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Gill tissue respiration in two species of crabs, Hemigrapsus nudus and Hemigrapsus oregonensis McCaughran, Donald Alistair 1962

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GILL TISSUE RESPIRATION IN TWO SPECIES OP CRABS, HEMIGRAPSUS NUDUS AND HEMICrRAPSUS OREGONENSIS DONALD ALISTAIR MeCAUGHRAN B.Sc, University of British Columbia, 1959 A THESIS SUBMITTED IN PARTIAL FULFILMENT OP THE REQUIREMENTS FOR THE DEGREE OP MASTER OP SCIENCE in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OP BRITISH COLUMBIA April, 1962 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r e xtensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allo;ved without my w r i t t e n permission. Department of Zoology  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. Date May, 1962 i A B S T R A C T Oxygen consumption of g i l l tissue of Hemigrapsus nudus and H. oregonensis has been measured at four levels of experimental salinity (35$, 75$, 100$, and 125$ sea water), three levels of experimental temperature (5°, 12,5° and 20°C) in a l l combinations for both winter and summer animals. Summer animals of both species were found to have higher weight-specific oxygen consumption than winter animals when measured at the average summer (20 C, 35$ sea water) and winter (5 C, 75$ sea water) f i e l d conditions. At other experimental conditions respiration rates of summer-adapted animals were found to be higher for the majority of conditions, but there were examples of equal seasonal rates and examples of winter rates exceeding those obtained from summer animals. Winter animals were found to be less temperature-dependent than summer animals. No laboratory compensation to temperature of biological significance could be found in either species. Hemigrapsus nudus demonstrated a higher weight-specific oxygen consumption than H. oregonensis. Both species showed with minor differences, similar responses to the various factors investigated. The weight-specific oxygen consumption data present indirect evidence which indicate that the g i l l s of both species are involved in regulating blood ions. The indications are that regulation of ions by the g i l l s is more pronounced in summer than in winter. The regression coefficient of weight-specific oxygen consumption as a function of whole body weight was found to have a value of -0.169. This value was similar for both species and not affected by experimental conditions. The differences in physiological response between species indicate that the less variable Hemigrapsus nudus may be a" select population which has migrated into this area from the outer coast and that H. oregonensis may be indigenous to this locality. i i TABLE OF CONTENTS INTRODUCTION MATERIAL AND METHODS 3 RESULTS 7 Seasonal Rate-Temperature Experiments 7 Effect of Season 8 Effect of Acute Temperature 9 Effect of Experimental Temperature 10 Effect of Experimental Salinity . 11 Interactions 12 Effect of Whole Body Weight 19 DISCUSSION 20 SUMMARY 29 LITERATURE CITED 32 i i i LIST OF FIGURES Relationship of weight-?specif ic oxygen consumption to time for g i l l tissue of Hemigrapsus nudus. Each point represents the amount of oxygen consumed per gram per hour during a ten minute interval. Two weights of crabs were used, 2.4 grams and 12.5 grams. The curves are eye-fitted . . . . . . . . . . 5a Seasonal Rate-Temperature curves, acutely measured for Hemigrapsus nudus and Hemigrapsus oregonensis. Summer animals were kept at 20°C and 35$ sea water and winter animals were kept at 5°C and 75$ sea water for 24 hours prior to experimentation. Summer and winter animals were also kept at their opposite seasonal conditions for 8 days. Each point represents the mean weight - specific oxygen consumption (z_) at that particular acute temperature . . . . . . . . . . . . . 7a Rate-Temperature curves acutely measured for Hemigrapsus nudus and Hemigrapsus oregonensis. Summer and winter animals were kept at 12.5°C and 35$ and 125$ sea water for 8 days prior to experimentation. Each point represents the mean weight-specific oxygen consumption (z) at that particular acute temperature, season and experimental salinity . . . . . 9a Main effect of Experimental Temperature (c) and Experimental Temperature - Season interaction (C 0) for Hemigrapsus nudus and Hemigrapsus oregonensis. Each point on the main effect curve i s the mean of a l l weight-specific oxygen consumption data measured at that particular experimental temperature. Each point on the Experimental Temperature - Season inter-action curves i s the mean of a l l weight - specific oxygen consumption data for that particular season and experimental temperature . . . . . . . . . 11a Main Effect of Salinity (A) and Season - Experimental Salinity interaction (0 A) for Hemigrapsus nudus and Hemigrapsus  oregonensis. Each point on the main effect curve is the mean of a l l weight-specific oxygen consumption data measured at that particular salinity. Each point on the Season -Experimental Salinity interaction curves i s the mean of a l l weight-specific oxygen consumption data measured at that particular season and salinity . . . . . . . . . . . . . . . . 13a Relationship of weight-specific oxygen consumption to experimental salinity for Hemigrapsus nudus and Hemigrapsus  oregonensis. Each point represents the mean of a l l weight-specific oxygen consumption data at that particular season, and experimental salinity measured at 20°C acute temperature . . . . . . . . . . . . . . . 14a iv 7 Acute Temperature - Experimental Temperature interaction (B C) for Hemigrapsus nudus and Hemigrapsus oregonensis. Each point represents the mean of a l l weight-specific oxygen consumption data measured at that particular acute temperature and experimental temperature . . . . . . . . . . . 15a 8 Experimental Salinity - Experimental Temperature, interaction (A C) for Hemigrapsus nudus and Hemigrapsus oregonensis. Each point represents the mean of a l l the weight-specific oxygen consumption data measured at that particular experimental salinity and experimental temperature . . . . . . 16a 9 Acute Temperature — Season interaction (B D) for Hemigrapsus  nudus and Hemigrapsus oregonensis. Each point represents the mean of a l l weight-specific oxygen consumption data measured at that particular acute temperature and season . . . 17a 10 Experimental Salinity - Experimental Temperature - Season interaction (ACD) for Hemigrapsus nudus and Hemigrapsus  oregonensis. Each point represents the mean of a l l weight-specific oxygen consumption data measured at that particular experimental salinity, experimental temperature and season . . 18a 11 Relationship of blood and urine total osmotic concentration (Dehnel, 1962; Stone, 1962) and g i l l tissue weight-specific oxygen consumption to Experimental Salinity for Hemigrapsus  nudus. Each point on the blood and urine curve represents osmotic concentration at that particular experimental salinity and experimental temperature. Each point on the g i l l tissue curve represents weight-specific oxygen consumption at that particular experimental salinity and experimental temperature measured at 10°C acute temperature 23a • LIST OP TABLES I Analysis of Variance for the means of weight-specific oxygen consumption data of Hemigrapsus nudus for the factorial design incorporating four levels of Experimental Salinity (A), four levels of Acute Temperature (B), three levels of Experimental Temperature (C) and two Seasons (D) . 8a II Analysis of Variance for the means of weight—specif ic oxygen consumption data of Hemigrapsus oregonensis for the factorial design incorporating four levels of Experimental Salinity (A), four levels of Acute Temperature (B), three levels of Experimental Temperature (C) and two Seasons (D) • * 8b III Seasonal compensation shown by g i l l tissue weight-specific oxygen consumption for Hemigrapsus nudus and Hemigrapsus  oregonensis at a l l combinations of Experimental Salinity (A), Acute Temperature, (B) and Experimental Temperature (C). The numbers in the table represent the type of compensation (Precht, 1951) shown by the tissue: type 5, indicates that the rate of respiration of summer-adapted animals is higher than that of winter-adapted animals; type 4, the seasonal rates are equal, and type 3, winter animals have higher seasonal rates . . . . . . . . . . . . . . . • . . 10a IV Temperature compensation in g i l l tissue weight-specific oxygen consumption rate between the three levels of Experimental Temperature at a l l combinations of Experimental Salinity, Acute Temperature and Season for Hemigrapsus nudus. The numbers in the table indicate the type of temperature compensation (Precht, 1951) shown by the tissue: type 5, the rate shown at the higher experimental temperature is higher than the rate shown at the lower experimental temperature; type 4, the rates at each experimental temperature are equal, and type 3, the rate shown by the lower experimental temperature is higher than the rate shown by the higher experimental temperature . . . . . . . . . 12a V Temperature compensation in g i l l tissue weight-specific oxygen consumption rate between the three levels of Experimental Temperature at a l l combinations of Experimental Salinity, Acute Temperature and Season for Hemigrapsus  oregonensis. The numbers in the table indicate the type of temperature compensation (Precht, 1951) shown by the tissue: type 5, the rate shown at the higher experimental temperature is higher than the rate shown at the lower experimental temperature; type 4, the rates at each experimental temperature are equal, and type 3, the rate shown by the lower experimental temperature is higher than the rate shown by the higher experimental temperature 12b A C K N O W L D G E M E N T A debt of gratitude is acknowledged Dr. P.A. Dehnel under whose direction this investigation was conducted. The time and assistance that were given during this study are very much appreciated. I am also indebted to Dr. P.A. Larkin for his valuable help with the statistical treatment of the results, and to Dr. C.V. Finnegan, Dr. tf.S. Hoar, and Dr. N.J. Wilimovsky for their c r i t i c a l reading of this study. This study was supported by the National Research Council of Canada and the National Science Foundation of the United States. I N T R O D U C T I O N The problem of assessing the effect of the environment on metabolism of marine invertebrates and their excised tissues has resulted in a comprehensive literature in recent years. Reviews of this work have been published by Bullock (1955) and Prosser (1955). The Decapod Crustacea have received significant attention in these studies as they are found adapted to a wide variety of habitats. Roberts (1957a) and Vernberg (1959) have shown latitudinal differences in the respiration rate of shore crabs. Roberts (1957a) found that Pachygrapsus crassipes collected at various localities along the California coast revealed differences in resting metabolism and he attributed these differences partly to physiological compensation to local temperature. Vernberg (1959) found the Q^Q'S °* tropical zone species to be similar to temperate animals when measured at intermediate and high temperatures but different when measured at low temperatures. Seasonal effects on respiration rate have been documented for Emerita by Edwards and Irving (1943a) and for two species of Hemigrapsus by Dehnel (i960). Flemister and Plemister (1951) working with Ocypode albicans demonstrated oxygen consumption to be lowest when osmotic stress was least, but Gross (1957) was not always able to demonstrate reduction in oxygen consumption with reduction in osmotic stress in Uca. Laboratory acclimation to temperature has been demonstrated in grapsoid crabs (Roberts, 1957a; Dehnel, I960). The effect of size on respiration rate has been well documented by Weymouth, Crimson, Hall, Belding, and Field (1944). They found the weight-respiration rate regression coefficient K (b in this study) to be 0.798 for the kelp crab (Pugettia producta) and 0.826 for a regression-line prepared from 54 species of Crustacea. The effects of size on crustacean metabolism have been compared with similar studies on other organisms in an excellent review by Zeuthen (1953). Several studies have combined factors in assessing environmental effects on metabolism. Broekema (1941) investigated the combined effect of temperature and salinity on the longevity of the shrimp Crangon crangon. She found the optimal salinity for two year old shrimp to change with temperature. Todd and Dehnel (i960) studied the influence of various temperature and salinity combinations on temperature tolerance of Hemigrapsus nudus and H. oregonensis. They found a seasonal difference and a difference induced by temperature and salinity acclimation in the heat tolerance of these two species. Dehnel (i960) found salinity to effect acclimation of respiration rate to temperature. He also found seasonal differences in the degree of acclimation to these factors. The effect of various environmental factors on the metabolism of isolated Decapod tissue i s only sparingly documented. Vernberg (1956) studied g i l l tissue and mid-gut gland respiration rate in nine species of crabs. He found g i l l tissue to respire at a greater rate in more active species, and the QQ2 values to be highest in terrestrial crabs and decrease progressively as the habitat approached the ocean depths. Mid-gut gland respiration did not demonstrate a similar relation to habitat but a correlation with activity was observed. Weymouth, ejb a l . (1944) have studied respiration of the mid-gut gland of Pugettia producta. They found the usual negative weight-specific regression and to be higher weight specifically for tissue compared with whole animal. Roberts (1957b) demonstrated acclimation to temperature in the - 3 -respiration rate of Pachygrapsus muscle between 8.5° and 23.5° C, but under similar conditions no acclimation could be demonstrated for brain. The majority of these studies, except the few mentioned above for whole animal, have been concerned with investigating the metabolic response to a single variable. There is l i t t l e documentation of multiple factor effect on a crab tissue. It was the object of the present study therefore, to investigate the combined effect, in a factorial design, of several variables (temperature, salinity, season, and whole body weight) on the oxygen consumption of the g i l l tissue of Hemigrapsus nudus and _H_. oregonensis and to compare the response to these factors with those shown by the whole animal. The influence of these factors and their interactions have been evaluated and discussed in terras of the function of the g i l l s in the metabolism of the whole animal. M A T E R I A L S AND M E T H O D S The two species of crabs were collected from Spanish Bank (Latitude, 49° 17'N; longitude, 123° 07'W), Vancouver, British Columbia. The physical and biological parameters of this area have been described (Dehnel, I960). He reports the average winter and summer temperatures and salinities of the collecting area to be approximately 5°C, 75$ sea water, and 20° C, 35$ sea water, respectively. These values were used in the experimental design and termed winter and summer baseline conditions. The 100$ sea water used in this work is based on a standard sea water of 17.65 °/oo chlorinity, 31.88 °/°° salinity. Summer experiments were conducted from July 1 to September 31, I960, and the winter experiments from January 1 to April 4, 1961. Three levels of experimental temperatures, 5°, 12.5°, and 20° C, and four levels of experimental salinities, 35%, 75%, 100%, and 125% sea water were used in a l l combinations at both seasons. The sea water was adjusted to the appropriate salinity by adding dechlorinated water or sea salt to 80% sea water. Salinity was measured on a conductivity bridge or by titration with AgNO^ , and a tolerance of - 5% was allowed. The term experimental is used throughout to describe the temperature and salinity of the sea water in which the animals were kept prior to measuring the respiration rate of the tissue. Crabs were collected in plastic buckets and covered with damp sea weed for transportation to the laboratory. To avoid introducing sex as another factor only male crabs were collected, and they ranged in weight from 0.60 to 20.0 grams for H, nudus and 0.60 to 10.0 grams for H. oregonensis. Animals for each experiment were kept in plastic containers (10^" x 13" x 4i") each holding 3.5 liters of sea water. The containers were aerated and kept in a constant temperature environment room a o.i° o in total darkness. The water was changed each day. At the baseline conditions the animals were held for 48 hours prior to experimentation in order to clear their guts and branchial chambers. At a l l other experimental conditions the animals were held for eight days. The animals were not fed during this time. Respiration rate of the tissue was measured using a Gilson Medical Electronics respirometer by the direct method of Warburg. The measurements were made using 50 to 70 milligram damp dried tissue samples. Tissue from equal weight crabs was combined until the desired sample weight was reached* This required approximately 8 grams of whole crabs per flask and 50 H. nudus and 65 BU oregonensis per experiment. The crabs were weighed on a Mettler "k" electrical balance to the nearest 0,01 gram. - 5 -The tissue was excised, damp-dried on f i l t e r paper and weighed to the nearest 0.1 milligram on a "Mettler H" electrical balance. The samples were then placed in 15 m i l l i l i t e r Warburg flasks containing 3 m i l l i l i t e r s of a saline solution prepared as follows: 850. ml. of 0.52 M NaCl 51.0 0.35 M MgCl2 35.0 0.35 M CaCl 2 35.0 0.42 M Na2S04 17.6 0.38 M H3BO4 1.3 0.48 M NaOH pH = 7.8 Throughout the dissection the flasks were kept in an ice-water bath. Respiration rate was measured at four acute (Warburg) temperatures (5°, 10°, 15°, and 20° C) for each experimental condition. The small drop-off in oxygen consumption with time (Fig, l) permitted use of the same tissue samples to measure the respiration rate at two acute temperatures. The f i r s t set of samples were respired at 5°C and then at 20° C and the second set at 10° C and 15° C, Five minutes were required to change the temperature of the respirometer water bath, and twenty minutes to equilibrate the tissue to the new temperature. Flasks were gassed with oxygen for 10 minutes during the equilibration period. Six hours from the time of dissection were required to measure "the rate of respiration at two acute temperatures. The use of one set~of tissue samples for respiration rate measurements at two acute temperatures was justified further by the fact that there was no change in slope of the Rate-Temperature (R,T.) curves due to a drop in oxygen consumption of the tissue at the second run temperatures (15° and 20° C). After each experiment the tissue and saline were placed in previously weighed aluminum f o i l containers and dried in an oven at 100° C for twenty-- 5a -Figure 1. Relationship of weight-specific oxygen consumption to time for g i l l tissue of Hemigrapsus nudus. Each point represents the amount of oxygen consumed per gram per hour during a ten minute interval* Two weights of crabs were used, 2.4 grams and 12.5 grams. The curves are eye-fitted. HEMIGRAPSUS NUDUS 300 -EXP TEMP. 20 ° C ACUTE TEMP 20 ° C o_a o o o o oo o o o o 2.4 gm o o o - • — r r BEGIN 0 2 MEASUREMENTS • • • * • • • —r*—w 12 5 gm ' T END 0 2 MEASUREMENTS 3 4 5 TIME IN HOURS - 6 -four hours* The dried pans were weighed to the nearest 0.1 milligram. Weight of the pan and the dried saline were subtracted from the total weight to obtain the dry weight of tissue. Oxygen consumption was expressed in micro-litres per gram of dried g i l l per hour at Normal Temperature and Pressure (N.T.P.). Respiration rates were plotted on double logarithm graph paper, microliters of oxygen per gram per hour on the ordinate and whole body weight on the abscissa. The data thus plotted formed a straight line. Therefore, the functional relationship was of the form: 0 2 = aWb where 0^ = micro-liters/gram of dry gill/hour W = whole body weight a and b are constants Therefore: log 0^ = log a + b log W Slopes of the regression lines of individual experiments were not different and the average slope was -0.169. The data were transformed to yield a series of 0^ values (Z) making every W equal to 4 grams by the following equation: Z = log 0 2 + b (log W - 4 log W) where Z = log 0^ when W = 4 grams An analysis of variance was performed on the Z values of each species. The Alwac III E Computer at the University of British Columbia was used to perform both the transformation and the analysis of variance. The analysis of variance programme required each treatment contain an equal number of observations. Therefore, fifteen values were chosen at random where sixteen or seventeen were obtained. Throughout this work the term main effects is used to describe the effects of single factors (e.g. salinity) on g i l l tissue respiration rate. The probability value at 0.01 is used as the level of significance. R E S U L T S SEASONAL RATE-TEMFEMTURE EXPERIMENTS: Baseline Conditions: The g i l l tissue respiration Rate-Temperature (R-T) curves measured over the physiological range of animals removed directly from f i e l d conditions (baseline conditions 5°C, 75% for winter and 20° C, 35% for summer) and of winter and summer animals held experimentally at the opposite season baseline conditions are presented in Figure 2. Hemigrapsus nudus: Hemigrapsus nudus demonstrates reverse seasonal compensation type 5 (Precht, 1951). This is shown by the fact that the R-T curve of winter animals held experimentally at summer conditions is lower on the ordinate by 40% at 5°C acute temperature and 54% at 20° C acute temperature than the summer baseline R-T curve, and the R-T curve of summer animals held experiment-ally at winter conditions is higher by 45% and 54% at 5° C and 20° C acute temperature respectively than the winter baseline R—T curve (Fig. 2). Hemigrapsus oregonensis: Hemigrapsus oregonensis also demonstrates reverse seasonal compensation (Fig. 2). The R—T curve of winter animals held experimentally at summer conditions is positioned 47% and 61% lower on the ordinate at 5°C and 20°C respectively than the R—T curve of summer baseline animals, and the R-T curve of summer animals held experimentally at winter conditions is positioned 10% and 50% higher at 10° C and 20° C acute temperature than the R-T curve of winter animals. At the 5° C acute temperature condition winter H. oregonensis demonstrates no seasonal compensation. - 7a -Figure 2. Seasonal Rate—Temperature curves, acutely measured for Hemigrapsus nudus and Hemigrapsus oregonensis. Summer animals were kept at 20°C and 35% sea water and winter animals were kept at 5°C and 75% sea water for 24 hours prior to experimentation. Summer and winter animals were also kept at their opposite seasonal conditions for 8 days. Each point represents the mean weight-specific oxygen consumption (jz) at that particular acute temperature. RATE - TEMPERATURE HEMIGRAPSUS NUDUS • SUMMER A SUMMER ot WINTER BASELINE 0 WINTER at SUMMER A WINTER BASELINE E 400 1 1 1 " RATE - TEMPERATURE HEMIGRAPSUS OREGONENSIS 6000 " at WINTER BASELINE at SUMMER B A S E L I N E 4001 i , , 5 10 15 20 A C U T E TEMP. ( ° C ) Interspecific Comparisons Hemigrapsus oregonensis shows a slightly greater seasonal reverse compensation than H. nudus at 10 , 15 and 20<C acute temperature. At 5 C acute temperature H. oregonensis demonstrates no compensation while H. nudus demonstrates a large degree of seasonal reverse compensation, H. nudus has a higher rate of respiration than H, oregonensis at a l l comparable conditions except 5° and 10°C acute temperature for the winter baselines. EFFECT OF, SEASON,(D): The total effect of season (mean of a l l summer respiration data compared to mean of a l l winter respiration data) is significant (Tables 1 and 2), Table 3 gives a seasonal comparison of the respiration rate means of each experiment in the design. The values given in Table 3 are types of compensation as given by Precht (1951). Type 5 indicates that the rate of respiration of summer adapted animals is higher than those adapted to winter conditions, type 4, the seasonal rates are equal and type 3, winter animals have the higher seasonal rate. Hemigrapsus nudus: G i l l tissue demonstrates reverse seasonal compensation in respiration rate (type 5) at a l l experimental and acute temperatures at 35% sea water experimental salinity,, As the experimental salinity is increased (75%, 100% and 125% sea water) the tissue demonstrates no compensation at many experimental conditions (type 4) and a few instances of partial compensation (type 3). There appears to be no trend with experimental and acute temperature in the type of compensation shown except that the only instance of partial compensation is observed at 5° C experimental temperature (Table 3). T A B L E 1 Analysis of Variance for the means of weight-specific oxygen consumption data of Hemigrapsus nudus for the factorial design incorporating four levels of Experimental Salinity (A), four levels of Acute Temperature (B), three levels of Experimental Temperature (C) and two Seasons (D), Source of Variation df Sum of Squares Mean Squares P A (Exp. Sal.) 3 0.015 0.0048 29.4 B (Acute Temp.) 3 0.692 0.2306 1401.6 C (Exp. Temp.) 2 0.004 0.0022 13.1 D (Season) 1 0.040 0.0405 245.9 AB 9 0.004 0.0004 2.5 BC 6 0.006 0.0009 5.7 CD 2 0.000 0.0001 .3 AC 6 0.026 0.0043 26.2 BD 3 0.005 0.0017 10.2 DA 3 0.036 0.0119 72.5 ABC 18 0.006 0.0003 1.9 BCD 6 0.001 0.0002 1.0 ACD 6 0.016 0.0027 16.4 DAB 9 0.002 0.0002 1.3 Other Interactions 18 0.065 0.0036 21.8 Total Treatment 95 0.858 0.0090 54.9 Error 1344 0.221 0.0002 Total 1439 1.079 - 8b -T A B L E 2 Analysis of Variance for the means of weight-specific oxygen consumption data of Hemigrapsus oregonensis for the factorial design incorporating four levels of Experimental Salinity (A), four levels of Acute Temperature (B), three levels of Experimental Temperature (C) and two Seasons (D). Source of Variation df Sum of Squares Mean Squares P A (Exp. Sal.) 3 0.048 0.0161 134.0 B (Acute Temp.) 3 0.671 0.2236 1860.9 G (Exp. Temp.) 2 0.003 0.0015 12.7 D (Season) 1 0.060 0.0603 501.5 AB 9 0.002 0.0002 1.8 BC 6 0.004 0.0007 6.1 CD 2 0.019 0.0093 77.6 AC 6 0.037 0.0062 51.7 BD 3 0.014 0.0048 39.7 DA 3 0.032 0.0106 88.0 ABC 18 0.009 0.0005 4.1 BCD 6 0.005 0.0009 7.2 ACD 6 0.005 0.0008 6.6 DAB 9 0.003 0.0003 2.6 Other Interactions 18 0.005 0.0003 2.3 Total Treatment 95 0.917 0.0097 80.4 Error 1344 0.162 0.0001 Total 1439 1.079 Hemigrapsus oregonensisi Hemigrapsus oregonensis also demonstrates reverse seasonal compensation in g i l l respiration rate at 35% sea water experimental salinity at a l l experimental and acute temperatures. There is also the trend for the tissue to show no compensation at the higher salinities (75%, 100% and 125% sea water). Partial compensation is shown at 12.5°C, 75% sea water. Interspecific Comparisons Both species exhibit reverse seasonal compensation at 35% sea water at a l l experimental and acute temperatures. There is no correlation between the experimental and acute temperature conditions and the "type of compensation shown at the higher ^ salinities (75%, 100% and 125% sea water). Therefore, no comparison can be made between species in this respect except that at 5°C experimental temperature reverse seasonal compensation is shown at a l l acute temperatures at 75% sea water by both species. At 100% and 125% sea water H. nudus demonstrates several instances of partial compensation at 5°C experimental temperature, whereas H. oregonensis does not at any experimental temperature. EFFECT OF ACUTE TEMPERATURE1 (B)s The effect of acute temperature (main effect) is assessed by comparing the four means of acute temperature derived by averaging a l l the data at each acute temperature. The acute temperature effect is significant for both species (Table 1 and 2). Figure 3 shows typical acutely measured rate-temperature curves. It is seen from these curves and from other data that experimental temperature, experimental salinity, and season affect the response the tissue shows to acute temperature change. These relationships will be discussed in the appropriate sections. - 9a -Figure 3. Rate-Temperature curves acutely measured for Hemigrapsus  nudus and Hemigrapsus oregonensis. Summer and winter animals were kept at 12,5°C and 35% and 125% sea water for 8 days prior to experimentation. Each point represents the mean weight-specific oxygen consumption (z) at that particular acute temperature, season and experimental salinity. HEMIGRAPSUS NUDUS EXR TEMP. 12.5 ° C 400l SUMMER O 3 5 % S . W . WINTER « |25%S.W. O ' • HEMIGRAPSUS OREGONENSIS EXR TEMP. 12.5° C" SUMMER o 35 % S.W. WINTER • I25%S.-W. 10 15 ACUTE TEMP (°C ) 20 - 10 -EFFECT OF EXPERIMENTAL TEMPERATURE (C)s The main effect of experimental temperature and Experimental Temperature-Season interaction are shown in Figure 4. The main effect is the comparison of the three means of experimental temperature. These means are derived by averaging a l l the respiration data obtained at each experimental temperature. Tables 4 and 5 give the type of compensation shown between experimental temperature at each acute temperature - experimental salinity combination for both seasons. Hemigrapsus nudus: The main effect of experimental temperature is significant (Table l ) . The significance is due to the 12.5°C mean being lower on the ordinate than the means of 5° and 20^ (Figure 4). Review of the compensation data in Table 4 shows that in most instances no compensation (type 4) was demonstrated, and where partial (type 3) and reverse compensation ("type 5) are shown the differences in values are small and randomly distributed. Therefore, no biological significance is placed on the experimental temperature effect. Hemigrapsus oregonensis: The main effect of experimental temperature is statistically significant (Table 2). Figure 4 shows H. oregonensis to demonstrate a small degree of partial compensation. Review of the total compensation data as presented in Table 5 shows H. oregonensis to demonstrate no compensation at the majority of experimental conditions. Partial and reverse compensation are also observed but the differences in the respiration rate values are small and randomly distributed so that no biological importance is placed on the effect of experimental temperature. - 10a -T A B L E 3 Seasonal compensation shown by g i l l tissue weight-specific oxygen ' consumption for Hemigrapsus nudus and Hemigrapsus oregonensis at a l l combinations of Experimental Salinity (A), Acute Temperature (B), and Experimental Temperature (C). The numbers in the table represent the type of compensation (Precht, 1951) shown by the tissue: type 5, indicates that the rate of respiration of summer-adapted animals is higher than that of winter-adapted animals; type 4 , the seasonal rates are equal, and type 3, winter animals have higher seasonal rates. Exp. Acute EXP. SALINITY (% S.W.) Temp. Temp. (°c) (°c) 35% 75% 100% 125% Hemigrapsus nudus 5 5 5 3 3 5 10 5 5 4 3 15 5 5 4 4 - 20 5 5 4 4 5 5 4 4 4 12.5 10 5 4 4 4 15 5 4 5 5 20 5 5 5 5 5 5 5 4 4 20 10 5 5 4 4 15 5 5 4 4 20 5 5 5 5 Hemigrapsus oregonensis 5 5 5 4 4 5 10 5 5 5 4 15 5 5 5 4 20 5 5 5 5 5 5 3 4 4 12.5 10 5 3 4 4 15 5 4 5 5 20 5 4 5 5 5 5 5 5 4 20 10 5 5 5 5 15 5 4 5 5 20 5 4 4 5 11 -Interspecific Comparison: Hemigrapsus oregonensis exhibits more instances of partial compensation and fewer instances of reverse compensation than H. nudus (Tables 4 and 5). The main effect (Figure 4) shows a small degree of partial compensation for H. oregonensis but shows no compensation for H. nudus. EFFECT OF EXPERIMENTAL SALINITY (A): The main effect of experimental salinity and the Season-Experimental Salinity interaction are plotted in Figure 5. The main effect is a comparison of the four means derived by averaging a l l the respiration data collected at each experimental salinity. Figure 6 represents respiration rate plotted against experimental salinity for a set of typical (20°C acute temperature) data. Review of a l l the salinity data (5°, 10° and 15°C acute temperature as well) show that four respiration rate-salinity relationships exist depending on the experimental condition: 1, no effect of experimental salinity; 2, increase in respiration rate with increase in experimental salinity; 3, decrease in respiration rate with increase in salinity; 4, a "V-shape" relationship where the respiration rate is lowest at 75% or 100% sea water. It is noted from Figure 6 that the response to salinity is influenced by the particular experimental temperature and season in which the respiration rate was measured. These factors will be discussed under interactions. Hemigrapsus nudus: The main effect of experimental salinity is significant (Table l ) . The respiration rate curve is "V-shaped" with the lowest rate of respiration between 75% and 100% and the highest rate at 35% sea water (Fig. 5). - 11a -Figure 4. Main effect of Experimental Temperature (c) and Experimental Temperature - Season interaction (C D) for Hemigrapsus nudus and Hemigrapsus oregonensis. Each point on the main effect curve is the mean of a l l weight-specific oxygen consumption data measured at that particular experimental temperature. Each point on the Experimental Temperature - Season interaction curves i s the mean of a l l weight-specific oxygen consumption data for that particular season and experimental temperature. 3000 EXP. T E MR (C) EXP. TEMP. - SEASON INTERACTION (CD ) H. NUDUS 2000 500 000 >» T3 o> 800 o 1 2000 o-A-• • O SUMMER » MAIN * EFFECTCl • WINTER H. OREGONENSIS 1500 1000 800 O SUMMER « MAIN * EFFECT (C) WINTER 12.5 EXP. TEMP. (°C) 20 - 12 -Hemigrapsus oregonensis; The main effect of salinity is significant (Table 2), The curve is slightly "V-shaped" with the lowest rate at 100% and the highest rate at 35% sea water. Interspecific Comparison: Hemigrapsus nudus shows less differences due to experimental salinity change in the main salinity effect than H. oregonensis. H. oregonensis shows the salinity curve of the main effect to decrease rapidly with experimental salinity increase between 35% and 100% sea water and then increase slightly to 125% sea water, whereas H. nudus shows much less drop in respiration rate from 35% to 100% but shows a greater increase in rate from 100% to 125% sea water than H. oregonensis. INTERACTIONS: The concept of interaction is explained clearly by Ostle (1954); he states (Pg. 345) that, "interaction is the differential response to one factor in combination with varying levels of a second factor applied simultaneously." The presence of an interaction destroys the additivity of the main effects; what is added by one factor at a certain level of the other factor is different than what is added at a different level of the other factor (Snedecor, 1956). Interactions between two factors are represented graphically by significant slope differences between lines when each line represents a level of one factor and the measured response (weight-specific oxygen consumption data in this study) is plotted on one axis and the second factor plotted on the other axis. FIRST ORDER INTERACTIONS: First order interactions are interactions between two factors. They are analyzed by comparing the interaction means which are derived by averaging - 13 -a l l the data for each level of one factor at each level of the other factor, When there are many factors in the design they are taken two at a time in a l l combinations. In this study there are four factors - -therefore, six f i r s t order interactions are possible. These are discussed in turn. SALINITY - ACUTE TEMPERATURE INTERACTION (AB): This interaction would appear graphically as four rate-acute temperature curves drawn from means derived by averaging the respiration data of each acute temperature at each salinity. The four rate-acute temperature curves represent each of the four salinity levels used in the design. The interaction would be significant i f significant slope differences occurred between the rate-temperature curves. No figure is given showing this interaction as i t is not significant for fl. oregonensis (P>O.Ol), but is significant for H. nudus (P<0.0l). The slope of differences shown by H. nudus are small and randomly distributed between acute temperatures. This interaction therefore, is not considered biologically significant. ACUTE TEMPERATURE - EXPERIMENTAL TEMPERATURE INTERACTION (BC): The interaction between acute and experimental temperature is shown in Figure 7 where means of the respiration data of each acute temperature at each experimental temperature are plotted for both species. Tables 1 and 2 show this interaction to be significant for both species. Review of Figure 7 however, shows the slope differences to be small and randomly distributed and, therefore, no biological importance can be placed on this interaction for either species. EXPERIMENTAL TEMPERATURE - SEASON INTERACTION (CD): The means of the respiration data for each experimental temperature at each season are plotted in Figure 4. - 13a -Figure 5* Main Effect of Salinity (A) and Season - Experimental Salinity interaction (D A) for Hemigrapsus nudus and Hemigrapsus  oregonensis* Each point on the main effect curve is the mean of a l l weight-specific oxygen consumption data measured at that particular salinity* Each point on the Season -Experimental Salinity interaction curves is the mean of a l l weight-specific oxygen consumption data measured at that particular season and salinity* 3000 SALINITY (A) SEASON - SALINITY INTERACTION (DA) H. NUDUS 2000 •5, 1500 h E CM O IO E E 1000 800 2000 1500 1000 800 35 _L SUMMER WINTER H. OREGONENSIS O SUMMER A MAIN ^ E F F E C T l A l l •WINTER 75 100 EXP SALINITY (%S.W.) 125 - 14 -Hemigrapsus nudus: No Season - Experimental Temperature interaction is demonstrated by H. nudus (Table l ) . Reference to Figure 4 shows no difference in the slopes of the two seasonal curves. Hemigrapsus oregonensis: The interaction between season and experimental temperature is significant (P<0.01, Table 2). Figure 4 shows the 12.5°C respiration mean for summer and winter to approach each other in value giving the interaction statistical significance. Comparison of the 5° and 20°C means shows the summer animals to be similar but the winter animals demonstrate the mean of 20°C data to be lower in value than the 5°C mean. SALINITY - EXPERIMENTAL TEMPERATURE INTERACTION (AC): This interaction i s shown in Figure 8 where the means of the respiration data of the four salinities at the three experimental temperatures are plotted. Both species show this interaction to be significant at the 0.01 level. The trend shown in Figure 8 is for the "V-shaped" relationship between respiration rate and salinity to become more distinct with increasing experimental temperature. Hemigrapsus nudus: The weight-specific oxygen consumption-salinity plot at 5°C experimental temperature shows a small "V-shapedM relationship. The respiration rate at 100% sea water i s lower than the rate at 35% and 125% sea water by 11% and 3% respectively. At the 12.5°C experimental temperature the lowest point on the curve is at 75% sea water. The 75% sea water respiration rate is 14% lower than the rate at 35% sea water and 43% lower than at the 125% sea water condition. At 20°C experimental temperature the respiration - 14a -Figure 6. Relationship of weight-specific oxygen consumption to experimental salinity for Hemigrapsus nudus and Hemigrapsus  oregonensis. Each point represents the mean of a l l weight-specific oxygen consumption data at that particular season, and experimental salinity measured at 20°C acute temperature• SALINITY - EXP TEMP - SEASON INTERACTION (A C D) H. N U D U S T3 3 0 0 0 2 0 0 0 1500 • 1000 8 0 0 6 0 0 E CM O I O 13 0 0 0 2 0 0 0 1500 1000 8 0 0 6 0 0 A 12.5 °C °C °c °c •c °c • 5 ° C • A •SUMMER 4,2.50 c O A OWINTER a 2 0 o c H. O R E G O N E N S I S A 12.5 °C 1 20 °C 35 75 100 E X P SAL IN ITY (% S.W.) 125 rate-salinity relationship demonstrates the most distinct "V-shaped" curve. The g i l l respiration rate at 100% sea water is lower than the rate at 35% and 125% sea water by 57% and 18% respectively. Hemigrapsus oregonensis: The relationship between weight-specific oxygen consumption and salinity at the 5°C experimental temperature is nearly linear. Respiration rate decreases 42% as the salinity increases from 35% to 125% sea water. At the intermediate experimental temperature a slight "V-shaped" relation-ship exists. The rate at 100% sea water is lower than the rate at 35% and 125% sea water by 5% and 12% respectively. A well-defined "V-shaped" curve is demonstrated at the high experimental temperature. The rate at 100% sea water is 82%. lower than the rate at 35% sea water and 16% lower than the rate at 125% experimental sea water. Interspecific Comparison: The interspecific differences in the three experimental temperature curves of respiration response to salinity change are due to differences in the 12.5° and 5°C experimental temperature curves. The 12.5° curve of H. nudus has its lowest rate at 75% sea water, whereas the curve of H. oregonensis has its lowest rate at 100% sea water. At 5°C experimental temperature H. nudus demonstrates a very slight "V-shaped" respiration-salinity relationship with the lowest rate at 100% sea water, whereas H. oregonensis demonstrates a linear decreasing respiration rate with increasing salinity. ACUTE TEMPERATURE - SEASON INTERACTION (BD): The means of the weight-specific oxygen consumption data of each acute temperature for summer and winter animals are presented in Figure 9. This - 15a -Figure 7* Acute Temperature - Experimental Temperature interaction (B C) for Hemigrapsus nudus and Hemigrapsus oregonensis. Each point represents the mean of a l l weight-specific oxygen consumption data measured at that particular acute temperature and experimental temperature* 4000 ACUTE TEMPERATURE - EXPERIMENTAL TEMPERATURE INTERACTION (BC) 400 5 I I L_ 10 15 20 ACUTE TEMP. (°C) - 16 -interaction is significant for both species (Tables 1 and 2), Hemigrapsus nudus: A comparison of the acute rate-temperature curves (Pig. 9) demonstrates the winter animals to be less temperature-dependent (curve of winter animals being more flattened) than summer animals. The summer animals show a respiration rate increase from 5°C to 20°C of 342%, whereas the winter increase in respiration rate is only 248%. Hemigrapsus oregonensis: Hemigrapsus oregonensis shows less temperature-dependence in winter than in summer (Fig. 9). The summer acute rate-temperature curve demonstrates an increase in respiration rate of 316% between 5° and 20°C, whereas the increase shown by winter animals is only 211%. Interspecific Comparison: The Acute Temperature - Season interaction is similar for both species. There is a small interspecific difference i n magnitude in the per cent difference from 5° to 20°C between summer and winter acute rate-temperature curves. The per cent difference shown by H. oregonensis is 150%, whereas the difference shown by H. nudus is 124%. SEASON - EXPERIMENTAL SALINITY INTERACTION (DA): The means of the respiration data for each experimental salinity at both seasons are plotted in Figure 5. This interaction is statistically significant for both species (Tables 1 and 2). Hemigrapsus nudus: Reference to Figure 5 shows a large seasonal difference in the effect of salinity on g i l l respiration rate. The summer curve is "V-shaped" with the lowest respiration rate at the 100% sea water experimental - 16a -Figure 8. Experimental Salinity - Experimental Temperature interaction (A C) for Hemigrapsus nudus and Hemigrapsus oregonensis. Each point represents the mean of a l l the weight-specific oxygen consumption data measured at that particular experimental salinity and experimental temperature. 2500 SALINITY - EXP. TEMP. I N T E R A C T I O N ( A C ) H. NUDUS 2000 1500 = 12001 E CD 0*18001 ro E E 15001 1200 1000 8001 35 A 12.5 °C • 5 °C A 12.5 °C • 20 °C H. OREGONENSIS A 12.5 °C 75 100 E X P SALINITY (%S. W.) 125 - 17 -condition. The winter curve shows a gradual increase in respiration rate with increase in experimental salinity. Hemigrapsus oregonensis: Summer H. oregonensis show a decrease in respiration rate with increase in salinity. The total decrease in respiration rate is 75%, when the rate at 35% sea water is compared with the rate at 125% sea water. The winter experimental salinity curve has i t s lowest respiration rate at the 100% sea water experimental condition. This curve differs from a typical "V-shape" at the low salinity condition (35%) where the respiration rate is lower than at 75% sea water, but higher than the 125% sea water condition (Fig. 5). SECOND ORDER INTERACTIONS: Second order interactions are interactions between three factors. If we use as an example •three factors each having three levels then a l l the data would be averaged to produce nine respiration means (3x3x3). A second order interaction can be graphed by the use of a three dimensional graph. The measured response (respiration rate) is plotted on the vertical axis and two of the factors on the horizontal axes and each of the levels of the third factor will be represented by a line on the graph. This interaction would be significant i f lines were different in slope. The interaction can also be observed graphically by making a number of graphs corresponding to the number of levels of one factor with each graph containing the measured response on one axis and a second factor on the other axis with each level of the third factor represented by a line. We are demonstrating the effect factor 3 has on the effect factor 2 has on the measured response to factor 1. We analyse for differences in differences. - 17a -Figure 9. Acute Temperature - Season interaction (B D) for Hemigrapsus  nudus and Hemigrapsus oregonensis. Each point represents the mean of a l l weight-specific oxygen consumption data measured at that particular acute temperature and season. 6000 _ 4000 E 2000} | 1000 800 A C U T E TEMP - S E A S O N I N T E R A C T I O N (BD) HEMIGRAPSUS NUDUS O O SUMMER • • W I N T E R 600 HEMIGRAPSUS OREGONENSIS 60001 .e 3 4000| "5> >> w u E 20001 "| 10001 8001 600' 10 15 A C U T E T E M P C O O O SUMMER • • WINTER 20 - 18 -SALINITY - EXPERIMENTAL TEMPERATURE - SEASON INTERACTION (ACD): The interaction of salinity, experimental temperature and season is observed as a seasonal difference in the difference in the response respiration rate shows to salinity change due to experimental temperature (Figure 10). Hemigrapsus nudus: The ACD interaction is significant for H. nudus (Table l ) . The summer and winter animals are different in the shape of the weight-specific-oxygen consumption—salinity curves due to changes in experimental temperature (Figure 10). The summer animals show larger differences between respiration rate-salinity curves with change in experimental salinity than winter animals. This is exemplified i f the 5° and 20°C experimental temperature lines are compared for summer and winter at 35% and 125% experimental salinity. The summer difference between the 5° and 20°C lines at 35% i s 470/11 O^gm/hr while the winter difference i s 450/A1; the difference at 125% is 360yUl in summer and 180/U in winter (Fig. 10). The shape of the respiration rate-salinity plots are also different in summer and winter. The summer 5° curve is linear and negative in slope, the winter 5° curve i s also linear but slightly positive in slope. The 12.5° curves also differ seasonally, the summer line i s "V-shaped** with the low respiration rate at 75% sea water, the winter curve is linear with a slightly positive slope. The 20°C plots are similar seasonally; they are "V-shaped" with the lowest rate of respiration at 100% sea water. Hemigrapsus oregonensis8 The ACD interaction is significant (p<0.0l) (Table 2). A comparison of the 5° and 20° curves at 35% and 125% shows the differences to be - 18a Figure 10. Experimental Salinity - Experimental Temperature - Season interaction (ACD) for Hemigrapsus nudus and Hemigrapsus  oregonensis. Each point represents the mean of a l l weight-specific oxygen consumption data measured at that particular experimental salinity, experimental temperature and season. RATE - SALINITY ACUTE TEMP 2 0 ° C H. NUDUS 2000 - 10001 T3 E o 60001 2000 • SUMMER OA.O WINTER • 5 A I 2 5 ° C 2 0 °C H. OREGONENSIS 1000 SUMMER OA.O WINTER • 5 ° C 4 12 5 ° C 20 °C 35 75 100 EXP. SALINITY (%S.W.) 125 - 19 -greater in the summer; the summer 5° and 20° differences at 35% and 125% sea water are 370^1/gm/hr and 52jX\ respectively while the winter differences are 28yUl and 12yil for the same conditions* The total curve shapes at the various experimental temperatures also differ. The summer 5° curve is linear with a negative slope (Pig. 10); the 5° winter curve is similar with less slope. The 12.5° lines are different seasonally; the summer line is "V-shaped" with the low rate at 75% sea water, the winter curve is roughly linear with high respiration rates at 75% and 125%. The 20° lines are similar seasonally; both are "V-shaped" with low rates at 100% sea water. Interspecific Comparisons; Both species demonstrate greater differences due to experimental temperature in summer than in winter. When the greatest difference in respiration rate at each salinity due to experimental temperature (greatest difference between curves) are summed and an average computed and compared interspecifically, H. nudus demonstratesthe greatest seasonal effect on the respiration rate-salinity relationship due to experimental temperature. The average greatest differences are 675/^1 02/gm/hr in summer and 348/X1 in winter for H. nudus and 493yUl in summer and 308yUl in winter for H. oregonensis. Other second order interactions (ABC, BCD and DAB) are not statistically significant for H. nudus, and even though they are of statistical significance for H. oregonensis they are considered to be of no biological significance as the differences in the means are small and random, EFFECT OF WHOLE BODY WEIGHT: At the 5% level of significance the differences in slopes of the regression lines of weight-specific oxygen consumption of the g i l l s as a - 20 -function of whole body weight do not differ. The average slope was found to be -0.169. This value is significantly different from weight corrected slopes of animals following a 2/3 (-0.333) or 3/4 (-0.250) rule. D I S C U S S I O N SEASONAL RATE-TMPERATURE EXPERIMENTS: Oxygen consumption of g i l l tissue measured over the physiological range shows summer animals to have higher rates at a l l acute temperatures than winter animals of both species (Fig. 2). This relationship, termed reverse compensation by Precht (1951), is only sparingly documented. Dehnel (i960) has shown the same relation to exist in a study of whole animal respiration working with H. nudus and H_j_ oregonensis. It is diff i c u l t to place any adaptive significance on reverse seasonal compensation. Prosser (1958) indicates that reverse compensation may be non-adaptive to temperature and may reflect a change at the enzymatic level where one enzyme may decrease in compensation for an alternate enzyme at different temperatures. Thus, the lower respiration rate of winter animals may be due to a different metabolic pathway being employed. The work of Prosser and Kanungo (1959) and Ekberg (1958) on goldfish and by Hoffman with Streptococcus cremonis as given in Prosser (1958) tend to support such an hypothesis. Work on the heat and cold depression ends of the R-T curves may reflect an extension of the physiological range which may be of adaptive importance. It is suggested therefore, that this work be extended to include a study of the effect on metabolism of extremes in temperature - 21 -and a study of the metabolic pathways involved in respiration. A review of the respiration data of winter and summer animals held at a l l combinations of temperature and salinity, including the winter and summer baseline conditions, shows a tendency at a l l experimental temperatures for a change from reverse seasonal compensation (type 5) to no seasonal compensation (type 4) and to a few instances of partial compensation (type 3). Such a tendency would seem to have l i t t l e biological importance, and no explanation can be given. Perhaps this phenomenon is reflecting the effect of salt concentration on respiratory enzyme activity. mgEMTURE AND SALINITY EFFECT: Temperature:-Crabs of both species were held for eight days at three different experimental temperatures (5°, 12.5° and 20°C) in combination with four experimental salinities (35%, 75%, 100%, and 125% sea water) at both seasons. The effect on respiration rate of experimental temperatures was assessed by measuring the weight-specific oxygen consumption of "the g i l l tissue at four acute temperatures (5°, 10°, 15° and 20°C). The data have been presented by the use of the Acclimated Rater-Temperature curve (Bullock, 1955) and the effect of experimental temperature has been found to be significant. The differences, however, are small and random and considered to be due to uncontrolled variation. The effect of experimental temperature, therefore, is considered to be similar to Precht*s type 4. This type of compensation has been documented by Haugaard and Irving (1943) for the cunner and by Scholander, Flagg, Walters, and Irving (1953) for several terrestrial insects. Dehnel (i960) has demonstrated the more common type 3 compensation to laboratory temperatures in a respiration study of the whole animal using Hemigrapsus nudus and H. oregonensis. - 22 Studies on other tissues of Hemigrapsus would probably show the typical partial compensation (type 3) as shown for whole animal respiration. Salinity:-The g i l l s of crabs offer a large exposed surface to the external medium. The g i l l , therefore, would seem to be an organ which is well suited to the function of osmotic regulation. In recent years "the osmotic function of the g i l l has been well documented: Gross (1957a) has demonstrated an exchange of salt and water in the g i l l chamber of Pachygrapsus; Koch, Evans, and Schicks (1954) have demonstrated active absorption of Na and CI ions by the g i l l s of Eriocheir sinensis; Green, Harsch, Barr and Prosser (1959) have shown that water and Na, K, Ca, Mg, and CI enter the g i l l s of Uca, and recently Flemister (1959) has presented histophysiological evidence to indicate that the g i l l s of Ocypode function in the active absorption of CI ions from the external medium. If the g i l l s are active in osmotic work we might expect to find an increase in g i l l tissue respiration with an increase in osmotic gradient. This relationship has been indicated many times in studies of whole animal respiration. Plemister and Flemister (1951) have shown oxygen consumption of Ocypode albicans to be least in isotonic media. Lofts (1956) has shown a salt marsh population of Palaemonetes varians to respire least at 26$ sea water, a salinity which is isotonic with the blood. An investigation of a sluice pond population, however, showed the respiration rate to be least in 6$ sea water which is much lower in salt concentration than the blood. Dehnel (i960) has shown Hemigrapsus nudus and H. oregonensis to respire most rapidly at low salinities, where the osmotic gradient is large, at any given temperature. These authors have suggested that the increase in oxygen consumption shown by these animals as the osmotic - 23 -gradient is increased may reflect osmotic work performed in order to maintain this gradient. There are objections to this hypothesis, however. Potts (1954) has shown on theoretical grounds, fortified with experimental evidence, that the work required to maintain a gradient of 0,314 M/l in fresh water was only 0,5% of the total metabolic energy for Eriocheir, Gross (1957) has also shown an increase in respiration rate correlated with an increase in osmotic gradient, but he suggests that the increase is due to an increase in activity. The oxygen consumption of g i l l tissue of Hemigrapsus nudus and H, oregonensis has been measured using animals held experimentally at four different salinities (35%, 75%, 100% and 125% sea water) in combination with three experimental temperatures (5°, 12,5° and 20°C) for summer and winter animals. Weight-specific oxygen consumption of the g i l l tissue of both species held experimentally at a l l the above combinations and respired at 10°C acute are presented with urine and blood osmotic concentrations at similar conditions in Figure 11, Both species are good regulators over the range of environmental salinities (25% to 75% sea water); beyond this range regulation begins to break down although the blood remains hypertonic to the medium up to 125% sea water. Figure 11 shows a large seasonal difference on the effect of experimental temperature on blood and urine concentration, Hemigrapsus nudus and H. oregonensis show less experimental temperature effect in summer than in winter (Dehnel, 1962; Stone, 1962), The urine and blood of summer crabs of both species are equal to each other in osmotic concentration but hypertonic to the medium over the range of experimental salinities 25% to 125% sea water. Winter H, nudus - 23a -Figure 11* Relationship of blood and urine total osmotic concentration (Dehnel, 1962; Stone, 1962) and g i l l tissue weight-specific oxygen consumption to Experimental Salinity for Hemigrapsus  nudus* Each point on the blood and urine curve represents osmotic concentration at that particular experimental salinity and experimental temperature. Each point on the g i l l tissue curve represents weight-specific oxygen consumption at that particular experimental salinity and experimental temperature measured at 10°C acute temperature. HEMIGRAPSUS NUDUS SUMMER • 3 * C 12 5 "C TEMR 1 | 75 100 EXP. SALINITY(% S.W.) 125 P000 3 25001; 2000.> 1500! 1000^ 2 0 *C 10* C J S . ! C ACUTE 3 3 2 0 0 0 ° 1500 1000 = 800 * - 24 -demonstrate the urine concentration to be hypotonic to the blood at a l l experimental salinities between 25% and 125% and at a l l experimental temperatures except 15°C where the urine is isotonic with the blood at concentrations less than 75% sea water. The urine concentration is also hypotonic to the medium above 90% sea water; below this level the urine is hypertonic to the medium. Winter H. oregonensis demonstrate the urine to be hypotonic to the blood at a l l experimental salinities and temperatures and hypotonic to the medium above 80% sea water medium concentration at 15 C, and 100% sea water medium concentration at 5 C; salinities below these medium concentrations the urine is hypertonic to the medium. When g i l l respiration is compared with blood and urine concentrations (Figure 11) a large seasonal effect is noticed. In the summer when the blood and urine of both species are essentially equal in osmotic concentration we find the g i l l respiration rate to be highest at the low experimental salinities and decreasing as the blood approaches isotonicity with the medium (experimental sea water 75% to 100%). As the salinity of the experimental sea water is increased and the blood tends toward a greater degree of hypertonicity the respiration rate of the g i l l of 12.5° and 20°C experimentally conditioned animals increases, and the 5 C animals continue to decrease. These data indicate that the increase in respiration rate of the g i l l may be due to an increase in osmotic gradient. The urine of summer animals appears not to function in osmotic balance (Figure l l ) . The data obtained for winter collected animals of both species, however, show the rate of respiration of the g i l l tissue to be quite variable and no significant correlation with experimental salinity is indicated. The g i l l s seem to be doing l i t t l e osmotic work. The urine of winter animals, however, is hypotonic to the blood and may be a major mechanism for - 25 controlling blood-salt concentration. Potts (1954) has stated that the production of a urine hypotonic to the blood yields a negligible saving of osmotic work for brackish water animals. However, his definition of brackish water is presumably based on approximately 50$ sea water. The production of a urine hypotonic to the blood may at times significantly reduce osmotic work performed by animals taken from this study area. Therefore, the reduction of respiration rate of the g i l l s of winter animals, particularly over the environmental salinity range, may reflect the reduction of osmotic work performed as a urine hypotonic to the blood is being produced. The data indicate that the g i l l s become more important in osmotic regulation in the summer when the gradient between blood and medium is maximum. It may be further speculated that in summer the g i l l s are adapted to the active absorption of salts from the environment. This is reflected in the weight-specific oxygen consumption of the tissue measured over a range of experimental salinities at various temperatures. In the winter less active absorption is necessary as the gradient between the environment and the blood is at the lowest value i t attains and the animals are producing a hypotonic urine. Therefore, the g i l l s are not as important in the regulation of blood salts during this season. The effect of experimental temperature on the respiration rate response to salinity is significant and similar for both species. Summer animals experimentally held at 12.5°C and 20°C have "V-shaped" curves with their highest respiration rate at 35$ experimental sea water and their lowest rates at 75$ and 100$ sea water respectively. Animals held at 5°C have a linear decreasing respiration rate with increased experimental salinity. This is probably explained by the fact that at 5°C experimental temperature - 26 -in the summer the crabs are approaching their lower lethal temperature. Mortality rate increases at 5°C as the salinity is increased, indicating that low temperature combined with high salinity presents a stress environment for summer—adapted crabs. It seems reasonable to assume that the linear decreasing curve shown by 5°C summer animals is reflecting the depressed metabolic rate as lethal conditions are approached. Winter H. oregonensis showsno consistent effect of experimental temperature on the respiration rate response to salinity, whereas H. nudus shows a slight increase in respiration rate with increase in experimental salinity as the experimental temperature decreases. No explanation for this phenomenon can be given at this time. Little biological importance is placed on the ordinal position of the three experimental temperature curves in relation to each other (see section Experimental Temperature). Salinity is the major factor in causing the large seasonal difference in respiration rate of the g i l l s ; the effect is probably due to the g i l l s adapting to the job of active absorption of salts in order to maintain a constant blood-salt level. Review of the present data indicates that temperature has l i t t l e effect in determining seasonal g i l l tissue respiration rates. SPECIES COMPARISON: The respiration rate of Hemigrapsus nudus g i l l tissue is in general higher than that of H. oregonensis. Dehnel (1962) indicates "that H. nudus has greater osmoregulatory ability than H. oregonensis. It seems reasonable that the g i l l s of a better regulator should have a higher weight-specific oxygen consumption. This tends to support further the idea of g i l l tissue reflecting osmotic work in terms of oxygen consumption. Gross (1957) and - 27 -Vernberg (1956) have found respiration and activity correlated and they have demonstrated respiration rate to be highest in crabs showing the highest activity rate. Since H. nudus is more active than H. oregonensis i t might be argued that the greater respiration rate shown by the g i l l s of H. nudus is due to activity. Review of whole animal respiration rates (Dehnel, I960) rules out this possibility as H. oregonensis has a respiration rate similar to, and in some experimental conditions, higher than H. nudus. The two species studied intraspecifically are very similar in their g i l l tissue respiration rate response to the various environmental parameters used. The response to acute temperature, experimental temperature, season and whole body weight is similar with only minor differences. The differences are believed to be due to uncontrolled variation. The response to salinity change is similar in the main effect, but response to salinity differs interspecifically with season. Summer animals of both species react similarly to changes in salinity, while winter animals show several differences. Winter Hemigrapsus oregonensis show no consistent change in respiration rate due to salinity change while winter H. nudus tend to increase respiration rate differentially with temperature as salinity is increased. The 20°C experimental temperature curve shows less increase in rate than the 5°C curve and the curves from 20°C acute temperature data show this tendency to a greater degree than 5°G acutely derived curves. At the present time no satisfactory explanation can be given. The interactions studied indicate that there are no major physiological differences between the two species. Hemigrapsus oregonensis generally shows more variation than H. nudus which indicates a more physiologically variable population. If, as Dehnel - 28 -(i960) suggests, H. oregonensis occupied this area originally and H. nudus has recently become established we might expect H. oregonensis to be more variable, as i t has been a select group of H, nudus which has been able to adapt to this estuarine area* RESPIRATION AND WHOLE BODY WEIGHT: The value of the regression coefficient _b has been the subject of much investigation and discussion i n recent years* Values of 1*0 to negative numbers have been found for this coefficient for various organisms (Zeuthen, 1953; Brody, 1945, and others), von Bertalanffy (1951) and von Bertalanffy and Krywienczyk (1953) have proposed several types of animals on the basis of the value of this coefficient as an inherent characteristic. Dehnel (i960) has found the regression coefficient (b) of weight-specific oxygen consumption of whole animals of both Hemigrapsus  nudus and H. oregonensis to vary between -0.685 and -0*333 depending on the experimental conditions. The g i l l tissue of both species of Hemigrapsus studied here demonstrated a more rigid weight-specific oxygen consumption -whole body weight regression coefficient* The value was -0.169 which is significantly different from the values shown for whole animals. The _b value of g i l l tissue was not influenced by experimental conditions which is a phenomenon unlike that shown by whole animal respiration. The value -0.169 shown by g i l l is different from any so-called types (Bertalanffy, 1951). It is believed that there is enough evidence available to indicate that no significance should be placed on the absolute values of this regression coefficient. This coefficient presumably assumes a value which is dependent upon inherent properties of the tissue, the environmental history of the animal, and the method employed in measuring the oxygen consumption. - 29 -S U M M A R Y 1* Oxygen consumption of g i l l tissue of Hemigrapsus nudus and H. oregonensis has been measured at four levels of salinity (35$, 75$, 100$ and 125$ sea water), three levels of experimental temperature (5°, 12.5°, and 20°C), at four acute (Warburg) temperatures (5°, 10°, 15°, and 20°C) in a l l combinations for both summer and winter animals. The data have been analysed and the effect of these various environmental parameters evaluated. 2» Acutely measured rate-temperature curves of winter and summer animals kept 24 hours at their respective seasonal temperature and salinity (baseline conditions) show summer animals to have higher weight-specific oxygen consumptions at a l l acute temperatures. 3. Effect of experimental temperature was found to be statistically significant but the acclimated rate-temperature curves showed no consistent change in ordinal position with experimental temperature change; no biological significance is placed, therefore, on the experimental temperature effect. These animals are considered to demonstrate no compensation to temperature and therefore, conform to type 4 of Precht (1951). 4. Rate-temperature curves of both species reveal the winter animals to be less temperature-dependent than summer animals. 5. Experimental temperature affects the response to salinity shown by g i l l respiration rate differently in the two species studied seasonally. Both species demonstrate the 5°C curve to decrease with increase in salinity change and the 12.5° and 20°C curves to be "V-shaped" during - 30 -the summer months. During the winter H. oregonensis show no consistent change in weight-specific oxygen consumption due to salinity increase at any experimental temperature, whereas H. nudus demonstrate respiration rate to increase slightly with increase in experimental salinity at a l l experimental temperatures when measured at high acute temperatures but show only the -5°C curve to increase with salinity at low acute temperatures. 6. Salinity data present indirect evidence as to the role of the g i l l in regulating blood salts. Summer animals of both species show an increase in respiration rate as the osmotic gradient between blood and environment increases. Winter respiration data of both species show l i t t l e change in respiration rate with increasing osmotic gradient. In the summer when the salinity of the environment is low and the animals are producing a urine isotonic to the blood, the g i l l s are active in absorbing salts from their environment in order to maintain blood osmotic concentration, whereas, in the winter when the salinity of the environment is high, osmotic concentration is largely maintained by a urine hypotonic to the blood. 7. Hemigrapsus nudus generally demonstrates a higher weight-specific oxygen consumption than H. oregonensis. It is suggested that this;is due to the g i l l s of H. nudus being adapted to greater osmotic work than H, oregonensis. 8. The regression coefficient of weight-specific oxygen consumption as a function of whole body weight was found to be similar in both species and to have a value of -0.169. This value was found to be constant regardless of experimental conditions. 9. No physiological differences have been found in this study to explain - 31 -the present distribution of these species. Hemigrapsus oregonensis shows a large variation in g i l l tissue respiration rate response to change in experimental variables. This suggests that H. oregonensis is indigenous to this area. The lesser variation in response shown by H. nudus suggests that this species is a select group of animals which may have migrated into this area from the outer coast. - 32 -L I T E R A T U R E C I T E D von Bertalanffy, L., 1951. Metabolic types and growth types. Amer. Nat.. 85: 111-117. von Bertalanffy, L., and J. Krywienczyk, 1953. The surface rule in crustaceans. Amer. Nat., 87: 107-110. Brody, S., 1945. Bioenergetics and Growth. New York: Reinhold Publishing Corp. 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 in the metabolism and activity of poikilotherms. Biol. Rev., 30: 311-342. Dehnel, P.A., 1960. Effect of temperature and salinity on the oxygen consumption of two intertidal crabs. Biol. Bull., 118: 215-249. Dehnel, P.A., 1962. Aspects of osmoregulation in two species of intertidal crabs. Biol. Bull.. 122: 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: 169-182. Ekberg, D., 1958. Respiration in tissues of goldfish adapted to high and low temperatures. Biol. Bull.. 114: 308-316. Flemister, L.J., and S.C. Flemister, 1951. Chloride ion regulation and oxygen consumption in the crab Ocypode albicans (BosqJ. Biol. Bull. t 101: 259-273. Flemister, S.C., 1959. His to physiology of g i l l and kidney of the crab Ocypode albicans. Biol. Bull., 116: 37-48. - 33 -Green, JDW., M. Harsch, L. Barr and C,L. Prosser, 1959. The regulation of water and salt by the fiddler crabs Uca pugnax and Uca pugilator. Biol. Bull.. 116: 76-87. Gross, W.J., 1957. A behavioral mechanism for osmotic regulation in a semi-terrestrial crab. Biol. Bull.. 113: 268-274. Haugaard, N., and L. Irving, 1943. The influence of temperature upon the oxygen consumption of the cunner Tautogolabrus adspersus Walbaum in summer and winter. J. Cell. Comp. Physiol., 21: 19-26. Kock, H.J., J. Evans and E. Schicks, 1954. The active absorption of ions by isolated g i l l s of the crab Eriocheir sinensis. Mededel. Vlaamse. Acad. Kl. l2±< Jarrgung XVI N1" 5. Lofts, B 0, 1956. The effect of salinity changes on the respiratory rate of the prawn Palaemonetes varians (Leach). J . Exp. Biol., 33: 730-736. Ostle, B,, 1954. Statistics in Research. Ames, Iowa: The Iowa State College Press. Potts, W.T.W., 1954. The energetics of osmotic regulation in brackish and fresh water animals. J. Exp. Biol., 31: 618-630. Precht, H,, 1951. Der Einfluss der Temperatur auf das Permentsystem, Verh. dtsch. zool. Ges., 1950, p 179. Prosser, C.L., 1955. Physiological variation in animals. Biol. Rev. 30: 229-262. Prosser, C.L., 1958. General Summary: The nature of physiological adaptation. Physiological Adaptation. Washington, D.C.: American Physiological Society. Prosser, C.L., and M.S. Kanungo, 1959. Physiological and biochemical adaptation of goldfish to cold and warm temperatures. II. Oxygen - 34 -consumption of liver homogenate, oxygen consumption and oxidative phosphorylation of liver mitochondria. J. Cell. Comp. Physiol., 54: 265-274. Roberts, J.L., 1957a. Thermal acclimation of metabolism in the crab Pachygrapsus crassipes Randall. I. The influence of body size, starvation, and molting. Physiol. Zool., 30: 232-242. Roberts, J.L., 1957b. Thermal acclimation of metabolism in the crab Pachygrapsus crassipes Randall. II. Mechanisms and the influence of season and latitude. Physiol. Zool., 30: 242-255. Scholander, P.P., W. Plagg, V. Walters and L. Irving, 1953. Climatic adaptation in arctic and tropical poikilotherms. Physiol. Zool., 26: 67-92. Snedecor, G.W., 1956. Statistical Methods. Ames, Iowa: The Iowa State College Press. Stone, D,, 1962. Effect of temperature and salinity on the osmotic concentrations of urine in two species of intertidal crabs. (M.Sc. thesis, University of British Columbia). Todd, M.-E., and P.A. Dehnel, 1960. The influence of temperature and salinity on heat tolerance in two grapsoid crabs, Hemigrapsus nudus and Hemigrapsus oregonensis. Biol. Bull., 118: 150-172. Vernberg, F.J., 1956. Study of the oxygen consumption of excised tissues of certain marine decapod C r u s t a c e a in relation to habitat. Physiol. Zool.. 29: 227-233. Vernberg, F.J., 1959. Studies on the physiological variation between tropical and temperate zone fiddler crabs of the Genus Uca. II. Oxygen consumption of whole organisms. Biol. Bull., 117: 163-184. - 35 -Weymouth, F.W., J.M. Crismon, V.E. Hall, H.S. Belding and J. Field II, 1944. Total and tissue respiration in relation to body weight: a comparison of the kelp crab with other crustaceans and with mammals. Physiol. Zool., 17: 50-71. Zeuthen, E,, 1953. Oxygen uptake as related to body size in organisms. Quart. Rev. Biol.. 28: 1-12. 

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