AND METHODS 5 RESULTS 11 Summer 11 Winter 13 Effects of Size, Sex, Crowding and Moulting . . 22 DISCUSSION 24 SUMMARY LITERATURE CITED 44 45 i i i LIST OF FIGURES 1 The influence of a previous history of 20°C, 35 per cent sea water on the 50 per cent survival values at 12 and 24 hours 14 2 The influence of a previous history of 20°C, 75 per cent sea water on the 50 per cent survival values at 12 and 24 hours 15 3 The influence of a previous history of 5°C, 35 per cent sea water on the 50 per cent survival values at 12 and 24 hours . 16 4 The influence of a previous history of 5°C, 75 per cent sea water on the 50 per cent -survival values at 12 and 24 hours 17 5 The effect of weight on the time survived in hours. 23 6 The influence of differing densities of crabs per 4 l i t e r s of water on the 50 per cent survival temperature 25 i v L I S T OP T A B L E S I The c h a n g e i n 50 p e r c e n t s u r v i v a l t e m p e r a t u r e s w i t h v a r i o u s t e m p e r a t u r e - s a l i n i t y c o m b i n a t i o n s . 21 I I A c o m p a r i s o n o f t o l e r a n c e s t o h i g h t e m p e r a t u r e i n s e v e r a l s p e c i e s 57 V ACKNOWLEDGMENT I t g i v e s me g r e a t p l e a s u r e a t t h i s t i m e t o a c k n o w l e d g e my i n d e b t e d n e s s and g r a t i t u d e t o Dr. P.A. D e h n e l , D e p a r t m e n t o f Z o o l o g y , u n d e r whose d i r e c t i o n t h i s i n v e s t i g a t i o n was c o n d u c t e d . The time and a s s i s t a n c e t h a t were g i v e n i n e v e r y p a r t o f t h i s s t u d y a r e v e r y much a p p r e c i a t e d . I n a d d i t i o n , i t has been a p l e a s u r e t o h a v e had t h e o p p o r t u n i t y o f t a k i n g p a r t i n t h e many s t i m u l a t i n g d i s c u s s i o n s d u r i n g t h e c o u r s e o f t h i s s t u d y . I am a l s o i n d e b t e d t o D r . P.A. L a r k i n , D e p a r t m e n t o f Z o o l o g y , f o r the v a l u a b l e h e l p w i t h s t a t i s t i c a l t r e a t m e n t o f t h e r e s u l t s . The r e s e a r c h was a i d e d i n p a r t by a S c h o l a r s h i p g i v e n by t h e B r i t i s h C o l u m b i a Sugar R e f i n i n g Company L i m i t e d and a S t u d e n t s h i p awarded by t h e N a t i o n a l R e s e a r c h C o u n c i l o f C a nada. INTRODUCTION Temperature tolerance as an experimental criterion for the demonstration of physiological change has found many uses and has been reported i n various ways throughout the literature. The majority of work on temperature tolerance has been done on f i s h , with relatively few experiments on invertebrates. In many cases the lethal point of animals was determined by the temperature at which death occured when the animal was subjected to slowly increasing temperatures (Gowanloch and Hayes, 1926; Piatt, Collins and Witherspoon, 1957). As various authors raised the temperature at differing rates, the results could not be compared adequately because the time over which the temperature was increased would be expected to have a narked effect on the f i n a l lethal point. Experiments involving a range of temperatures at the upper temperature limits of the species, where either the time to death or per cent survival at intervals was noted when animals were held at a constant temperature, provided a more accurate determination of the lethal temperature as well as allowing comparisons among the species (Pry, Brett and Clawson, 1942; Brett, 1952; Spoor, 1955). Acclimation to low and high temperatures has been demonstrated i n many animals on the basis of laboratory acclimation, season, microgeography and latitude. In this discussion, acclimation i s used to include a l l types of - 2 -"demonstrable compensatory change" (Bullock, 1955). Acclimation has been demonstrated through a higher rate function at any intermediate temperature by low acclimated animals when compared with high acclimated ones. Dehnel and Segal (1956) in laboratory studies on the American cockroach, Periplaneta americana, found a higher rate of oxygen consumption i n equal weight nymphs and adults after acclimation to 10°C than when compared with 26°C acclimated animals, when measured at 20°C. Previously the culture had been maintained for at least three generations at a constant temperature, 27°C. Edwards and Irving (1945). showed the presence of seasonal acclimation i n the sand crab, Emerita talpoida. At a l l experimental temperatures below 20°C, oxygen consumption of animals in summer was less than that in winter. Ohsawa and Tsukuda (1956) found seasonal acclimation of response to temperature (exuding of the body) in the periwinkle, Nodilittorina granularis. Microgeographical acclimation has been demonstrated i n the limpet, Acmaea limatula. Samples taken from lower intertidal levels had a higher rate of heart beat than those from higher levels (Segal, 1956). Mayer (1914) has shown that at different latitudes there i s a similar rate function within a species at the environmental temperatures of the individuals, even though these temperatures are different. The rate of pulsations of the b e l l of Aurellia aurita was similar i n animals off Nova Scotia and off Florida with a temperature difference - 3 -of 15°C. Scholander, Flagg, Walters and Irving (1953) compared many poikilotherms, mostly interspecific but closely related species, from Arctic and tropic regions to find the general consistency of acclimation when rates of oxygen consumption were investigated. Dehnel (1955) demonstrated a higher rate of growth i n gastropods from high latitudes when compared with low latitude populations. Another facet of acclimation i s i t s effect on the temperature tolerance of a species. A previous temperature history at the upper levels of the physiological temperature range i s known to raise the thermal resistance. Conversely, low temperature acclimation w i l l decrease the tolerance to high temperatures. The rates of gain and loss of heat tolerance appear to show a consistant trend i n diverse groups of animals. In a l l cases, gain of heat tolerance i s much more rapid than i t s loss. Brett (1946) demonstrated that the goldfish, Carassius auratus, required a total of thirty days to acclimate to 28°C when brought from 4°C i n 8°C steps, but that acclimation occured at different rates, requiring twenty days from 4°C to 12°C, but only three days from 20°C to 28°C. Loss of heat tolerance i n the crayfish, Orconectes rusticus, was not completed by the end of the sixteenth day when the crayfish were transferred from 22° - 23°C to 4°C. The heat tolerance lost after thirteen days at 4°C was regained in about 24 hours (Spoor, 1955). Ohsawa (1956a) using the periwinkle Nodilittorina granularis stated, "The acclimatization to higher temperatures in the - 4 -winter snails i s established more readily than the acclimatization to lower temperatures in the summer animals." The effects of salinity changes on temperature tolerance have been studied less extensively. Broekema (1941) demonstrated that the temperature and salinity relations were closely dependent upon each other i n the shrimp Crangon crangon when survival in various combinations of the two was noted; a low salinity was endured better when the temperature was high. Wikgren (1953) showed that there was almost a continuous loss of ions from the crayfish Potamobius f l u v i a t i l i s as the temperature dropped to,2°-3°C from 17°C when the animals were transferred from tap water to d i s t i l l e d water. There was much less loss of ions, apparently more efficient osmoregulation, at higher temperatures (17°C and above). The lobster, Homarus  americanus, was shown to have a higher lethal point when both the salinity and temperature were high in the period of acclimation. A decrease i n sal i n i t y resulted i n a lowering of thermal resistance (McLeese, 1956). The two species of shore crabs studied in this investigation, Hemigrapsus nudus and H. oregonensis, are subjected to wide ranges of temperature and salinity conditions i n their natural environment. Experimental parameters of temperature and salinity were based on average seasonal conditions, to determine i f these seasonal changes, summer and winter, cause a resulting change in the upper - 5 -temperature tolerance of the two species. Also, animals were acclimated to various combinations of temperature and salini t y , to ascertain any resulting changes i n the temperature tolerance within either season. Because of the morphological similarity and like habitat in this area of these two species, i t was hoped that physiological differences, i n this case resistance to high temperatures, could be demonstrated. This was found to be the case. Additionally, effects of size, crowding, sex and moulting on the tolerance were noted. MATERIAL AND METHODS The experimental animals, Hemigrapsus nudus and H. oregonensia,were collected from Spanish Bank Beach on the south shore of Burrard Inlet. The Eraser River flows into Georgia Strait near this point, to account mainly for the recorded fluctuations in salinity occuring in this area. The beach has a gradual slope and changes i t s contour, from sand to rocks and again to sand. The lower sandy area i s exposed only on the lower tides. The crabs are found in the rocky intermediate area, the main population of H. nudus distributed higher than that of H. oregonensis. Individuals of both species, however, can be found almost from highest occurence to lowest.'' - 6 -The salinity i s quite uniform throughout the winter, ranging from 70 per cent to 30 per cent sea water (based on 100 per cent sea water as .31.88 °/oo). In the spring, with the greater influx of fresh water from the Fraser River, the salinity begins to drop and has been recorded as low as 10 per cent i n the summer months. At the same time as the salinity i s decreasing, temperature increases, and the relatively static summer state results where the average salinity i s about 35 per cent. N' Daily fluctuations in the salinity also occur, as the incoming tide carries a less saline water along the shore i n the collecting region. A visual demarkation, several hundred yards from shore, between the muddier, less saline Fraser River water and the more saline water results. The two species of crabs are found in the spaces under rocks where a pool of water or mussel shells (Mytilus edulis) provide a suitable micro-environment. Both species are found commonly under the same rock. H. oregonensis are found also in damp sand under rocks, often partially buried." Both males and females were used i n the temperature tolerance experiments, when preliminary experiments showed no difference in the resistance of the sexes. Gravid females or individuals missing any appendages were discarded. A complete weight range was collected except for very large animals, those weighing more than about 7.0 grams. The crabs were transported to the laboratory i n canvas buckets with damp seaweed. The experiments reported here were conducted - 7 -from 1957 to 1959 inclusive. In this investigation, holding experiments refer to those where the temperature tolerance of the crabs i s determined without any previous laboratory acclimation. The animals were tested after at least 24 hours in the laboratory from the time they were collected, during which time they were held at temperature and sali n i t y conditions which approximated those i n the f i e l d . This time period allowed the gut to be cleared partially so that at the high test tolerance temperatures, deposition of faeces and urine did not foul the water. In acclimation experiments, on the other hand, animals were held at previously determined acclimation conditions which differed from environmental circumstances i n at least one factor. In these experiments the crabs were acclimated for at least one week in the laboratory. Preliminary experiments indicated that acclimation was complete i n less than seven days; the tolerance of the two species, therefore, was tested from seven to a maximum of twenty-one days i n some experiments. Crabs were not fed at any time during the experiments, and were kept in darkness. If the acclimation temperature differed greatly from the environmental temperature, the crabs gradually warmed or cooled t i l l the desired temperature was reached, usually a period of two hours. Plastic dishes containing just under 4 l i t e r s of water v/ere used for the experimental animals. Holding or acclimation temperatures were either - 3 -5°C or 20°C, -1°C, based on average winter and summer temperatures. The salinities were either 35 per cent or 75 per cent sea water, again average seasonal conditions. Hence, four experimental combinations were used. About thirty-five crabs were placed in each container. Animals were changed to water of appropriate temperature and salinity, usually once per 24 hours, over the acclimation period. Temperature tolerance tests were conducted by floating the plastic containers in water baths in which the experimental temperature did not vary more than -0.1°C. The appropriate salinity, corresponding to that used during the acclimation or holding period, was employed for each tolerance test. Five animals per dish (per 4 l i t e r s of water) were tested at 1°C intervals, from 23°C to 35°C. The animals weighed from approximately 0.2 grains to 6.0 grams, so that the five crabs chosen for each test temperature ranged in weight from small to large. At each temperature both males and females were tested. Only animals with a f u l l y hardened carapace were used in the experiments. Crabs were brought to the test temperature from acclimation temperatures over a period of two to four hours at which time the experiment was begun. Preliminary experiments indicated more time was unnecessary as a more gradual warming period did not alter the results. The experiment was conducted for 24 hours and at each temperature dead animals were removed at 12 hours; live ones were changed to clean water. A l l - 9 -animals were dried with gauze and weighed to the nearest 0.1 gram. Animals were considered dead only when a l l movement ceased. Immediately prior to death, the sole motion visible was a slight pumping of water through the g i l l chambers. Using this criterion as a death point, at no time did animals revive when returned to lower temperatures. Crabs which moulted during the 24 hours of the experiments were discarded. One set of experiments was used to test the effect of different numbers of crabs per dish on the thermal resistance. A l l experimental conditions were as described above except for the varying densities of animals at each test temperature. The relationship of weight and time to death was determined by checking at hourly intervals over the 24 hour period and removing and weighing any dead animals. There was a change of water at 12 hours as noted in the above experiments. Data were plotted on arithmetic paper as i t was f e l t this type of plot gave the most accurate representation of the temperature tolerance curve. Each point, indicating per cent survival at any given temperature, represented an average of a number of duplications (each five animals per dish) for those particular conditions. In no instance was this average based on less than six repetitions resulting in a total of at least thirty animals; usually more were used. The total graph then, was based on a minimum of one - 10 -hundred eighty animals. No attempt was made to derive a formula for the f i t t i n g of each curve, but the points were merely joined. The 50 per cent survival temperature was determined from the place at which the survival curve crossed the line representing 50 per cent survival of the animals. Por the s t a t i s t i c a l analysis of the results, an analysis of variance was done for the complete summer data, that i s , the four combinations of temperature and salinity for both species at 12 and 24 hours, mainly to determine i f a species difference existed. The difference i n tolerance between the species was less during the summer months, so that a significant difference here definitely indicated one for the complete data, both summer and winter. Por individual comparisons such as the difference i n tolerance between the species at a given temperature-salinity combination, the Student " t " test was used to compare the 50 per cent survival temperatures. Another method was to compare the total area under the curve for any two graphs, contrasting the total cumulative per cent survival. The s t a t i s t i c a l probabilities resulting from the " t " test using this method were approximately the same as when 50 per cent survival temperatures were compared. Since the 50 per cent survival point i s a value commonly used i n the literature, this was chosen as a basis for comparison rather than the total cumulative survival value. In a l l cases where a s t a t i s t i c a l l y significant difference has been - 11 -d e m o n s t r a t e d u s i n g t h e " t " t e s t , t h e l e v e l o f s i g n i f i c a n c e i s b a s e d upon t h e 0.05 p r o b a b i l i t y p o i n t , a l t h o u g h t h e p r o b a b i l i t y i n some i n s t a n c e s i s l e s s t h a n t h e 0.01 l e v e l . RESULTS SUMMER Av e r a g e c o n d i t i o n s o f s a l i n i t y and t e m p e r a t u r e c h o s e n t o r e p r e s e n t t h e summer months were 20°C and 35 p e r c e n t s e a w a t e r . P e r c e n t s u r v i v a l c u r v e s f r o m the t o l e r a n c e e x p e r i m e n t s w i t h t h e s e c o n d i t i o n s w e r e c o n s i d e r e d as t h e b a s e l i n e c u r v e s f o r c o m p a r i s o n v / i t h a c c l i m a t i o n e x p e r i m e n t s where b o t h s p e c i e s were a c c l i m a t e d t o 20°C, 75 p e r c e n t s e a w a t e r ; 5°C, 35 p e r c e n t s e a w a t e r ; and 5°C, 75 p e r c e n t s e a w a t e r . T e m p e r a t u r e s a t w h i c h 50 p e r c e n t s u r v i v a l o c c u r e d f o r 12 and 24 h o u r s were u s e d as a b a s i s f o r c o m p a r i s o n s . H o l d i n g E x p e r i m e n t : 20°C and 55 p e r c e n t s e a w a t e r R e s u l t s o f t h e b a s e l i n e e x p e r i m e n t s showed t h a t H. o r e g o n e n s i s was more r e s i s t a n t t h a n H. nudus a t b o t h 12 and 24 h o u r s . The t e m p e r a t u r e a t w h i c h 50 p e r c e n t s u r v i v a l was f o u n d f o r H. o r e g o n e n s i s was 33.43°C f o r 12 h o u r s and 33.16°C f o r 24 h o u r s . P o r H. nudus t h e v a l u e s were 32.95°C f o r 12 h o u r s and 32.50°C f o r 24 h o u r s ( P i g . 1) . A c c l i m a t i o n E x p e r i m e n t : 20°C and 75 p e r c e n t s e a w a t e r B o t h s p e c i e s showed a v e r y marked i n c r e a s e i n t o l e r a n c e a f t e r a c c l i m a t i o n t o 20°C and 75 p e r c e n t s e a w a t e r . T h i s - 12 -combination of temperature and sali n i t y differed from the base line conditions only i n the salinity, having been changed from 35 per cent to 75 per cent sea water. H. oregonensis again showed more tolerance than H. nudus with values of 34.50°C for 12 hours and 34.38°C for 24 hours, an increase of 1.07°C and 1.22°C respectively over the base line values. 50 per cent survival temperatures for H. nudus were 33.62°C and 33.45°C for 12 and 24 hours, an increase of 0.67°C and 0.95°C (Pig. 2). The increase found in both species was significantly different from the base line values, P<.05. It i s important at this point to note that acclimation to the above conditions provided the most favourable background for withstanding the high test tolerance temperatures. Acclimation Experiment: 5°C, 55 per cent sea water Low temperature and low salinity combined appeared to be slightly less favourable for survival during the acclimation period as the number of deaths i n both species was greater than with any of the other combinations, but with fewer deaths among the H. nudus. Temperature tolerance experiments showed the tolerance to be much the same as the base line values, with no significant difference for 50 per cent survival, 33.28°C and 33.11°C for H. oregonensis and 33.38°C and 32.64°C for H. nudus for 12 and 24 hours (Pig. 3). Acclimation Experiment: 5°C, 75 per cent sea water Acclimation to these conditions did not result i n much change i n the tolerance when compared with base line - 13 -conditions. It i s possible that the time allowed for acclimation was not of sufficient duration, since animals collected in the winter when these conditions were found in the f i e l d did show a lowering of the tolerance. No change, however, in the temperature at which 50 per cent survival occurred was detected in the summer animals after seven days acclimation compared with a total of thirteen days acclimation. The temperatures for 50 per cent survival for H. oregonensis were 33.20°C and 32.73°C and for H. nudus, 33.05°C and 32.58°C for 12 and 24 hours (Pig. 4). These values did not have a significant difference from those found in the base line experiments. When these animals however, were compared with animals acclimated to 20°C, 75 per cent sea water (Pig. 2), significant difference i n tolerance was found, P< .05; summer animals, then, when acclimated to both high and low temperatures with high salinity, demonstrated a reduction of thermal tolerance with the low temperature acclimation. WINTER The winter base line represented the average conditions in the winter months. Winter animals were acclimated to the three remaining combinations of 20?C, 35 per cent sea water: 20°C, 75 per cent sea water: and 5°C, 35 per cent sea water. Holding Experiment: 5°C. 75 per cent sea water The 50 per cent survival temperatures were 32.59°C and 32.43°C for H. oregonensis and 32.00°C and 31.79°C for - 14 -F i g u r e 1. The i n f l u e n c e o f a p r e v i o u s h i s t o r y o f 20°C, 35 p e r c e n t s e a w a t e r on t h e 50 p e r c e n t s u r v i v a l v a l u e s a t 12 (•) and 24 (o) h o u r s . B o t h s p e c i e s i n summer and w i n t e r a r e shown. E a c h p o i n t r e p r e s e n t s t h e a v e r a g e s u r v i v a l o f a t l e a s t t h i r t y a n i m a l s . 100 90 80 70 60 50 40 30 20 10 0 _J > > ID CO LU O cn LU 100 Q. 9 0 8 0 7 0 6 0 5 0 40 30 20 10 0 HEMIGRAPSUS NUDUS HEMIGRAPSUS OREGONENSIS SUMMER BASE LINE SUMMER BASE LINE 20°C,35%SEA WATER 20 °C, 3 5 % SEA WATER ® ® • > : -1 \ \ * - 12 HOURS 24 HOURS — • \ \ \ \* - l2 HOURS ...... : \\ \\ in 32.90 ? ? 32.93 i I I I i i i i i 33.16 ' i i i i i i \\\\ ' ? 33.43 • • HEMIGRAPSUS NUDUS WINTER 20 ° C , 3 5 % SEA WATER HEMIGRAPSUS OREGONENSIS WINTER 20 °C, 3 5 % SEA WATER ®—®—®—® N\ \ 1 : 1 _ Q_\ \ \<*-l2 HOURS 24 HOURS—*\ \ \ 1^ —12 HOURS 24 HOURS —1>\ I \\ 32.22? ? 32.54 1 1 1 1 1 ' • ' 111 3 3 . 0 4 ? ? 33.38 • i i i i i i i 28 29 30 31 32 33 34 35 28 29 30 31 32 33 34 35 TEST TEMPERATURE °C - 15 -Figure 2. The influence of a previous history of 20°G, 75 per cent sea water on the 50 per cent survival values at 12 (•) and 24 (o) hours. Both species in summer and winter are shown. Bach point represents the average survival of at least thirty animals. 100 90 80 70 60 50 40 30 20 10 0 < > > cr Z) CO UJ ( J cr UJ Q_ HEMIORAPSUS NUDUS SUMMER 20°C,75% SEA WATER 100 90 80 70 60 50 40 30 20 10 0 HEMIORAPSUS OREGONENSIS SUMMER 20 'C, 73 % SEA WATER 24 HOURS -'34.50 HEMIGRAPSUS NUDUS WINTER 20 °C, 75% SEA WATER HEMIGRAPSUS OREGONENSIS WINTER 20 °C, 75% SEA WATER ® — © — © — e — o \ 9 — ® — ® — a — ® ^ - ® t \ \\ \\ n 1 I HOURS 24 HOURS — M I I 1 HOURS 24 HOURS -»\ I \ ,1,1 33.49 + j 34.32 1 1 1 1 1 • 33.66 i J34.40 l i I I i i i i 28 29 30 31 32 3334 5 28 29 30 31 32 33 34 35 TEST TEMPERATURE °C. - 16 -Figure 3. The influence of a previous history of 5°C, 35 per cent sea water on the 50 per cent survival values at 12 (•) and 24 (°) hours. Both species in summer and winter are shown. Each point represents the average survival of at least thirty animals. 100 90 , 80 70 60 50 40 30 20 I ° or z> CO HEMIGRAPSUS NUDUS SUMMER HEMIGRAPSUS OREGONENSIS SUMMER 5°C, 35% SEA WATER 5°C,35% SEA WATER - e—® —ft e — » c — 9 A \ 1 12 HOURS 24 HOURS - M 1 \ \ « - 12 HOURS 24 HOURS "•ll \ \ 32.64 T ? 33.38 II I 33.11 # 33.28 LU O cn UJ D- 100 90 80 70 60 50 40 30 20 10 0 HEMIGRAPSUS NUDUS HEMIGRAPSUS OREGONENSIS WINTER WINTER 5 ° C, 35 % SEA WATER 5 °C,35% SEA WATER At • : . \ V \ 9 \ \ \ 24 \ « - l 2 HOURS HOURS \W-I2 HOURS 24 HOURS \ 9 : , V \ 1 V » 28.68 ff 28.81 1 1 1 1 1 1 l i 30.13 W 30.18 • i i i i i i i 2 8 2 9 3 0 3 1 3 2 3 3 3 4 3 5 28 29 30 31 32 33 34 35 TEST TEMPERATURE °C. - 17 -Figure 4. The influence of a previous history of 5°C, 75 per cent sea water on the 50 per cent survival values at 12 (•) and 24 (o) hours. Both species in summer and winter are shown. Bach point represents the average survival of at least thirty animals. > or CO HEMIGRAPSUS NUDUS SUMMER 5 ° C , 7 5 % SEA WATER UJ O t r w 100 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 10 0 HEMIGRAPSUS NUDUS WINTER BASE LINE 5 ° C , 7 5 % SEA WATER 9 9 9 9 24 HOURS ,<•- 12 HOURS HEMIGRAPSUS OREGONENSIS SUMMER 5 ° C , 7 5 % SEA WATER • • 32.73 • • 33.20 J I I I L HEMIGRAPSUS OREGONENSIS WINTER BASE LINE 5 ° C , 7 5 % SEA WATER -®-24 HOURS 12 HOURS 28 29 30 31 32 33 34 35 28 29 30 31 32 33 34 35 TEST TEMPERATURE °C. - 18 -H. nudus for 12 and 24 hours respectively (Pig. 4). The difference between these winter values and those obtained for summer animals acclimated to these same conditions (winter) i s significant, P<.05. Acclimation Experiment: 20°C, 35 per cent sea water Crabs collected during the winter months and acclimated to the above conditions, which corresponded to the summer base line combination, were found to have survival values quite similar to those found i n the summer months. The 12 and 24 hour 50 per cent survival temperatures were 33.38°C and 33.04°C for H. oregonensis. and 32.54°C and 32.22°C for H. nudus, the greatest difference being only 0.41°C from the summer months (Pig. 1). This meant that in less than one week the winter animals were able to approach the degree of resistance found in the summer animals which may have had several months under these conditions i n the f i e l d . There was a significant difference in the 50 per cent survival points between winter animals acclimated to summer base line conditions, and the winter base line. Acclimation Experiment: 20°C, 75 per cent sea water Acclimation tor:-these conditions provided the most suitable environment to withstand the test tolerance temperatures; there was a marked increase in the temperature at which 50 per cent survival occured, from the base line. Again, the animals acclimated in the winter approximated the tolerance found i n summer animals acclimated to these same conditions, and were significantly different from the - 19 -winter base line. In one instance,H. nudus at 12 hours, the tolerance was even greater than that found in summer animals acclimated to these same conditions. The 50 per cent survival values for H. oregonensis were 34.40°C and 33.66, and were 34.32°C and 33.45°C for H. nudus for 12 and 24 hours (Pig. 2), an average increase of about 2°C over the base line. Acclimation Experiment; 5°C, 35 per cent sea water Animals acclimated to the low salinity, low temperature combination showed the greatest difference, both from the summer counterpart and the winter base line. The 50 per cent survival temperatures for H. oregonensis were 30.18°C and 30.13°C for 12 and 24 hours and for H. nudus 28.81?C and 28.68°C (Pig. 3). This was a drop of approximately 2.5°C for H. oregonensis from the winter base line conditions and 3°C from the survival temperatures obtained from summer animals acclimated to these same conditions. The drop in the survival temperatures for H. nudus was even greater, being about 3.5°C and 4.5°C respectively. When winter animals acclimated to both high and low temperatures with low salinity are compared, the low temperature acclimation again results in a significantly different reduced tolerance (P<.05). During the acclimation period, the number of deaths was greater in both species than at any other temperature-salinity combination, a similar result to that found with the summer animals. Contrary to H. nudus being the slightly more resistant during the acclimation period i n the summer animals, - 20 -that species was less resistant than H. oregonensis in the experiments involving winter animals. As seen from the results of the above experiments, the 50 per cent survival temperatures for.both 12 and 24 hours were higher for H. oregonensis than for H. nudus i n a l l except one case when the value for H. oregonensis was only 0.10°C lower. Usually the 50 per cent survival temperature for H. oregonensis was higher by a much greater amount than this, being i n some cases over a degree. Analysis of variance on summer data indicated that H. oregonensis was the more resistant at lethal temperatures, P< .01. The lower resistance i s surprising in H. nudus as the species i s more commonly found at a higher tide level, and i t would be expected, a p r i o r i , that a greater tolerance for high temperatures would be of more advantage to that species. Also, the similarity of the 50 per cent survival temperatures for 12 and 24 hours within each species was consistant throughout. At each test temperature the values were very close, and in many cases were identical. Table I summarizes the results. Salinity had a pronounced effect on the amount of variabi l i t y that was obtained in a series of replications of one of the experimental conditions. Whether the acclimation temperature was 20°C or 5°C, but particularly with the low acclimation temperature, the low salinity (35 per cent sea water) resulted i n much more variable survival percentages at the test tolerance temperatures than those found with - 21 -TABLE I The change in 50 per cent survival temperature with various temperature-salinity combinations SUMMER WINTER H. nudus H. oregonensis H. nudus H. oregonensis 20°C, 35$ 12 hr. 32.95°C* 33.43* 32.54 sea water 24 32.50* 33.16* 32.22 33.38 33.04 20°C, 75$ 12 hr. 33.62 sea water 24 33.45 34.50 34.38 34.32 33.45 34.40 33.66 5°C, 35$ 12 hr. 33.38 sea water 24 32.64 33.28 33.11 28.81 28.68 30.18 30.13 5°C, 75$ 12 hr. 33.05 sea water 24 32.58 33-20 32.73 32.00* 31.79* 32.59* 32.43* * Base line tolerances in the two seasons, summer and winter. - 22 -the higher salinity. Also, the temperature at which 100 per cent survival occurred was a much more tenuous point with the lower salinity. This can easily be seen in any of the low salinity graphs, as there i s a gradual slope to the f i n a l breaking off point, whereas with the 75 per cent salinity, the break i s abrupt, directly from 100 per cent. It should be noted that the difference in temperatures between the points where 100 per cent or close to 100 per cent survival i s found for 24 hours and complete mortality i s very slight. Effects of Size, Sex, Crowding, and Moulting A l l weights of crabs did not appear to have the same resistance to the high test tolerance temperatures. Smaller animals, in both species, seemed slightly more tolerant than larger ones. There was no direct relationship between weight and time to death, but results indicated that i f any animal lived, i t was usually less than 1 gram. This seemingly greater tolerance in the smaller crabs was prevelant when crabs from a l l temperature-salinity combinations were tested. One graph was chosen to demonstrate this phenomenon (Pig. 5). As mentioned previously, both males and females were used in the experiments when the sex was found to have no influence on the resistance to the high temperatures. To the contrary, crowding, that i s more than 5 animals per 4 l i t e r s of water, caused a marked lowering of resistance to high temperatures. About 0.333 l i t e r s of sea water per 1 gram live animal weight was found to be satisfactory. When less water was allowed, by including more animals per dish, dying - 23 -Figure 5. The effect of weight on the time survived in hours at 31°C in Hemigrapsus nudus. The animals had a previous history of 5°C, 75 per cent sea water. Each point represents one animal. The regression line i s eye-fitted. H E M I G R A P S U S N U D U S WINTER B A S E L I N E 5 ° C , 7 5 % S E A W A T E R T E S T T E M P E R A T U R E 3TC i i 1 i i 1 1 — r I 2 3 4 5 6 7 8 W E I G H T IN G R A M S - 24 -animals affected the water even though they were removed immediately upon death so those remaining were more apt to die (Pig. 6). Thus, with 5 animals per dish, the 50,per cent survival temperature for 12 hours was 32.95°C, with 10-15 animals per dish, 30.68°C, and with 19-28 animals, 27.86°C. This crowding effect existed only at the high test tolerance temperatures. As was mentioned in the previous section, when there were at least 35 animals per dish at the acclimation temperature of 20°C, ho mortality due to crowding was noted. Since only crabs with a f u l l y hardened carapace were used in the experiments, the effects of moulting on the, temperature tolerance were determined from those animals which moulted during the 24-hour course of the experiment. These animals invariably died, possibly because of increased permeability of the carapace, together with the high temperature, were beyond the limits of tolerance. DISCUSSION Acclimation to a favourable high temperature with resulting increase in the high temperature tolerance of the species, has been demonstrated in animals many times (Sumner and Doudoroff, 1938; Brett, 1946; Mellanby, 1954; Spoor, 1955; McLeese, 1956). The two species of crabs Hemigrapsus nudus and H. oregonensis, studied here, proved to be no exception. Temperature tolerance i n conjunction with salinity - 25 -Figure 6. The influence i n Hemigrapsus nudus of differing densities of crabs per 4 l i t e r s of water on the 50 per cent survival temperature at 12 hours with a previous history of 20°C, 35 per cent sea water: 5 animals per dish (o), 10 to 15 animals per dish (•), 19 to 28 animals per dish ( A ) . Each point represents the average survival of at least thirty animals. HEMIGRAPSUS NUDUS SUMMER BASE LINE 20°C,35% SEA WATER TEST TEMPERATURE °C. - 26 -has been studied less extensively, and i t was of interest to find that the salinity had a very marked effect on the temperature tolerance of the species. Although results from the survival curves may differ i n summer animals as opposed to winter animals, the trend with regard to^tolerance in the particular temperature-salinity combination was the same in both seasons and both species, but there was a definite species difference. During the summer months, the environmental temperature i s high and the salinity i s low. When both species of winter animals are acclimated to these conditions, i t was found that these acclimated animals had a tolerance at high temperatures which was approximately as great as that found in the summer animals. Likewise, these winter animals when acclimated to the high salinity as well as to the high temperature, demonstrated i n both species, again a temperature tolerance very similar to that found in summer animals acclimated to these same conditions, by far the most favourable combination to resist heat death. F i r s t of a l l , i t i s evident that these animals can regain their tolerance to high temperatures very rapidly^ after a low temperature history^by acclimation in the upper part of the physiological temperature range. This rapid gain of heat tolerance has been documented many times. Mellanby (1954) reported that the heat coma point i s shifted with experimental acclimation at the upper end of the tolerable range in the mealworm, Tenebrio molitor and mosquito, - 27 -Aedes aegypti. The gain in tolerance was rapid as twenty hours acclimation produced as much change as longer acclimation periods. Spoor (1955) found complete gain of heat tolerance in the crayfish in less than 24 hours with an approximate 18°C change i n temperature (4°C to 23°C). Ohsawa (1956a) determined that gain i n heat tolerance by the periwinkle was complete i n less than 48 hours when acclimated to 30°C from 10°C. One exception to a f a i r l y rapid gain i n heat tolerance i s a recorded time of about twenty-two days for total acclimation in the American lobster when transferred from 14.5°C to 23°C (McLeese, 1956). McLeese suggested that the reason for this unusual length of time i s that the lobster has a long latent period before onset of demonstrable acclimation, i n this case ten days. A rapid gain of heat tolerance would be of extreme advantage to most inter t i d a l animals. In those animals permanently submerged, a quick augmentation would appear to be of l i t t l e direct advantage as bodies of water, even i f relatively small, only very slowly change i n temperature. Por intertidal animals, the increase i n temperature may be as much as 10°C over a period of a few hours in the tidal rhythm. At Spanish Bank, the collecting area for H. nudus and H. oregonensis, tide pools have been recorded at 26°C to 28.5°C. In this case, a rapid gain in tolerance, particularly over a period of a few hours would be most advantageous." Probable increase in salinity in tide pools during the low tide period in summer months aids the animal - 28 -in resisting the high temperatures as experimental results have indicated that high salinity provides the most favourable environment. Regarding the low temperature series, the results can not be interpreted as readily. The seasonal difference i s marked when compared with the seasonal change i n the high temperature series. The change was consistantly to a lower temperature for the 50 per cent survival value i n the winter animals. Por the winter base line, 5°C, 75 per cent sea water, a drop of about 1°C, from summer animals acclimated to these same conditions, was found i n both species at 12 and 24 hours. With the low salinity , low temperature combination, loss i n heat tolerance i s much greater. In this low temperature series i t i s clear that the seasonal alteration in the f i e l d environment produces a noticeable reduction of tolerance in the winter animals to high temperatures. Seasonal change i n heat tolerance of a species has been recorded many times. Edwards and Irving (1943). found a 10°C difference in the death point of Emerita talpoida with summer animals higher than winter ones. Brett (1944) found a change i n lethal temperature i n Algonquin Park fishes from spring to f a l l . In most cases, the species studied have been acclimated either solely to various temperatures in the laboratory and the temperature tolerance studied, or the tolerance has been tested at/diff er ent seasons with no laboratory acclimation. An example of seasonal comparisons in thermal resistance with the same acclimation - 29 -temperature i n summer and winter i s a study on planaria (Schlieper and Biasing, 1953 as reported i n Pry, 1958) and the rainbow trout (Keiz, 1953). Both species were more tolerant of high temperatures i n the summer months. Ohsawa (1956a) found that summer periwinkles were active at a higher temperature than winter animals which were tested over the sair.e range; p r i o r to the test, the summer animals had a 43 hour history at 10°C which corresponded to winter f i e l d conditions. In both studies on the periwinkle and the crabs investigated here, winter animals were acclimated to summer conditions v/ith r e s u l t i n g summer tolerances, but not the r e c i p r o c a l . Studies on several species of fresh water f i s h (Hart, 1952), demonstrated that winter animals i n some species had a lower l e t h a l temperature than summer animals, aft e r acclimation to the same temperature i n the respective seasons. As i n a l l acclimation experiments, the animals of the low temperature series were kept one week before survival was determined at the test tolerance temperatures. There was no change i n the tolerance ever the testing i n t e r v a l , seven to thirteen days. This period of approximately two weeks was i n agreement with that found necessary by other investigators to demonstrate laboratory acclimation to low temperature. It was evident from experiments on animals collected i n winter months that summer animals acclimated to winter f i e l d conditions of temperature and s a l i n i t y i n no way approached the reduced tolerance found i n winter animals. Likewise, summer animals acclimated to low temperature plus low s a l i n i t y had a much greater - 30 -acclimation condition i n winter animals. Insufficient length of time for acclimation i s an obvious possibility, but i t would be thought that some change would have shown i n the tolerance over the period of two weeks. A long latent period before the onset of acclimation to the low temperature i s feasible as a latent period of 10 days was reported i n acclimation to a higher temperature i n the lobster (McLeese, 1956). Another possibility i s that some other factor or a combination of factors influences the acclimation to low temperatures but not to the reverse, as winter animals which were acclimated to summer conditions gave approximately the same results as summer animals. Dehnel (1958) has shown that in these two species of crabs, H. nudus and H. oregonensis, that a simulated seasonal variation of light i n the laboratory has a .pronounced effect on metabolism measured by oxygen consumption. It i s feasible that summer animals v/ould never have the same temperature tolerance as that found in the winter animals solely by acclimating them to winter temperature and salinity conditions. Hoar (1956) from experiments on goldfish '.'..concluded that the seasonal variations in thermal tolerance previously noted i n f i s h maintained under constant temperature conditions are photoperiodically controlled...". It i s hoped that future experiments w i l l determine this relationship more clearly i n these crabs. Since two weeks acclimation in the summer animals did not bring about a change over the testing period, i t i s important to note that animals cannot be said to acclimate or not acclimate to a parameter - 31 -merely because there i s no evident change. In nearly a l l instances low salinity resulted in a lower value for the 50 per cent survival temperature than with high salinity, whether tie temperature was'5°C or 20°C. Gross (1957) gives values for H. nudus and H. oregonensis, calculated from Jones (1941), which indicate the osmotic gradient maintained by these two species when placed i n various concentrations of sea water. The crabs are isotonic to the external environment at 100 per cent sea water (based on 34.6 °/oo). At 75 per cent sea water there i s an extremely slight gradient maintained, about 2.5 per cent sea water in H. oregonensis and 8.0 per cent in H. nudus. The gradient maintained at 35 per cent sea water, however, i s about 36 per cent i n both species. These values cannot be applied directly to the present experiments, but certainly they can indicate the trend. The maintenance of this gradient presumably results i n more work being done by the animal at the lower salinity, and i n a greater stress placed on i t than at 75 per cent sea water where the gradient i s only a few per cent. Other proof of more osmotic work being performed at the lower salinity in these crabs i s a 22 per cent increase in respiratory rate at 35 per cent over that found at 75 per cent sea water i n a 1.0 gram animal measured at 10°C (Dehnel, unpublished). It i s probable that the lower 50 per cent survival temperatures with the lower salinity i s due to the increased metabolic work necessary at that salinity. The lethal or near lethal temperatures i n the test tolerance experiments presumably alone are causing a marked strain - 32 -on the metabolic activity of the crabs and the additional strain of maintaining a large gradient between the blood and the external medium i n the low salinit y , results in the animal dying at a lower temperature. In the present investigation, low salinity, combined with low temperature, particularly in the winter animals, was the most disadvantageous acclimation combination for withstanding the test tolerance temperatures. Wikgren (1953) showed a greater loss of ions when the temperature decreased in the crayfish when transferred from tap to d i s t i l l e d water. He f e l t that this greater ion loss at the lower temperatures could be assigned to depressed absorption of ions and chloride. A similar relation between salinity and temperature was found by Broekema (1941) i n the shrimp, Crangon crangon. The salinity optimum for length of l i f e depended on the temperature with a downward shift in the salinity optimum with a increase i n temperature. High temperatures generally were less favourable for survival than low, but low salinity was endured better with a higher temperature. This was similar to the pattern in the two species of crabs studied in these experiments; with low salinity, temperature tolerance was increased when there was a previous high temperature history rather than a low, particularly i n winter animals. In addition Broekema showed that the difference between the internal blood concentration and external concentrations i n the shrimp was greater at high than at low temperatures and suggested that at low temperatures the limits of l i f e are - 33 -exceeded sooner than when the temperature i s high. Jones (1941) demonstrated a higher osmotic pressure in the blood of H. nudus and H. oregonensis with a higher temperature, signifying an increase i n the degree of regulation. Thus, with a difference i n temperature acclimation at the low salinity i t i s probable that the difference i n sustained gradients could carry through to the temperature tolerance tests, and thereby affect the lethal point. Jones, however, showed a great variation in the osmotic pressure of the blood when these crabs were near death from air exposure and f e l t that this tended' to refute a certain lethal osmotic pressure as the primary cause of death. Broekema's findings, demonstrating that shrimp i n a high salinity lived longer at a low temperature, at f i r s t appear contrary to the results of this investigation where the most favourable history forresisting high temperatures was one of both high temperature and high sali n i t y . It must be remembered, however, that the criterion for optimal conditions were quite different, a b i l i t y to resist lethal temperatures i n this investigation, whereas i n Broekema's experiments the length of l i f e was the factor. The results cannot be directly compared, but i t i s f e l t that they both show the same trend, particularly with respect to low sa l i n i t i e s . Kinne (1958) reported the shifting of heat tolerance in three species of animals, the polychaete Nereis diversicolor, the amphipod Gammarus duebeni, and the isopod Sphaeroma hookeri. - 34 -The salinity of the pond from which a l l three species were collected was about 12 °/oo, and i n a l l three species when kept at sa l i n i t i e s below this, heat resistance i s lowered. Increased heat resistance results in animals from the higher s a l i n i t i e s , above 12 °/oo. Kinne suggested that the alteration of water and ion balance with increased water content at extremely low sa l i n i t i e s decreases the heat tolerance, whereas the lowered water content at higher temperatures favourably affects the resistance to high temperatures. The range of sa l i n i t i e s chosen were well beyond the limits of regulation at least i n Gammarus duebeni from values given by Verwey (1957) and at the upper s a l i n i t i e s in Nereis diversicolor (Smith, 1955b). It would be interesting to know i f the change in heat tolerance would be the same when the animals were kept at sa l i n i t i e s within the physiological regulatory range. It could be expected that such a similar change would exist so that the variation in water content of the animal would be extremely slight or n i l . If such were the case, another explanation than change in water content for the difference i n heat tolerance in animals from low and high s a l i n i t i e s would be necessary. Since the salinity of the pond where the animals were collected was only 12 °/oo, this would result in a f a i r l y large sustained gradient at least in two of the three species for which data are available (Smith, 1955b; Verwey, 1957), and the resulting stress from the extra gradient necessary at even lower s a l i n i t i e s could cause a weakening at lethal - 35 -temperatures more quickly. The higher sal i n i t i e s presumably within limits would approximate more nearly natural conditions i n the body fluids of the animals, to provide a more favourable environment for survival. Another species studied by Kinne (1956);, the hydroid Cordylophora caspia, has slight osmoregulatory a b i l i t i e s , and again was found to withstand a high temperature better at a high salinity than at lower ones. Possibly the explanation here would be that the higher salinity i s nearer the concentration of body fluids of the hydroid, resulting in a more favourable environment to resist a stress, in this case, temperature. The geographic limitation of the Polychaete Nereis i s related to the salinity-temperature balance of i t s environment. The range of Nereis diversicolor in the Baltic Sea apparently i s limited by the colder parts of the year when i t could not withstand the low salinity expressed as a chlorinity of 4 grams per l i t e r i n which i t i s able adequately to regulate in the warmer summer months (Smith, 1955a). Studies on Neanthes l i g h t i indicated that i t was able to survive in fresh water in Lake Merced, California, because of i t s viviparous mode of reproduction where the young are sufficiently developed at birth that they are able to osmoregulate and that the species i s not exposed to severe winter cold. Laboratory experiments showed that the ab i l i t y to regulate in water of a chlorinity of 1.0 to 0.09 grams per l i t e r was inhibited at 1.5°C but possible at 13°C in worms tested in vitro (Smith, 1957). - 36 -Animals acclimated to temperatures in the upper part of their physiological temperature range show a remarkable similarity in lethal temperatures. A few comparisons for 12 hours median tolerance among animals acclimated to approximately 20°C and a favourable salinity i f marine forms are given i n Table II. Por the most part the lethal temperature i s at least 30°C. In the species difference found in this investigation, the greater thermal resistance of H. oregonensis i s surprising since i t occupies a lower level on the beach. Within the same species, Lit torina litorea, the individuals collected at high tide level had greater resistance to lethal temperatures than those collected a few yards away at low tide level (Gowanloch and Hayes, 1926). Ohsawa (1956b) determined species difference for the periwinkles Nodilittorina granularis and N. v i l i s . The latter occurs in the upper zone of the wide range occupied by N. granularis, which was found to be the more tolerant at lethal temperatures, a similar result to that found with H. nudus and H. oregonensis. In most animals then, and possibly a l l , a previous high temperature history increases heat tolerance and a low one decreases i t . Along with this i s the influence of salinity where low s a l i n i t i e s almost invariably are endured better at a higher temperature, and high temperature, high salinity combined being most favourable for withstanding upper lethal temperatures. Low salinity, low temperature generally i s least favourable for length of l i f e and for - 37 -TABLE II A comparison of tolerances to high temperature i n several species. Species 12 hour median tolerance, °C Source Hemigrapsus nudus 33.62 H. oregonensis 34.50 Orconectes rusticus 36.4 0. propinquus 35.0 Cambarus fodiens 35.0 Homarus americanus 28.4-30.5 Hyalella azteca 33.0 Ameiurus nebulosus 33.4 Carassius auratus 34.5 Spoor, 1955 Bovbjerg, 1952 Bovbjerg, 1952 McLeese, 1956 Bovee, 1949 Brett, 1944 Pry, Brett and Clawson, 1942 - 38 -heat tolerance. The effect of size and sex on heat tolerance was found to vary in different animals reported i n the literature. In Hemigrapsus nudus and H. oregonensis, smaller animals seemed to be slightly more resistant to the high test tolerance temperatures than larger individuals. There was no difference in tolerance between the sexes. Edwards and Irving (1943). found no difference in sex i n Emerita talpoida but larger animals seemed slightly more resistant than small ones. In lethal temperature experiments on young speckled trout, Salvelinus fontinalis, size did not affect the time to death at high temperatures in the same age group (Pry, Hart and V/alker, 1946). Hoar. (1955) found an increased tolerance at 1°-2°C in goldfish with an increase i n size from 3 to 7.5cm. McLeese (1956) concluded that in the size range of lobsters studied, from 21 to 28cm, there i s identical response to lethal temperatures. In the crayfish, sex and size did not affect heat tolerance (Spoor, 1955). Kinne (1958) showed a decreased tolerance in female Gammarus duebeni to high temperatures, and increased tolerance in smaller (younger) animals. In Sphaeroma hookeri, smaller males appeared more resistant than larger ones. There was apparently no sex difference i n this species. Thus, i t i s seen that no conclusions can be drawn regarding the effect of size or sex on heat resistance. It seems entirely random whether these factors have no effect, increase tolerance, or decrease i t . The amount of water available to each animal during - 39 -the experiments testing heat tolerance was found to influence markedly that tolerance even though animals were removed at death. At least 0.333 l i t e r s of water per gram liv e animal weight per 12 hours was necessary to prevent death from overcrowding. Most investigators testing the tolerance of a species allow much less water than this. Gowanloch and Hayes (1926) tested 10 to 15 periwinkles per 250cc of water (.250 l i t e r s ) , less water than that found necessary in the lethal temperature experiments on these crabs. Spoor (1955) allowed less than 50cc (.05 l i t e r ) of water per gram of animal weight when testing 5 to 10 crayfish i n a one l i t e r Erlenmeyer flask. Brett (1952) used about 0.395 l i t e r s per gram of animal using 1.0 gram as an average weight, similar to the calculated necessary minimum for the crabs. McLeese (1956) used 10 lobsters at each constant temperature. Taking a value of 400 gramsas an average weight of the experimental animals, only about 13.5cc (0.0135 l i t e r s ) at the most was allowed per gram. This was very much less than would be expected i f the amount for the Decapod Crustacea found i n this investigation can be related. Also, the values given for the lethal temperatures are surprisingly low, both in comparison with other Crustacea and particularily with the values obtained by Huntsman (1924) for Stages IV and V in the lobster. An estimated lethal temperature of about 34°C i s at least 3.5°C greater than that obtained by McLeese under the most favourable acclimation conditions. Too many animals per volume of water may account in part for results. In many cases i n the literature, - 40 -the volume of water used in the experiment i s not indicated, but presumably tests were conducted to ensure a satisfactory density of animals. It i s probable that the volume needed varies somewhat i n different species. Moulting during the 24 hours of the experiment was found to have an adverse effect on high temperature tolerance in H. nudus and H. oregonensis. Crayfish moulted successfully i n temperatures from 12°C to 36°C; the stage in the moult cycle had no effect upon heat tolerance (Spoor, 1955). To the contrary, McLeese (1956) showed that the average survival time for soft-shell lobsters was less than that for hard-shelled individuals. Again nothing definite can be concluded about a relationship between moulting and lethal temperatures as results from various workers give diversified conclusions. It i s f e l t that confusion s t i l l exists in the literature with respect to acclimation, acclimatization and adaption. Bullock (1955) did much to alleviate this i n his review paper, but there i s no consistency as yet to the term used or to meaning. Here the term acclimation i s used i n preference as did Bullock, but with a l l three terms having the same meaning. The classic example given to show acclimation i s a higher rate of oxygen consumption by low temperature acclimated animals when compared with high temperature acclimated animals at any temperature over the physiological temperature range. If the rate i s lower or the same i t i s generally conceded that acclimation has not occured. Thus, H. nudus and H. oregonensis have a seasonal change in respiratory rate (Dehnel, unpublished) - 41 -with the winter rate lower than that i n the summer in rate-temperature graphs. This i s a typical case of non-acclimation. Prom the work on high temperature tolerance i t was found that these animals had a change in their lethal temperature, upward with a high temperature history, downward with a low temperature history. This change i s called acclimation, practically without exception, i n the literature, which includes Bullock's review paper. There i s an obvious inconsistancy i n what i s considered a valid criterion to indicate that acclimation has occured. In these two species, two different measurements of physiological processes showed a change; one type of measurement i s accepted as indicating the presence of acclimation, the other i s not. To determine the presence of acclimation, particularly over a change of seasons, only one factor must have changed as i t i s possible that two factors such as temperature and salinity could vary fortuitiously so that a measured criterion such as lethal temperatures would be the same i n animals from two environments indicating no acclimation when i t actually had occured. Thus, high temperature, low salinity i n one season of the year and low temperature, high salinity i n another season and varying i n a particular way, might give the same survival values when the animals were actually acclimated to both temperature and salinity. The change in light intensity annually definitely influences both temperature tolerance (Hoar, 1956) and respiratory rate (Dehnel, 1958) - 42 -and i t i s possible this must be considered when testing seasonal acclimation. It i s possible that a wrong impression could be obtained from high and low temperature acclimated animals when tested at a temperature intermediate between the two acclimation temperatures. Since loss and gain of heat tolerance can have differing rates (Spoor, 1955) with a few hours in some cases being sufficient for total acclimation, the degree to which the cold acclimated animal increases any physiological rate could be proportional to the amount the warm acclimated animal decreases i t s rate when both are tested at an intermediate temperature giving the same value. Another facet i s the term "cold resista,nt" used to indicate that animals with a previous temperature history i n the lower physiological temperature range have extended their l i f e processes to cover a lower range of temperatures although further up the scale the physiological processes i n high and low acclimated animals are the same. This also, generally, is excluded from true acclimation and yet i t f i t s any definition given for acclimation — "demonstrable compensatory change" (Bullock, 1955) or "...some adjustment to enable i t to meet unfavourable conditions of i t s physical and chemical environment, and any such adjustment may be considered as an acclimatization..." (Heilbrunn, 1952). Thus, to summarize, i t i s f e l t that acclimation to various factors whether i n the laboratory or in the natural environment of the animal i s much more widespread than has formally been conceded i f various measurements of acclimation - 43 -are considered as equally valid. Acclimation, so far as i s known, i s always i n the favour of the animal; that i s , i f a physiological change occurs at a l l , i t always allows for more chance for survival. Differing rates of loss and gain of tolerance to high and low temperatures, heat or cold depression at certain temperatures as well as the interactions of the many environmental factors must be ascertained quantitatively before acclimation can be f u l l y understood. - 44 -SUMMARY 1. The influence of seasonal change and laboratory acclimation to various temperature-salinity combinations, on heat tolerance was determined for two species of grapsoid crabs, Hemigrapsus nudus and H. oregonensis. 2. There was a seasonal change in 50 per cent survival in both species when base lines from summer and winter were compared. 3. A definite species difference i n tolerance to high temperatures was found to exist, but both species reacted similarly to any particular temperature-salinity combination. 4. Acclimation to a high temperature generally increased the resistance to lethal temperatures whereas acclimation to low salinity generally decreased i t . High temperature, high salinity was the most favourable combination to withstand the high test tolerance temperatures. 5. Gain in heat tolerance whether the sa l i n i t y was low or high was rapid, less than one week. 6. Winter tolerances with both low and high s a l i n i t i e s i n the low temperature series were not demonstrated in the laboratory with summer animals. Various reasons are suggested which might explain this apparent discrepancy. 7. Moulting during the test tolerance experiments adversely affected the resistance. The number of animals per dish at each test temperature had a pronounced effect on tolerance. The sex of the crabs did not effect the survival, but smaller animals appeared to be slightly more resistant. - 45 -LITERATURE CITED Bovbjerg, R.V., 1952. Comparative ecology and physiology of the crayfish Orconectes propinquus and Cambarus fodiens. Physiol. Zool., 25: 34-56. Bovee, E.C., 1949. Studies on the thermal death of Hyalella azteca Saussure. Biol. Bull., 96: 123-128. Brett, J.R., 1944. Some lethal temperatures of Algonquin Park fishes. Pub. Ont. Pish. Res. Lab., No. 63: 5-49. Brett, J.R., 1946. Rate of gain of heat tolerance i n goldfish (Carassius auratus). Pub. Ont. Pish. Res. Lab., No. o T : 5-28. Brett, J.R., 1952. Temperature tolerance in young Pacific salmon, Genus Oncorhynchus. J. Pish. Res. Bd. Can., 9: 265-323. Broekema, M.M.M., 1941. Seasonal movements and the osmotic behaviour of the shrimp, Crangon crangon L. Arch. Neerl. Zool., 6: 1-100. Bullock, T.H., 1955. Compensation for temperature i n the metabolism and activity of poikilotherms. Biol. Rev., 30: 311-342. Dehnel, P.A., 1955. Rates of growth of gastropods as a function of latitude. Physiol. Zool., 28:- 115-144. Dehnel, -P.A., 1958. Effect of photoperiod on the oxygen consumption of two species of intertidal crabs. Nature, Lond., 181: 1415-1417. Dehnel, P.A. Effect of temperature and salinity on the oxygen consumption of two species of intertidal crabs. Unpublished. Dehnel, P.A., and E. Segal, 1956. Acclimation of oxygen consumption to temperature in the American cockroach (Periplaneta americana)• Biol. Bull., I l l : 53-61. Edwards, G.A., and L. Irving, 1943. The influence of temperature and season upon the oxygen consumption of the sand crab, Emerita -talpoida Say. J. Cell Comp. Physiol., 21: Tb'9-182. Pry, P.E.J., 1958. Temperature compensation. An. Rev. Physiol. 20: 207-224. - 46 -Fry, F.E.J., J.R. Brett and G.H. Clawson, 1942. Lethal limits of temperature for young goldfish. Rev. Can. de Biol., 1: 50-56. Fry, F.E.J., J.S. Hart, and K.F. Walker, 1946. Lethal temperature relations for a sample of young speckled trout, Salvelinus fontinalis. Pub. Ont. Fish. Res. Lab., No. 66: 9^35. Gowanloch, J.N., and F.R. Hayes, 1926. Contributions to the study of marine gastropods. I. The physical factors, behaviour and intertidal l i f e of Littorina. Contr. ( Canad. Biol., N.S., 3: 135-165. Gross, W.J., 1957. An analysis of response to osmotic stress i n selected Decapod Crustacea. Biol. Bull., 112:. 43-62. Hart, J.S., 1952. Geographic variations of some physiological and morphological characters in certain freshwater f i s h . Pub. Ont. Fish. Res. Lab., No. 72: 1-79. Heilbrunn, L.V. 1952. An outline of general physiology. Philadelphia: W.B. Saunders Co. 818 pps. Hoar, W.S., 1955. Seasonal variations i n the resistance of goldfish to temperature. Trans. Roy. Soc. Can., 49: 25-34. Hoar, W.S., 1956. Photoperiodism and thermal resistance i n goldfish. Nature, Lond., 178: 364-365. Huntsman, A.G., 1924. Limiting factors for marine animals. 2. Resistance of larval lobsters to extremes of temperature. Contr. Canad. Bi o l . , N.S., 2: 91-93. Jones, L.L., 1941. Osmotic regulation in several crabs of the Pacific Coast of North America. J. Comp. Cell Physiol., 18: 79-91. Keiz, G. , 1953. Uber die Beziehungen zwischen Temperatur-Akklimatisation und Hitzeresistenz bei eurythermen und s'tenothermen Fischarten (Squalius cephalus L. und Trutta iridea W. Gibb). Naturwissenschaften, 40: 245-250. Kinne, 0., 1956. Uber den Einfluss des Salzgehaltes und der Temperatur auf Wachstum, Form und Vermehrungbei dem Hydroidpolypen Cordylophora caspia (Pallas), Thecata, Clavidae. Zool. Jahrb., Allg. Zool. u. Phys., 66: 565-638. - 47 -Kinne, 0., 1958. Adaptations to salinity variations - some facts and problems. Reprinted from Physiological Adaptations (Amer. Physiol. Soc., Wash., D.C), 92-106. Mayer, A.G., 1914. The effects of temperature upon tropical animals. Pap. Tortugas Lab., 6: 3-24. Mellanby, K., 1954. Acclimatization and the thermal death points in insects. Nature, Lon., 146: 165. McLeese, D.W., 1956. Effects of temperature, salinity, and oxygen on the survival of the American lobster. J . Pish. Res. Bd. Can., 13: 247-272. Ohsawa, W., 1956a. The experimental acclimatization i n the temperature response relation and the heat tolerance of the periwinkle, Nodilittorina granularis (Gray). J . Inst. Polytechnics, Ser. D, 7: 197-217. Ohsawa, W., 1956b. The species difference in the concentration and temperature-response relations and the heat tolerance of periwinkles. J. Inst. Polytechnics, Ser. D, 7: 219-227. Ohsawa, W., and H. Tsukuda, 1956. The seasonal variation in the temperature response relation and temperature tolerance of the periwinkle, Nodilittorina granularis (Gray). J. Inst. Polytechnics Ser. D, 7: 173-188. Piatt, R.B., C.L. Collins and J.P. Witherspoon, 1957. Reactions of Anopheles quadrimaculatus Say to moisture, temperature and light. Ecol. Monog., 27: 303-324. Scholander, P.P., W. Plagg, V. Walters, and L. Irving, 1953. Climatic adaption in Arctic and tropic poikilotherms. Physiol. Zool., 26: 67-92. Segal, E., 1956. Microgeographic variation as thermal acclimation i n an intertidal Mollusc. Biol. Bull., 111: 129-152. Smith, R.I., 1955a. On the distribution of Nereis diversicolor in relation to salinity i n the vi c i n i t y of Tvarminne, Finland, and the Isefjord, Denmark. Biol. Bull., 108: 326-345. Smith, R.I., 1955b. Comparison of the level of chloride regulation by Nereis diversicolor in different parts of i t s geographical range. Biol. Bull., 109: 453-474. - 48 -Smith, R.I., 1957. A note on the tolerance of low sa l i n i t i e s by Nereid polychaetes and i t s relation to temperature and reproductive habit. Ann. Bio l . , 33: 93-107. Spoor, W.A., 1955. Loss and gain of heat-tolerance by the Crayfish. Biol. Bull., 108: 77-87. Sumner, F.B., and P. Doudoroff, 1938. Some experiments on the temperature acclimatization and respiratory metabolism in fishes. Biol. Bull., 74: 403-429. Verwey, J., 1957. A plea for the study of temperature influence on osmotic regulation. Ann. Bio l . , 33: 129-149. Wikgren, B., 1953. Osmotic regulation i n some aquatic animals withLspecial reference to the influence of temperature. Acta. Zool. Pennica, 71: 1-102. 

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