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A study of the osmoregulatory role of the antennary glands in two species of intertidal crabs Stone, Dmitry David 1962

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A STUDY OP THE GSMOREGtlLATOHY HOLE OP THE ANTENNARX GLANDS IN TWO SPECIES OP INTERTIDAL GBABS by DMITRY DAVID STONE B. A,, University of B r i t i s h Columbia, 1 9 5 4  A THESIS SUBMITTED IN PARTIAL FULFILMENT OP THE REQUIREMENTS FOR TIE DEGREE OP MASTER OP SCIENCE  in the Department of ZOOLOGY  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1$$Z  In presenting  t h i s thesis i n p a r t i a l f u l f i l m e n t of  the r e q u i r e m e n t s f o r an advanced degree a t t h e 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 r e f e r e n c e and for extensive g r a n t e d by  study.  I f u r t h e r agree t h a t p e r m i s s i o n  c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may  the Head o f my Department o r by h i s  be  representatives.  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 a l l o w e d w i t h o u t my w r i t t e n  Department o f  Zoology  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada. Date  May,  Columbia,  19 62  permission.  X  ABSTMCO? Two species of inter t i d a l crabs, KesalKrapsus oregonensis and H. nudus ©cour in large numbers at Spanish Bank, Vancouver, British Columbia.  1'he area Is characterized by sea water of  high teaperature and low salinity In suoaer and  ION  temperature  and high salinity In winter. The Grabs oeatoregulate strongly in low s a l i n i t i e s , keeping blood considerably hypertonic to the external medium. They do not regulate strongly In salinities higher than those normally found In the f i e l d In winter (70«-80jS sea water). This study attempts to establish the role of the excretory organs (antennary glands) In osmoregulation.  Effects on their  function of seasonal adaptation, temperature, osmotic stress and body size  were  also Investigated.  Experimental teraperatures of 5 ° , 1 5 ° and 2 5 ° C and ©edia of 6%, 1 2 $ , 25%, 75%$ 1 0 0 $ , 1 2 5 $ , 1 5 0 $ and 1 ? 5 # sea water were used ( 1 0 0 $ sea water:31.B8$« salinity).  Animals were brought  Into the laboratory and equilibrated In 75% s©a water for 3 6 - 4 8 hours at the experitaentsl temperature.  After equilibra-  tion, groups of 1 0 - 1 5 animals were transferred to each of the experimental salinities.  After 3 , 2 4 and 48 hours, 1 0 urine  samples were drawn from each group, sealed in separate capillary  1 tubes and quick-frozen.  Osmotic concentration was aeasured by  the method of aeltlng point determination. Identical series of experiments were carried out, ouraaer and winter.  Procedures  ii  differed only in that summer animals were damp-dried and weighed "before sampling. For each species and experimental temperature, a series of urine osmotic response curves was drawn. Data for summeradapted animals at 15° G and winter-adapted animals at 5° C were used for most comparisons. mean f i e l d temperatures.  These approximated seasonal  Osmotic gradients between urine and  media at 4-8 hours formed the basis for comparison of seasonallyadapted responses. Data were analysed for salinity and temperature effects and seasonal differences by means of Student's t " test, which w  was used also to evaluate differences between urine and blood concentrations.  Differences attributable to weight, and inter-  specific differences in X3/B ratios were analysed by means of Wilcoxon's Matched-Pairs Signed-Hanks test. Concentration of urine was found to f a l l in dilute, and rise i n concentrated media at rates, directly related to osmotic stress, which declined with time and were influenced by the seasonal adaptation of the animals and the experimental temperature.  Mew equilibria were generally established by 48 hours,  at levels, particularly in concentrated media (above 100$ sea water), which were considerably higher i n summer- than in winteradapted animals. Hyper-osmotic regulation was achieved i n summer-adapted animals with the production of blood-lsotonic urine, implicating extra-renal mechanisms.  In winter-adapted animals, hyper-osmotic  Hi  regulation was enhanced by production of blood-hypotonic urine. Summer-adapted animals appeared to resist blood change in 100-150$ sea water by producing blood-hypertonic urine, and although this resistance was maintained longest in 100$ and 125$ sea water, blood soon became hypertonic.  In general, cool-  ing retarded, and warming stimulated salt absorption and regulation.  Winter-adapted animals in high salinities did not effect-  ively resist blood change, and both urine and blood quiokly became hypertonic. Effects on urine concentration of cooling or warming summer-adapted animals and warming winter-adapted  animals were  significant only in low and intermediate s a l i n i t i e s . Body size had, in some cases, significant effects on urine concentration.  Small H . nudus. taken from summer f i e l d condi-  tions, had urine significantly hypertonic to that of large animals.  This was also true of H . oregonensis at 1 5 ° in concen-  trated media. In winter-adapted  animals^ H* oregonensis had total osmotic  U/B ratios significantly higher (nearer unity) than Hi nudus for the whole range of experimental salinities at 5  0  C.  In suramer-  adapted animals at 15° 6* ff/fc ratios approached unity in both species. Seasonal adaptation of osmoregulatory mechanisms i n both species altered the balance of active processes so that urine was lower, both in absolute concentration and relative to blood, in winter than in summer.  •11  ACKNOWLEDGEMENTS  The author wishes t o thank Dr. P. A. Dehnel f o r h i s guidance i n the planning and execution of t h i s study, and f o r h i s i n v a l u a b l e c r i t i c i s m of the manuscript i t s preparation.  In a l l stages of  Technical a s s i s t a n c e o f Mrs. Maureen Douglas  i s g r a t e f u l l y acknowledged.  A p p r e c i a t i o n I s expressed t o  Drs. J . R. Adams, C. V. Finnegan and ¥. S. Hoar f o r t h e i r c r i t i c a l reading of the manuscript.  T h i s study was aided by  grants from the N a t i o n a l Research C o u n c i l of Canada and the N a t i o n a l Science Foundation  of the United S t a t e s .  iv  CONTENTS  INTRODUCTION  . . . . .  • .  1  MATERIAL AND METHODS  RESULTS  4  . 10  .  10  .EFFECTS OF S A L I N I T Y SEASONAL EFFECTS OF SALINITY  . . . . . . . . . . .  17  SEASONAL EFFECTS OF TEMPERATURE TEMPERATURE EFFECTS AT GIVEN S A L I N I T I E S  14  . . . . 19  EFFECTS OF TEMPERATURE ON RATES OF URINE AND BLOOD CONCENTRATION CHANGE I N HIGH S A L I N I T I E S  . 20  EFFECTS OF S I Z E ON OSMOTIC RESPONSE  24  RELATIONSHIPS BETWEEN URINE AND BLOOD CONCENTRATIONS  26  DISCUSSION  28  SUMMARY  48  LITERATURE  CITED  51  V  LIST  1.  Urine  c o n c e n t r a t i o n changes  Hemigrapsus mental 2.  Urine  concentration  inities Urine  oregonensis  s a l i n i t i e s at  oregonensis  3.  at  exposed 5°  4.  In  a  to  a range  of  experi-  C  10a  changes to  summer-adapted  exposed  15°  i n winter-adapted  range  of  H.  experimental  sal-  C  11a  concentration  nudus exposed at  OF F I G U R E S  changes  to a range  of  i n summer-adapted H . experimental  salinities  15° C  12a  Urine  concentration  nudus  exposed  to  changes  a range  in winter-adapted  of  experimental  H.  salinities  13a  . at 5 ° C, . . . . . . • 5.  6.  Osmotic gradients  between  u r i n e and media  summer- and w i n t e r - a d a p t e d  crabs  to  salinities  a range  Effect in  of  orabs  of  experimental  temperature exposed  to  exposed  on 48-hour u r i n e  selected  salinities  for for  48  hours 14a  concentration 19a  vi  LIST OF TABLES  1. Comparison of 48-hour urine concentration with concentration of experimental media, summer and winter, at 5 ° , 1 5 ° and 2 5 ° C  12b  2. Comparison of 48-hour urine concentrations of Hemigrapsus oregonensis with those of H. nudus at 5 ° » 1 5 ° and 2 5 ° C (Absolute values given In Table 4) . . . .  14b  3. Comparison of 48-hour urine concentrations of summeradapted animals (S) at 1 5 ° C with those of winteradapted animals (¥) at 5 ° C  15a  4. Comparison of 48-hour urine concentrations of summeradapted (S) and winter-adapted  (W) animals at 5 ° , 1 5 °  and 2 5 ° C  l6a  5 . Comparison of 48-hour urine concentrations at 5 ° » 1 5 ° and 2 5 ° C, In selected media.  20a  .  6. Differences i n urine concentration between large ( L ) and small (S) summer-adapted animals  (Wllcoxon's  Slgned-Sank Test). . . . . . .  24a  7. Comparison of 48-hour urine and blood concentrations in summer- and winter-adapted  animals at 5 ° , 1 5 ° and  25° C i n selected media  .  26a  8. Wilcoxon's Signed-Rank Test ( 2 t a i l e d ) f o r i n t e r s p e c i f i c differences i n TJ/B r a t i o s ; U/B r a t i o s converted to % and paired f o r same conditions  27a  INTRODUCTION Osmotic regulation i n aquatlo animals has been reviewed by Krogh (1939) and Beadle (1957) and discussed i n d e t a i l more recently by Prosser and Brown (I96I).  Beadle (1957  Pg. 3 3 5 ) ,  commenting on the evolution of osmotic regulation i n Crustacea, postulated "that i n marine crabs there are at least two  sets of  active prooesses at work, i n the g i l l s and i n the excretory organs, which are responsible f o r the Ionic imbalance between blood and sea water."  He suggested that adjustments i n the rates  of these processes could have l e d , i n appropriate environments, to the evolution of both hypo- and hyper-osmotic regulation. The method of urine formation  i n various Crustacea has been the  subject of considerable research (Pioken, 1 9 3 6 ; Maluf, 1 9 4 1 ; Robertson, 1957; Martin, 1957).  Riegei and Kirsohner, i960; and the review of Osmotic behavior of nine species of eastern  P a c i f i c brabs was  investigated by Jones ( 1 9 4 1 ) , who  categorized  Hemigrapsus oregonensis and j j . nudus as hyper-osmotlc regulators, without the capacity for hypo-Osmotic regulation i n high s a l i n ities.  The mechanisms by which crabs e s t a b l i s h and  osmotic and  maintain  lonio gradients between t h e i r i n t e r n a l and external  environments have been studied (Hagel, 1 9 3 4 ; Green, Harsch, Barr and Prosser, 1955).  Carolnus maenas. a crab showing no hypo-  osmotic regulation, Prosser and Brown (I96I Pg. 1 4 ) postulate three mechanisms which play a part i n hyper-osmotlc regulation: "low permeability to water and s a l t s , increased f l u i d output, p a r t i c u l a r l y of urine, and active salt absorption from the  medium." The participation of the antennary glands in hyperosmotic regulation in pachygrapsus crassipes, a doubly regulating intertidal species, i s suggested by grosser, Green and Chow ( 1 9 5 5 ) .  Gross ( 1 9 5 2 ) suggests that active absorption of  water may be a method of hypo-osmotic regulation.  The antennary  glands have been considered to be more important In ionic than total osmotic regulation (Prosser, Green and Chow, 1955J Green, Harsch, Barr and Prosser, 1 9 5 9 ; Prosser and Brown, 1 9 6 1 ) . Much o f the evidence for this view has been drawn froo the work of Nagel ( 1 9 3 4 ) , Webb (1940), Robertson ( 1 9 4 9 ) and Parry (1954). In a seml-terrestrlal crab, Coenoblta perlatus4 Gross and Holland { i 9 6 0 ) demonstrated behavioral mechanisms for regulation of osmotic concentration of the blood.  The antennary glands In  this species were shown to contribute only to the regulation of potassium and not at a l l to total osmotic regulation.  The ratio,  Urine/Blood concentration (U/B ratio) for specific ionB In a variety of both regulating and adjusting Crustacea, indicates that the antennary glands can act selectively to control certain ionic imbalances between blood and external media (Peters, 1935; Picken, 1936} Prosser, Green and Chow, 1955; Gross, 1959; Gross and Holland, i 9 6 0 ; Prosser and Brown, 1961). Temperature acclimation of rate functions in polkilotherms has been documented (Bullook, 1955; Prosser, 1 9 5 5 ) , and the effects of temperature ( on osmotio regulation in aquatic organisms have been investigated and reviewed (Wlkgren, 1 9 5 3 ; Verwey, 1 9 5 7 ) .  The effect of external salinity on animal aotiv-  - 3 *  i t y , particularly cn osmotic "behavior and the flux of water and ions between body fluids and media, and concomitant weight changes have been studied (Jones, 1 9 4 1 ; Robertson, 1 9 ^ 9 , 1953J Gross, 1 9 5 ^ , 1955» 1957a? Prosser, Green and Chow, 1 9 5 5 ; Dehnel, i 9 6 0 ) . Broekema (1941) studied the combined effects of temperature and salinity on seasonal migrations and osmotic relationships in the shrimp, Crangon crangon.  In this species, and in  Carolnjdes (Caroinus) maenas (Broekhuysen, 1 9 3 6 ) , i t was found that both very low and very high salinities were best tolerated at high temperatures.  Blood concentration in C. crangon held  in a constant salinity of 15$o was shown to f a l l as the temperature deoreased by 10° C.  In animals held in sea water of  25$o, a similar decrease in temperature resulted in a rise i n blood concentration.  The effect of both these changes was to  reduce the difference between internal and external concentrations, as the animals were isotonio in salinities of 21$o to 23%Q, depending on temperature.  Similar temperature effects  and salinity tolerances were postulated for other Crustacea which show seasonal changes in distribution, from cold high salinity situations in winter to warm, low salinity situations in summer. By extension, this may also include intertidal species which do not migrate but are exposed in their usual habitat to similar seasonal changes in temperature and salinity (Verwey, 1 9 5 7 ) . The osmotio behavior of f|. oregonensis and H. nudus. has  - 4-  been studied with respect to blood responses to a range of experimental temperatures and s a l i n i t i e s (Dehnel, 1 9 6 2 ) .  These  two species are established i n a geographic area with seasonal temperature and s a l i n i t y c h a r a c t e r i s t i c s s i m i l a r to those discussed by Broekema (1941) and Verwey (1957).  The a c t i v i t y  of the antennary glands i n osmotic regulation, as r e f l e c t e d In the t o t a l osmotic concentration of urine from s i m i l a r l y exposed animals, i s herein considered as a further contribution to the understanding of the physiology of these animals.  Evidence  w i l l be presented that the osmoregulatory responses of these species change s i g n i f i c a n t l y with seasonal f i e l d and s a l i n i t y , and that hypo-osmotic  temperature  regulation does occur to  some degree at least In summer-adapted animals, which agrees i n part with the findings of Gross ( 1 9 5 7 a ) .  Further, Interspecific  differences In osmotic response and U/B relationships w i l l be presented which may be related to the d i f f e r e n t  intertidal  l e v e l s c h a r a c t e r i s t i c a l l y occupied by the two species i n the study area and to t h e i r usual geographic d i s t r i b u t i o n . MATERIAL AND METHODS Two species of shore Crabs were used i n these experiments, Hemigrapsus nudus (Dana) and H. oregonensis (Dana).  The animals  were c o l l e c t e d at two seasons, summer and winter from the Intert i d a l zone at Spanish Bank, Vancouver, B r i t i s h Columbia.  The  habitat and the seasonal fluctuations i n temperature and s a l i n i t y have been described (Dehnel, i 9 6 0 ) .  - 5 -  The animals were collected in plastic pails and covered with damp seaweed.  In the laboratory, they were:rinsed with  stock 50$ to 80$ sea water and distributed into plastic trays measuring 12 X 9§ X 4 inches. Usually, four groups of animals were selected, each containing a similar weight range.  Depend-  ing on size, experimental salinity and temperature, the number per tray varied from 10 to 15 animals.  Each group was intended  to provide three sequential sets of 10 separate urine samples. In order to bring the animals to a oommon osmotio level and to clear their intestines, each group was totally immersed in 4.0 l i t e r s of 75$ sea water.  This has been selected as a  suitable intermediate salinity for equilibration (Dehnel, 1962). The trays were covered with perforated l i d s and placed in a darkened refrigerator or constant-temperature water bath, set at the ourrent experimental temperature.  The animals were not  fed and the water was renewed once every 24 hours, both in the i n i t i a l equilibration period and during the experiments. Most of the ohange in osmotio concentration of the blood in both species occurred in the f i r s t 24 hours following the transfer of animals to new conditions of temperature and salinity (Dehnel, 1962). Based on this, an ample osmotic equilibration period of 36 to 48 hours in 75$ sea water was selected for these experiments.  Following the equilibration period, each group of  animals was transferred to 4.0 l i t e r s of water at experimental temperature and salinity conditions.  It was necessary to aerate  the trays at high experimental temperatures and both high and low s a l i n i t i e s .  - 6-  The experimental temperatures used In both summer and winter series were 5 ° , 1 5 ° and 2 5 ° C.  The experimental  s a l i n i t i e s were 6 $ , 1 2 $ , 25$> 75%, 1 0 0 $ , 1 2 5 $ , 1 5 0 $ and 1 7 5 $ sea water, based on 1 0 0 $ sea water: 31.88$o s a l i n i t y and 17.65$© ehlorinlty.  These s a l i n i t i e s were obtained either by d i l u t i n g  stock sea water with deohlorinated tap water or by d i s s o l v i n g In i t an appropriate amount of "Sea S a l t " , a product of the L e s l i e Salt Co. of San Francisco.  S a l i n i t i e s were determined  by means of a conductivity bridge, and a l t e r n a t i v e l y by t i t r a t i o n with AgNO^. For the summer series of experiments, weights ranged from O.29 grams f o r both species, to about 1 5 . 0 grams f o r H. nudus and 1 0 . 0 grams f o r H. oregonensis.  For most of t h i s series, a l l  animals were weighed Immediately p r i o r to urine sampling. For the winter s e r i e s , to f a c i l i t a t e sampling, the smallest animals used were about 1 . 0 gram and the animals were not weighed. Only males were used and care was taken to avoid molting animals. During the summer, when daytime c o l l e c t i o n was possible, groups of 10 animals of each species were set aside f o r immediate sampling.  During the winter, night c o l l e c t i o n s were  c a r r i e d out, and urine samples were not taken from animals r e moved d i r e c t l y from f i e l d conditions.  In a l l other respects,  the procedure followed i n both seasons was the same. In the summer experiments, the following procedure was used.  A f t e r 3 , 24 and 48 hours holding i n the experimental  conditions, a group of animals was removed.  Each was damp-  dried with cheese-cloth and weighed to the nearest 0 . 0 1 gram  - 7 -  on a M e t t l e r ftodel K7T e l e c t r l o b a l a n c e .  The animals were  then p l a c e d i n numbered paper cups and covered. U r i n e sampling was accomplished by means o f g l a s s capillary  tubes, 0.40 mm.  i n s i d e diameter and 1| inches l o n g ,  drawn t o a f i n e t i p and i n s e r t e d i n t o a small rubber p i p e t t e bulb.  The t i p o f each tube was broken o f f at a diameter  a b l e t o the s i z e o f the animal to be sampled.  suit-  The c r a b s were  h e l d In the f i n g e r s and g e n t l y but t h o r o u g h l y b l o t t e d d r y w i t h Kleenex t i s s u e .  They were then manipulated under a b i n o c u l a r  microscope, so that the t i p of a f l a t t e n e d and b l u n t e d n e e d l e , mounted on the microscope stage, c o u l d be I n s e r t e d under one o f the o p e r c u l a c o v e r i n g the u r i n a r y pores.  As the operculum was  r a i s e d , the t i p o f t h e o a p i l l a r y tube was i n s e r t e d beneath i t . T h i s u s u a l l y oaused t h e c r a b t o e x p e l s u f f i c i e n t u r i n e f o r a sample to be drawn i n t o the tube.  I f necessary, gentle pressure,  e x e r t e d d o r s o - v e n t r a l l y on the body o f t h e crab would cause expulsion of urine.  Care was taken t o a v o i d t i s s u e damage and  c o n t a m i n a t i o n o f the samples by water from the g i l l  chambers  o r gut f l u i d , which was o c c a s i o n a l l y e x p e l l e d through the mouth. I f a sample were l o s t , a second one c o u l d g e n e r a l l y be o b t a i n e d from t h e other s i d e o f t h e animal.  U s u a l l y , an excess o f u r i n e  was drawn i n t o the tubes and the samples reduced t o approximately 3.0 mm3  o r a l e n g t h o f about 5.0 ram i n t h e s t r a i g h t bore, by  p r e s s u r e on the b u l b .  The very f i n e t i p was then broken o f f t o  reduce the s u r f a c e t e n s i o n h o l d i n g the sample i n the end o f t h e tube, and the sample p o s i t i o n e d c e n t r a l l y by withdrawing the  - 8 -  tube from the bulb while maintaining a slight pressure with the fingers.  The entire tapered end of each tube was then  broken off squarely with foroeps.  The tubes were sealed  immediately with "Seal-Ease", placed in numerical order on labelled microscope slides and the samples quick-frozen on dry ice.  Samples were then transferred to a brine tray held at  -15° C, and kept in a frozen state u n t i l needed.  The animals  were returned to the experimental conditions after the 3-hour and 24-hour samplings, and discarded after 48 hours. The same sequence of experiments was followed in both the summer and winter series: one species, H. nudus. was subjected to the 8 experimental salinities at 5 ° C, followed by 2J« oregonensis.  The refrigerator or water bath was raised  f i r s t to 1 5 ° C, then to 2 5 ° C, and the same sequence repeated each time. Some experiments were repeated entirely or in part, both as a check on the technique and a verification of results; Measurement of the total osmotic concentration of urine samples was accomplished by the method of melting point determination described by Jones ( 1 9 4 1 ) and modified by Gross ( 1 9 5 4 ) * The data were analysed for salinity effects, temperature effects and seasonal differences by means of Student's "t° test. The same test was applied to the differences between mean osmotic concentrations of blood (Dehnel,,1962) and urine from similarly treated animals.  Differences In urine osmotic concentration  due to weight, and Interspecific differences In U/B ratios were  evaluated by means of Wllcoxon's Matched-Pairs Signed-Banke test.  Unless otherwise stated, s t a t i s t i c a l significance i s  attributed to P values <,  0.01.  Throughout this presentation, certain other terms are used with a specific connotation.  Following Dehnel ( i 9 6 0 ,  Pg. 223) aoollmatlon refers to "intra- and inter-specific compensatory changes whether these changes be phenotyplc or genotypio."  Unless otherwise specified, experimental conditions  refers to any of the 24 possible temperature-salinity combinations to which the animals were subjected in order to determine urine osmotic response.  Osmotic regulation i s considered to  be the maintenance of some degree of imbalance between the total osmotic concentrations of the blood and the external medium, regardless of the concentrations of the constituent ions and non-electrolytes.  Ionic regulation i s considered to be the  maintenance of an imbalance between blood and medium levels of particular ions. Volume regulation as such has not been discussed, but i s recognized as a corollary of osmotic regulation in some soft bodied invertebrates (Soheer, 1948).  In others,  lt i s suggested that osmotic and volume regulation may be separate funotions (Prosser and Brown, I 9 6 I ) .  The term osmotio  concentration refers to the total osmotic concentration of blood, urine or the medium. The equilibration or holding period is the time during which animals were held in 75$ sea water at experimental temperatures  in order to clear their intestines  and bring their body fluids to a common osmotic level.  The  - 10  -  term gradient refers to a difference in total osmotic or particular Ionic concentration between body fluids or a body f l u i d and an external medium, whioh are separated by a d i f f e r entially permeable membrane. It may be experimentally imposed and transitory, or established and maintained by the osmoregulatory activity of the organism conoerned. RESULTS  EFFECTS OF SALINITY; The osmotic responses of H. oregonensis and H. nuduq to a range of experimental s a l i n i t i e s , expressed as urine concentration ($ sea water), are shown in Figures 1 to 4.  Data for  summer experiments, carried out at 1 5 ° C, and winter experiments, at 5 ° C, are presented. These temperatures are approximations of the seasonal f i e l d means (Dehnel, i 9 6 0 ) .  In each figure,  urine concentration at time zero i s the mean of a l l three-hour values used in the figure.  This i s the point of divergence  (steady state value) for purposes of discussion.  It w i l l be  noted that the 3, 24 and 48-hour values for animals in 75$ sea water were obtained after 36 to 48 hours equilibration in that medium. Fluctuations in the 75$ sea water curves are presumed to represent normal changes in steady state urine.  Res-  ponses common to the four sets of curves are the rise In urine concentration with time in high s a l i n i t i e s , and the f a l l in low salinities, at rates directly related to the gradients between media and the steady state urine concentrations* The sensitivity  - 10a -  F i g u r e 1.  U r i n e c o n c e n t r a t i o n changes i n summeradapt ed Hemigrapsus oregonensis exposed to a range of experimental a t 15°  C  salinities  HEMIGRAPSUS U J I 8 0 I  SUMMER  -  OREGONENSIS  15 ° C  150 %  <  <I60  •  125 %  • 100%  4 0 ' 2 4  Tl M E  (HOURS)  4 8  - 11  -  of both species to external sea water concentrations i s demonstrated by the separation of response curves at 24 and 48 hours.  Regulation of urine osmotic concentrations is demon-  strated by the leveling of response curves for the media within the physiological limits of the meohanlsm. Hemigrapsus oregonensis: Summer animals (Fig. 1 ) , did not survive 48 hours In 175$ sea water.  Regulation in lower salinities was demonstrated  by the leveling of the curves after 24 hours.  Only in 125$ sea  water did the urine concentration curve show an Increased slope after 24 hours.  This might have been due to experimental var-  iation in the 24 and 48-hour determinations, as, even in 150$ sea water, the leveling was distinct. Winter animals (Fig. 2) survived 48 hours in 175$ sea water, probably reflecting adaptation to high salinity field, conditions.  Regulation of urine concentration was again marked,  particularly in high s a l i n i t i e s .  The curves for 1 2 5 $ , 150$ and  175$ sea water flattened after 24 hours.  In hypotonic media,  the curves, after f a l l i n g markedly between 3 and 24 hours, flattened and demonstrated hyper-osmotic regulation. The absolute difference between 48-hour urine concentrations in 6$ and 150$ sea water was 125$ In summer and 88$ i n winter animals.  The major portion of this difference was contributed  by animals from high salinities, whose summer urine concentrations were significantly higher than corresponding winter ones. It is shown that i n summer, H. oregonensis  maintained  - 11a -  Figure 2 .  Urine concentration changes in winteradapted H. oregonensis exposed to a range of experimental s a l i n i t i e s at 5° C.  HEMIGRAPSUS 2 1 8 0  WINTER  -  OREGONENSIS  5 °C  UJ  < •  175 %  •  150 %  •  125 %  •  100 %  <  UJ CO  <I20  or  • 2755%% •  40 24 TIME  4 8  (HOURS)  12  %  urine concentrations hypertonic to high salinity media. This was very l i k e l y due to the continued absorption of salts by gill,and gut tissues and a concomitant loss of water to the external media through the integument.  In winter, in high sal-  i n i t i e s , 48-hour urine tended to be hypotonic to blood and media. In both seasons, hyper-osmotic regulation occurred i n low salini t i e s , even i n 6$ sea water, which Is probably near the lower lethal limit for the species. Reference to Table 1 gives the significance of observed departures of urine concentration from isotonicity with the media.  It should be noted that In summer animals, a l l 48-hour  urine concentrations for the three experimental  temperatures,  and salinities from 25$ to 150$ sea water were significantly different from those of the media. hour urine concentrations at 5 °  c  In winter animals, the 48-  in 100$ and 125$ sea water,  at 1 5 ° C in 75$ and 150$ sea water, and at 25° G in 75$ sea water did not differ significantly from the media. Hemigrapsus nudus: Summer animals (Pig. 3) survived 48 hours in 150$ but not 175$ sea water, as did H. oregonensis.  The curves for a l l ex-  perimental salinities were again well separated* indicating sensitivity to external osmotic concentrations.  The leveling  of the curves and the existenoe of substantial gradients between urine and the media demonstrate hyper-osmotlc regulation In dilute media, active absorption and reabsorptlon i n concentrated media and the approach of urine to new equilibria.  Soma evidence  - 12a  Figure 3 .  -  U r i n e c o n c e n t r a t i o n changes i n summeradapted. H. nudus exposed to a range o f experimental s a l i n i t i e s a t 1 5 °  G.  H E M I G R A P S U S £ 1 8 0 1  S U M M E R  -  N U D U S  15 ° C  150  4 0  I  1  2 4  TIME  4 8  (HOURS)  %  - 12b -  Table 1 : Comparison of 48-hour urine concentration with concentration of experimental media, summer and winter, at 5 ° , 1 5 ° and 2 5 ° C.  Summer Species! T(°C)  100%  j 126*  Nudus  5  p<o.ooi  P<0,001 I P<0.001 I P 0,010 I N,S.  Or eg.  5  P<0,001  P<0.001 I P<0,001  P 0,001  Nudus  15  P<0,001  P<0.001  P<0,001  P 0.005  Greg,  15  P<0,001  P<0.001 I P<0,001  P  0,001  fNudus  25  P<0.001 I P<0.001 I P<0,001 ! 1 P<o.oo5 |P<0,001 P<0.001 i f . .,-1. . •• I r Winter  P  0,001  P<0.001  P  0.001  P< 0,001*  [P<0,001 ; P<0,001 ! ? P<0,001 I P<0.001  N.S,  P  N,S,  N,S,  Oreg.  25  •  Nudus [Oreg,  8  .'.  1  I.D, I  I.D.  I I.D,  I  T  f"  0,001  p<0*005 P<0*010 4  'Nudus i 15  *P<0,001 ? P< 0,001  P<0 001  Oreg,  fp<o,ooi I N.S,  P<0,001  Nudus '{•25 H  |P<0.001 } P<0,001  P<0,010  Oreg,  j P<0,001  t  ! 5  1  I.D. =  25  Insufficient data.  .1  0,010 j N.S* • I P 0,001 j N.S, P  ?  f  P 0.001 j P<0,002 P<0.001 I p o.ooi i P<0.001 1  -  13 -  of efforts toward hyperosmotic regulation is derived later, from U/B relationships. In winter animals (Fig. 4 ) , the separation of ourves, especially i n 75$ and 100$ sea water was not as clear as i n the summer experiments. The animals survived 48 hours i n 150$ but not 175$ sea water.  The lower survival limit roBe above the 6$  summer level, and the 48-hour urine concentration i n 1 2 $ sea water was significantly lower than the comparable summer value. Flattening of the 12$ curve was less pronounced than i n summer. In 1 0 0 $ , 125$ and 150$ sea water i n summer, 48-hour urine was hypertonic to the media. As in H . oregonensis. this might be due to Continued salt absorption and water loss.  In winter,  at high s a l i n i t i e s , blood concentrations were a l l hypertonic but urine concentrations became hypotonic to the media. Due to high winter mortality i n 6 $ sea water, the absolute difference between 48-hour urine concentrations i n 12$ and 150$ sea water was considered. winter.  This was 9 2 $ i n summer and 86$ i n  As in the case of H . oregonensis most of the difference  was due to higher summer urine concentrations i n media above 100$ sea water. Interspecific Comparison: In summer, H. nudus showed a greater leveling than H. oregonensis of urine concentration curves i n high salinities. This suggests that H* nudus in high salinities.  potentially i s the better regulator  In low salinities, the a b i l i t i e s of both  - 13a -  Figure 4 .  Urine concentration ohanges in winteradapted H. nudus exposed to a range of experimental salinities at 5° C.  - 14 -  species to hyper-osmoregulate were similar. In winter, H. oregonensis exceeded H. nudus i n a b i l i t y to regulate in salinities lower than 75$ sea water. Reference to Table 2 shows that In winter, at 5 ° C, the 48-hour urine concentrations of the two species differed significantly in media below 75$ sea water, with those for H- oregonensis being higher.  In summer, at 1 5 ° C, they differed  significantly only In 1 0 0 $ sea water, with H. nudus having the higher value. SEASONAL EFFECTS OF SALINITY.; The a b i l i t i e s of the two species to establish and maintain osmotio gradients between their urine and the external media were observed to change from summer to winter.  In order to  evaluate seasonal effects, a set of gradient curves was derived from the data of Figures 1 to 4 and presented i n Figure $.  The  points on which the curves are drawn were obtained by subtracting the osmotio concentration of the experimental medium from each 4-8-hour urine>osmotic concentration. Experimental temperatures for summer and winter animals were 1 5 ° C and 5 ° C respectively.  The significance of the observed differences between  summer and winter urine i s given i n Table 3» Winter urine of both species (Figi 5 ) tended to be slightly hypertonic to comparable summer urine in s a l i n i t i e s up to 75$ sea water, but was significantly hypotonic in more concentrated i  media.  Reference to Figure 5 shows a marked divergence of  - 14a -  F i g u r e 5«  Osmotic gradients between u r i n e and media f o r summer- and winter adapted crabs exposed f o r 48 hours to a range of e x p e r i mental s a l i n i t i e s .  70 HEMIGRAPSUS OREGONENSIS • • SUMMER (I5°C)  _ 60 -  •  WINTER ( S°C)  HEMIGRAPSUS  g 0 5  Q UJ  NUDUS  O  O SUMMER (I5°C)  O  O WINTER (5°C)  40  (0 UJ  z 30 or 3  20 UJ  IUJ GO  10  UJ O <  ISOTONICITY  or  "-•-'V  -10  \  -20 J L 6 12 25  \  \  \  \  O  50  MEDIUM  75  100  CONCENTRATION  125 (%SEA  150 WATER)  175  - 14b -  Table 2: Comparison of 48-hour urine concentrations of Hemigrapsus oregonensis with those of H. nudus at 5©, 150 a 250 C. (Absolute values~"given In Table 4). a  n  Concentration of Media {% S . W . ) T(°C) 6$  -  12$  25$  75$  100$  125$  N.S.  N.S.  N.S.  N.S.  N.S.  -  P< 0.001 P<0.001 N . S .  N.S.  N.S.  N.S.  N.S.  N.S.  N.S.  *  P<0.001 N . S .  N.S.  S  5  ¥  5  3  15  N.S. N.S.  N.S.  W  15  -  N.S.  P<0.001 P<0.001 N . S .  S  25  •  -  N.S.  w  25  N . S . P<0.001 N . S .  N.S.  N.S.  150$  175$  -  P<c0.005 N . S .  P<0.001 P<0.001 N . S .  P< 0.001  - 15  -  summer and winter gradient curves for both species In media above 75$ sea water.  Summer animals exhibited positive urlne-  to-mediura osmotic gradients in excess of 4 5 $ sea water in low salinity media and of 12$ to 21$ i n high salinity media.  Winter  gradients in low salinities remained in the same range as summer ones, but in the higher s a l i n i t i e s , gradients from + 1 . 0 $ to -18$ sea water were shown. Hemigrapsus oregonensis; Significant seasonal differences in 48-hour urine osmotic concentrations were found in media of 2 5 $ , 7 5 $ , 100$ and 125$ sea water, but not in 12$ sea water (Table 3 and Fig. 5 ) .  In  media of 1 2 $ , 25$ and 75$ sea water, winter urine was hypertonic to summer urine. Above 7 5 $ , winter urine became hypotonic to summer urine, and above 100$ sea water, to the media as well. Urine from winter crabs in 1 0 0 $ and 1 2 5 $ sea water was nearly Isotonic with the media, but from crabs i n 150$ sea water, It was significantly hypotonic to the medium. Urine from summer crabs was significantly hypertonic to the media in a l l experimental salinities (Table 1 ) .  In 100$ and 125$ sea water, summer  urine concentrations showed the greatest departure from comparable winter values (Table 4). Hemigrapsus nudus: The results for H. nudus in general paralleled those for M* oregonensis. with the difference that in 12$ sea water, summer  - 15a  -  Table 3: Comparison of 48-hour urine concentrations of summer-adapted animals (S) at 1 5 ° C with those of winter-adapted animals (W) at 5 ° C.  Urine Concentration {% S.W.) Exp. Sal. S 71  12^  100$  12% W  s  w  S  W  S  59  76  78  92  95  121  w 97  .3 . . W 138  117  Nudus P<0.010  67  74  N.S.  74  87  N.S.  88  96  P<0.001  Pf0.001  112  139  101  120  Greg. N.S.  P<0.010  P<0.010  P<0.001  P<0 010 o  » 16 -  urine was significantly hypertonic to winter urine (Table 3 and Pig. 5 ) .  In 25$ and 75$ sea water, urine of summer and In media above 75$ sea water,  winter animals was similar.  summer urine remained significantly hypertonic to both winter urine and the media, while winter urine, which was not significantly different from the medium in 100$ sea water, was significantly hypotonic in 125$ *id 150$ sea water (Tables 1 and 3 ) . a  Interspecific  Comparison:  In summer, both species exhibited similar osmotic U-M gradients i n media of 6$ to 75$ sea water.  Urine from H. nudus  was slightly hypertonic to that of H. oregonensis in these experimental s a l i n i t i e s .  In 100$ sea water, H. nudus urine was  significantly hypertonic to that of H, oregonensis but approached the same concentration i n 125$ sea water (Table 2 ) . Winter urine osmotic concentrations for the two species differed  sig-  nificantly only in 12$ and 25$ sea water (Table 2 ) . Poor survival of H. nudus in 6$ sea water precluded comparison in this medium. In a l l media except 150$ sea water, in which the urines had similar concentrations, H. oregonensis tended to have the higher values. Both species were found to be oapable of significant hyper-osmotlo regulation i n low salinities at both seasons. The hypertonloity of 48-hour summer urines to high salinity media, though of undoubted occurrence, appears to be largely an absorption phenomenon, as the animals do not normally encounter summer salinities much above 35$ sea water (Dehnel, i 9 6 0 ) .  A slight  16a -  Table 4 : Comparison of 48-hour urine concentrations of summer-adapted (SI and winter-adapted (W) animals at 5 ° , 1 5 ° and 25° C.  Urine Conoentratl.on ($ S.W.) Exp.Sal.12$ S N  1  5  s  64  s  w 59  71  N.S. 0  5  58  74  w 78  S 91  N.S, 78  87  0  15  15  71  45  76  P<0.010  ;  67  74  5^ N.S.  N  25  60  48  95  25  71  57  P<0.010  73  76  74  92  76 N.S.  96  94 N.S.  88  77  S 123  125$ S  W 97  138  150#  W  S 117  P<0.010  P<0.010  121  138  101  120  P<0.010  P<0.010  121  138  91  111  P<0.010  P<0.010  112  139  91  ?<0.010  P<0.010  89  123  138  84 N.S.  93  78  P<0.010  97  112  P<0.010  P<0.010  109  135  88  P<0.010  W  155  1^6  N.S. mo-  -  -  «a>  -  112  P<0.010  N.S.  76  95  N.S.  N.S.  N.S.  P<0.010  o  84  W  N.S.  P<0.010 P<0.010 N  100$  75$  112  P<0,010  174  138  P<0.010  **, 17 —  oapaoity to regulate in high salinities for short periods i s , however, indicated and w i l l be discussed. Production of hypotonic urine Is carried on by winter animals of both species in high s a l i n i t i e s . SEASONAL EFFECTS OF TBMPEBATUBE •: Table 4 gives 48-hour urine concentrations at 5 ° , 1 5 ° , and 25° C for summer and winter animals held in experimental salini t i e s of 1 2 $ to 150$ sea water.  The significance of observed  differences between the mean values i s Included. Hemigrapsus oregonensis: At 5 ° C, In salinities below 75$ sea water, winter animals showed significantly animals.  higher urine concentrations than summer  In 75$ sea water, urine concentrations were alike,  while in higher salinities* summer values signlfioantly exceeded winter values.  At 1 5 ° C, In low s a l i n i t i e s , no significant d i f -  ference was shown between summer and winter urines. In 75$ sea water and higher concentrations, a l l summer values were significantly higher than their winter counterparts. values significantly  At 2 5 ° C, summer  exceeded winter values In a l l salinities  but 25$ sea water, in which they were the same. A rlee in experimental temperature from 5 ° to 1 5 ° C was accompanied by a lowering from 75$ to 25$ sea water of the external concentration in which the seasonal means approached equality.  - 18 -  Hemigrapsus nudus: At 5 ° C, i n salinities of 75$ sea water and less, no significant differences were observed between summer and winter urine concentrations.  In 100$ and 125$ sea water, summer values  significantly exceeded winter values.  In 150$ sea water, a  similar but not statistically significant difference was observed. This lack of significance could be linked to a relatively high variance in the summer data for this salinity (S«=9.46), and to a reduced sample Size caused by mortalities (Ne6.0).  At 1 5 ° and  2 5 ° C, summer and winter urine concentrations were not significantly different in 25$ and 75$ sea water, but in both lower and higher s a l i n i t i e s , summer values significantly exceeded winter values. Interspecific Comparison: Out of fifteen comparisons of seasonal mean urine concentrations carried out for each species, H. nudus showed no significant difference i n seven cases, and H. oregonensis. in four cases.  No s t a t i s t i c a l Importance can be attributed to this  ratio however, as the probability of Its occurrence i s above 5 0 $ . At best, there i s a suggestion that H. oregonensis tends to show seasonal differences in urine osmotic concentration over a wider range of conditions than H. nudus.  The principal differences In  response between the two species occur In media of 1 2 $ to. 75$ sea water,  -  19 -  TEMPERATURE EFFECTS AT GIVEN SALINITIES; Data were selected from Table k to show the effect of increasing temperature on 48-hour urine concentration for animals in given media.  Low ( 1 2 $ ) , intermediate ( 7 5 $ ) and  high ( 1 2 5 $ sea water) salinities were chosen, and for each of these, the value at 5 ° C was compared with those at 1 5 ° C and 25° C, and the value at 1 5 ° C with that at 2 5 ° C.  Significant  results are shown in Figure 6 and Table 5» It should be noted that the effects of temperature on osmotic concentration of urine, where they existed, were significant in low and intermediate s a l i n i t i e s , but not in high s a l inities. Hemigrapsus oregonensis; In 1 2 $ and 75$ sea water, winter animals showed a significant decline i n urine concentration when experimental temperature was raised from 5 ° to 1 5 ° C (Fig. 6 ) . No further significant changes occurred with the rise from 1 5 ° to 2 5 ° C. Summer animals i n 1 2 $ sea water showed a significantly higher urine concentration at 2 5 ° C than at 5 ° C. Urine concentration at 1 5 ° C did not differ significantly from the values at 5 ° or 2 5 ° C. In 125$ sea water.neither summer nor winter urine concentrations changed significantly with changes in experimental temperature. Hemigrapsus nudus: In 1 2 $ sea water, winter animals of this species also showed a significant decline in urine concentration with an  - 19a -  Figure 6.  Effect of temperature on 48-hour urine concentration in crabs exposed to selected salinities.  - 20 -  Increase In experimental temperature from 5 ° *o 1 5 ° C. In 75$ sea water, a similar decline occurred with the Increase of experimental temperature from 1 5 ° to 25° G. Summer animals showed no significant change with increased experimental temperature in any of the three media (Table 4 ) . As in H. oregonensis . ehanges in experimental temperature had no significant effect on the urine concentration of animals i n 125$ sea water. Interspecific Comparison: In winter animals of both species i n 12$ sea water, and in H. oregonensis i n 75$ sea water as well, urine concentrations were significantly higher at 5 ° than 15° C (Pig. 6 ) . £[• Btidus showed no significant change between 5  P  Winter  and 1 5 ° C but  urine concentrations at these temperatures were significantly higher than at 25° C. In the latter species - summer animals t  showed no significant changes In urine concentration with Increased temperature, while in H. oregonensis i n 12$ sea water, a significant upward trend was observed with an Increase i n experimental temperature from 5 ° to 25° G. EFFECTS OF TEMPERATURE GM RATES OF URINE AND BLOOD CONCENTRATION CHANGE IN HIGH SALINITIES: Because salinity determinations on body fluids were made only at 3 , 24 and 48 hours, rates of concentration change cannot precisely be gauged from the response curves.  However, an  indication of temperature effeots can be obtained by considering  20a  -  Table 5 : Comparison of 48-hour urine concentrations at 5 ° , 1 5 ° and 25° e , In selected media.  Exp. Temp. ro)  Media  f  Cone. ($s.w..) 12$  VS  75$  0  15  125^  12$  N-W  P<0.001 N.S.  o-s  P<0.001 P<0.001 N.S. N.S.  0-W  N.S.  N.S.'  vs  25°  75$  125$  5° 12$  vs  75$  25° 125$  N.S. N.S. P<0.001 N.S.P<0.001 P<0.001 N.S.  N.S.  N.S. £<0.001 P<0.001 N.S.  N.S. N.S. P<0.005 N.S. F<0.001 N.S.  N.S.  - 21  the Intervals in which rising blood and urine concentrations passed isotonicity.  Both blood (Dehnel, 1962) and urine in  summer-adapted Hemigrapsus were hypertonic to a l l experimental media at 5 ° , 1 5 ° and 2 5 ° G at 48 hours.  In winter-adapted  animals, blood of both species was hypertonic to a l l s a l i n i t i e s . Urine of H. oregonensis was Isotonic to 100$ sea water and hypertonic to higher salinities. Urine of H. nudus was hypotonic to 100$ sea water and higher salinities (Pigs. 1 to 5 and data not presented).  Blood and urine concentrations rose from equil-  ibrated values at time zero and passed isotonicity at times directly related to the season, to concentrations of the media and experimental temperatures. At 5  0  C, the blood of winter-adapted animals reached iso-  tonic ity with 100$ sea water in the interval between 0 and 3 hours, with 125$ and 150$ sea water, between 3 and 24 hours, and with 175$ sea water, between 24 and 48 hours.  At I 5  0  and 2 5 ° C  the only change was In 175$ sea water, where isotonicity was reached earlier, between 3 and 24 hours;  At 5 °  urine was  Isotonic to 100$ sea water by 3 hours, and did not become hypertonic to any higher salinity at 5 ° , 1 5 ° or 2 5 ° C. In summer-adapted animals, at I 5  0  C in 100$ sea water,  Isotonicity was reached in blood at 3 hours* and in urine between 0 and 3 hours*  Very slight hypotoniclty of blood to urine and  medium at 3 hours suggested a weak effort to regulate blood concentration In this salinity, which disappeared by 24 hours. Iri 125$ sea water, blood reached isotonicity between 3 and 24 hours,  - 22 -  and urine, in the next interval.  Again, slight hypotonicity  of blood to urine and medium suggested weak regulation. In 150$ and 1 7 5 $ sea water, both urine and blood passed isotonicity between 3 and 2k hours.  In animals cooled to 5 ° C, blood and  urine in 1 0 0 $ , 1 2 5 $ and 150$ sea water reached the concentration of the media between 1 3 and 2k hours.  In 1 0 0 $ sea water, 3-hour  blood was hypotonic to urine and medium, in 125$ sea water, tothe medium only, and in 150$ sea water, to both again. Blood concentration appeared to be regulated successfully In 100$ sea water for over 3 hours, and with partial success (with production of blood-hypertonic urine) for kQ hours.  In 1 2 5 $ sea  water, regulation appeared to be somewhat successful up to 24 hours, at which time blood was s t i l l slightly hypotonic to urine, but not to the medium.  Regulation was shown in 150$ sea  water for over 3 hours but It was not evident at 24 hours. When warmed to 2 5 ° C, these animals showed blood Isotonicity in 100$ sea water between 0 and 3 hours (earlier than at 1 5 ° C) and in 125$ sea water, between 3 and 24 hours, as at 1 5 ° C. Urine became isotonic in 100$ sea water between 3 and 24 hours, later than blood, and at 3 hours i t was hypotonic to blood*  At 24  and 48 hours, blood was hypotonic to urinej but not to the medium.  This i s some evidence of an effort towards  regulation.  hypo-osmotio  In 1 2 5 $ , 150$.and 1 7 5 $ sea water, urine passed iso-  tonicity between 3 and 2k hours with no instance of blood-hypertonic urine being formed.  23 -  Hemigrapsus nudus: Winter-adapted animals showed rates of blood concentration change like those of H. oregonensis at a l l temperatures and salinities except at 25° C in 175$ sea water where the animals died sometime after 3 hours, and at which point blood was not yet Isotonic.  Urine did not become hypertonic at any temperature  In media of 100$ sea water or higher. In summer-adapted animals, at 1 5 ° C, both blood and urine became isotonic with 100$ sea water between 0 and 3 hours, and with 125$ and 150$ sea water, between 3 and 24 hours.  In 100$  sea water, 3-hour urine was hypertonic to blood (U/B=1.06) suggesting a slight effort toward hypo-osmotic regulation of blood concentration.  In animals cooled to 5° C, blood isoton-  i c l t y with 100$, 125$ and 150$ sea water was reached between 3 and 2k hours.  Urine isotoniolty was reached earlier In 100$  sea water, and at 3 hours, a U/B ratio of 1.06 obtained, suggesting, as at 1 5 ° C, slight regulation in this medium. In 125$ and 150$ sea water, 3-bour U/B ratios were < 1 . 0 .  In 175$ sea  water, urine reached isotoniolty between 3 and 2k hours.  In  summer-adapted animals warmed to 25° C blood passed isotoniolty In 100$ sea water between 0 and 3 hours, In 125$ sea water, between 2k and 48 hours, and In 150$ sea water between 3 a»d 24 hours. Urine became isotonic between 0 and 3 hours in 100$ and between 3 and 24 hours In 125$ and 150$ sea water.  Urine was hypertonic  to blood In 100$ sea water at 3 , 24, and 48 hours, and In 125$  -  zk -  sea water at 3 and 2k hours, suggesting a considerable e f f o r t to regulate blood concentration i n these s a l i n i t i e s ;  In 1 5 0 $  sea water, however, the 3-hour urine was hypotonic to blood, with no evldenoe of regulation of blood concentration by the antennary glands. EFFECTS OF SIZE OM OSMOTIC RESPONSES: In order to determine whether small and large animals d i f f e r e d s i g n i f i c a n t l y i n urine concentration under Identical conditions, the mean urine concentrations of the smallest and largest summer animals were compared (Table 6 ) .  This was done  f i r s t by grouping the urine concentrations o f the four or more smallest, and an equal number of largest animals used i n each determination, where weight data were c o l l e c t e d , and determining the two mean values.  These were treated as a pair of  samples from one normally d i s t r i b u t e d population.  Such p a i r s  were calculated f o r both species, from urine concentrations of f i e l d animals and experimental animals a f t e r 3 , 2k and kQ hours In experimental conditions, where at least 8 samples were used f o r the determinations.  From the r e s u l t s , a table of differences  was determined by subtracting the mean urine concentration of the largest from that of the smallest animals.  I t was then  possible to group negative and p o s i t i v e differences according to experimental s a l i n i t y , f o r each species.  Groups of eight or  more p a i r s were selected, i n which a l l or most of the differences had the same sign.  An evaluation of the r a t i o s of p o s i t i v e to  is  Table 6 :  24a «-  Differences i n urine concentration "between large (L) and small (S) summer-adapted anipals. (Wilcoxon's Signed-Rank T e s t ) .  No.of O.C.S.-G.C.L. Source of Pairs x w't'(G:m. )Balrsr Differences' ; S T(°C) jfe.W. S L No.+- No.N  F,  F  2.0*  0  F  F  1.8  N  15°  6-25  2.5  6.4  ,> 8  0  15°  100-175  1.8  3.6  8  N  25°  6-73  2.0  5.7  8  0  25°  6-75  1.5  3.6  14  9  5-  N  25°  100-150  1.4  4.7  9- •  7  2  0  25° .100-175  I ?  4.0  12  11  1  2  7.8 '4.5  .-  Smallest Sum- of P r o b a b i l i t y Signed Ranks  8*  8  0  0  P » 0.010  8  7  1  -6  p" > 0 . 0 5 0  1 .  7  ,. +2 .  P *: 0 . 0 2 0  8  0  ,  2,  ,  6  0 . ,  . *  \  P a 0.010  +12.5  P >. 0 . 0 5 0 •,  -35.5  P > 0.050  -5-5 -8  ' , P. > 0 . 0 2 0 ,  P > 0.010  F - F i e l d Conditions (Summer) O.C.S. » Mean osmotio concentration of urine of smallest animals O.C.L. - Mean osmotio concentration of urine of largest animals  - 25 -  negative differences i s given in Table 6. The Wllcbxon test takes into account the size of the differences between the mean urine concentrations of small and large animals and tests the null hypothesis that either group has an equal chance to have the higher concentration. The implications of the significant weight effects are not obvious.  Some differences, while not  significant at the 0 . 0 1 level, have P values < 0.05 and may suggest a tendency. Small summer-adapted ]|. nudus. taken from f i e l d conditions, tended to have significantly higher urine concentrations than large animals.  A similar, but not s t a t i s t i c a l l y significant  tendency was shown by H. oregonensis.  In high experimental  s a l i n i t i e s , the same tendency i s suggested at 1 5 ° and 25° C for H. oregonensis. and 25° C for H. nudus. At 1 5 ° C, large H. nudus. in low experimental salinities, tended to have higher urine concentrations than small animals.  At 25° C,, in low s a l i n i t i e s ,  neither species showed significant differences in urine concentration attributable to differences in weight. There i s an apparent anomaly i n the fact that small J2« nudus under summer f i e l d conditions of low salinity and high temperature showed significantly higher urine concentrations than large animals, while in experimental conditions approximating those of the f i e l d , the observed differences were reversed but not significant at the 0 . 0 1 level.  This was probably due to  the equilibration of experimental animals i n 75$ sea water before  -26  exposure to low s a l i n i t i e s i n the laboratory, and the emplri o a l grouping of data from a range of experimental s a l i n i t i e s f o r comparison with those from more constant f i e l d conditions. RELATIONSHIPS BETWEEN URINE AMD BLOOD CONCENTRATIONS: Consideration Is given In Tables 7 and 8 to the r e l a t i o n ship between urine and blood concentrations i n animals exposed to the same experimental conditions. Blood data were provided by the experiments of Dehnel ( 1 9 6 2 ) .  Forty-eight-hour urine  values were, taken from Table 4, 3-hour and 24-hour values from raw data.  Table 7 gives t o t a l osmotic Urine/Blood r a t i o s of  previously equilibrated animals of both species, held f o r 48 hours i n media of 1 2 $ , 75$ and 125$ sea water, summer and winter. Also included, are Intraspeoific Urine minus Blood concentration differences, and a s t a t i s t i c a l evaluation of their significance by means of Student's " t test. 9  Table 8 gives the significance  of i n t e r s p e c i f i c differences In U/B r a t i o s of JJ. nudus and M» oregonensis  i n both seasons, over the entire range of exper-  imental conditions. In order to apply the Wilcoxon t e s t , a l l U/B r a t i o s were converted to percentages and paired.  Arbitrarily,  for each experimental temperature, a l l values f o r H. oregonensis were subtracted from the corresponding values f o r If. nudus.  The  differences were ranked and the ranks given the signs of the differences.  The p o s i t i v e and negative ranks were summed, and  the smaller sum provided an entry Into the tables of p r o b a b i l i t y .  - 26a  Table 7 ; Comparison of 48-hour urine and blood concentrations i n summer- and winter-adapted animals at 5 ° » 1 5 ° and 2 5 ° i n selected media.  Summer  Media Con o. (# S.W.)  12$  T(°C)  U-B  U/B  125*  75* P V  f  ; U/B  U-B  U/B  P  U-B  P  Nudus 5  0.84 -11.7 p<o. oio; 0.97 -2.'.'-4 N.S.  Oreg. 5  0 . 9 0 - 6.2 N.S.  Nudus 15  1.01  0.9 N.S. •!« 0.99 -0.6 N.S.  Oreg. 15  1.02  1.3 N.S.  Nudus 25  0.98  -1.0 N.S.  Oreg. 25  0.98  -1.1 N.S.  Nudus 5  0 . 7 5 - 1 9 . 3 : P<0.001 0 . 8 3 - 1 9 . 8 P<0.001 0 . 8 3 -24.3 P < 0 . 0 0 1  Oreg. 5  0.83 -15.1  P<0.001 0 . 9 5 - 5 . 0 N.S.  0 . 9 3 - 9 . 4 P<0.005  Nudus  0 . 6 7 - 2 1 . 9 P<0.001 0 . 9 7 - 2 . 5 N.S.  0 . 7 7 - 3 3 . 6 P<0.001  15  Oreg. 15  0.63 -31.1  Nudus 25  0 . 6 3 -28.3  Oreg. 25  0.69  & 0.99 -1.3 N.S. •  0.94, -8.3' N.S.  -  0.98  -  ***  3 . 3 N.S.  0 . 9 5 -4.6 P<0.001 0.96 - 5 . 7 P<0.010 $ 0.98 -1.7 N.S. 1.04  3 . 3 N.S.  0 . 9 8 -2.4 N.S. 1.00 -0.2 N.S.  P<0.001 0 . 7 7 - 2 2 . 9 P<0.001 0 . 8 6 - 1 7 . 7 P<0.001  -  *  -25.I  * « 24-hour blood values U/B = Urine/Blood r a t i o U-B » Urine-Blood gradient  0.87 -12.7 0.87 -12.1  ->  0.82 -.24.4 P<0.001  P<0.001 0 . 8 8 -1.5.4 P < 0 . 0 0 1  * 2?  Summer U/B ratios are shown i n Table 7 to approach unity in both species. In most of the selected conditions, blood was more concentrated than urine. Where U/B ratios > 1 . 0 , the departure from unity was not significant.  Blood was significantly  hypertonic to urine i n H. oregonensis in 75$ and 125$ sea water at 1 5 ° C and in H. nudus, only i n 12$ sea water at 5 ° G. Winter U/B ratios l a both species in the selected conditions were a l l lower than comparable summer ratios.  The absolute d i f -  ferences i n concentration between urine and blood increased to s t a t i s t i c a l l y significant levels i n most of these conditions. The increases were due to a generally larger net deorease i n urine concentration than blood concentration from summer to winter i n similar experimental conditions. Blood values were significantly higher than urine values In a l l conditions except for JJ. oregonensis. 5 ° G, and H. nudus. 1 5 ° C, in 75$ sea.water. Table 8 shows that at 5 ° C, H. oregonensis had higher U/B ratios than H. nudus over the entire range of experimental s a l inities.  The difference was due to relatively lower blood con-  centrations in H. oregonensis at this temperature.  The discrep-  ancy between ratios was significant In winter and approached significance i n Bummer animals. s a l i n i t i e s , H* nudus  At 15° and 25° G, in the same  ratios were higher, both in summer and i n  winter, but the difference was significant only i n winter animals at 25° G.  - 2?a -  Table 8: Wilc6xon s Signed Rank Test (two tailed) for Interspecific differences in U/B ratios; U/B ratios converted to $ and paired for same conditions. r  Souroe of Pairs  Ho. of Smallest£ of  U/B  Season  Pairs  Trend  T(°G) % S.W. 5°  Winter  22  +38.5  0>N  P<0.01  23  -77.4  K>0  P>0.05  19  ^19.5  N>0  P<0.01  5°  20  +43.5  0>N  P>0.02  15° 6-175$  22  +88.5  0>N  P>0.0^  25°  18  -45.5  N>Q  P>0.0'5  15°  25°  Summer  Ranked Dlffs.  Probability  6-175$  :  '  1  - 28 .-  DISCUSSION Hyper-osmotic  regulation of blood concentration i n  ![• oregonensis and H. nudus was demonstrated by Jones (1941). Gross (1957a) was also able to show some degree of regulation i n these species.  hypo-osmotic  Recent work (Dehnel, 1962)i  c a r r i e d out for a f u l l year, has demonstrated  that the osmo-  regulatory a b i l i t i e s of the two species changed s i g n i f i c a n t l y from summer- to winter-adapted animals.  The r e s u l t s presented  here support and complement the l a t t e r findings with d e t a i l s of urine osmotic responses. EFFECTS OF SALINITY: From an equilibrated or steady state at time zero (Figs. 1 to 4), the urine osmotic response ourves f a l l i n low and r i s e in high s a l i n i t i e s at rates which i n general decline with time and appear to reach new e q u i l i b r i a - with media wlifchin the p h y s i o l o g i c a l l i m i t s of the species. Blood response curves f o r Hemigrapsus (Dehnel, 1962)  and Pachygrapsus  exhibit similar patterns.  In the l a t t e r investigation, samples  were drawn at shorter i n t e r v a l s ( 1 , stepwise changes towards new  (Gross, 1957a)  3 , 6 and 12 hours), so that  steady states are evident.  As i n  the present case, most of the changes were complete by 24 hours immersion In the media.  In Emerlta, an adjuster, Gross (1957a)  showed that a l l blood changes were complete a f t e r only two hours In a comparable range of experimental s a l i n i t i e s . The antennary glands of Pachygrapsus  have been shown to  -  29  -  funotion mainly In the regulation of p a r t i c u l a r blood ions but not of t o t a l blood osmotic concentration (Jones, 1 9 4 1 ; Hobertson, 1 9 4 9 ; Prosser, Green and Chow, 1 9 5 5 ; Gross, 1 9 5 7 a , 1959).  This conclusion was based on the isosmoticity of blood  and urine i n a variety of temperature and s a l i n i t y combinations, and on high U/B  r a t i o s f o r magnesium (Gross, 1 9 5 9 ) .  The prawns  Palaemonetes varlans. Leander serratus and L. s q u l l l a . In f  t  d i l u t e media, produce urine isotonic to blood (Panikkar, 1 9 4 1 ) . Parry ( 1 9 5 4 )  showed Mg and SO^ to be lower in blood than i n  urine i n L. serratus.  In both species of Hemigrapsus. summer-  adapted animals at least have t o t a l osmotio U/B r a t i o s close to unity over the entire range of experimental temperature and s a l i n i t y (Table 7 ) .  At the same time, large osmotio gradients  between external media and body f l u i d s (48-hour blood and urine) were obtained i n s a l i n i t i e s below 75$ sea water ( F i g . 5 ) *  The  animals are thus shown to be e f f e c t i v e l y regulating hyperosmotically.  There was no s i g n i f i c a n t weight increase suoh as  would accompany a large i n f l u x of water from the hypotonic media (Dehnel* 1 9 6 2 ) .  A f t e r an i n i t i a l rapid drop-in urine concen-  t r a t i o n * the rate of salt loss was reduced a f t e r 24 hours, so that a new  state of equilibrium was approached.  The demon-  ' strated hypertonioity of 48-hour urine i n summer animals exposed to concentrated media (Fig* 5 ) can have l i t t l e adaptive importance, since s a l i n i t i e s higher than 35$ sea water are not as a rule encountered i n t h i s geographic area.  Webb ( 1 9 4 0 ) postulates  that salt absorption i s a continuous process under normal  - 3 0  conditions.  -  The continued increase in urine concentration  may thus be attributed to the activity of salt absorbing tissues in the gut and g i l l s which, when adapted to high temperature and low salinity, continue to act as i f they were aiding hyperosmotic regulation, within physiological limits, of blood concentrations.  If this i s the case, the leveling of response ourves  in high salinities may be the result of interference by Increased blood ion concentrations, with the absorptive mechanism.  Until  the flux of sodium, magnesium, calcium, potassium, chloride and sulfate ions between concentrated media and the urine and blood is known, and the presence and Influence of adaptive extravascular salt pools is established, a more complete explanation of this summer phenomenon cannot be undertaken. Three major differences distinguish the urine osmotic responses of summer- and winter-adapted animals of both species, at their respective temperatures, to the same range of experimental s a l i n i t i e s .  The f i r s t i s that over a given range of  external concentrations, winter animals showed a smaller range of wine concentration than summer animals.  This was markedly  true for H. oregonensis (Figs. 1 to 4). Hemigrapsus hudus. In winter, showed a reduced tolerance for very low external salinity.  Such a reduction, expressed by high mortality, was also  shown for C. crangon. a migratory shrimp (Broekema, 1941).  The  second difference was that total osmotic U/B ratios for winter animals were in most cases Significantly lower than summer ratios, that i s , urine was more hypotonic to blood over the range of  -31  -  experimental salinities (Table 7 ) .  This suggests winter  participation of the antennary glands i n hyper-osmotlc regulation.  The significance of low U/B ratios i s not easy to see  in relation to the third and most important difference between winter and summer responses:  the production In winter of hypo-  tonic urine in external salinities above 75$ sea water for H. nudus and above.100$ sea water for H. oregonensis. While hypo-osmotio regulation of blood concentration has been well documented for a number of Crustacea from aquatic, intertidal, semi-terrestrial and terrestrial habitats (Jones, 1 9 4 1 ; Broekema, 1 9 4 1 ; Prosser, Green and Chow, 1 9 5 5 ; Cross, 1957a  and b; Riegel, 1 9 5 9 ) * It was not found In Hemigrapsus by  Jones ( 1 9 4 1 ) , whose results have been widely cited. (1957a)  Gross  however, held that some degree of hypo-osmotic regulation  of blood concentration occurred in Hemigrapsus from California, giving a value of up to 33$ perfect regulation for 20 hours in 150$ sea water*  This value was obtained by dividing the sus-  tained gradient between blood and medium at 20 hours by that at time zero.  Heoent work (Dehnel, 1 9 6 2 ) has shown that both  species of Hemigrapsus; equilibrated In 75$ sea waterj cannot maintain blood hypo-tonlcity when transferred to experimental salinities of 1 0 0 $ to 175$ sea water*  Present results have  indicated that although true regulation of blood concentration was not established in hypertonic mediaix increases in ooncentration may be resisted to some degree.  It was shown that urine  may differ in concentration from both blood and media, and that  — 32 —  seasonal changes occurred i n urine as well as blood osmotic responses. Summer-adapted Hetalgrapaus i n the f i e l d maintained t h e i r body f l u i d s considerably hypertonic to summer s a l i n i t i e s ( 2 5 $ to 3 5 $ ) .  TJrine was  shown to be nearly isotonic with blood.  Similar osmotic behavior i s found i n Carolnus  i n d i l u t e sea  water and Srloohelr i n fresh water (Krogh, 1 9 3 9 ) .  Webb (19*K))  has suggested that active water uptake i s suspended and ion exohanges i n g i l l s and antennary glands are i n t e n s i f i e d under these conditions.' The low permeability c h a r a c t e r i s t i c of the exoskeleton  of regulating forms (Gross, 195?a) would aid the  animals i n r e s i s t i n g the i n f l u x of excess water with increasing osmotic gradients.  '  When exposed to increased or decreased  . experimental  s a l i n i t i e s , summer-adapted animals behave, osmotioally, some-, what as i f they were s t i l l i n "normal" summer conditions, a l though the concentration of t h e i r body f l u i d s follows changes In the external medium*  In low s a l i n i t i e s , both species main-  tained hypertonlolty of blood and urine (Figs 3 and 5 and Dehnel, 1962).  This was  done without the production of  blood-hypotonic  urine, and might have been accomplished as Webb ( 1 9 4 0 ) Another p o s s i b i l i t y i s that during the experimental  suggests.  period,  s a l t s are mobilized from adaptive extra-vascular pools, whose existence was postulated by Hukuda ( 1 9 3 2 ) and v e r i f i e d i n Pachygrapsus by Gross ( 1 9 5 8 , 1 9 5 9 ) .  No experiment as yet has  - 33 -  been conducted to e s t a b l i s h the presence of these pools i n In 100$  Hemigrapsus.  sea water, H, nudus showed a s i g n i f i c a n t l y  larger gradient between 48-hour urine and the medium than Hi oregonensis. but at higher s a l i n i t i e s , there was no s i g n i f i c a n t difference (Table 2 ) .  Urine was s i g n i f i c a n t l y  blood i n H. oregonensis i n 75$ so i n H. nudus (Table 7 ) .  -hypotonic to  and 125$ sea water but was not  Urine osmotic response curves (Figs.'-l  and 3) rose past Isotoniolty In most cases by 24 hours* hours, in s a l i n i t i e s up to 150$  At 48  sea water, new e q u i l i b r i a were  approached, while i n 175$ sea water, l e t h a l blood concentration was reached before 24 hours (Dehnel, 1962), at whloh time In the present experiments, urine was equivalent to 192$ sea water i n H. oregonensis.  I t i s apparent from the above changes that  osmoregulatory mechanisms which are adapted to high-temperature low-salinity conditions, seem to continue to operate In a simi l a r way even when exposed to high experimental s a l i n i t i e s at summer temperature.  Summer-type regulation, characterized by  active Ion absorption and reduced water loss, and presumably accompanied  by ion reabsorption In the antennary glands, Is  l a r g e l y extra-renal and does not change after a period as short as 48 hours i n experimental conditions.  Parameters to which the  regulatory mechanisms may become acclimated are temperature, salinity(and T/S combinations), l i g h t and desiccation, brought about by the seasonal progression of low tides from night In winter, to day i n summer.  The e f f e c t of temperature on osmotic  responses of seasonally adapted animals w i l l be considered i n a l a t e r section.  - 34 -  The e x c r e t i o n of blood-hypotonic urine as a means of maintaining blood concentration above that of t h e medium has been w e l l documented (see below).  Winter-adapted Hemigrapsus  i n the f i e l d showed blood concentrations hypertonic to 70-80$ sea water (Dehnel, 1962).  Urine data f o r winter animals from  the f i e l d are not a v a i l a b l e , but a f t e r 51 hours (48 e q u i l i b r a t i o n plus 3 experimental) i n 75$ sea water a t 5° C  H. nudus  had higher blood concentrations than H. oregonensis (Dehnel, 1962)  while t h e i r u r i n e concentrations were a l i k e and hypotonio  to the blood.  For comparison w i t h summer data, these values  have been considered t o approximate  the u r i n e and blood r e l a t i o n -  ships i n winter animals from f i e l d c o n d i t i o n s . I n summer animals from f i e l d c o n d i t i o n s , H. nudus had both blood and u r i n e conc e n t r a t i o n s higher than H. oregonensis. U/B  Mean w i n t e r and summer  r a t i o s f o r H. nudus were 0.88 and O.95,  and f o r H. oregon-  e n s i s 0.93 and O.98, w i t h u r i n e - t o - b l o o d gradients of 13$ and 6$ and 7$ and 2$ r e s p e c t i v e l y . Winter animals of both s p e c i e s , i n experimental media below average w i n t e r sea water concentration (70-80$) regulated t h e i r blood concentration w i t h the production of blood-hypotonlc urine.  Absolute 48-hour blood and u r i n e concentrations were  s i g n i f i c a n t l y higher In H. oregonenals than H. nudus i n 12$ and 25$ sea water, and 6$ sea water was t o l e r a t e d by H. oregonensis only ( F i g s . 2 and 4, Table 2, data not presented and Dehnel, 1962).  The l a r g e r blood-to-medium g r a d i e n t s shown by H. oregon-  ensis suggest a more a c t i v e i o n absorbing mechanism i n t h i s  -  35  -  s p e c i e s , perhaps c o r r e l a t e d w i t h i t s c h a r a c t e r i s t i c a l l y estuarine d i s t r i b u t i o n .  The a c t i v e absorption of Ions from  hypotonic media has been demonstrated  i n a v a r i e t y of r e g u l a t -  ing Crustacea, among them, a c r a y f i s h , Astaous. and the crabs Carolnus and E r i o c h e i r . the l a t t e r r e l a t e d to Hemigrapsus (Schwabe, 1933; Brown, 1 9 6 1 ) .  Nagel, 1 9 3 4 ; Krogh, 1939;  c i t e d by Prosser and  I n crabs, the g i l l s have been recognized as major  s i t e s of absorption (Nagel, 1 9 3 4 ;  Gross, 1 9 5 7 a ) . Excess water can  enter the animals by d i f f u s i o n and a c t i v e a b s o r p t i o n , together w i t h Ions, through the g i l l s .  U r i n e , i f formed by  filtration,  at f i r s t may be i s o t o n i c w i t h the blood and be rendered hypot o n i c by the reabsorption of s p e o i f i c Ions. of  As long as the l o s s  ions i n the u r i n e Is at l e a s t balanced by a c t i v e absorption  from the d i l u t e media, the animals can achieve and maintain osmotic e q u i l i b r i u m (Webb, 1 9 4 0 ) .  Increased u r i n e output i n  d i l u t e media has been shown to a i d i n e l i m i n a t i o n of excess water i n Carclnus (Prosser and Brown, 1 9 6 I ) .  I t has not been demon-  s t r a t e d i n the present data but may be Important In Hemigrapsus as w e l l . In media more concentrated than normal winter sea water, at 5 ° C, both species maintained blood h y p e r t o n l c i t y and continued to e x c r e t e , at 48 hours, u r i n e hypotonio t o the blood and the media.  As was the case i n summer-adapted animals, the w i n t e r  balance between a c t i v e processes i n the g i l l s and antennary  glands  appeared t o be maintained during short-term exposure of the animals to concentrated sea water.  The general u r i n e - t o - b l o o d  - 36 -  relationship established in average winter sea water conditions was retained, although absolute body f l u i d concentrations changed (Table 7).  Gradients between blood and media tended to  decrease with increasing external concentration above 75$ sea water (Dehnel, 1962) to a minimum In 175$ sea water for J3. nudus and 125$ for H. oregonensis.  Above 125$ sea water In the latter  species, they again Increased.  Urine-to-medium gradients on  the other hand tended to Increase with increasing external concentrations, from a minimum In 100$ sea water to a maximum i n 175$ sea water, for both species (Fig. 5).  Henal excretion of  magnesium and retention of sodium and potassium in dilute media has been demonstrated in Pachygrapsus (Prosser, Green and Chow, 1955; Gross, 1957$), and the extra-renal excretion of sOdlura In concentrated media has been suggested.  Until ion determinations  are available for local Hemigrapsus blood and Urine, the precise a c t i v i t i e s of the antennary glands cannot be established. EFFECT OF TEMPERATURE ON OSMOTIC RESPONSES OF SEASONALLY-ADAPTED ANIMALS: Broekema (1941) reported that Crangon crangon maintained in sea water of 29$o showed a gradual f a l l in blood concentration as experimental temperature was allowed to rise with the seasonal change from spring towards autumn (blood-medium gradient gradually Increased). A reversal of these changes occurred when the experimental temperature was allowed to f a l l between autumn and winter.  This species, in Dutch waters, winters offshore In water  -  37  -  of relatively high salinity and migrates shoreward into more bfaokish conditions in spring and early summer.  Survival at  low temperature was correlated with high salinity and high temperature Increased tolerance to low salinity.  Other species,  with a reverse migratory pattern, appeared to tolerate low s a l i n i t i e s better at low temperatures.  These included a spider  P-rab, Hyas araneus. a shrimp, Crangon allmanl and a prawn, Pandalus montagui.  A third group* represented by the orab  Rhlthrqpanopeus harr 1,8,1 and the amphipod Qammarus duebenl had tolerances similar to Hyas but did not migrate seasonally (Verwey, 1 9 5 7 ) .  The two species of Hemigrapsus combine toler-  ances similar to C . crangon. and non-migratory habits.  In gen-  eral, high temperatures increase and low temperatures decrease metabolic rates.  Dehnel ( i 9 6 0 ) suggested that low salinities  at high temperatures may impose a greater stress than high ones. This i s compatible with observed osmotio gradients maintained by these species between blood and media and urine in high and low s a l i n i t i e s . Winter-adapted animals of both species in dilute ( 1 2 $ ) sea water showed significantly greater 48-hour hypertonicity at 5 ° C than at 1 5 ° C (Fig. 6 ) .  With the rise in temperature,  the U/B ratio decreased because urine concentration decreased more than blood. A rise in experimental temperature from 1 5 ° G to 2 5 ° G oaused no further significant change in urine concentration.  Blood data from equivalent animals are not available  for comparison.  In 75$ sea water, at 5 ° C , winter animals of  -  38  -  both speoies showed similar 48-hour urine concentrations but H. nudus had a higher blood concentration, hence a smaller U/B ratio (Table 7 ) .  At 1 5 ° C, H . nudus urine remained unchanged*  blood concentration dropped, the U/B ratio rose, and regulation weakened. In H. oregonensis. however, blood did not change but urine concentration decreased giving a lower U/B ratloi  The  gradient between urine and medium dropped from 21$ to 2$ sea water.  Thus, in experimental conditions approximating winter  f i e l d temperature and salinity, H . oregonensis responded to a rise in temperature, increased permeability and metabolic rate, by a drop i n urine concentration, while maintaining blood at the level found at 5 ° C.  This is probably achieved by increased  reabsorption in the antennary glands.  A further rise in temp-  erature to 2 5 ° C caused no significant change in urine concentration, but a rise in the U/B ratio from O.77  to O.87,  accompanied by a decrease in blood concentration.  was  This species  regulates less strongly in 75$ sea water as temperature increases. *  n  E» nudus. the rise in temperature from 1 5 ° to 2 5 ° C resulted  in a significant decrease in urine, but not blood, concentration, hence a lower U/B  ratio  (Pig. 6 ) .  The 48-hour U/B ratios for  the two speoies in 75$ sea water were identical at 2 5 ° C but &• nudus had urine and blood values about 7$ sea water higher than H. oregonensis. indicating stronger regulation. Urine and blood concentrations were alike for summeradapted H. oregonensis in 12$ sea water, at 1 5 ° C , and the gradient between these fluids and the medium was $$% sea water.  - 3 9-  Cooling the animals to 5 ° C reduced this gradient hy 10$ sea water for urine and 2$ for blood.  Blood osmotic concen-  tration was regulated nearly as strongly as at 1 5 ° C, with a slight but not significant indication that the antennary glands were involved.  Urine and blood concentrations were similar at  2 5 ° C but the gradient between them and the medium Increased, Indicating that summer adaptation favours stronger regulation at high temperatures and low s a l i n i t i e s and emphasizes, the resemblance of the temperature and salinity tolerances of this species to thosa of G. crangon.  .  Summer-adapted H. nudus at 5° C in 25$ and 12$ sea water, showed significant, and in 6 $ sea water, slight, hypotoniclty of urine to blood (Table 7 ) , suggesting here also that the antennary glands are taking part In the elimination of excess water and reabeorptlon of needed ions. At 1 5 ° C and 2 5 ° G, in dilute media, urine concentrations were not significantly d i f ferent from those at 5 ° C, but blood-td-urlne gradients were slightly redueed, suggesting that cooling of summer-adapted animals In low salinity conditions reduced their capacity for salt absorption and stimulated greater reabsorptlon in the antennary glands to compensate, The effects of cooling or warming summer-adapted animals and of warming winter-adapted  animals were pronounced only i n  low experimental s a l i n i t i e s . In high s a l i n i t i e s , similar changes In temperature caused no significant change in urine concentration i n either species.  It Is probable that high  - 40 -  s a l i n i t i e s pose less of an osmotic problem than low s a l i n i t i e s (see gradients, F i g . 5)» and that temperature changes consequently do not a l t e r the balanoe between absorptive and reabsorpt l v e a c t i v i t i e s as much i n high as i n low s a l i n i t i e s . I n comparing summer and winter mean urine concentrations i n the range of experimental s a l i n i t i e s and at 5 ° , 1 5 ° and 25° C, (Table 4) i t should be noted that each of the lower  temperatures  i s foreign to one of the seasonally-adapted groups, and that 25° C i s f o r e i g n to both.  Therefore, differences between the  r e s u l t s given i n Tables 3 and 4 are attributable to temperature e f f e c t s on the seasonal groups, , EFFECT OF TEMPERATURE ON RATES OF URINE AMD BLOOD CHANGE IN HIGH SALINITIES: Local Hemigrapsus. a f t e r e q u i l i b r a t i o n i n 75% sea water, showed r i s e s i n urine and blood concentration when placed i n sea water of 100$ or higher concentration.  Rates of change were  related to the season and experimental s a l i n i t y and temperature. These animals do not regulate i n the usual sense (Dehnel, 1962) at 48 hours, but give some evidence of r e s i s t i n g for a time upward changes i n blood concentration.  The antennary  glands  appear to be implicated In t h i s resistance, and t h e i r a c t i v i t y , with that of absorptive tissues, i s modified by changes i n experimental  temperature.  Hemigrapsus oregonensis: Winter urine did not become hypertonic to concentrated  .. 41 -  media, so that only blood changes are considered.  A rise in  experimental temperature from 5 ° to 1 5 ° or 25° 0 shortened the time taken for blood to become isotonic to 175$ sea water. Presumably, salt absorption was enhanced i n 100-150$ sea water as well, but this effect was concealed in the relatively long Intervals between samples. In summer-adapted animals at 1 5 ° C, blood reached isotonic ity with 100$ sea water more slowly than In winter-adapted animals at any temperature used.  The higher adaptation tem-  perature appeared to favour stronger regulation. In 125$ sea water, urine changed more rapidly than blood, and the antennary glands appeared to function In retarding blood change. s a l i n i t i e s , however, no such retardation was evident. the animals to 5 °  G  In higher Cooling  reduced absolute urine and blood concen-  trations at 3 hours, suggesting that the lower temperature retarded a l l active processes involved in regulation.  Warming the  animals from 1 5 ° to 25° C raised 3-hour blood values by 8-22$ in 125-175$ sea water, while urine values increased less.  These  increases suggest that temperature elevation affects salt absorption more than salt excretion. Hemigrapsus nudus: Winter-adapted animals at 5° 9 i n high salinities did not produce blood-hypertonic urine. Blood changes i n 100-175$ sea water were not obviously accelerated by Increased experimental temperature.  - 42 -  In summer-adapted animals at 1 5 ° C, blood concentration exceeded 100-175$ sea water by 3 hours. Blood-hypertonic urine, suggesting an attempt at regulation, was produced for 24 hours in 100$ sea water, but apparently not in higher s a l inities.  Cooling the animals 1 5 ° to 5 ° C tended to retard salt  absorption, so that 3-hour blood concentration was lower at the lower temperature.  Salt excretion was less affected, and urine  was hypertonic to blood for 48 hours in 100$ sea water and for over 3 hours in 125-175$ sea water.  In a l l media, however, both  urine and blood passed isotonicity around 3 hours and attempts at regulation were weak. Warming the animals from 1 5 ° to 25° C tended to raise urine concentration.  Although blood was not  kept hypotonic to 100$ sea water for as long as 3 hours, i t was less hypertonic at the higher temperature at each sampling time. In 125$ sea water, blood was hypotonic for over 24 hours with the production of markedly hypertonic urine.  This suggests  that the higher temperature enhanced regulation.  In 150$ sea  water both urine and blood changes were more rapid at 25° C and no regulation was evident. It appears that, as in Pachygrapsus. wherever a degree of regulation i s found * i t commences very soon after exposure of the animals to the experimental conditions. E F F E C T S OF S I Z E ON OSMOTIC  RESPONSE:  L i t t l e work has been done on the effect of body size on osmotio behavior In invertebrates.  Broekema (1941) compared  bloodeconcentrations of "young" and "old" C. crangon and found  -  43  -  no significant; difference between them i n media of 1 9 . 3$o and 25.3$o at 20° C and in 34.7#o at 4 ° - 5 ° C.  Dehnel ( 1 9 6 2 ) found  no significant differences In blood concentration between small and large JJ. nudus and H. oregonensis either from f i e l d conditions or from a range of experimental conditions similar to that used in the present investigation. Urine concentration i n summer-adapted R> nudus taken from f i e l d conditions was significantly higher in small than in large animals (Table 6).  A similar, but insignificant trend was  shown in H. oregonensis. This may be an expression of proportionally higher rates of ion absorption In small animals, which allow more Ions to pass out i n the urine while adequate blood hypertonicity i s maintained.  Equilibration i n 7 5 * sea water at  1 5 ° G reduced osmotic stress, and In fi. nudus at least, caused different responses in large and small animals when they were placed in low salinity media.  In these conditions, at 1 5 ° and  2 5 ° C, the urine of large animals was slightly hyper-tonic to small animals.  In experimental salinities up to 7 5 * sea water  at 25° C, H. oregonensis showed no significant size effect* concentrated media, at 1 5 ° C, however, small H.  In  oregonensis  tended to have urine significantly hypertonic to that of large animals. at 2 5 ° C.  A similar but insignificant relationship was shown It seems that neither size group regulates success-  fully against high external salinities, but small animals, by producing urine closer to blood concentration show a greater potential for regulation than large animals.  This i s true i f  - 44 -  a criterion of hypo-osmotic regulation i s the production of blood-hypertonic urine.  The response of H. nudus to high  external salinity at 2 5 ° C differed from that of H, oregonensis. The apparent size effect i s the same In both speoies. Hemigrapsus nudus produced blood-hypertonlc urine in concentrated media to a degree not shown by U/B ratios, and not shown by H. oregonensis. Hypertonlcity of urine to blood i s not sufficient to make this s p e c i e a successful hypo-osmotic regulator, but i t suggests the presence of a greater potential than that possessed by H. oregonensis. RELATIONSHIP  BETWEEN  U R I N E AND BLOOD CONCENTRATION:  Total osmotic U/B ratios in H. oregonensis and H. nudus changed seasonally in animals from the same experimental conditions (Table 7 ) .  Summer-adapted animals, which in the f i e l d  regulated strongly against low salinities, produced blood-isosmotic urine.  In low, intermediate and high experimental s a l i n -  ities at 5 ° , 1 5 ° and 25° C, U/B ratios approached unity in both species.  Exceptions occurred in H. nudus in 12$ sea water at  5 ° C, and in H. oregonensis in 75$ and 125$ sea water at 1 5 ° C, where urine was signlfloantly hypotonic to blood (Table 7 ) . In the f i r s t ease, active ion absorption from the medium may be slowed down by the low temperature, necessitating a greater recovery of ions from the urine and oausing a reduced total osmotic U/B ratio.  The same order of difference between blood  and urine occurred in H. nudus in 6 $ and 25$ sea water as well, at this temperature.  In the second case, H. oregonensl.s in  concentrated sea water had blood hypertonic to the media.  The  - 4-5 -  production of blood-hypbtonic urine must have l i t t l e value In regulation at these s a l i n i t i e s , and the s t a t i s t i c a l significance attributed to i t i s partly the result of low variance in the salinity determinations on blood and urine. The disappearance of significant differences between urine and blood at 25° G, suggests that they are more apparent than real at 1 5 ° C . In experimental conditions, winter U/B ratios for both species depart considerably from unity in high and low salinities (Table 7) In 75$ sea water, where osmotic conditions approach the seasonal norm, differences between urine and blood for H . oregonensls at 5° C and H . nudus at 15° C were not significant.  Significant  differences between urine and blood in low salinities were associated with strong hyper-osmotic regulation, and in high salinities with a possible persistence of a seasonally acclimated balance between ion absorption i n the g i l l s and reabsorption in the antennary glands. Interspecific comparison of U/B ratios showed s t a t i s t i c a l l y significant temperature effects in winter-adapted but not summer-adapted animals*  At 5  D  C for the salinity range 6$ to  175$ sea water, and for combined 3 , 24 and 48-hour data* winteradapted ]i# oregonensis showed a signifleant tendency to have U/B ratios nearer unity than H i nudus (Table 8 ) i At 1 5 °  ratios  tended to be nearer unity in H i nudus. but were not signifleant.  At 25° C , ratios for H i nudus again tended to be nearer unity, showing significance at the 0.01 level; 1  At 5 ° C , the tendency  for U/B ratios to be signifioahtly higher in H . oregonensis was  - 46 -  based on a large proportion of oases in which the urine of this speoles was hypertonic to urine of comparable H. nudus. At the same time, no trend was evident In blood concentration differences.  At 2 5 C, the tendenoy for H. nudus to have higher 0  U/B ratios was based on the fact that H. nudus urine and H» oregonensis blood were more frequently higher in concentration than the same fluids in the other species.  The inter-  specific difference in U/B ratios for winter-adapted animals at 5 ° C suggests that reabsorptlve processes in the antennary glands are more active in H. nudus than H. oregonensis at that temperature.  Increasing temperature has a different effect on the  two species, so that at 2 5 G 0  t  H. oregonensis shows on the  average larger urine-minus-blood gradients than H. nudus.  This,  coupled with higher blood concentrations suggests that active absorptive sites are more temperature-sensitive in H. Qregonensla. In summer-adapted animals, total osmotic U/B ratios differed less between species than i n winter-adapted animals (Table 8 ) . At 5  0  G, the tendency for ratios in H. oregonensis to be higher  than in H. nudus was based on the fact that blood in H. nudus and urine in H. oregonensis were more frequently higher in concentration than Conversely. A rise In experimental temperature to 1 5 ° C increased the number of Instances, in these comparisons, In which urine of H. nudus was hypertonic to that of H. oregonensis.  Total osmotic U/B ratios In the latter were slightly higher  than in H. nudus.  Gradients between urine and blood, probably  correlated with more active ion reabsorptlon, were greater In  - 47 -  H. nudus. At 25° G, the tendency was reversed and insignificant, and on the average, blood and urine differences between the two species were slight.  - 48 -  SUMMARY. 1.  Two species of shore crabs, Hemigrapsus oregonensis and  H. nudus, from the Vancouver area, were equilibrated In 75$ sea water for 3 6 to 4 8 hours and exposed to a range of experimental salinities from 6 $ to 175$ sea water at 5 ° , 1 5 ° and 25° C.  Urine samples were drawn after 3 , 2 4 and 4 8 hours, and  their osmotio concentration ($ sea water) was measured by the method of melting point determination. Duplicate series of experiments were carried out, using animals adapted to summer and winter f i e l d conditions.  A l l summer animals were weighed  during the experiments, and representative groups were sampled at the time of collection. 2.  Urine concentration was found to f a l l in dilute, and rise  in concentrated media, at rates directly related to the gradients between media and equilibrated urine concentrations, and Influenced by the seasonal adaptation of the animals and the experimental temperature.  The rate of concentration change in  a given medium was not continuous between time zero and 4 8 hours, but in most instances became slower after 2 4 hours.  New equil-  ibria were generally established by 4 8 hours. 3.  Hyper-osmotlo regulation In summer-adapted animals was  achieved with the production of blood-isotonic urine, Implicating extra-renal mechanisms.  In winter-adapted animals, the  production of blood-hypotonlo urine indloated the participation of the antennary glands in hyper-osmotic regulation.  - 4-9 -  4. Evidence i s presented which suggests that in summer animals, adapted to f i e l d conditions of low salinity and high temperature, the antennary glands function to some degree i n retarding increases in blood concentration in media of 100$ to 150$ sea water.  In general, cooling retarded, and warming  stimulated salt absorption and regulation. The period of resistance to blood change, where demonstrated, xvas longest in 100$ or 125$ sea water and shorter in higher s a l i n i t i e s . Hemigrapsus nudus appeared to have a greater potential for hypoosmotic regulation than H. oregonensis. 5.  The antennary glands were not shown to funotion in resist-  ing upward changes in blood concentration in winter animals, adapted to f i e l d conditions of high salinity and low temperature. 6. Changes in experimental temperature revealed  interspecific  and seasonal differences in 48-hour urine concentration. Summer-adapted H. oregonensis in dilute (12$) sea water showed significantly higher urine concentration at 25° C than at 5° C, but H. nudus showed no temperature effects In low, intermediate or high s a l i n i t i e s . showed significant  Winter-adapted animals of both species decreases in urine concentration in low and  intermediate, but not high s a l i n i t i e s , when the experimental temperature was increased. 7. In both speoies, summer and winter adaptation tended to favour stronger hyper-osmotic regulation at the respective seasonal temperatures than at temperatures foreign to the seasons.  -  8.  50 -  Body size was shown to have, in some circumstances, a  significant effect on urine osmotic concentration.  Small  H. nudus. taken from summer f i e l d conditions, had significantly higher urine concentration than large animals, while H. oregonensis did not show similar tendency.  In concentrated media,  small H. oregonensis at their seasonal temperature had urine significantly hypertonic to that of large animals. 9.  In winter-adapted animals, H. oregonensis had total osmotio  U/B ratios significantly higher (nearer unity) than H. nudus for the whole range of experimental salinities at their seasonal temperature.  Increasing the experimental temperature caused a  rise In the ratios in H. nudus so that at 2 5 ° C, they were significantly higher than those in H. oregonensis. In summer-adapted animals, H. Oregonensis had higher ratios at the seasonal and lower temperature but at 2 5 ° C, unity.  H. nudus again had ratios nearer  In summer animals however, these tendencies were not  statistically significant. 10.  Seasonal adaptation of osmoregulatory mechanisms i n  Hemigrapsus i s shown to alter the balance of active processes so that for a given range of experimental conditions, urine i s lower i n winter animals than in summer animals both In absolute concentration and relative to the blood.  -  51  -  LITERATURE CITED Beadle, L. C., 1 9 5 7 .  Comparative physiology: osmotio and  ionic regulation i n aquatic animals* 19:  Ann. Rev. P h y s i o l . ,  329-358.  Broekema, M., K. M., 1 9 4 1 .  Seasonal movements and the osmotic  behavior of the shrimp Crangon crangon ( L . ) . Arch. Neerl. Zool., 6 : 1 - 1 0 0 . Broekhuysen, G. J . ,  1 9 3 6 . On development, growth and d i s t r i b -  ution of Careinus maenas ( L * ) . Arch. Neerl. Zool*, 2 : 257-399.  Bullock, T. H., 1 9 5 5 .  Compensation f o r temperature i n the  metabolism and a c t i v i t y of polkilotherms. 30:  311-342.  Dehnel, P. A., i 9 6 0 .  Effect of temperature and s a l i n i t y on the  oxygen consumption 118:  B i o l * Rev.,  of two i n t e r t i d a l orabs.  Biol. Bull.,  215-249.  Dehnel, P. A., 1 9 6 2 . Aspects of osmoregulation i n two species of i n t e r t i d a l crabs. Gross, ¥. J . ,  1952.  invertebrates.  B i o l . B u l l , * ( i n press).  Studies of response to stress i n selected Ph.D. Dissertation, University of C a l i f -  ornia at'Los Angeles. Gross, W . J . ,  1 9 5 4 . Osmotic responses i n the slpunoulid,  Dendrostoimuro. zpsterlculum. Gross, W . J . ,  1955.  J .  Exp. B i o l . , 3 1 :  402-423.  Aspects of osmotio regulation In crabs  showing the t e r r e s t r i a l habit.  Amer. Nat., 8 9 : 2 0 5 - 2 2 2 .  - 52  Gross, W. J . , 1 9 5 7 a .  -  An analysis of response to osmotic stress  :r In selected decapod Crustacea. Gross, W. J . , 1 9 5 7 b .  Biol. Bull., 112:  43-62.  A behavioral mechanism for osmotic reg-  u l a t i o n i n a s e m i - t e r r e s t r i a l crab.  113:  Biol. Bull.,  268-274.  Gross, W. j . , I 9 5 8 .  Potassium and sodium regulation i n an  i n t e r t i d a l crab. Gross, W. J . , 1 9 5 9 .  B i o l . B u l l . , 114: 3 3 4 - 3 4 7 .  The e f f e c t of osmotic stress on the i o n i c ,  exchange of a shore crab.  B i o l . Bull., 116:  Gross, W. J . , and P. V. Holland., i 9 6 0 .  Water and ionic reg-  u l a t i o n i n a t e r r e s t r i a l hermit crab. 33:  P h y s i o l . Zool.,  21-28.  Hukuda, K . ,  1932.  media.  Change of weight of marine animals i n d i l u t e  J . Exp. B i o l . , 9 : 6 1 - 6 8 .  Jones, L. L., 1 9 4 1 .  Osmotic regulation i n several crabs of the  P a c i f i c Coast of North America. 18:  248-257.  J . C e l l . Comp. Physiol.,  79-92.  Krogh, A., 1 9 3 9 .  Osmotic regulation i n aquatic animals.  Cambridge at the University Press. Kaluf, N. S. H., 81:  1941.  Urine formation In c r a y f i s h .  Biol. Bull.,  134-148.  Nagel, H., 1 9 3 4 .  Osmoregulation i n Crustacea.  Physiol., 2 1 : Panlkkar, N. K . ,  Parry, G., 1 9 5 4 .  468-491.  1941.  Chlrooeohalus.  Zeitschr. Vergl.  Osmotic behavior of f a i r y J . Exp. B i o l . , 1 8 :  shrimp,  110-114.  Ionic regulation i n the palaemonid prawn  Palaemon (=Leander) serratus. J . Exp. B i o l . , 3 1 :  601-613.  -  Peters, H., 1935*  53 -  Uber den E i n f l u s s des Salzgehaltes lm  Aussenmedlum auf den Bau und die Funktion der : Exkretlonsorgane dekapoder Crustaeeen.  (Nach Untersuohungen an  Potamoblus f l u v i a t i l u a und Homarus v u l g a r i s ) .  2eitschr.  f . Morph. u. Okol. d. T i e r e . , 3 0 : 355~381. Ploken, L . E . R., 1 9 3 6 . The mechanism of urine formation i n invertebrates. Arthropods.  I The excretion mechanism i n c e r t a i n  J . Exp. B i o l . , 1 3 : 3 0 9 - 3 2 8 .  Prosser, C. L . , and F. A. Brown J r . , 1 9 6 1 . Comparative Animal Physiology.  Philadelphia, Saunders.  Prosser, C. L., C. L . Green, and T. J . Chow, 1 9 5 5 . Ionic and osmotio concentration In blood and urine of Paohygrapsus orasslpes acclimated to different s a l i n i t i e s . 109:  99-107.  Riegel, J . A., 1 9 5 9 . Some aspects of osmoregulation species of sphaercmid isopod Crustacea. 116:  Biol. Bull.,  In two  Biol. Bull.,  272-284.  Riegel, J . A,, and L . B. Kirsohner, i 9 6 0 .  The exoretion of  i n u l i n and glucose by the c r a y f i s h antennal gland.  Biol.  B u l l . , 118: 2 9 6 - 3 0 7 . Robertson, J . D., 1 9 4 9 . ebrates.  Ionic regulation i n some marine i n v e r t -  J . Exp. B i o l . , 2 6 : 182-200.  Robertson, J . D., 1 9 5 7 . Osmotic and ionic regulation i n aquatic invertebrates.  Recent Advances i n Invertebrate Physiology.  University of Oregon Publications. Scheer, B. T., 1 9 4 8 .  Comparative Physiology.  Hew York, Wiley.  - 54 -  Schwabe, E . , 1 9 3 3 . Uber d i e Osmoregulation versohledener Krebse (malacostracen). Verwey, J . ,  Z e i t s o h r . v e r g l . P h y s i o l . , 1 9 : 183-236.  1 9 5 7 . A p l e a f o r the study of temperature  on osmotic r e g u l a t i o n . Webb, D, A., Roy.  Ann.  influence  B i o l . , 3 3 : 129-149.  1 9 4 0 . I o n i c r e g u l a t i o n i n Oaroinus maenas.  Soc.  London, S e r i e s B.,  Proc.  129: 107-136.  Wikgren, Bo-Jungar, 1 9 5 3 . Osmotic r e g u l a t i o n i n some a q u a t i c animals, with perature.  s p e c i a l reference  Acta-  Z o o l . Fennica,  to the i n f l u e n c e of tem7 1 : 1-102.  

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