li«P^:;- ^^ "^^ CONTRACT REPORT M-77 1 STUDIES ON THE TIME COURSE OF SALINITY AND TEMPERATURE ADAPTATION IN THE COMMERCIAL BROWN SHRIMP PENAEUS AZTECUS IVES A. Venkataramian, vc?, ^, LaK-.P'tu^ ratncia Dies' John D, Valleau, Gordon Gunfcer Gulf Coast Rssearch Laboratory Ocean Springs, Miss. 39564 September 1977 L-:- .1 n . m!(MSi i «^M i r ■silUUtlimMiiiiu rr^;;ri-ed tri Office, Chief <~'^ Prifiinpp,- Washington. M.S nAr\y/ Mohuoi-jd ijy Hydraulics Laborato, U. S, Army Engineer Waterways Experimen. P. O. Sox 631, Vicbbura, Miss. 3918- / y Destroy this report when no longer needed. Do not return it to the originator. -■• -•> -rt- Unclassified SECURITY CLASSIFICATION OF THIS PAGE (When Data Enlarad) REPORT DOCUMENTATION PAGE READ INSTRUCTIONS BEFORE COMF>t,ETING FORM 1. REPORT NUMBER Contract Report H-77-1 2. GOVT ACCESSION NO 3. RECIPIENT'S (fATALOG NUMBER 4. TITLE Cand Subl/tle; STUDIES ON THE TIME COURSE OF SALINITY AND TEMPERATURE ADAPTATION IN THE COMMERCIAL BROWN SHRIMP PENAEUS AZTECUS IVES 5. TYPE OF REPORT & PERIOD COVERED Final report 6. PERFORMING ORG. REPORT NUMBER 7. AUTHORr*; A. Venkataramiah G. J. Lakshmi Patricia Biesiot John D. Valleau Gordon Gunter 8. CONTRACT OR GRANT NUMBERfsJ DACW 39-73-C-0115 9. PERFORMING ORGANIZATION NAME AND ADDRESS Gulf Coast Research Laboratory Ocean Springs, Mississippi 39564 10. PROGRAM ELEMENT. PROJECT. TASK AREA a WORK UNIT NUMBERS 11. CONTROLLING OFFICE NAME AND ADDRESS Office, Chief of Engineers, U. S. Army Washington, D. C. 20314 12. REPORT DATE September 1977 13. NUMBER OF PAGES 370 : °~ : m ■ r^ i _□ I '^ I <=! I ° I a : m i CD : CD U. MONITORING AGENCY NAME a ADORESSCf/ dl/Zerent from Controlling Olllce) U. S. Army Engineer Waterways Experiment Station Hydraulics Laboratory P. 0. Box 631, Vicksburg, Mississippi 39180 tS. SECURITY CLASS, (ol thia report) Unclassified 15«. DECLASSIFI CATION/ DOWN GRADING SCHEDULE 16. DISTRIBUTION STATEMENT (ol thie Report) .Approved for public release; distribution unlimited. '7. DISTRIBUTION STATEMENT (ol the abstract entered In Block 20, II dlllereni from Report) , 18. SUPPLEMENTARY NOTES 19. KEY WORDS (Contln\ie on reverse aide it neceaaary and Identity by block number) Aquatic ecosystem Crustacea Environmental effects Salinity effects Shrimps Temperature effects 20. ABSTRACT (Caatiaue aix reverma alda It naceaaary mtd Identity by block number) The time course of salinity and temperature adaptation in brown shrimp Penaeus aztecus was determined by analyses of certain behavioral and physiolog- ical responses, the respiratory rates and osmotic and ionic regulation. The animals were transferred separately from a background salinity (S) lS°/c,o (control) and temperatures of 18°, 25° and 32°C to different test conditions for salinity adaptation. The test salinities were 2, 5, 10, 15, 25 and 36°/oo (Continuedl DD , JAN 73 '*'•> EDITION OF t HOV 65 IS OBSOLETE Unclassified SECURITY CLASSIFICATION OF THIS PAGE (When Date Entered) Unclassified SECURITY CLASSIFICATION OF THIS PAGEfHTian £>•(• EnloredJ 20. ABSTRACT (Continued). and temperatures were 18°, 25° and 32°C. The duration of the tests was for one week (168 hours). The behavior of the experimental shrimp was influenced at 25°C, which was their normal habitat temperature, primarily by salinity changes. At other temperatures the behavior was affected by the interaction of salinity and temperature. The blood osmotic and chloride ion regulation was hyperos- motic below and hyposmotic above the control salinity level. The rate of sa- linity adaptation was determined on the basis of steady-state levels in oxygen uptake and in blood osmotic or chloride concentration levels. At 25°C there was a positive interaction in the various phases of adaptation between the res- piratory rates on the one hand and the osmotic and chloride ion gradients on the other hand in the respective salinities. This interaction was not consis- tent at other test temperatures. On the basis of osmotic and chloride ion steady-state levels, salinity adaptation was faster at 25°C than at either 18° or 32°C; also salinity adaptation occurred in a wider range of 2 to 36°/oc>S within a week. The salinity range of adaptation decreased from 5 to 25°/oo at 18°C and from 10 to 25°/ooS at 32°C. Within those salinity ranges the rate of mortality was usually low. Next to 25°C the salinity adaptation and survival rates were more favorable in 18°C than in 32°C. However, at 18° and 32°C the steady-state levels in the respiratory rates and in the blood osmotic (or chloride ion) concentrations appeared at different periods after the transfer was made. Consequently there was no synchrony between these responses at 18° or 32°C as opposed to 25°C test conditions. The respiratory rates at 18°C reached steady-state levels faster than the osmotic or chloride ion concentra- tions. On the contrary, at 32°C the steady-state levels appeared in the blood salt levels faster than in the respiratory rates. These discrepancies might have occurred partly due to the temperature-related differences in the be- havioral pattern of shrimp. Normally the shrimp exhibited prolonged hyper- activity at 32°C and inactivity at 18°C which naturally influenced the respiratory rates both quantitatively and in relation to the time scale. In this report the implications of deriving conclusions on the state of salinity or temperature adaptation by taking individual physiological responses (i.e. respiratory rates) as an exclusive criterion are discussed. In brown shrimp, salinity and temperature requirements are shown to be size-dependent. The optima for subadult shrimp (95 mm mean length) seem to exist above 10°/ooS, preferably between 15 and 25°/ooS, and below 25°C. In contrast the juveniles (70 mm mean length) of our previous studies have shown preference to salini- ties lower than 17°/oo and to temperatures slightly higher than 26°C. The possible existence of seasonal salinity and temperature optimal rhythms is discussed in relation to the life cycle of brown shrimp. Magnesium, calcium and potassium levels of the blood increased with salinity increases. Changes in test temperatures (18° or 32°C) affected the normal regulation pattern of these ions exhibited at 25°C. The physiological or behavioral responses were not significantly affected when minor changes occurred in these ionic ratios. Major changes, however, produced some physical abnormalities and high death rates. The effects became greater at temperatures higher than 18°C. Below 35 percent of the normal calcium levels the shrimp started dying; death rates increased with decreased calcium and increased temperature. Complete removal of magnesium from the test salinity was relatively less harmful than the re- duced calcium levels. Reduced potassium killed even fewer shrimp, but pro- duced a high incidence of abdominal (tail) cramps in shrimp. Low blood potassium levels and low temperature combinations seemed to cause the cramping in shrimp. Unclassified SECURITY CLASSIFICATION OF THIS PAGECHTion Data Enlerod) PREFACE This report was prepared under Contract No. DACW 39-73-C-0115 with the Gulf Coast Research Laboratory, Ocean Springs, Mississippi, for the U. S. Army Engineer Waterways Experiment Station (WES). The study was an extension of a previous investigation undertaken on the recommenda- tion of the OCE Estuarine Ecological Consultants Board in an Interim Report entitled "Effects of Engineering Activities on Coastal Ecology," September 1969. The investigation was carried out under the general direction of Dr. Gordon Gunter, Director Emeritus of the Gulf Coast Research Labora- tory. Dr. A. Venkataramiah was the Principal Investigator, and he was assisted by Dr. G. J. Lakshmi, Patricia Biesiot, and John D. Valleau. The contract was monitored by Mr. F. A. Herrmann, Jr., Assistant Chief of the Hydraulics Laboratory, under the general supervision of Mr. H. B. Simmons, Chief of the Hydraulics Laboratory. Directors of WES during the conduct of this study and the prep- aration and publication of this report were BG E. D. Peixotto, CE, COL G. H. Hilt, CE, and COL John L. Cannon, CE. Technical Director was Mr. F. R. Brown. TABLE OF CONTENTS Page PREFACE 1 LIST OF TABLES 7 LIST OF FIGURES 8 I: INTRODUCTION 21 II: MATERIALS AND METHODS 33 Experimental Design 33 Experimental Animals 33 Laboratory Holding Procedure 34 Temperature Acclimation Procedure 35 Preparation of Salinity Media 35 Preparation of Deviated Ion Media 37 Behavior 38 Behavior in Media of Deviated Ion Ratios 38 Blood Sampling 40 Blood Analyses 41 Osmotic concentration 41 Blood chloride 41 Other electrolytes 41 Determination of Oxygen Consumption 42 Flow-through respirometry 42 Oxygen consumption in media with deviated ions . . 46 Statistics 46 III: RESULTS 48 Effect of Salinity and Temperature on Behavior and Survival 48 Effect of salinity change 49 Effect of temperature background 52 Effect of salinity and temperature change 53 Blood Osmoregulation During the Time Course of Adaptation 56 Effect of 25°C acclimation on osmoregulation ... 66 Effect of 32°C acclimation on osmoregulation ... 67 Effect of 18°C acclimation on osmoregulation ... 68 Page Time Course of Blood Ion Regulation 69 Effect of 25°C acclimation on chloride regulation 69 Effect of 32°C acclimation on chloride regulation . . 73 Effect of 18°C acclimation on chloride regulation . . 77 Time Course of Regulation of Other Ions 77 Calcium ion regulation 81 Magnesium ion regulation 88 Potassium ion regulation 99 Effect of Salinity Change on Osmotic and Ionic Concentration 106 Osmoconcentration 113 Chloride concentration 120 Calcium concentration 129 Magnesium concentration 139 Potassium concentration 145 Osmotic and Ionic Regulation in Relation to the Isosmotic Line 152 Osmoregulation 152 Chloride regulation 154 Calcium regulation 154 Magnesium regulation 157 Potassium regulation 159 Interaction of Salinity and Temperature on Osmotic and Ionic Regulation 159 Osmoregulation 159 Blood chloride ion 165 Calcium ion 169 Magnesium ion 169 Potassium ion 174 Effect of Sex on Osmotic and Ionic Regulation 178 Oxygen Consumption in Time Course of Adaptation 186 Effect of 25°C acclimation on oxygen consumption 186 Page Effect of IS^C acclimation on oxygen consumption 200 Effect of 32°C acclimation on oxygen consumption 213 Effect of Salinity Change on Respiration 226 Effect of 25°C acclimation 226 Effect of 32°C acclimation 232 Effect of 18°C acclimation 234 Interaction of Salinity and Temperature on Respiration 236 Effect of Temperature Background on Adaptation .... 241 Effect of Acclimation and Test Temperature 251 Effect of Sex on Oxygen Consumption 255 Behavior and Survival in Salinities with Deviated Amounts of Cations 255 Behavior in control salinity 257 Effect of deviated sodium 257 Effect of reduced potassium 259 Effect of reduced calcium 260 Effect of reduced magnesium 260 Effect of Deviated Cation Concentrations in Low Salinities on the Behavior and Survival 261 Effect of deviated sodium 261 Effect of reduced potassium 263 Effect of reduced calcium 263 Effect of reduced magnesium 263 Effect of Acclimation to Media with Deviated Ions on Tolerance in Extreme Salinity and Temperature .... 264 Oxygen Consumption in Deviated Ion Media 265 Oxygen consumption in synthetic seawater 265 Oxygen consumption in reduced calcium 267 Effect of total elimination of magnesium 270 Effect of reduced potassium on oxygen consumption 272 Metabolic rates in relation to temperature .... 272 Metabolic rates in relation to ionic changes . . . 275 IV: DISCUSSION 277 Time Course of Salinity Adaptation Temperature Influence on Osmotic and Chloride Regulation Temperature Influence on the Steady-State Levels Salinity and Temperature Requirements in Relation to Size Osmoregulation and Energy Relations Metabolic Compensation to Temperature Change . . Regulation of Other Cations V: SUMMARY VI: EPILOGUE VII: ACKNOWLEDGMENTS VIII: LITERATURE CITED APPENDIX A: Definition of Terms Acclimation Brackish water Estuaries Euryhaline Hyperosmotic Hyposmotic Ionic regulation Isosmotic Metabolic rates Nongenetic adaptations Osmoregulators Osmotic concentration Salinity Serum Standard metabolism Weight specific metabolic rate APPENDIX B: Tables I-IX. Mean blood osmotic concentration jl^ S. E X-XVIII. Mean blood chloride concentration + S. E Page 277 282 283 284 287 290 291 297 300 302 Al Al Al Al Al Al A2 A2 A2 A2 A2 A3 A3 A3 A3 A4 A4 Bl B2 Bll Page XIX-XXVII. Mean blood potassium concentration +_S.E B20 XXVIII-XXXVI. Mean blood calcium concentration ji^S.E B29 XXXVII-XLV. Mean blood magnesium concentration jt^S.E B38 XLVI-LIV. Mean oxygen consumption ^ S. E B47 LV-LVII. Mean oxygen consumption +_ S. E. in media having variation in cation concentration B56 LIST OF TABLES Table No. Page 1 Effect of salinity and temperature change on the mortality rate of Penaeus aztecus 51 2 Sex effect on blood osmoregulation in Penaeus aztecus 181 3 Sex effect on blood chloride regulation in Penaeus aztecus 182 4 Sex effect on blood calcium ion regulation in Penaeus aztecus 183 5 Sex effect on blood magnesium ion regulation in Penaeus aztecus 184 6 Sex effect on blood potassium ion regulation in Penaeus aztecus 185 7 Level of significance of oxygen consumption rates in Penaeus aztecus between sampling intervals . . . 190 8 Level of significance of oxygen consumption rates in Penaeus aztecus between sampling intervals . . . 193 9 Level of significance of oxygen consumption rates in Penaeus aztecus between sampling intervals . . . 196 10 Leve^ of significance of oxygen consumption in Penaeus aztecus between sampling intervals . . . 204 11 Level of significance of oxygen consumption rates in Penaeus aztecus between sampling rates . . 207 12 Level of significance of oxygen consumption rates in Penaeus aztecus between sampling intervals 210 13 Level of significance of oxygen consumption rates in Penaeus aztecus between sampling intervals 217 14 Level of significance of oxygen consumption rates in Penaeus aztecus between sampling intervals 220 15 Level of significance of oxygen consumption rates in Penaeus aztecus between sampling intervals 223 16 Effect of salinity change on the respiratory rates of Penaeus aztecus in the process of adaptation to salinity and temperature 231 17 Effect of salinity change on the respiratory rates of Penaeus aztecus in the process of adaptation to salinity and temperature 233 18 Effect of salinity change on the respiratory rates of Penaeus aztecus in the process of adaptation to salinity and temperature 235 7 Table No. Page 19 Effect of temperature change on the respiratory rates of Penaeus aztecus in the process of adaptation to salinity and temperature 240 20 Effect of temperature change on the respiratory rates of Penaeus aztecus in the process of adaptation to salinity and temperature 242 21 Effect of temperature change on the respiratory rates of Penaeus aztecus in the process of adaptation to salinity and temperature 243 22 Effect of temperature background on the respiratory rates of Penaeus aztecus tested at normal temperature (25°C) conditions 247 23 Effect of temperature background on the respiratory rates of Penaeus aztecus tested at 32°C conditions 249 24 Effect of temperature background on the respiratory rates of Penaeus aztecus tested at 18°C conditions . . . 250 25 Sex effect on the oxygen consumption of Penaeus aztecus in the process of salinity and temperature adaptation 256 26 Effect of deviated ions on the mortality rates of Penaeus aztecus 258 27 Effect of deviated ions on the mortality rates of Penaeus aztecus 262 28 Effects of acclimation of Penaeus aztecus to salinities of 5 and 10°/oo with deviated ionic ratios and testing in normal but extreme salinites 2.5 and 42.5°/oo 266 Figure No. LIST OF FIGURES 1 Temperature acclimation tanks with temperature control setup 36 2 Flow-through respirometry apparatus 43 3 Effect of salinity and temperature change on the behavioral responses in Penaeus aztecus 50 4 Changes in the blood osmotic levels of Penaeus aztecus in the process of salinity adaptation at 25°C. the control conditions were 15°/ooS and 25°C 57 Figure No. Page 5 Changes in the blood osmotic levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The control conditions were 15°/ooS and 25°C 58 6 Changes in the blood osmotic levels of Penaeus aztecus in the process of salinity adaptation at 18 C. The control conditions were 15°/ooS and 25°C 59 7 Changes in the blood osmotic levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The sample size was 44 in the control condi- tions 15°/ooS and 32°C 60 8 Changes in the blood osmotic levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 32°C 61 9 Changes in the blood osmotic levels of Penaeus aztecus in the process of salinity adaptation at 18 C. The control conditions were 15°/ooS and 32°C 62 10 Changes in the blood osmotic levels of Penaeus aztecus in the process of salinity adaptation at 18°C. The control conditions were lS°/ooS and 18°C 63 11 Changes in the blood osmotic levels of Penaeus aztecus in the process of salinity adaptation at 25 C. The control conditions were 15°/ooS and 18°C 64 12 Changes in the blood osmotic levels of Penaeus aztecus in the process of salinity adaptation at 32 C. The control conditions were 15°/ooS and 18°C 65 13 Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 25°C 70 14 Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 32 C. The control conditions were 15°/ooS and 25°C 71 15 Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 18 C. The control conditions were 15°/ooS and 25°C 72 Figure No. Page 16 Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The control conditions were 15°/ooS and 32°C 74 17 Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 32°C 75 18 Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 18°C. the control conditions were 15°/oc5S and 32°C 76 19 Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 18°C. the control conditions were 15°/ooS and 18°C 78 20 Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 25 C. The control conditions were 15°/ooS and 18°C , . 79 21 Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The control conditions were 15°/ooS and 18°C 80 22 Changes in the blood calcium levels of Penaeus aztecus in the process of salinity adaptation at 25 C. The control conditions were 15°/ooS and 25°C 82 23 Changes in the blood calcium levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The control conditions were 15°/ooS and 25°C 83 24 Changes in the blood calcium levels of Penaeus aztecus in the process of salinity adaptation at 18°C. The control conditions were 15°/ooS and 25°C 84 25 Changes in the blood calcium levels of Penaeus aztecus in the process of salinity adaptation at 18°C. the control conditions were 15°/ooS and 18°C 85 26 Changes in the blood calcium levels of Penaeus aztecus in the process of salinity adaptation at 25 C. The control conditions were 15°/ooS and 18°C 86 10 Figure No. Page 27 Changes in the blood calcium levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The control conditions were 15°/ooS and 18°C 87 28 Changes in the blood calcium levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The control conditions were 15°/ooS and 32°C 89 29 Changes in the blood calcium levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 32°C 90 30 Changes in the blood calcium levels of Penaeus aztecus in the process of salinity adaptation at 18°C. The control conditions were 15°/ooS and 32°C 91 31 Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 25°C 92 32 Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The control conditions were 15°/ooS and 25°C 93 33 Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 18°C. The control conditions were lS°/ooS and 25°C 94 34 Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The control conditions were 15°/ooS and 32°C 96 35 Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 32°C 97 36 Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 18°C. The control conditions were 15°/ooS and 32°C 98 37 Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 18°C. The control conditions were 15°/ooS and 18°C 100 11 Figure No. Page 38 Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 18°C 101 39 Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 32°C. The control conditions were 15°/ooS and 18°C 102 40 Changes in the blood potassium levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 25°C 103 41 Changes in the blood potassium levels of Penaeus aztecus in the process of salinity adaptation at 32°C. the control conditions were 15°/ooS and 25°C 104 42 Changes in the blood potassium levels of Penaeus aztecus in the process of salinity adaptation at 18°C. The control conditions were 15°/ooS and 25°C 105 43 Changes in the blood potassium levels of Penaeus aztecus in the process of salinity adaptation at 32°C. the control conditions were 15°/ooS and 32°C 107 44 Changes in the blood potassium levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 32°C 108 45 Changes in the blood potassium levels of Penaeus aztecus in the process of salinity adaptation at 18°C. The control conditions were 15°/ooS and 32°C 109 46 Changes in the blood potassium levels of Penaeus aztecus in the process of salinity adaptation at 18°C. the control conditions were 15°/ooS and 18°C 110 47 Changes in the blood potassium levels of Penaeus aztecus in the process of salinity adaptation at 2S°C. The control conditions were 15°/ooS and 18°C Ill 48 Changes in the blood potassium levels of Penaeus aztecus in the process of salinity adaptation at 32°C. ^The control conditions were 15°/ooS and 18°C 112 12 Figure No. Page 49 Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 25°C 114 50 Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 25°C 115 51 Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 18 C. The control conditions were 15°/ooS and 25°C 116 52 Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 32 C. The control conditions were 15°/ooS and 32°C 117 53 Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 25 C. The control conditions were 15°/ooS and 32°C , . 118 54 Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 32°C 119 55 Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 18 C. The control conditions were 15°/ooS and 18°C 121 56 Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 18°C 122 57 Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 32 C. The control conditions were 15°/ooS and 18°C 123 58 Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 25°C 125 59 Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/°oS and 25°C 126 13 Figure No. Page 60 Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 25°C 127 61 Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 32°C 128 62 Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 32°C 130 63 Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 32°C 131 64 Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 18°C 132 65 Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 18°C 133 66 Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/°oS and 18°C 134 67 Comparison of blood calcium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B) , and 32°C (C) . Control conditions were 15°/ooS and 25°C .... 135 68 Comparison of blood calcium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B) , and 32°C (C) . Control conditions were 15°/ooS and 32°C .... 137 69 Comparison of blood calcium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B) , and 32°C (C) . Control conditions were 15°/ooS and 18°C .... 138 70 Comparison of blood magnesium ion levels of Penaeus aztecus during adaptation to various salinities at 25 C. The control conditions were 15°/ooS and 25°C. . 14*^ 14 Figure No. Page 71 Comparison of blood magnesium ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 25°C. . 141 72 Comparison of blood magnesium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 25°C. . 142 73 Comparison of blood magnesium ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 32°C. . 143 74 Comparison of blood magnesium ion levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 32°C. . 144 75 Comparison of blood magnesium levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/°°S and 32°C. . 146 76 Comparison of blood magnesium levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B) , and 32°C (C) . The control conditions were 15°/ooS and 18°C . . 147 77 Comparison of the blood potassium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B), and 32°C (C) . The control conditions were 157°oS and 25°C 148 78 Comparison of the blood potassium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure) , 25°C (B) , and 32°C (C) . The control conditions were 15°/ooS and 32°C 150 79 Comparison of the blood potassium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B) , and 32°C (C) . The control conditions were 15°/ooS and 18°C 151 80 Effect of salinity and temperature change on the blood osmoregulation of Penaeus aztecus in relation to the isosmotic line 153 81 Effect of salinity and temperature change on the blood chloride ion regulation of Penaeus aztecus in relation to the isosmotic line 155 82 Effect of salinity and temperature change on the blood calcium ion regulation of Penaeus aztecus in relation to the isosmotic line 156 15 Figure No. Page 83 Effect of salinity and temperature change on the blood magnesium ion regulation of Penaeus aztecus in relation to isosmotic line. Animals were tested from control conditions of 15°/ooS and 18°, 25°, and 32°C 158 84 Effect of salinity and temperature change on the blood potassium ion regulation of Penaeus aztecus in relation to the isosmotic line. Animals were tested from control conditions of 15°/ooS and 18°, 25°, and 32°C 160 85 Comparison of the time course of osmoregulatory res- ponses in Penaeus aztecus in relation to the tem- perature change. Animals were tested from control conditions of 15°/ooS and 25°C 161 86 Comparison of the time course of osmoregulatory responses in Penaeus aztecus in relation to the temperature change. Animals were tested from control conditions of 15°/ooS and 32°C 162 87 Comparison of the time course of osmoregulatory responses in Penaeus aztecus in relation to the temperature change. Animals were tested from control conditions of 15°/ooS and 18°C 164 88 Comparison of the time course of chloride ion regu- lation in Penaeus aztecus in relation to tempera- ture change. Animals were tested from control conditions of 15°/ooS and 25°C 166 89 Comparison of the time course of chloride ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 32°C 167 90 Comparison of the time course of chloride ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 18°C , . 168 91 Comparison of the time course of calcium ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 25°C 170 92 Comparison of the time course of calcium ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 32°C 171 16 Figure No. Page 95 Comparison of the time course of calcium ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 18°C 172 94 Comparison of the time course of magnesium ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 25°C 173 95 Comparison of the time course of magnesium ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 32°C 175 96 Comparison of the time course of magnesium ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 18°C 176 97 Comparison of the time course of potassium ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 25°C 177 98 Comparison of the time course of potassium ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 32°C 179 99 Comparison of the time course of potassium ion regulation in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 18°C 180 100 Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 25°C. The control conditions were 15°/ooS and 25°C .... 187 101 Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 32°C. The control conditions were 15°/ooS and 25°C .... 188 102 Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 18°C. The control conditions were 15°/ooS and 25°C .... 189 103 Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 18°C The control conditions were 15°/ooS a^d 18°C .... 201 104 Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 25°C. The control conditions were 15°/ooS and 18°C .... 202 17 Figure No. Page 105 Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 32°C. The control conditions were 15°/ooS and 18°C 203 106 Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 32°C. The control conditions were 15°/ooS and 32°C 214 107 Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 18°C. The control conditions were 15°/ooS and 32°C 215 108 Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 25°C. The control conditions were 15°/ooS and 32°C 216 109 Comparison of the oxygen consumption rates of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure) , 25°C (B), and 32°C (C) . Animals were tested from control conditions of 15°/ooS and 25°C 227 110 Comparison of the oxygen consumption rates of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B), and 32°C (C) . Animals were tested from control conditions of 15°/ooS and 32°C 228 111 Comparison of the oxygen consumption rates of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B), and 32°C (C) . Animals were tested from control conditions of 15°/ooS and 18°C 229 112 Comparison of the time course of oxygen consumption responses in Penaeus aztecus in relation to temperature change. The control conditions were 15°/ooS and 25°C 237 113 Comparison of the time course of oxygen consumption responses in Penaeus aztecus in relation to temperature change. The control conditions were 15°/ooS and 32°C 238 114 Comparison of the time course of oxygen consumption responses in Penaeus aztecus in relation to temperature change. The control conditions were 15°/ooS and 18°C 239 18 Figure No. Page 115 Effect of control temperature on the time course of oxygen consumption rates in Penaeus aztecus during salinity adaptation at 25°C 244 116 Effect of control temperature on the time course of oxygen consumption rates in Penaeus aztecus during salinity adaptation at 32°C . . . . . 245 117 Effect of control temperature on the time ' course of oxygen consumption rates in Penaeus aztecus during salinity adaptation at 18°C 246 118 Comparison of the oxygen consumption rates of Penaeus aztecus during the first 10 hours of adaptation and the new steady-state levels. From the control conditions 15°/ooS and 18°C, the shrimp were transferred for salinity adaptation at 18°C (section A of the figure], 25°C (B), and 32°C (C) 252 119 Comparison of the oxygen consumption rates of Penaeus aztecus during the first 10 hours of adaptation and the new steady-state levels. From the control conditions 15°/ooS and 25°C, the shrimp were transferred for salinity adaptation at 18°C (section A of the figure), 25°C (B), and 32°C (C) 253 120 Comparison of the oxygen consumption rates of Penaeus aztecus during the first 10 hours of adaptation and the new steady-state levels. From the control conditions 15°/°oS and 2&^ 5^'^C the shrimp were transferred for salinity adaptation at 18°C (section A of the figure), 25°C (B), and 32°C (C) 254 121 Oxygen consumption rates of Penaeus aztecus in 15°/ooS synthetic seawater of normal ionic composition at 18°, 25°, and 32°C. The control conditions were 15°/ooS normal seawater and 25°C 268 122 Oxygen consumption rates of Penaeus aztecus in 15°/ooS with 25 percent calcium ion concentration 269 123 Oxygen consumption rates of Penaeus aztecus in 15°/ooS with 0 percent magnesium 271 124 Oxygen consumption rates of Penaeus aztecus in 15°/ooS with 30 percent potassium concentration 273 19 Figure No. Page 125 Effect of temperature change on the oxygen consumption rates of Penaeus aztecus in deviated ion media 274 126 Comparison of the effects of deviated ionic ratios on oxygen consumption rates of Penaeus aztecus at 18°, 25°, and 32°C 276 20 STUDIES ON THE TIME COURSE OF SALINITY AND TEMPERATURE ADAPTATION IN THE COMMERCIAL BROWN SHRIMP PENAEUS AZTECUS IVES I: INTRODUCTION This report is concerned primarily with the time course of acclimation to salinity and temperature changes by brown shrimp Penaeus aztecus Ives. Some observations on the behavioral re- sponses of the shrimp in salinity media with deviated (modified) ion concentrations are also reported. These experiments are a contin- uation of studies originally begun in 1970 to determine salinity and temperature relations in brown shrimp. The U.S. Army Corps of Engi- neers sponsored the studies with the objective of understanding the impact of engineering works on the ecology of coastal waters. During the last century estuaries have become vulnerable to in- tensive human activities such as the enormous expansion of industries, the installation of power plants, increased intensity of fishing and recreational activities, and pollution. The Army Corps of Engineers has been involved with flood control, intracoastal waterways and other aids to navigation, construction of dams for diverting and storing water, building levees and spillways, and filling and dredg- ing operations. These operations naturally affect the normal pro- cesses of the estuaries and alter the physical attributes like sa- linity and temperature. Gunter (1967) described one instance in Louisiana estuaries where some of these operations eliminated the gradual transition from fresh water to salt water. Consequently, the distribution pattern of local flora and fauna including large animals and birds had undergone great changes. In addition to affecting natural salinity and temperature re- gimes, dredging and filling alter the characteristics of the 21 interface between the water and bottom where high biological activity is concentrated. Bottom living animals and grassbeds are removed in one area and are covered over in a different place. The heat gener- ated from power plants is dispersed by mixing with the main body of water in several estuaries; but this practice has produced some un- expected vertical stratification problems involving salinity and tem- perature as described by Cronin (1967). Estuaries are essential for the completion of the life histories of a vast majority of shallow-water marine animals including those of most commercial fisheries on the Gulf coast. Commenting upon the im- portance of estuaries, Pearse and Gunter (1957) stated that "the young of many animals usually thought of as marine, require areas of low salinity for nursery grounds." They added further that "the dis- tribution and abundance of the blue crab and of the commercial shrimp of South Atlantic and Gulf coasts are dependent on the presence of estuarine areas. The early stages of penaeid shrimp apparently re- quire oceanic water, but the older larvae must reach bay waters or perish." In addition to the migratory species, estuaries also host their own indigenous populations of copepods, planktonic forms, several species of mollusks, fishes, and palaemonid shrimp of the grass beds. In view of the biological and commercial importance of estuaries, a team of consultants for the Corps of Engineers, Drs. Eugene Cronin, Gordon Gunter, and Sewell Hopkins, analyzed the problem and made rec- ommendations to the Corps to sponsor some research program on the salinity problems of some coastal water species. Among the more important recommendations were: 1. To determine the distribution, abundance, rates of growth, and total production of some animal and plant species in 22 relation to quantitative and qualitative changes in salinity in their natural environment. To conduct laboratory studies on the survival or mortality of "most important species" in a range wide enough to in- clude lethal extremes. Rates of respiration in different salinities and other physiological evidence of stress should certainly be studied, as should rates of growth and repro- duction in various nonlethal salinities. Adaptability to changing salinity should be included in laboratory experi- mentation (Ref. Effects of Engineering Activities on Coastal Ecology, U.S. Army Corps of Engineers, August 1971), During the first phase of the studies carried out at this labora- tory, the survival, growth, ionic regulation, and metabolic rates of brown shrimp were determined in relation to salinity and temperature changes. The present project is a continuation of phase one. Brown shrimp were selected as the experimental species because of their euryhalinity, their abundance in the commercial fisheries, and their biological importance in the food chain of the estuarine community. Their wide distributional range enhances their value as a key species. They extend southward along the Atlantic coast from North Carolina to Florida and westward to the Yucatan Peninsula in the Gulf of Mexico, with the exception of the southwest Florida estuaries. They are now the most abundant commercial shrimp. Selection of this species for study has definite advantages over others with a more localized dis- tribution. It should be noted, however, that the data obtained for brown shrimp may not be applicable directly to the white and pink shrimp except in a broad sense. This is because of the apparent differences in the salinity and temperature requirements within the three species. Gunter et al. (1964) have shown the presence of a positive correlation between the salinity concentration and the abundance of three species in the Gulf of Mexico. The authors found that the greatest amount of white shrimp are produced in the low saline waters of Louisiana, the greatest amount of brown shrimp in the saltier bays of Texas and the 23 greatest amount of pink shrimp around south Florida where the salinities are almost oceanic. In nature most postlarval P_. aztecus are exposed to lower and more fluctuating estuarine temperatures than those encountered by white shrimp. Aldrich et al . (1968) ob- served in the laboratory that the temperature optima of the two species differed. The variations in the environmental requirements of the three species indicate that they should be studied separately. Despite their commercial importance brown shrimp, or for that matter any one of the penaeid species, failed to attract the atten- tion of physiological ecologists until recently. Widespread studies on the general biology of the North American white shrimp were initi- ated in 1930 by Weymouth, Lindner and Anderson (1933) . At that time, 95% of the total shrimp catch consisted of white shrimp. The main emphasis of those studies was to learn about the life history (spawning, embryology, larval history, growth and longevity) , the migratory pattern, the effect of fishing, and the abundance of the species in relation to salinity and temperature conditions. Experi- ments with tagging techniques revealed data relating to the growth and seasonal migration. Among other investigators who originally supplied information on the biology of American penaeid shrimp, work of the following should be noted: Spaulding (1908), Viosca (1920), Burkenroad (1934, 1939), Pearson (1939), Anderson et al. (1949) and Gunter (1950). Among early workers Burkenroad (1934, 1939) gave the most ex- tensive data on brown shrimp. His information related partly on the salinity preference of this species as being different from the white shrimp. He showed that brown shrimp are less abundant in less sa- line and more abundant in more saline coastal waters. Gunter (1950) confirmed this point and added specific data. Burkenroad also pointed out that the sexually mature adult brown shrimp in Louisiana waters are found beyond the inner littoral zone. Gunter (1967) ranked white shrimp, brown shrimp, and pink shrimp in order of their 24 preference to low-salinity water. The distribution of P. aztecus shows that it is a warmwater species. The northern limit of penaeid shrimp is approximately Cape Hatteras, North Carolina, near the north- ern limit of the Carolinian (subtropical) zone of biogeography. These animals are quickly killed by cold waves (Gunter and Hilde- brand 1954) while the hardier crustacean cohort, the blue crab Callinectes sapidus, with a northern distribution to Canada, has not been recorded as a casualty. Gunter (1950) had observed cor- relation between the seasonal variations in the abundance of penaeid shrimp (and other invertebrates) and the annual temperature cycle but not with salinity changes or any other phenomenon. Therefore, he concluded that temperature is apparently a much more important factor than salinity in the general cyclic movements of marine animals in the South Atlantic and the Gulf of Mexico. In a later publication Gunter (1967) stated that although salinity is a limit- ing factor in the geographic distribution of penaeid shrimp, it is certainly not the sole limiting factor. From controlled experiments we have observed that it is indeed not a single environmental factor which apparently governs the dis- tribution of brown shrimp but a combination of factors. Two of these, namely salinity and temperature, counteract, support, and modify the physiological effects of each other. During these studies three different size groups of brown shrimp (13-20 mm, 21- 45 mm, and 50-75 mm length ranges) were tested in eleven salinities in a 0.34 to 59.5°/ooS range, at temperatures of 21°, 26° and 31°C (Venkataramiah et al. 1974). Over this range none of the experi- mental shrimp died in salinities from 8.5 to 34.0°/oo and conse- quently this range was designated as the normal salinity tolerance range. The normal salinity tolerance range decreased progressively as the test temperatures were altered from 26°C to 21° and 31°C. Background temperature is an important factor in the survival of shrimp when both salinity and temperature changes occur. Brown 25 shrimp acclimated at 31°C withstood such sudden changes better than those acclimated at 21°C. In growth studies conducted simultaneously, several groups of postlarvae were acclimated separately in 8.5, 17.0, 25.5, and 34.0°/ooS in combination with 21°, 26°, and 31°C tempera- tures for three weeks. Later they were raised for six weeks in their acclimated conditions. In each condition the animals received daily feeding rations at 4.5, 6.2, 8.1, and 11% of their biomass. Food consumption was highest at 31°C, but the best conversion ratios were obtained at 26°C. At 21°C the survival, food conversion efficiency and growth rates were lowest of all temperatures. Growth and survival rates were influenced by the interaction of salinity and temperature. By virtue of their euryhalinity, young brown shrimp were found to grow in salinities ranging from 8.5 to 34.0°/oo, but growth rates were best in the near-optimal conditions of 8.5 and 17.0°/ooS and 26°C. Also in these concentrations the animals survived temperature changes better than in higher salinities. Similarly, salinity vari- ations were better tolerated in normal temperature conditions (26°C) that in either 21°C or in 31°C. In estuaries frequent salinity changes from low to high and vice versa can occur at almost any time but the major temperature changes are seasonal. For this reason, apparently, the compensatory means to tolerate adverse salinity conditions are better developed in brown shrimp than are those for temperature (Venkataramiah et al. 1974, pp. 62-63) . In the absence of an effective mechanism for temperature regulation, the shrimp seem to have a limited capacity to tolerate temperature changes. This was shown by the high incidence of muscle paralysis, convulsions, necrosis, and loss of diurnal rhythmicity in animals exposed suddenly to low temperatures (Venkataramiah et al . 1974, pp. 31-36). Recently we observed the occurrence of abdominal cramps in brown shrimp that were confined to 18°C (unpublished results) In adjusting to the ill effects of extreme salinity and tempera- ture changes, penaeid shrimp usually show a number of behavioral and 26 physiological reactions. Sometimes the behavior may help to improve the effectiveness of the physiological regulation. One of the im- portant examples of their behavior is the seasonal southward migra- tion of penaeid shrimp from Georgia to Florida (Burkenroad 1949; Lindner and Anderson 1956; McCoy and Brown 1967) and from Texas to Mexico (Gunter 1962) during the cold season and their return in the spring. These coastal movements are only extensions of seasonal movements out of the bays and shallow offshore waters in the fall and return in the spring which Gunter showed in a series of studies in Louisiana and Texas for the major motile species of organisms in the northern Gulf. Presumably, similar seasonal movements take place in all bays and shallows of the temperate zone. In the laboratory, brown shrimp try to escape from low tempera- ture (Aldrich et al. 1968) or from extreme salinity and temperature conditions (Venkataramiah et al. 1974) by burying in the substratum. In addition to burying behavior when faced with stress shrimp also run and swim vigorously or jump out of the tanks. Along with the escape responses, the shrimp would seem to resort to simultaneous physiological regulation. Ionic and osmotic regulation are two important aspects of the physiological regulation. Ionic regulation in aquatic animals occurs essentially between the external medium and the blood and between the blood and tissues. In the process of ion regulation brown shrimp showed significant differences between the ionic composition of the blood and that of the external medium (Venkataramiah et al. 1974). Sodium and chloride comprised the major blood ions. In a test salin- ity range of 8.5 to 34.0°/oo these ions were maintained well above the concentration levels of the external media in the low-salinity range (8.5 and 17.0°/ooS). In the high salinity range (25.5 and 34.0°/ooS) the ions were at a lower level. On the other hand, the magnesium concentration was much below the level of external sa- linities throughout, while calcium was well above the level. Shrimp 27 acclimated at 21°C regulated relatively better in 17.0°/ooS than in 8.5°/ooS. At 26°C and 31°C the regulation was comparatively more efficient throughout the salinity range. Osmoregulation is a capacity found in all estuarine animals. On the basis of chloride ion concentration, which normally comprises about 50% of the total blood osmoconcentration, the euryhaline brown shrimp exhibited hyperosmotic regulation in low salinities (8.5 and 17.0°/oo), hyposmotic regulation in high salinities (25.5 and 34.0°/oo) and isosmotic with 17°/ooS or slightly above. Osmoregulation is sub- jected to temperature influence similar to ion regulation. In our previous studies on salinity tolerance brown shrimp were acclimated to 8.5, 17.0, 25.5, and 34.0°/ooS. The organisms were found to take advantage of the acclimation by expanding their salinity tolerance range into the media adjacent to that of the acclimation. For instance, acclimation to 8.5°/ooS helped them to penetrate to a lower salinity of 3.4°/oo without any mortality. Normally in a direct transfer from 17°/ooS about 50% of the shrimp died in 3.4°/ooS. Shrimp acclimated to 34.0°/ooS had similar advantages of penetrating a higher concentration of 42.5''/ooS without any apparent ill effects. In the same fashion the shrimp acclimated to 21°C survived in greater numbers and in a wider salinity range when tested at 21°C than in 31 °C. The animals acclimated to 31 °C suffered the same disadvantage at 21°C; but the effect was lesser than that experienced by those acclimated at 21°C and tested at 31°C. The animals acclimated at 31°C survived better at 26°C than at 31°C (Venkataramiah et al. 1974). Therefore, acclimation seems to be a physiological phenomenon that permits the organisms to compensate for alternatives in the environmental variables. Such compensations promote an increased capacity to survive in an unfavorable environment. Regulation is presumably a faster process than acclimation, resulting from routine 28 activities of specific, pre-existing regulatory organs (Kinne 1971). On the other hand, acclimation results in actual changes in the re- sponse mechanisms. It requires time to develop and involves all levels of organismic functions and structures. Thus regulation and acclimation are not alike. The capacity for osmoregulation in the estuarine invertebrates depends on the time course of the salinity or temperature changes. A number of euryhaline invertebrates have been shown to exhibit higher capacities upon gradual changes (step- wise transfer or slow variation) from normal to extreme conditions. The time course of nongenetic capacity adaptation to salinity or temperature seems to occur in three phases: immediate response, stabilization, and a new steady state (Kinne 1971) . Immediate responses begin seconds or minutes after the transfer of the animals into the test conditions. Frequently the responses involve over- or under-shoots in activity, metabolic rates, or in other performances. Changes may also occur in the behavior and other physical conditions. The process of stabilization may require hours, days, or weeks. Dehnel (1962) found that in the crab Hemi- grapsus oregonensis the stabilization of blood osmoconcentration is a function of salinity and temperature. At 15°C stabilization to a 6-100% seawater range was completed within 24 hours but not to a 125-150% seawater range. When the temperature was lowered to 5°C stabilization occurred within 24 hours to a salinity concentration range of 6-150% seawater. In the same species (H. oregonensis), Gross (1963a, b) noticed a measurable acclimation to 51°/ooS in more than five days and a strong acclimation in 22 days. In Carcinus maenas transferred from 25.9°/oo to H.8°/ooS the blood concentra- tion became diluted within 26 hours and then remained constant (Duval 1925) . However, comprehensive information on the new steady state of performance is lacking except for a few species. The crab H. ore- gonensis exhibited greater capacity for osmoregulation in high sa- linity after acclimation for more than 20 days to about 51°/ooS than 29 the individuals previously kept in about 34°/ooS. Similar responses were found in Penaeus aztecus acclimated to both salinity and temper- ature variables by changing these factors gradually from control (Ven- kataramiah et al. 1974). We have no knowledge of the rates at which young brown shrimp can acclimate to salinity and temperature changes; but this information is important for the U.S. Army Corps of Engineers in maximizing their planning and permitting activities. On the basis of our previous studies, recommendations have been made to the Corps of Engineers to the effect that their floodwater discharges should not suddenly lower the salinities and temperatures in coastal waters at the time of postlarval migration (Ref. Venkataramiah et al. 1974, pp. 124-125). In view of the ecological implications it was decided to study the capacity for acclimation in brown shrimp P. aztecus under conditions of sudden changes in salinity and temperature in- stead of gradual changes. Besides the salinity and temperature variation the estuarine invertebrates may also face a situation where the ion composition of the medium and the relative proportion of other solutes may under- go significant modifications. Lethal salinity effects due to changes in relative proportions of solutes seldom occur in pure marine hab- itats. However, in fresh and brackish waters, particularly in 5 and 8°/ooS ranges, such variations may reach critical levels. For this reason this salinity range has been considered by some workers as a significant ecophysiological boundary line, characterized by the presence of a minimum number of species (Kinne 1971) . Lobza (1945) observed pronounced ion ratio changes to occur in salinities below 4°/oo. Sudden changes in important hydrochemical characteris- tics have been reported by Kirsch (1956) in the water samples ob- tained near the Bute and Knight inlets (British Columbia) between 4°/oo and 7°/ooS. The ion ratios in North Caspian Sea salinities 30 change in relation to the flow magnitude of River Ural; below 7°/ooS ion rations change, possibly by the removal of certain ions from the solution (Vinetskaya 1959) . Usually there is a higher ratio of carbonate and sulfate ions to chloride and of calcium ions to sodium in estuarine waters than in seawater. Extreme evaporation may lower these ratios (Clarke 1924) . Ionic changes may also occur depending upon local soil type of the river, the vegetation and fauna, and industrial or other prod- ucts discharged into the waters. Changes in ionic composition of the ambient medium have been shown to modify salinity and temperature tolerance. High potassium content reduced the salinity tolerance in the fresh water amphipod Dikerogammarus haemobaphus (Birshtein and Beliaev 1946). On the contrary, addition of potassium improved tolerance to higher sa- linities in mysids Mesomysis kowalevskyi (Karpevich 1958) . The estuarine turbellarian Gunda ulvae suffered extensive water uptake and salt loss in fresh water and in brackish water of low salt con- tent unless both media had a supranormal calcium content (Pantin 1931a, b; Weil and Pantin 1931) . In the mollusk Mytilus edulis addition of calcium and magnesium increased thermostability while addition of potassium decreased it (Schlieper and Kawalski 1956) . Addition of potassium and calcium increased cold resistance in the oligochaete Enchytraeus albidus while addition of calcium and mag- nesium decreased heat tolerance (Kahler 1970). Although our laboratory findings show that young brown shrimp can tolerate a salinity range of 8.5°/oo to 34.0°/oo and that by acclimation the range can be expanded from 3.4°/oo to 42.5°/oo, in nature they are mainly confined to less saline waters in which the ion ratios are likely to deviate from normal as shown above. In Louisiana waters Wengert (1972) found that brown shrimp of 11 to 100 mm long occur most abundantly in salinities between 0.99°/oo and 3.00°/oo. Postlarval brown shrimp (9 to 20 mm) are found in 31 the Mississippi Sound in salinities between 2°/oo and 25°/oo; but they are most common in salinities below 15°/oo, with the greatest abundance in 2°/oo to 5°/ooS (Perry et al. 1974). However, it has also been observed that within this low concentration range brown shrimp are unevenly distributed. Food availability is suggested as one of the reasons for the unevenness but it does not seem to be the exclusive limiting factor. Therefore, it is necessary to deter- mine if the deviation in ion ratios of the coastal waters has any significant effect on the physiological responses and pattern of natural distribution. The effects of ion ratio changes were also studied on the behavior and survival of Penaeus aztecus. 32 II: MATERIALS AND METHODS Experimental Design The time course of adaptation in brown shrimp, Penaeus aztecus, was studied by analyses of physiological responses to salinity and temperature variations. Respiratory rates and blood osmotic and ionic regulation were used as physiological parameters. Brown shrimp were acclimated for one week to either 25°C (laboratory temperature), IS^C or to 32°C in a control salinity of 15°/oo. The animals were then directly transferred to salinities 2, 5, 10, 15 (control), 25, and 36°/oo for adaptation in combination with 18°, 25°, and 32°C. The test salinities and temperatures approximate the ranges of spring and summer estuarine conditions. Respiratory rates were measured at in- tervals of 0, 1, 2, 4, 6, 10, 24, 48, 72, 96, 120, 144, and 168 hours. Blood sampling was conducted at the same intervals except for addi- tion of a 16-hour sample and ommission of the 120- and 144-hour samples. It was presumed that in the process of adaptation the major physiolog- ical changes would take place within one week (168 hours). Males and females were tested separately to determine the effect of sex on the physiological responses. Experimental Animals Juvenile brown shrimp ranging from 85 to 100 mm total length were obtained from local bayous or bays and from bait shrimp dealers. The animals were collected from waters at a salinity range of 10 to 20°/oo. Although the shrimp occurred in a 14° to 30°C range, most were taken from 23° to 29°C. The animals were transported from the collection area to the laboratory in 20-gallon (75.7 L) styrofoam ice chests and provided with continuous oxygen. The use of pure oxygen during transport seemed to help the animals have a fast recovery from trawling stress as well as from the overcrowding effect in the bait tanks. 33 Laboratory Holding Procedure In the laboratory the shrimp were stocked in four raceway tanks. Each tank was 10 feet (3.05 m) long, 4 feet (1.22 m) wide, and 2 feet (0.61 m) high and held about 450 gallons (1703 L) of water. Under ambient temperature (25°C) conditions, a maximum of 1000 shrimp were stocked in each raceway tank. Stocking densities were changed with holding temperature conditions. At high temperature (32°C), the den- sity was lowered to 600 shrimp per tank, while at 18°C the density could be increased to more than 1000 animals. Often there was an initial high mortality during the first 48 hours, primarily due to physical stress either in trawling or in transportation. The initial mortality ranged from 10 to 25% of the total stock per day. By the third day the rate decreased to 5% under normal conditions and then to 1% on succeeding days. The phenomenon of mass molting which occurred sometimes had resulted in heavy mor- talities. In a single night as many as 15 to 20% of the normal ani- mals would molt and die. Although some of these deaths occurred from natural stress of temperature and salinity changes, a great majority of the shrimp were vulnerable to attacks by the other shrimp at this time and were killed. In general there was a low incidence of mass molting at 18°C and a high incidence at 32°C. The shrimp were allowed to adjust to laboratory conditions at 15°/ooS and 25°C for about 48 hours. They were then transferred to the respective control temperatures 25°, 18°, or 32°C for acclimation. The temperature was changed gradually from ambient to the control within 24 hours. After the shrimp were kept for seven days in the control or acclimation conditions, they were tested. Animals in the laboratory conditions or under acclimation re- ceived dry food pellets at a rate of approximately 5% of their body 34 weight per day. Occasionally fresh shrimp meat was added. Feeding was suspended 24 hours before testing and during the test period. Temperature Acclimation Procedure The temperature acclimation was carried on for the most part in a specially constructed temperature-controlled filter system (Fig. 1) in addition to thermostatically controlled Instant Ocean culture tanks (Aquarium Systems, Inc) . The system consisted of four 250-gallon (946 L) circular fiberglass tanks (#1-4 in the figure) connected in line with a 250-gallon rectangular plywood tank (#5). The shrimp were held in the circular tanks while the rectangular tank served for filtration and for temperature control. Heating or cooling units were placed in compartments A, B, or D as needed. Seawater was continuously pumped from compartment D to the animal tanks through a polyvinyl chloride (PVC) pipe (#15) fixed about six inches above the tanks. The water in excess of a fixed level in the animal tanks drained through a center standpipe (#6) . The drainage water flowed through a PVC pipe below the tanks (#16) and was airlifted into compartment A. The heavier particulate matter, especially sand, dropped out in compart- ments A and B. Fine particulate matter was filtered out in compart- ment C. The heated or cooled water was pumped into the animal tanks from compartment D. Temperatures were successfully maintained to within +].°C in this system. At 32°C about 150 shrimp were acclimated in each of the four tanks but the number was increased to 200 at 18°C. Preparation of Salinity Media Raceway tanks received filtered water directly from Davis Bayou adjacent to the Laboratory grounds. Salinities were adjusted to the Reference to trade names in this report does not imply endorsement of the products. 35 n Oi H Oi J z ►J o J w M < I H UJ n O) o 0. ^ t ) ffl M tf a. I£ M M S U u rr. F- H •^ ,1 M C ) U1 to H > n -> -> n fl Q H > fii 2 [1] U W ,1 H n « ct < rf. <: u > (y. m > X IX a Oi fii H M en u u V H ,1 H n H H H )— < t 1 a < < < a. 3 P-. < u U: 3 O •1. Z O (0 r2 :«; r> V, u < K F- Ul y. QC O :j _] H S o u a: u u o a z < e-< < m u o u u > a: u o z 3 §^ 0) t/l o u ^-> C o o 3 ■p rt t-i (U g^ ■p in o ■p u o cd o 3 P n) 0) H 0) 3 (30 •H 36 desired concentrations by adding synthetic sea salt [Instant Ocean, Aquarium Systems, Inc.) or by diluting with dechlorinated tap water. Salinity media were prepared with natural seawater of 30°/ooS. The acclimation salinity (15°/oo) was prepared by diluting the sea water with dechlorinated tap water but the test salinities 2, 5, 10, 15, and 25°/oo were made by diluting the seawater with deionized water (100,000 ohm/cm resistance). The other test salinity, 36°/oo, was prepared by concentrating seawater with synthetic sea salt (Instant Ocean, Aquarium Systems, Inc.). Salinity concentrations were measured with a T/S Refractometer (Model 10402, American Optical Corp.). The instrument was periodi- cally checked for accuracy by comparison with a portable Induction Salinometer (Model RS-7A, Industrial Instruments, Inc.) using stan- dard seawater as reference (obtained from lAPO Standard Seawater Service, Charlottenlund Slot, Charlottenlund, Denmark). Salinities were checked daily and adjusted at two-day intervals. Preparation of Deviated Ion Media For these studies, artificial sea salts were used which had been specially blended by excluding one major cation such as a Ca-free mix, a Mg-free mix, a K-free mix, and a 50% Na-free mix. Each of the four sea-salt mixes had only one cation missing, the other ions being pres- ent in the normal ratio. The salt of each eliminated cation was then added to the appropriate cation-free mix in specific proportions to make up salinity media in which the cation deviated from the ratio of the normal amount present in seawater. Osmotic concentration of each salinity medium was adjusted to that of normal seawater by the addition of TRISMA-8.3 [Tris (hydroxymethyl) aminomethane buffered to a pH of 8.3]. 37 Behavior The behavior and survival rates of brovm shrimp in each of the test conditions mentioned previously were monitored for one week. Ten-gallon aquaria with undergravel filters and sand substrates were used for the observations at 25° and 32 °C; the tanks were set in a water bath to maintain the high temperature. Thermostatic aquaria of 50-gallon capacity were used for testing at 18°C. They also had undergravel filtration and sand substrates. A partition was inserted in these tanks so that the shrimp were actually held in 25 gallons of seawater. Ten shrimp (average length 94.4 mm and average weight 6.4 g) were tested in each experimental condition. They had been exposed to the control (25°C, 15°/ooS) or to one of the acclimation conditions (18° or 32°C, 15°/oo) as previously described. The shrimp were directly transferred and were not fed for the duration of test- ing. Timers on the light switches were set to give 12 hours each of alternate light and darkness. Observations of behavior were contin- uous for the first few hours after transfer and were made every four hours, excluding the nighttime, after the second day. Mortalities were removed as they occurred. Those shrimp which molted in the test aquaria and were attacked and killed by other shrimp in the same tank were not counted as deaths due to the salinity and/or temperature change unless they had shown signs of distress before molting. Behavior in Media of Deviated Ion Ratios Behavior and survival of brown shrimp exposed to media of de- viated ionic ratios were studied in two series of tests. In the first series shrimp were acclimated to artificial seawater with the normal proportion of all cations and then tested in seawater with deviated ion ratios. In the second series the shrimp were acclimated to de- viated ion media and then tested in extreme salinity concentrations with normal ion ratios at extreme temperatures. For both series the 38 shrimp were acclimated to the respective media for one week at 25°C. The animals were fed daily during acclimation but were starved during the test period. Plastic boxes of 7 L capacity were used as test containers. Aeration was continuous but no substrate or filtration was provided. For the first series, brown shrimp were acclimated separately to salinities of 5, 10, and 15°/oo. The shrimp acclimated to 15°/ooS were tested in media of 15°/ooS changing the concentration (in per- centage) of one cation at a time. The test solutions were prepared so that different media contained the respective cations in the following concentrations: 85, 95, 120, and 150% Na; 5, 10, 15, 25, and 35-6 Ca; 0, 1, 4, and 6% Mg; and 10, 30, 40, 50, and 60% K. Testing was done at 25''C in salinity media of 10°/oo having concentrations of 85% Na; 15 and 25% Ca; 0 and 4% Mg; and 40, 50, and 60% K. Animals acclimated to 5°/ooS were tested in media with concentrations of 85% Na; 15% Ca; 0% Mg; and 60% K at a salinity of 5°/oo and temperature of 25°C. Ten shrimp were tested in each of the above conditions. Tliey were rinsed with 500 ml of distilled water before transfer to the test media. Behavior and survival rates were monitored for 24 hours. For the second series of tests, shrimp were acclimated sep- arately in media at 5 and 10°/ooS in which the percentage of one cation was changed: 120% Na; 15% Ca; 6% Mg; or 40% K. After acclimation the shrimp were directly tranferred to normally composed artificial seawater of 2.5 and 42.5°/ooS at temperatures of 18° and 32 °C. Acclimation of large numbers of brown shrimp to the deviated ionic media was difficult. Mortalities occurred during acclimation from 50% up to almost 100% in some cases. Very few shrimp survived 39 acclimation to the 10°/ooS with 40% K or 6% Mg concentrations; so testing of shrimp from those backgrounds could not be done. Too few shrimp survived in 5°/ooS with 40% K concentration to test at both 18° and 32°C; therefore, tests were omitted at 18°C. The tests on shrimp from the other deviated ion backgrounds were conducted using as many experimental animals as possible from among the sur- vivers of acclimation. Blood Sampling Blood samples were taken from the test shrimp at intervals described previously and analyzed for total osmotic concentration and for chloride, potassium, magnesium, and calcium. The shrimp were sacrificed during the sampling operation. Water samples were taken at the start of each test. Since blood sampling was done at close intervals it was always difficult to select animals of the same size range in each condition. The fact that shrimp were sampled by sex made it more impracticable. In order to overcome these problems 6 to 10 animals of the same sex were initially sorted out into perforated plastic boxes. The boxes were transferred into the test tanks (250 gallon capacity) and retrieved at set intervals. Before sampling each shrimp was wiped with a towel moistened in de- ionized water to remove external salt and then dried with tissue paper. Blood was taken directly from the heart and from the ventral sinus. Dispo Pipets (American Hospital Supplies) with sharply ta- pered ends (1 mm diameter) were used to collect the blood. Care was taken to prevent puncturing the hepatopancreas. Normal samples were light to dark blue in color but those with contamination were dis- colored. Such samples were discarded. The samples that were rela- tively transparent were also discarded since such samples apparent- ly came from freshly molted shrimp. Each sample consisted of blood pooled from two to four shrimp of the same sex. More animals from 32 °C acclimation were needed for 40 the blood pooling than from either 18° or 25°C. Five samples were taken at each time interval in each test condition except in some extreme conditions where heavy mortalities precluded the use of more than one or two samples per time period. The samples were centri- fuged in a refrigerated centrifuge (-4°C) for 20 minutes at 3000 rpm and the clear serum preserved for analysis. Blood Analyses Osmotic concentration The osmotic concentration of serum and water samples was mea- sured on an Advanced DigiMatic Osmometer (Model 3D, Advanced Instru- ments, Inc.). Each determination required 0.2 ml of sample fluid. The apparatus reads the osmotic concentration in mOsm/kg. It is accurate up to + 2 mOsm if the sample concentration is less than 500 mOsm but above 500 mOsm the accuracy increases to I'o of the reading. Repeatability is within 1%. The instrument was periodi- cally calibrated with sodium chloride standard within the 100 to 1000 mOsm/kg concentration range. Blood chloride Blood chloride concentration was estimated on a Buchler-Cotlove Chloridometer (Model 4-2008, Buchler Instruments, Inc.). The instru- ment operates on an amperometric end-point principle and shuts off automatically at a preset increment of indicator current. Each sample contains 0.1 ml of serum, 4 ml of nitric-acetic acid reagent (O.IN) and four drops of gelatin reagent. Under normal conditions the instrument is accurate up to 0.1% mEq/L. Estimations were made on a 5-6400 mEq/L (high setting) range. Other electrolytes Potassium, magnesium and calcium ion concentrations were de- termined on the Atomic Absorption Spectrophotometer (Model 305, 41 Perkin-Elmer) . Samples were prepared by diluting 0.1 ml aliquots in various proportions so that the sample ionic concentrations were within the working range of the spectrophotometer. The diluted samples were analyzed at different wavelengths. Potassium concen- tration was estimated by emission at a wavelength of -3^ my (visible range) . Magnesium and calcium ion concentrations were determined by absorption at wavelengths of 285.9 my (ultraviolet) and 210.9 my (visible) , respectively. The instrument was calibrated with chemical standards supplied by Harleco and was set to read the concentrations in ppm; the readings were converted to mEq/L. Both standards and samples were diluted with 0.1% lanthanum oxide to reduce the chemical interference. The interference was particularly high in the case of calcium ion. Determination of Oxygen Consumption The oxygen consumption rates were determined in a flow-through respirometry apparatus using the Winkler method for oxygen analysis (Standard Methods, 12th Edition, 1965) and galvanic cell oxygen ana- lyzer techniques. Flow-through respirometry The flow-through respirometry apparatus consisted of four water reservoirs (R,, R2, Rt, and R^) , 12 respiratory chambers and an oxy- gen analyzer (Fig. 2) . The salinity and temperature control con- ditions were maintained in reservoir 4 and test conditions were main- tained in reservoirs 1 and 3. Reservoir 2 served as a thermal water bath to maintain test temperatures. The solution was filtered and aerated in reservoirs 1 or 4 and siphoned into a constant level chamber in reservoir 2. From the constant level chamber water passed into two distribution chambers. Each distribution chamber supplied water to six respiratory chambers. Water which entered the constant level chamber in excess of the constant level mark 42 D ■P oj U 03 oj u o •H a, ^-1 =S O u I 3 O 0) •H overflowed into reservoir 3. Both the constant level chamber and distribution chambers served to maintain constant flow rates into the respiratory chambers. The flow rates out of the respiratory chambers were regulated by 20-gauge hypodermic needles. From the chambers the salinity medium entered reservoir 3. The needles were fixed to a flow control board at the same level. Flow rates can be altered by changing the level of the control board or by changing the gauge of the needles. Disposable syringe bodies (10 ml) containing a small amount of filter material (Metaframe Spic and Spun balls) were placed in the line before the hypodermic needles. The filter material trapped the particulate matter coming out of the respiratory chambers and was replaced daily. The untrapped material may block the needles, oc- casionally resulting in the death of some test animals. The dissolved oxygen levels were determined by diverting the flow from the respiratory chambers to the oxygen probe chamber by means of a 3-way stopcock. The stopcock was installed between the syringe body and the needle. Reservoir 3 received water from the constant level chamber, respiratory chambers, and/or probe chamber. This water was periodically returned to the respective reservoir (either R, or R^) through a small pump operated by a magnetic relay switch. The volume of the respiratory chambers was adjusted to about 400 ml and the flow rates to 800 ml per hour. These rates were established on the basis of the size of the test shrimp. In de- termining the flow rates, care was taken to maintain the dissolved oxygen levels in the respiratory chambers well above the critical levels. At room temperature (circa 25°C) the level is in the vi- cinity of 2.0 ml O^/L. It is higher at 32°C. At 18°C the O2 satu- ration is always maintained at higher levels than the actual con- sumption. 44 A thin layer of sand was added in the respiratory chambers to meet the thigmotactic requirements of the shrimp. On smooth bot- tomed surfaces shrimp do not rest but tend to swim almost constantly. Addition of sand naturally reduced their activity to normal. Test animals were introduced into eleven chambers, keeping the twelfth chamber empty as a control. Feeding was suspended from one day prior to the experiments through the rest of the experimental period. The test animals were allowed to become accustomed to the res- piratory chambers for about 12 to 16 hours. During this period they were maintained in conditions similar to those of their control and received seawater from reservoir 4. After the initial adjust- ment period the animals were gradually exposed to the test media supplied from reservoir 1. The time taken for replacing the control medium by the test solution varied depending on the test salinity and the temperature change. Salinity changes took place in 15 to 60 minutes. Temperature changes from 25° to 18° or 32°C occurred in less than two hours. However, extreme temperature changes from 18° to 32°C or vice versa required up to three hours. The first hour readings were taken as soon as the test salinity and temperature conditions appeared in the respiratory chambers. Zero hour readings at the three different test temperatures represent the oxygen consumption in the respective control conditions of 15°/oo and 25°C or 18° or 32°C. Respiratory rates (ml O2/L) were determined on the basis of differences in dissolved oxygen levels recorded between the animal chambers and the reference chamber. For the sake of comparison of the responses exhibited by shrimp of different weights, the res- piratory rates were converted to ml 02/L/g. The oxygen levels were analyzed with a galvanic cell oxygen analyzer calibrated against the Winkler method. It should be added, however, that the readings ob- tained with the oxygen analyzer at 18° and 32°C were not reproducible probably due to the fact that the instrument was not temperature 45 compensated. At those temperatures, samples were analyzed by the Winkler method. The diurnal rhythm of brown shrimp was taken into consideration while determining the respiratory rates. Being nocturnal, the ani- mals exhibit higher respiratory rates at night than in daytime. As such, the nocturnal rates do not meet the criteria set forth for standard metabolic rates by Gordon (1972). He stated that the ideal standard metabolic rate should be an animal's metabolism under the simplest and least physiologically demanding conditions. Therefore, the oxygen consumption rates were measured between 1000 and 1200 hours when the animals were least active. Oxygen consumption in media with deviated ions Procedures for oxygen sampling in the deviated ion media were essentially the same as in the previous oxygen study with some changes. Shrimp were acclimated for one week at 25°C and 15°/ooS as before. They were then separately tested in 15°/ooS at 18°, 25°, and 32°C, with the following ion concentrations: 25-6 Ca, 0% Mg, and 30% K. The total test time was reduced to 24 hours and samples were taken at 1, 2, 3, 4, 6, 10, and 24 hours. Shrimp were transferred directly from acclimation tanks to the test chambers at 0 hour. Additional chambers were added in the system and supplied with normal seawater to act as controls. Statistics Data from male and female shrimp were analyzed separately but were combined for the general results: mean, standard deviation, standard error. The t tests were done to determine the effect of sex on mean blood ionic levels and osmoconcentration and on oxygen consumption using the IBM 1130 Scientific Subroutine Package. The t statistics for hourly oxygen consumption comparisons were computed following the formula given by Sokal and Rohlf (1969). 46 In addition to reporting the actual mean values obtained at the different time intervals sampled, the technique of the moving average (Longley-Cook 1970) was utilized. This is one method to determine the trend in a time series; it tends to smooth out short-term variations in the data giving it a smoother curve. The advantage of using this technique instead of fitting a curve freehand is that it is free from operator bias since a formula is used. In a series of data Xj, xj, Y -4- Y +Y Y +Y +Y X3...X , the moving average is calculated by 1 2 5, 2 5 4, etc. " 3 3 The average value over a number of recordings is then substituted for each individual value. 47 Ill: RESULTS Effect of Salinity and Temperature on Behavior and Survival When an organism encounters an unfavorable environment it may resort to one or more behavioral or physiological means or to both simultaneously in order to avoid the harmful effects. In nature behavioral regulation takes many forms in relation to time courses, such as diurnal movements and seasonal migrations. Spontaneous escape responses are another form of behavioral adjustment to a harmful situation. During the first phase of the studies on salinity-temperature relations of brown shrimp, the spontaneous behavior was studied closely CVenkataramiah et al. 1974, pp. 29-36). The animals were exposed to a spectrum of salinities ranging from 0.34°/oo to 59.5°/ooS, which was wider than in the present experiments. Effects of temperature changes were observed at 21°, 26°, and 51 °C. The responses were classified, on the basis of their activity level, as normal activity, hyperactivity, inactivity (or resting) and "de- pression" (stillness to the point of nonreaction) . The activity noticed among the control shrimp (17.0°/oo and 26°C) was designated as normal. In other salinities, which were classified as critical or lethal depending on the survival, the animals were initially hyperactive. Ther, they became inactive or rested. At this point they either resumed normal activity or en- tered into a state of depression. Hyperactivity included vigorous escape responses such as swimming, walking, digging or jumping out of the tanks. Animals normally recovered from the inactive phase but rarely from depression. The effect of hyperactivity is re- flected distinctly in physiological responses , as will be shown later. Perhaps it was during the resting or inactive phase of A8 normal activity that stabilization in the regulation took place. During depression often there was a failure of the regulatory pro- cesses. Burying has been recognized not only as a diurnal rhythmic activity but also as a means of escape from too dilute or too saline waters or from too cold or warm conditions. Burying was normal when induced by one of these factors but was uncoordinated when the two factors were changed simultaneously. In response to salinity or temperature stress, the shrimp sometimes congregated in small groups of three or four (Fig. 3a), the significance of which is not well understood but which appears to be part of their responses to stress. Effect of salinity change In the present studies behavior was observed in 2, 5, 10, 15 (control), 25, and 36°/ooS in combination with 18°, 25° (ambient), and 32 °C. Additional observations on behavior that were not re- ported earlier will be presented here. Behavior in the test con- ditions was compared with that of the normal behavior in control conditions (15°/ooS and 25°C) . Normal behavior includes burying on a diurnal basis and other routine activities such as walking, swimming, or digging. About half of the animals buried in 15°/ooS and 25 °C conditions during the day, the number varying from time to time. Some shrimp remained motionless for several hours. Others walked or swam short distances. There were no mortalities among the animals acclimated and tested in 15°/ooS and 25°C (Table 1). A temperature increase to 32 °C caused nine out of ten shrimp to bury in 15°/ooS. Change of temperature to 18°C made them less active and rarely induced burying. Survival rates were also normal at these temperatures. Salinity change from control 15°/oo influenced the behavior and survival even though the test temperature (25 °C) was the same as that of acclimation (25°C). Transfer to any of the other sa- linities--2, 5, 10, 25, or 36°/oo--resulted in hyperactivity as an immediate response. The extent and duration of the hyperactivity 49 a. Note the grouping together of three or four shrimp in response to salinity and temperature stress. Normal shrimp were not observed behaving in this manner b. After losing coordinated loco- motor movements, this shrimp was seen spiralling in the water and then in this nose-diving posture. It held the posture for about 30 seconds, beating its pleopods rapidly, before falling over »-^ c. Abdominal cramping condition: Normal shrimp (bottom), half cramped (middle), and fully cramped (upper) condition Figure 3. Effect of salinity and temperature change on the behavioral responses in Penaeus aztecus I/) 3 o (D ■(-J N 03 V) 3 0) nj (D Cu 4-1 O (D ■M nJ Di X ■t-i O o c oj J= u 3 ■!-> 03 U t/) (U H LO CM c 0) o ^1 •H 3 +-> ■1-1 03 03 B U U • H 0) o I— 1 fX O E a (D < H + + CM + o + + CM + + + + 4-> CIh t/1 E U CO LO (M CO •—I o 03 •H P.-T3 c C o •H o m X ^ CM ■* (^ N- ^- N- (2) OOOOOOOOOO CDtJ-CVJOOOCD^OJOOO (DiDCDtOioinintoi?)^ o o o o ^ 00 Q 00 10 CD CD in O O 6 O Q CD ^ OJ O ® CD CD CD t£) ID 00 se X X 1 03 — 1 o 03 O CD -H .— 1 C ^- ^ > O (D o 0) C+-I CL LO q 13 — 1 O lU M-i -H -O X O CD +-J fn +J on M 03 03 H T3 O level ions w oncent d Stan pply t CD O 4-> CJ C 03 •H -H O 03 +-> -a e -H }^ O C t/l - ^ e o o .— > -H on to O !-i S ^ O ^03 — 1 O XI C M T3 O !h ^-^ O CM e O M +J -H O -1-1 C M 4-) o — 1 C O O P^ 43 O O fn 'H CD O Jh ^H UJ -H C/l (/) 00 OS q CD dj • H 13 O i-i (h tu 03 O CL, 03 ujsoLu-AinvnotNSO (£> 03 cn !/) (\) f^ o CO ^ (/) a e t/1 o U> U> U) 1^ 00006 ^ fd o <» ^ If) if> in ^ '*" 6-^ ^ CVJ O ;0 U> (£ 00 J O in 25 o e o ■o o o o c o o nS u 00 ujsoui— Aiiivnoirjso 58 ""o^o 600000 o o LusQuj— xinvnoi^so 59 I o u ^ D< O U x: +-' c •H tn 3 u C o (J 3 0) u N O ■H (N t/) o dj o ^ (D — .C o O H LO •H r-< 4-1 O • -a -H +-> -a ca c o c o o o o UJSQUJ- Ainvioi^so 03 ■(-> C Oh ■ H rt -o to cO 60 X C -i-i 03 -H X C U -H .— ( ctf • CO a> o 3 to 00 to •H 0) 60 o oooooooo CMOI-OJOCDUJ't n C) n CI ^ o <5 C3 C> 10 ^ J f> ai 0) (T> O) U) CO CD 00 CO o o o cl> o o o 00 CC * C\l O CO lO f^ f^ f^ h- r^ (o to s o o o 00 U3 ^ ID ID ID oooAooocS 6 cb 6 cb o o o o o lD^CVjOa3lD<3-C\100U5"a-C\JOCpy?^CM lO IT) LO ujsom-AinviOI^SO in 1^ CM en 5 o o o o _ 00 CD ^ 00 O U3 (D ID CD CD (/) (1) o u O o +-> ca C LO t/) o (D to 3 N Cu o 0) 0) o c o o 0) •H u +-> o O 00 e -I O 4-) -T3 O C o o OJ r- c c 03 to (U OX) c U 00 uMOi" -AinvnoMSO 03 o3 03 X +-) •H C ■H rH OJ to O to to 0 o o u 62 t-C^tn 1— -c—znu — > —I 1 T" o o o o § o C5 C) n o o c; M o CO ^0 lO 'J- CM o O) i^ V r^ r- U3 ID 10 1^ h- h- r- CO w s «3 O O "O' CO — u 0666660 CD ^ c t/1 CO LusoLu-AinviOlMSO 3 -l a, CO 4= C (/5 <4H 0 0 •H ■* ■M ^ t/l ■H I—* T) (/) (U C 03 > 0 3 (D CJ .— ( 0) I— ( M U 0 •H •H U W +-> ■(-) 0 c CD H 0 .—* CO c; iX 0 B CU a T? -C 1 (LI 0 UJ ^ 00 5 •M r— ( K c ■P ■H 03 W C CU 0 bO •H C ■(-> 03 03 x: +J u 03 -d 03 0 r-t io O O O O Q O O O 00 to ^ c^ O CO r>- u3 ic (D U3 (£1 in ujsouj-A±nvnowso •H 03 65 Effect of 25°C acclimation on osmoregulation In shrimp acclimated to and tested at 25 °C the immediate shock effects were observed in all salinities other than in control 15°/ooS (Fig. 4) . The shock effect was observed both in behavior and in the blood osmoregulation. In salinities lower than 15°/ooS the shrimp lost salts to the external media (efflux] . In higher salinities the animals gained salts from the media (influx) . In test salinities adjacent to 15°/ooS immediate responses were exhibited for only a brief period of about two hours. In extreme concentrations of 2°/oo and 36°/ooS the initial responses were prolonged for about four to six hours. Usually the greatest changes in the blood concentration occurred during the immediate regulation phase. The initial osmoregulatory pattern in shrimp acclimated to 25°C and tested in 32°C (Fig. 5) or in 18°C (Fig. 6) was similar to those tested at 25°C in the respective salinities. One difference, how- ever, was that the salt loss or gain occurred longer in 18° or 32°C than in 25°C. Another difference was that the shrimp lost salts even in the control salinity 15°/oo because of their experiencing a tem- perature change from acclimation. Between the two test temperatures, the changes continued longer in 18°C than in 32°C. At 18°C the rapid salt exchange transpired for two hours in 15°/ooS, four hours in 25°/ooS and from 16 to 24 hours in the other salinities (Fig. 6) while at 32°C rapid salt exchange ceased after two to four hours (Fig. 5). Stabilization of the blood osmoregulation at 25°C started within two to six hours, and a new steady state was reached between the fourth and seventh day in all salinities. On the basis of moving averages the steady-state levels appeared even earlier, by the sec- ond day in 5, 25, and 36°/ooS. The wide fluctuations in standard deviation values at 2, 5, and 36°/ooS (Fig. 4) reflect the individual osmoregulatory variations. The new steady-state levels were main- tained at higher levels in 25 and 36°/ooS than in 15°/ooS and lower in 2, 5, and 10°/ooS. 66 The stabilization process commenced within hours after transfer to 32°C and new steady-state levels were established by the third or fourth day. These levels in 2, 5, and 15°/ooS were similar to those at 25°C, while in 25 and 36°/ooS the levels were slightly higher. However, it should be mentioned that as a result of temperature change from 25°C stabilization and steady-state processes were de- layed. The delay was even longer at 18°C. It was reported above that the duration of salt loss was longest in 18°C. Also in this temperature the extent of initial loss was greatest. However, the regulatory capacity subsequently improved and part of the salt losses were recovered. New steady-state levels were established after the third or fourth day in most salinities. Another major deviation in 18°C from 25° and 32°C test temperatures was the presence of greater ionic fluctuations by individual shrimp, particu- larly in 2, 5, and 10°/ooS. This conclusion was based on the high standard deviation values. Effect of 32"'C acclimation on osmoregulation The blood osmotic concentration levels in brown shrimp accli- mated to 32°C and 15°/ooS are shown in Figures 7 to 9. The control shrimp (32 °C) maintained slightly higher osmoconcentration levels than their counterparts acclimated and tested in 25°C (657 mOsm com- pared with 643 mOsm, respectively) . However, the response pattern was similar in both groups with respect to the test salinities. In 2, 5, and 10°/ooS there was an initial loss of salts while in 25 and 36°/ooS there was a salt gain (Fig. 7). The duration of initial osmoregulatory changes in 32°C test temperature was longer than in animals acclimated and tested in 25°C. The immediate responses con- tinued from four to six hours in all salinities except in 25°/ooS. When the shrimp were transferred to other test temperatures 25°C (Fig. 8) and 18°C (Fig. 9) , the duration of the initial salt changes continued from four to six hours in most of the conditions. However, 67 the shrimp in 18°C failed to stabilize the blood osmoconcentration in some of the salinities for a whole day. Stabilization and new steady-state levels seemed to have occurred in 32°C within the first day in 10, 15, and 25°/ooS. The new steady- state levels were also maintained at relatively higher levels than in shrimp from 25°C acclimation temperature. In 2, 5, and 36°/ooS the process of stabilization was still in progress at the end of the ex- periments. In test temperatures 25° and 18°C stabilization in osmoregulation and new steady states were attained in 10, 15, and 25°/ooS. The steady- state levels decreased gradually with temperature from a highest in 32°C. In addition to the above salinities steady-state levels were also established in 2 and 5°/ooS at 25°C but not in 36°/ooS. Al- though the shrimp in IS^C were somewhat successful in controlling the salt loss in 2°/ooS, apparently they failed to reach a steady state. The low sample size in this medium was a result of heavy mortality. Effect of 18°C acclimation on osmoregulation The osmoregulatory trends of shrimp acclimated to 18°C and tested in 18°, 25°, and 32°C are shown in Figures 10 to 12. The mean control osmoconcentration values of 18°C acclimated shrimp were the highest (674 mOsm) of all test temperatures (Fig- 10)- As in the previous two groups there was a steep loss or gain of salts following the transfer to various salinities at 18°C. The duration of salt exchange between the shrimp and the external media increased with the deviation of test salinities from 15°/ooS. Tlie salt exchange occurred for less than two hours in salinities close to control while in 36°/ooS it continued for a maximum of ten hours. The initial osmoregulatory pattern at 25°C (Fig. 11) and 32°C (Fig. 12) remained essentially the same as in 18°C except that in 5 and 10°/ooS the animals continued to lose the blood salts longer than in other concentrations. 68 Stabilization of the blood salt regulation occurred in most of the test conditions within six hours after transfer. At 18°C steady states occurred in 10, 15, 25, and 36°/ooS after six hours and in 2 and 5°/ooS after four days. At 25°C new steady -state osmotic levels appeared in all salinities except 5°/oo within one day. In salinities below 15°/ooS, considerable individual fluctuations (Ref. the standard deviation values) were seen in the regulation process. On the basis of the actual mean values it was hard to decide whether the animals attained new steady-state levels within seven days. But the moving averages indicate the possibility of complete acclimation to these media. At 32°C steady-state levels did not appear within one week in 2 and 36°/ooS. Steady-state levels were reached in other salin- ities within a day. In 2°/ooS greater fluctuations continued in the regulation of individual shrimp for six hours after the transfer. There was also a high mortality in 2°/ooS. Time Course of Blood Ion Regulation Regulation of inorganic blood ions was followed in Penaeus aztecus during the time course of salinity and temperature adaptation. Among the ions analyzed were blood chloride, calcium, magnesium, and potas- sium. Effect of 25°C acclimation on chloride regulation The chloride ion regulation of shrimp acclimated in 15°/ooS and 25°C was studied in the time course of salinity adaptation at test temperatures 25°C (Fig. 13), 32°C (Fig. 14), and 18°C (Fig. 15). The shrimp acclimated to and tested in 25 °C experienced an initial rapid chloride ion exchange in 5 and 25°/ooS for about an hour or two. In 2 and 36°/ooS the ion exchange continued for nearly six hours. The high standard deviation values in 2 and 36°/ooS indicated the large regulatory fluctuations between individual shrimp. In 32° and 18°C the initial salinity-related response pattern was similar as in 69 16 24 48 72 96 168 TIME — hours Figure 13. Changes in the blood chloride levels of Penaeus aztecus in the process of salinity adaptation at 25°C. The control conditions were 15°/ooS and 25°C and the control sample size was 53 shrimp 70 00 U) Oh rt T3 rt to X (T> •M ^ •H ? <4-l O 8 to f t/l u «> 1 0) o u s fH 1- C <1> 03 o o o O 00 U) ro Od OJ _J 1 I 1 I I I I 1 1 1 A VV^ ^tr Hcvj O O Q O 00 fO C\J O (NJ o o o o C\J O 00 U3 ^ — OJ CVJ — ~ to CVJ o M 03 10 0) oJ C 0) cl, -a c <4-l O o o !/l .—I -H O 0) Jh > -p 1} c ^ o o CD -O (U •H X U H o o u •T) rg o Kl o r— 1 ■!-> X3 03 (D C r- O +-» •H C 03 0) M C 03 X U 0) U 3 DO -|/b3UJ_3aid0nH0 oj o 00 CD to ro cj CM O ro 6 6 6 6660660000 CsJQOO«)'r a> •rH c- -a c o o —I o > -p —I O O tJ m •H ^ o I—) x • o u o T3 00 O —I O ^ -M (D C JZ o +-> 'H 4-> C nS •H 4-> o bO C rt u •H IX, 25 °C, but the duration of the immediate response phase was longer. For instance in salinities next to 15°/ooS this response phase con- tinued for about two hours at 32 °C and for about four hours at 18°C as opposed to one hour at 25°C. In 36°/ooS the chloride influx con- tinued for almost ten hours before any stabilization was commenced. Stabilization started in most of the salinities at 25° and 32°C test temperatures within ten hours. In 18°C, although major changes occurred from six to ten hours, changes of smaller magnitude contin- ued from 16 to 24 hours. At 25 °C not only was the steady state reached sooner than at 18°C, but the fluctuations in the ionic reg- ulation were minimal. However, at 18°C it should be noted that in 2 and 36°/ooS the ion regulation was less steady. In 2°/ooS the shrimp were continuously losing ions to the medium but the rate of loss after the third day was not significant. The ion regulation was unsteady in 36°/ooS. Apparently no steady-state levels appeared in either salinity. At 32°C a steady-state chloride level appeared in 25°/ooS slower than in 5, 10, and 15°/ooS. At 18° and 32°C, great ionic regulatory fluctuations were noticed in 2 and 36°/ooS media between the different time intervals (Figs. 14 and 15) and between the individual shrimp. The relatively stable ionic regulation at 25°C indicated that at other temperatures (18° or 32°C) the shrimp were unlikely to handle the chloride regulation problems successfully even after one week particularly in 2 and 36°/ooS. Effect of 52°C acclimation on chloride regulation In brown shrimp acclimated to and tested at 32°C, chloride in- flux occurred for a maximum of two hours in 25 and 36°/ooS media (Fig. 16). In 5°/ooS the salt loss continued for about four hours. At lower temperatures of 25°C (Fig. 17) or 18°C (Fig. 18), the dura- tion of salt influx in 25°/ooS was also the same as in 32°C. But in 73 UJ 8 Ob ^ CSJ oooooqooooooo 'd-cvJOOo^DC^JQoo^o^^c\JOOO^) " * rorOfo tOtOCMCVi lO to ro c^ CM '*■ o o o o o o CO ID 'J- fM O CO U3 CO c\j CM CM eu — ~ o o o o o CD ID •^i- CM O CO 00 CM CM CM CM o o o CO CD "^ CM CM CM Oh B •H I u -a '^ X +-> t/l •H nj C 3 •H —I tsl CM 03 O 3 -T3 C 03 C_> 0 CM C o3 n/t>3iJLi-3aidonH0 ?: ii-V-;ir r^V OOQOQOOOO6cbpQO iO io *" ^ 'd-'^-^^^rOrorotOrOCJ ^ 1 (£> X O) +-> •H OJ C 1^ •H 2 f-H 'S- t/i ^ (/> <+-i >- O 3 o t/) .c lA to 1 o u UJ U o "? O CnI J-i ro 1- CO o O o o O 8 O o CO CD ^ CM CD ro CO CVJ CM CM CM K) o o CO (D CM CM o O O O O O O O o o t CM CJ CO CD ^ CM a CO CD CM CM rO CM CM CM CM ro CM CM o ■U 3 O c o 0) o CM -o r^ •H 0) ? ^ JZ O H ^ " X CM o • 3 u o -a o .c O LO CD 1 III S n/b3Lu-3amo"iH0 10 ID C u 3 (30 * M-l O if) -p -H O o -a 0) •H 4:: U H O ^H 4:: • U U o X) 00 O —I o 1— I p X3 aj <1> C J=. O P -H P C ct3 •H +-> CI, to 0) W) nj C , 05 X X P U -H C CO 05 f— I 10 (U f-i 3 n/b3UJ-3aidO"lHD (h 76 36°/ooS the initial salt influx could not be controlled for about four hours at 25°C and for 24 hours at 18°C. At 25°C the ionic regulatory fluctuations were lowest of all the test temperatures. However, in dilute media the ion loss continued from four to six hours. The same types of responses were also seen at 18°C. At 18°C the chloride ion regulation seemed to stabilize within four to six hours in all media except 2 and 36°/ooS. At 25° and 32''C steady-state chloride ion levels appeared in 5, 10, 15, and 25°/ooS within the test period. The steady-state levels occurred from the first day in 10, 15, 25, and 36°/ooS and after two days in 2 and 5°/ooS. In 18°C evidently the salinity adaptation was incomplete except in 10 and 15°/ooS. Effect of 18°C acclimation on chloride regulation The brown shrimp acclimated to and tested in 18°C (Fig. 19) ini- tially lost chlorides in the dilute media. But part of these initial losses were made up during the stabilization process and the new levels were nearly on par with those in 25°C (Fig. 20) or 32°C (Fig. 21). At 18°C the initial responses continued in the various salinities from one to six hours (Fig. 19). In 10, 15, 25, and 36°/ooS a steady state was reached by six hours, while in 2 and 5°/ooS it was much later, by the fourth day. New steady states were obtained in all salinities except 2, 5, and 36°/oo within one day. In 5 and 36°/ooS steady states appeared after four days at 32°C while in the other salinities, these levels appeared after one day. At 32 °C great fluctuations were present in the chloride ion regulation in 2 and 36°/ooS between the time inter- vals and the individual shrimp. Time Course of Regulation of Other Ions Calcium, magnesium, and potassium ions represent a minor portion of the blood osmoconcentration in comparison with chloride and sodium. 77 OOOO OOOOOOOOOOQQOO CMOOOCD ri-C^OOOtO'^CVJOOOl£)'^SQ°0 rOfoCvJcvj rQtOrocvjcj^'iJ-'ttorOrorOrooj o o 00660^ lOfOCVJCNJC\J(NJ(M~f'^rOOJOJcjCMCM 6 6 6 6 CM O CO CO to to C\J C\J X 2 M n3 (1) s 03 o (D O O ^H O -M fH C CL, O o s 0) -d o f-l -H o -a -^ c X o o o ^ e 0 0 r-H ^1 .-H ■M 0 ^ 0 CO 1 h- c H 10 • 000 C 00 nj •-! X U +-> • C J 8§^8oo SSSS^ojoS CD u O o O CO u ^ +-> -a i£> n) a> X 1^ ■M 00 C •H ^ r— * ^1- CJ \2 M-i 3 o c> to 1 to (/5 U III (L) o 2 t+-i O <£ O CJ t/l i-H rH O (D ^ > ■U 0) rH O o 0> ^ iri. t — ' ^'1 V— - in .r :L: ^ < -^t — , Q 6 6 6 6 6o 6666 0660^66666 ^ W O 00 U) 'J'tO rroJocOcCoa6lO^<>JC3CO(£>5}^ OJ CVJ OJ — — — oj ojOOcj — ~tO(M<\IOjCVJCO~~ — 1 ^f » --c / ^ H r [ ^ - Mf7">H m / < ^ 6 6 6 6 6 6 6 6 oj oj cvj cj ~ — — 7^6 6 6 6 6 CM CM 1 /b3Uj-wni3"lV3 oj ■(-> O -H T3 C O O O O .— I X> —I o ■M G O C O •H 0) (/) JZ d) H c cS • ,G U U o LO rg rg ■!-> (U C M O 3 -H W) -P •H 03 82 m 1 a, S nj 13 03 X ■M ■H ^D C ■H ^ T— t CO 'l- 4-1 O ^ 1 O LO UJ ^ rsi 2 ■T3 1- X 03 .1/^1 . I I I Lj I 1 I *"] 1 1 I—/ 1 Jt- — i — Jk — Jk — i — i — =t — ^ QOOOQ OOOQOO O O Q O Q Q O O. Q Q _1 1 1 1 1 O- 1 x_ QOqOO Q QCi OJ OJ •H -a c o o O I U -|/b3UJ-y\ni0nV0 •H OJ O X -H H o o o r\i JO CD 03 4J C O C -H •H +-> 03 to -M D 00 iH 03 a •H [1< 83 6 6 6 6 Q 6 6 6 6 6 q 6 6 6 6, o 6 c^ o CO <£> ^^cvjOobsc Qfloyj^j-oJrtob r\i)d — — OJOJMJCi — ^ tr, C^ r\l rii f>J Ki — (Ni OJ fO c\j cvj di cvj cd CD e o I LJ I (/) 4-1 O V) o OJ LO O OJ 0) o (U 4-> N 03 (/) ;3 ni C > c c X o ■p o c aj •H JH +-> 3 o 4-1 SI 03 f> 3 0) 03 C (D a. o o5 T3 o3 fl. ■=• tn LO o I LJ > 0) s 3 03 O -a o o 13 c o o XI i-H o ■P c o o 0) H C 03 • X U U o 00 LO P rsi o3 D C !h O 3 -H 00 +-> ■ H CS tU P 85 03 QQQOOQOQ OOQ ^c\jocou3obtO';f(\joooa> OJCJCVJ — — CvjCOCJOJCvJ — ~ Q O Q O O O. O JCM--~ ~~ ^ cd o 00 cr> 'tf- c\J CM CO CO — — — — 12 o CO CD 4_) a> •H CJ c t^ 1-H an 03 ^ !/) fH (U +-> rH c o td u 3 •H OJ O ^ r— 1 H 03 O -o u o 0 o LO ^H CM 43 •(-> m o: x; +-I c o c ■H •H 4-1 Cfl 1/1 +-> 03 +-> CI, (O CO a> 13 03 ^ O) •H ^ C ■H ^ OJ 03 e t/1 u> 3 2 O en 0) U o (- O o O 00 •T3 X 03 O Q O ^ cj p 95 S£ CO cvj Q Q Q Q O 00 U) ^ CO d CO CO CO CO CO O Q Q O Q O O CD OOOtD^COOOO — rocjcocococo — - se I O Q O Q Q O Q ^ CJ O CO CO ^" CO CJ CO CO — ~ — ~ — ^' cJ CO CO CO c£S 't ?J gj go /b3iju- O O O Q O ^ ^ CO O CD CO ^ t^ CO CJ ~ ~ ~ CO 3 LO t) rH OJ ■M a> Nl ^-1 n1 ^1 0) •p f-H c o B o 3 •H wnioivo calcium ion concentration in all the test conditions. Stabilization of calcium ion regulation started on the first day in all temperatures. New steady-state levels appeared within the test period in 18°C in all salinities except 25°/ooS, a medium which was close to the control sa- linity. By the time the tests were concluded, the process of stabili- zation was still in progress in most of the salinities at 25° and 32°C except in some of the dilute media. A steady-state level was not reached even in the control salinity 15°/oo in 25° or 32°C. Also the regulatory fluctuations were large at 25° and 32°C, both among indi- vidual shrimp and between sampling intervals. Brown shrimp acclimated to and tested at 32°C showed relatively fewer variations in their initial regulatory responses (Fig. 28). But more variations were present when tested at 25° or 18°C (Figs. 29 and 30, respectively). Even though stabilization started within a few hours, new steady states were attained in only some of the conditions. At 32°C, which was their acclimation temperature, steady-state levels appeared in none of the salinities. At 18°C steady-state levels appeared in all salinities except 2°/ooS while at 25°C the levels appeared in 5, 10, and 15°/ooS. The low sample size in 2°/ooS after 16 hours was a result of the high mortality rate. Magnesium ion regulation Brown shrimp acclimated to 25°C were tested at 25°C (Fig. 31), 32°C (Fig. 32), and 18°C (Fig. 33). The control mean (15°/ooS and 25 °C) of magnesium ion concentration was higher at 25 °C than at the other acclimation temperatures. There was a sudden initial increase in the magnesium ion concentration in all the test salinities ex- cept 2°/ooS (Fig. 31). Within two hours the ion concentration began to decrease in 2, 5, and 10°/ooS. A similar pattern was seen at 32°C (Fig. 32). Magnesium ion regulation appeared to be more effi- cient at 18°C (Fig. 33), particularly in the dilute media. The regu- latory efficiency of shrimp acclimated to 25 °C decreased in 25 and 88 -*^T 1 1 1 1 — I I 1 1 — ~ O O Q _. _ _ _ IDI-CVJOOD 00 ^ y.yj \^ \ju 0Jcvjc>Jcvj~ rOtotOfocvj O Q Q O O OS jg 5r. _. O ^ CM O CV) O 0) (U O tNl r— I -H '-I (/I E O 3 ^ to o c o o ^ c 0) o U 00 nj T3 ■!-> 0) -H ^ d 3 -H OO •—! ■H 03 y >_i (O ^^r^iotototo roc\ic-> 00 rt C T3 U X n/t>3uj-wnionvo (Nl 4) D •H c •H 4-1 O 90 So q o C5^ ^ CV O 00 CO o) -a ■t-j nJ (U r-H isi 0) -l/b3Uj-^Mni3-|V3 CJ (L> -^ JZ nj H o -a • o u o o f— ( 00 x: rt 4-) c c o OJ 4-> X 03 t~0 -H 0) 03 !-i I/) •H 91 100 ,M 80 60 40 -4^A-A ^^ 4^ 2%. —45 20 -~«4— r ''^ Ui E S 2 (/) IJ 2 O < 100 80 60 • 1= .45^-_ i ■■ I'i 1 5 i -4 - 3 1 f" ! 1 '1 012 4 24 48 72 96 0 12 4 72 96 TIME -hours TIME -hours Figure 31. Changes in the blood magnesium levels of Penaeus aztecus in the process of salinity adaptation at 25°C"! The control conditions were 15°/ooS and 25°C and the control sam- ple size was 54 shrimp 92 6 6 6 6 6 Q q 6 o x: I UJ ^ ro to CO CO CM ~ Q Q Q Q 6 . CJ CO ^ O U> A 6 6 6 6 6 6 6 2)^cjocx)a3'3-co cb 6 6 6 o 6 6 6 6 6 6^6 6 6 'T CO O 00 li> •^ N CO (/) 0) aj C OJ B. 4-1 O I— I 0) e H u 3 o •H Ln O 03 ■M Oh 03 C TJ •H o! bO-H C C 03 -H 4= -H U 03 I/) • <+H rg o (1) ;« !-i a> D O W) o •H ^ -|/b3uj — wniS3N9VW 93 -V-rAV T 1 1 r- ooooooQQo 0606 6 66666666666qq <^ ro ro oj CM CM ~ M>-cD*^»3-rOfoCNJcjcvj~ 6 6 6 6 Q 6 6 6 6 I UJ OQOOO— — — — — — — — -— —- — — _— — — _, Q O Q Q Q O Q i> 6 6 CM CO — — 1/1 t/1 o o u fn o (Nl CD ■M C oj C •H CO o (/) o 3 -^ O 0 (D LO ■l-J I— I N n) o U o (/) C o 1/1 C 0 4-1 -H O TS c (« o ,-( u > -H e c 3 o !/) c ,c -a u O o O 00 OJ 10 4-1 (u a, DO 03 C T3 CO 03 1^ (U I/) 3 U-i 00 O n/ b3iu— i^niS3N9Vl^ 94 36°/ooS at the three test temperatures and in 2 and 5°/ooS at 25° and 32°C. In 2, 5, 10, and 15°/ooS stabilization in the magnesium ion regulation started within two to six hours at 25 °C (Fig. 31) . In high salinities, especially 36°/ooS, it was a much slower process. Stabilization progressed at the same rates in 32°C (Fig. 32) and 18°C (Fig. 33) . In 25 and 36°/ooS media, individual variations in the ion regulation were very high as shown in the standard deviation values (Figs. 31, 32, and 33). The shrimp acclimated and tested at 25°C apparently reached new steady-state levels in all but 36°/ooS (Fig. 31). At 32°C, however, the animals were still in the process of stabilization in 10, 15, 25, and 36°/ooS by the time the tests were concluded (Fig. 32) ; in 2 and 5°/ooS steady-state levels were apparently reached. In 18°C it was doubtful whether steady-state levels were attained in salinities other than 2°/ooS (Fig. 33). The shrimp acclimated to 32°C had the lowest control mean ion concentration of the acclimation temperatures. The initial response of shrimp acclimated to 32°C was a sudden increase in the magnesium ion concentration. The responses continued from four to six hours in 2, 5, 10, and 15°/ooS and longer in 25 and 36°/ooS at the three test temperatures (Figs. 34, 35, and 36). Stabilization of ion regulation at 32°C started within six hours in 2, 5, 10, and 15°/ooS, within a day after the transfer in 25°/ooS, but apparently not in 36°/ooS (Fig. 34). At 25°C a similar pattern was noticed in the respective salinities (Fig. 35) . In 18°C the shrimp started the stabilization process on the first day itself in salinities from 2 to 25°/oo (Fig. 36) . In 36°/ooS the process commenced two days later. At 32°C new steady-state levels were attained in all salinities ex- cept in 36°/ooS, while at 25°C these levels were seen in 2 and 10°/ooS only. At 18°C steady-state levels appeared in 5, 10, and 15°/ooS but not in other concentrations. 95 o o o q CO o ^ oj 96 ID oj a UJ oj Ob lO lO J CD 't 00 O s 3 •H (D C W) CO E O O u CO Mh 0 0 0 LO 0 T3 0 C 4-> \o (U cd to oj ■ +-> U ctj eg tJ to ctj X C •P cij ■H 0 c CO u LO ^0 (U 3 CO ~-^ !/) O LO ^-^ -H o en (h (/) 0) O o X (/) 0) -o u o o ^ fH (1) Dh a, c CO 0) +-1 CO c CL, O C C O •H O CO S X 3 1 — I C e 3 •H OJ c 97 I I ' I ) I 1 I I o-J I I 1 jc I 1 1—1 l—Jr 1 A — i: — at — i- q Q 6 O O O 6 O Q Q O Q 6 Q O O O, Q Q Q Q Q q ^OflOtO^CjO OOID'J-CO CVjQOOUJtPJ CJ Q (D iO ^ (Si B 3 l/b3Lu-wniS3N9VW ■H t4-l 0 tu O C 98 The brown shrimp acclimated to and tested at 18°C (Fig. 37) ex- hibited the same initial response pattern as the shrimp acclimated to 25°C showed with respect to different salinities. This was also true in animals tested at 25°C (Fig. 38) and 32°C (Fig. 39). Within two to six hours after transfer, stabilization commenced in most of the salinities. However, in 36°/ooS it was a very slow process, taking place after the third day at 18°C and 32°C (Figs. 37 and 39, respec- tively) and on the fourth day at 25°C (Fig. 38). In the course of stabilization individual shrimp showed greater fluctuations in 25°C than in 18° or 32°C. Also the fluctuations were higher in 25 and 36°/ooS than in lower concentrations. New steady-state levels ap- peared in 2, 5, 10, and 15°/ooS at the three test temperatures but not in 25 or 36°/ooS. Potassium ion regulation The brown shrimp acclimated to 25°C and tested in 25°C (Fig. 40), 32°C (Fig. 41), and 18°C (Fig. 42) experienced an initial drop in the potassium ion concentration in 2, 5, 10°/ooS and sometimes in 15°/ooS. In 25 and 36°/ooS there was a simultaneous increase. The duration of the initial drop varied from one to two hours in 5 , 10, and 25°/ooS and from six to ten hours in 2 and 36°/ooS regardless of water temperature. The stabilization process started within the first day in most of the conditions. The process continued through the fourth day until steady-state levels were established. During stabilization, fluctuations in potassium regulation increased in test temperatures 25° and 32°C. New steady-state levels were observed in most of the conditions between the fourth and seventh day. However, except on the basis of moving averages, it was not possible to conclude from actual values whether new steady states were obtained in 10 and 15°/ooS at 25°C (Fig. 40) and in 2 and 5°/ooS at 32°C (Fig. 41). 99 CD U3 Q Q OJ O O OQQOOO. OQO CD <\JO00a5^C^O00t£> CM CM ~ — — ~ — o o o o o CVJ O CO (£) ^ O. O Q O O cvJ ^ 00 to «^ n/b3UJ-iAjniS3N9VlAJ o o o o O 00 O) '^ 3 o 3 o I UJ c u O o ■H 00 ■M ^ nj +-) +-" -13 rt • X 6 in o m u o t/l o O 03 3 o LO 0) (Nl N •H C 3 o 0) N 3 C D- > 4-1 0 u o s c/t) <4-i 00 ^^ O •— I o \D {/) Ti t^ (U nJ T3 > C (U CO CO I— I o O LO E ^^ og 3 o •H LO C (/I 1— I 'iH 0) C e 3 0) o o 3 bO (1> (U -a 03 <-" o o s C 3 t/l c o 0) 4-1 C O 03 03 o e -t ^ ■ ' r. T 4<- K . . , .1 , . 1 O O Q O O O CVJ O CO 10 d <1> 03 C a, o u CO 3 u LO c/^ o o 0) ■M LO CS CM O < 03 • ^ U CLiO 03 CO -T3 ^ 03 -13 X C •M 03 0) 03 e 03 -\ (/) o LD o C VI •H 1/1 0) (D C 03 U 00 to 4) P ro &, O •H +-1 -d c c o ■H O T3 OJ 03 OJ U c n/b3Lu-iMniS3N9VW 101 I O Q "^ ^ ^ ^ _- O Q c\J O oct CM CO - S'' 6 Q 6 Q 6 6, 6qq6q66q6 OJ OJ 7 o \* t l-€I]J>-t % ; o o o -I/b3Ui-^MnlS3NSVW ^-1 o 3 o (/I .— I !/) (1) 00 X r-H •H c 01 •H X) C Jh LO 03 (/) .— 1 U 3 M-i C ^ O o O • H 6 tn •!-> i-H • H X 0 TD ao > C •tH 4-1 ID c c C o (U W) o (N c 03 C •H C •H t/1 O (/) N c o! • H rt 4-> t/l X Ci, u 03 (U T1 I-H , o3 g- o^ X 03 1^ 4-1 •H m - t 700 _l i 680 g 660 640 620 600 580 560 540 520 500 480 460 -Z/- ACCL 2500 TEST ZS'C 36%, 0 12 ■ // '. -L. _!_ 145 140 135 - 75 70 10 16 ■ 24 48 72 96 TIME -hours 168 Figure during 49. Comparison of the blood osmotic levels of Penaeus aztecus adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 25°C 114 940 920 900 880 860 840 820 800 780 760 E 740 O ^ 720 >- t 700 _l < ^ 680 660 640 620 600 580 560 540 520 500 480 460 en -//- ACCL 25«C TEST SZ'C 36%, /> ^' y / 25%, ■V/ '^Z'^-. \\( L "•• .AyNTROL ^A^^ii Y> -«. 10%, 5%, r ■ ■ ■ _i_ -//- _i_ _!_ - 145 140 - 135 - 130 125 - 120 115 110 105 100 95 90 85 80 75 70 0 12 10 16 24 48 72 96 TIME -hours 168 Figure 50. Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 25°C 115 940 920 900 880 860 840 820 800 780 760 g 740 (/) O E 720 t voo _l < _l 680 O g 660 640 620 600 580 560 540 520 500 480 460 -//- ACCL 25'C TEST 18"^ ,^'"^-— « .^' - 145 140 135 257o, ■ // \r / CONTROL 15%, I0%< V " ■•//., ■-•-// • 5%, O /o^ 2%. / Z^- -1 1 1 I i_ -/f- 130 125 120 115 5? _J 110 < o •s. v> o 105 100 95 90 80 75 70 0 1 2 10 16 24 48 72 96 TIME-hours 168 Figure 51. Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 25°C 116 9 411 920 900 - 880 - 860 840 - 820 800 780 760 E 740 m O f 720 >- H H 700 < o V) o 680 660 640 620 600 580 560 540 520 500 480 460 -//- ACCL SZ-'C TEST 32">C 36%o , ./''' /A 25%, J5%g,. CONTROL ; — ^->^^ ^/--- t — 10%. — ^^ V V .,^-^^ 5%, 2%. -L. _l I I L 0 12 10 16 24 48 72 96 TIME— hours 140 135 130 - 125 120 i 115 >- 110 ^ < -I o 105 V) o 100 95 90 - 85 80 75 70 168 Figure 52. Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 32°C 117 940 920 900 880 860 840 820 800 780 760 E 740 O I 720 >- Zi 700 < O 680 CO o 660 640 620 600 580 560 540 520 500 480 460 Figure during -//- ACCL 32»C TEST 25»C 36% ^^^ //^' -I 25%. 140 135 130 125 120 115 110 -, i CONTROL -//- .v/- \ V, \ ^- \ \ V // — -•' 15%, I0%« 5%o 2%o / ...^' -//- _i I i_ 105 100 95 90 85 80 75 70 O O 0 12 10 16 24 48 72 96 TIME-hours 168 53. Comparison of the blood osmotic levels of Penaeus aztecus adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 32°C 118 940 920 900 880 860 840 820 800 780 760 S ^"0 E I 720 < 700 _l O 2 680 to o 660 640 620 600 580 560 540 520 500 480 460 -// ACCL 32»C TEST I8"C \ J6%. / i /^^' 257o, CONTROL I iV. Z I57oo ■ * ^ ^ ^ ^ ^ ^j^ ^ ^ ^ ^ l^^< ...//.. J0!/» \ «v y> . ,^' 27« V \ -//- 140 135 130 125 120 115 110 O z 105 to o 100 95 90 80 75 70 0 12 10 16 TIME-ht)ur* 24 48 72 96 168 Figure 54. Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 32°C 119 presumably because of temperature change from acclimation. The salt levels were, however, partially restored from the second day onward. The effective new steady-state levels at 32° and 25°C approximated closely those of shrimp acclimated and tested at 25°C (Fig. 49). The osmoregulatory efficiency was disturbed further at 18°C (Fig. 54). Rapid initial changes in the blood osmoconcentration occurred in all salinities lasting from ten hours in 2°/ooS up to one full day in 36°/ooS. However, the regulatory efficiency improved in the meantime and the salt concentration levels were stabilized. But the steady- state levels deviated considerably from those tested at 32°C. The osmoconcentration pattern of animals acclimated to 18°C was essentially alike when tested in 18°C (Fig. 55), 25°C (Fig. 56), and 32°C (Fig. 57). The fluctuations in osmoconcentration at 18°C were relatively lower than in the shrimp acclimated to 25°C or 32°C and tested in 18°C (Figs. 51 and 54, respectively). New steady-state levels were established in all salinities from the fourth day. Osmo- regulation was apparently more effective in salinities from 10 to 25°/oo at 25°C than at 18° or 32°C. Only minor osmotic changes ap- peared at 25°C in 10, 15, and 25°/ooS. In 36°/ooS the salt influx was lower than at 18° or 32°C. For some reason the regulatory pro- cess in 5°/ooS appeared atypical. Oxygen consumption responses also corresponded with the osmotic concentration in 5°/ooS. The shrimp experienced more changes in osmoconcentration at 32°C (Fig. 57) than at 18° or 25°C, characterized by a greater salt influx in 25 and 36°/ooS. Steady-state levels were not established in 2 and 36°/ooS within the one-week period. Tlie regulatory efficiency of shrimp acclimated to 18°C was affected less in 32°C than those accli- mated to 32°C and tested in 18°C (Fig. 54). Chloride concentration The chloride ion concentration in these experiments was found to constitute approximately half of the total blood salt concentration. 120 -f/- AccL la-c TEST I8"C 140 - 135 130 125 - 120 115 110 105 O •s. 100 O 95 90 85 80 75 70 16 "■ 24 48 72 96 TIME-hours 168 Figure during 55. Comparison of the blood osmotic levels of Penaeus aztecus adaptation to various salinities at 18°C. The control conditions were 15°/°°S and 18°C 121 940 920 900 880 860 840 820 800 780 760 E 740 - w O E 720 I >- t 700 z O 660 640 620 600 580 560 540 520 500 480 460 -y/- ACCL IS'C TEST 25»C 36%, 140 135 130 125 120 115 110 105 < _) O CO 100 ° . 95 90 85 80 75 70 0 1 2 -f/- 10 16 24 TIME -hours 48 72 96 168 Figure 56. Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/°°S and 18°C 122 E O E I o w o 940 ACCL le'C // 1 920 - TEST 32-C 900 . 880 - ,.--'" - 860 " / 840 - / 820 800 t 780 * - 760 i 1 740 720 1 ^ 257oo 700 1 / 680 L--'--.__. ^/, CONTROL ^^ 660 "••■••■••• ^"— — ^^'' 640 \ loy?,"... 620 600 580 \\ ■I r-/ 560 540 V \ 520 \ Z' 500 \ — '-v ; 480 - 460 1 I 1 1 1 1 1 /i 1 1 1 1 lJ - 140 135 130 - 125 - 120 115 110 105 < _l O irt O 100 95 90 - 75 70 0 12 10 16 24 TIME — hours 48 72 96 168 Figure 57. Comparison of the blood osmotic levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 18°C 123 Therefore, total salt and chloride ion levels were generally maintained parallel to each other. The chloride ion concentration was hyposmotic in 2, 5, and 10°/ooS and hyperosmotic in 25 and 36°/ooS in relation to the ionic level among shrimp in 15°/ooS. This trend was consistent, regardless of acclimation or test temperature. Animals acclimated and tested in 25°C showed relatively few variations in the chloride ion con- centration levels in 5, 10, 15, and 25°/ooS (Fig. 58). It would indi- cate that at normal temperature (25°C) the concentration of chloride ions was less affected by salinity change within the 5 to 25°/ooS range. In 2 and 36°/ooS the changes were much greater. However, the mortality rate in 2 and 36°/ooS was zero. Large fluctuations in the chloride ion concentrations occurred be- tween the first and 96th hours at 32°C (Fig. 59). The fluctuations were even greater at 18°C (Fig. 60) . The salinity-related response pattern at 32°C was similar to that at 25°C. There was a rapid initial chloride ion loss at 18°C in 15°/oo and lower salinities. Part of the initial ionic losses at 18°C were eventually recovered. As a result, at 18° and 32°C the new steady-state levels in all salinities except 36°/oo were either equal to those at 25°C or slightly higher, as in 2 and 10°/ooS at 18°C. The shrimp acclimated and tested at 32 °C experienced large fluc- tuations in chloride concentration, particularly in 2, 5, 25, and 36°/ooS (Fig. 61). Also, the resistance to ion influx in 25 and 36°/ooS was less effective than in shrimp acclimated and tested in 25°C (Fig. 58) . Consequently at 32°C the steady-state levels in 25 and 36°/ooS were less effective than in shrimp acclimated and tested in 25°C (Fig. 58) . Consequently at 32°C the steady-state levels in 25 and 36°/ooS were held at higher levels than in the other group. In 2, 5, and 10°/ooS, although initial fluctuations were present in the levels, the final steady-state levels did not vary from those of 25°C acclimation. Shrimp acclimated to 32°C were obviously more efficient in controlling the chloride ion concentration in 25°C 124 460 440 420 400 380 360 _l 340 liJ E 320 I tiJ Q 300 280 260 240 220 200 180 160 140 -VA- ACCL ZS^C TEST ZS'C — '/A -^ /, '>^^ CONTROL V f'/^ '//-- 0 12 -//- 10 16 24 TIME —hours 48 72 96 36 /o< 25%, 15%, 10%, 5%< 2% 160 150 140 130 120 110 vO UJ 9 cr 100 o _l X o 90 80 70 60 50 168 Figure 58. Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 25°C 125 460 440 420 400 380 360 340 320 liJ a 300 (r. o X o 280 260 240 220 200 180 160 140 -//- ACCL 25"C TEST 32°C -t. V 36%< .^' '-VA^^X' 25%< CONTROL 157c ^> ^^ ^^ '' •/A- I0%o 5 /oo 2%o. -L. -L. 0 12 10 16 TIME -hours -//- 160 150 140 130 120 110 100 Q (r o _i X o 90 70 60 50 24 48 72 96 168 Figure 59. Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 25°C 126 460 440 420 400 380 360 -I 340 tr liJ E 320 I g 300 3 280 I o 260 240 220 200 180 160 140 -ff- ACCL 2'b'^ TEST I8°C V/ 0 12 10 V/- 16 TIME -hours 24 48 72 96 36%, 160 150 140 130 120 110 J^ UJ 100 o X o 90 80 70 60 50 168 Figure 60. Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 25°C 127 460 440 420 400 380 360 ;^ 340 w ill E 320 I UJ 9 300 a. o X 280 260 240 220 200 180 160 140 -//- ACCL 32"^ TEST 32"C 36 Ax 25%. - .(' //- k \ V -//- 160 150 140 130 120 110 J* bJ 9 100 g X o 90 80 70 60 - 50 0 12 10 16 24 48 72 96 TIME-hours 168 Figure 61. Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 32°C 128 Fig. 62) than in 32°C, especially in low salinities. AT 18°C shrimp suf- fered heavy chloride losses in 2 and 5°/ooS (Fig. 63). After the second day, however, the ions were recovered to a considerable extent in 5°/ooS but not in 2°/ooS. There was also a high mortality rate in 2°/ooS. The brown shrimp which were acclimated to 18°C appeared to have effectively controlled the chloride ion levels in 18°C (Fig. 64), 25°C (Fig. 65), and 32°C (Fig. 66). Also the control shrimp in 15°/ooS and 18°C maintained the highest mean chloride concentration (298 mEq/L) of all the control temperatures. The mean control levels decreased with increased acclimation temperatures- -292 mEq/L at 25°C (Fig. 58) and 283 mEq/L at 32°C (Fig. 61) . The initial rapid ionic losses observed in 2, 5, and 10°/ooS at 18° and 32°C were recovered partly by the second day. The steady-state chloride levels at 32°C were established more closely to that of shrimp in 15°/ooS than at 18°C. In 2S°C the chloride ion changes were minimal in both dilute and saline media with respect to changes at 18° and 32°C. At 25°C there was no apparent attempt to increase the blood chloride ion concentration from the initially low- ered levels in the dilute media as occurred in 18° and 32°C. Calcium concentration Calcium ion concentration increased with the test salinities but the raise was not consistent in relation to salinity increases in the range of 5 to 15°/ooS. Brown shrimp acclimated and tested at 25°C (Fig. 67b) exhibited a hyposmotic calcium ion concentration in 2°/ooS and hyperosmotic concentrations in 5, 25, and 36°/ooS in relation to the level in the control salinity. In 10 and 15°/ooS the concentrations were isosmotic. In 25 and 36°/ooS the ion levels increased more at 32°C (Fig. 67c) than at 25°C but decreased at 18°C (Fig. 67a). Calcium ion concentration was hyposmotic in 2, 5, 10, and 15°/ooS at both 18° and 32°C. In 5°/ooS the calcium level was almost equal to the control level at 32°C and exceeded it at 25°C. 129 460 440 420 400 380 360 _l 340 UJ 6 320 I UJ 9 300 q: O ^ 280 o 260 240 220 200 180 160 140 -/A- ACCL 32X TEST 25-C ■•^.^ 36 /©« isiJk, 0 12 _, 1 // 1_ 10 16 24 TIME - hours 160 150 140 130 120 110 100 q: o 90 80 70 60 50 48 72 96 168 Figure 62. Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 25°C. The con- trol conditions were 15°/ooS and 32°C 130 460 440 420 400 380 360 ^ 340 UJ E 320 UJ S 300 c o -I X o 280 260 240 220 200 180 160 140 ■VA- ACCL 32"C TEST \ex .^ 36%« ■ 90 _l_ 0 12 10 -//- _1_ 160 150 140 130 120 J« 110 UJ a 100 -I X o - 80 70 . 60 16 TIME -hours 24 48 72 96 168 50 Figure 63, Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 18°C. The con- trol conditions were 15°/°oS and 32°C 131 460 440 420 400 380 360 _j 340 \ ^ 320 I 9 300 (T O -I 280 X o 260 240 220 200 180 160 140 -y/- ACCL I8°C TEST 18° 36%, 0 12 10 ' //- 16 TIME— hours -L. . 150 140 130 120 110 liJ 9 100 tr o _i X o 90 80 70 60 50 24 48 72 96 168 Figure 64. Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 18°C 132 460 440 420 400 380 360 _, 340 UJ 320 E Q o I 300 280 260 240 220 200 180 160 140 -/f- ACCL I8°C TEST ZS'C r /h-^. y' 36 fo< 25%. ■/A-, ^ CONTROL ■yp \ -y/-"r..7!""'~ — ""^ " '5°/'" I07o. ,5>. 0 12 -//- 150 140 130 120 110 s? UJ 9 q: 100 o _l X 90 80 70 60 50 10 16 TIME-hours 24 48 72 96 168 Figure 65. Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 18°C 133 4 6 0 440 420 400 380 360 _j 340 UJ E 320 I g 300 3 280 X u 260 240 220 200 180 160 140 -//- ACCL I8°C TEST ZZ'C ^ ^5S5bs— -I 130 / ---- y/ — 25%, 15%, //■ — ' '/>"' \OJJp I0%o • •' 5%^ 2% ■y/- 150 140 120 - 110 O 100 o -I I o - 90 - 80 - 70 60 50 0 12 10 16 24 4f TIME-hours 72 96 168 Figure 66. Comparison of the blood chloride ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 18°C 134 0 12 16 24 TIME -hours Figure 67. Comparison of blood calcium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the fig- ure), 25°C (B), and 32°C (C) . Control conditions were 15°/ooS and 25°C 135 The shrimp acclimated to 32°C (Fig. 68 a, b, c,) maintained a higher mean control ion concentration (22.8 mEq/L) than those acclimated to 25°C (19.7 mEq/L). At 32°C (Fig 68c) the calcium ion was controlled effectively on the first day with little variation. On the second and third days, however, the variations increased considerably. The cal- cium ion levels were hyposmotic in 2 and 5°/ooS, hyperosmotic in 25 and 36°/ooS, and isosmotic in 10°/ooS with respect to the ionic concentra- tion of shrimp in 15°/ooS. At 25°C (Fig. 68b) the calcium ion level was hyperosmotic only in 36°/ooS. Furthermore, greater ionic fluctua- tions were observed in animals tested at 25° and 18°C than in the control shrimp; also the hyposmotic and hyperosmotic ion concentration patterns were similar in the various salinities. However, at 18°C (Fig. 68a) the salinity-related calcium curves were widely separated from each other as opposed to 32°C (Fig. 68c) . The animals tested at 25°C (Fig. 68b) represent an intermediate trend between 18° and 32°C. The wide separation of the curves may indicate a possible interaction between salinity and temperature on calcium content. In shrimp acclimated and tested at 18°C (Fig. 69a) the initial calcium concentration levels were maintained below the control 15°/ooS level. The ion concentration increased above the 15°/ooS level after ten hours in 36°/ooS and after two days in 25°/ooS. As a result, in both salinities the calcium ion concentration became hyperosmotic. In other media it was hyposmotic throughout and levels were lower than in 25° and 32°C (Figs. 69b and 69c, respectively) and in the corre- sponding salinities. The response pattern at 32°C was similar to that in 18°C, except that the final steady-state levels at 32°C were rela- tively higher. At 25°C the ion levels in salinities 25°/oo and below were hyposmotic while the calcium increments in 2, 5, and 10°/ooS were disproportionate. The variations in the calcium con- centration levels between the salinities were lowest at 32°C, ex- cluding 2°/ooS. The variations increased progressively as the test temperatures were lowered to 25° and 18°C. 136 -^/^ Ul f 22.0 2 20.0 2 18.0 16.0 14.0 12.0 •//• B _l I L V^ 30.0 28.0 - TEST 32*^ 26.0 24.0 22.0 t- 20.0 18.0 16.0 ACCL 32'>C 36^^ / __25%. , ^„_,^^r ^....J-Q^-;.*-' ■ tf"";"^^^C9NTR9L ^ -—^r^. -f*-^^ "°'°° \ ^^. ■ " ■ — ,- 0 12 10 ' // ' ' ' ^ 16 24 48 72 96 TIME -hours 90 J < (J 80 70 60 50 130 120 110 100 90 80 70 168 Figure 68. Comparison of blood calcium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the fig- ure), 25°C (B), and 32°C (C) . Control conditions were 15°/ooS and 32°C 137 30.0 28.0 26.0 24 .0 22.0 20.0 18.0 16.0 14.0 ^:^^^-----I2^Z3fe^^it^C ^ I07oo 90 80 70 10 16 TIME-hours 24 48 72 96 Figure 69. Comparison of blood calcium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the fig- ure), 25°C (B), and 32°C (C) . Control conditions were 15°/ooS and 18°C 138 Magnesium concentration Magnesium ion concentration in general increased with the sa- linity of the medium. In 2, 5, 10, and 15°/ooS the increase was generally uneven and lacked a sequence while in 25 and 36°/ooS it was disproportionately high. The ion concentration levels in brown shrimp acclimated and tested at 25°C (Fig. 70) were hyposmotic in 2 and 5°/ooS, hyper- osmotic in 25 and 36°/ooS, and isosmotic in 10°/oo and 15°/ooS. The ion concentration in 25°/ooS increased gradually to over 200% above the control level and to more than 700% in 36°/ooS without reaching steady-state levels. The response sequence remained nearly the same at 32°C (Fig. 71). One difference was that the animals lost a little magnesium in 10°/ooS and gained a little in 5°/ooS. Steady-state levels did not appear at 32°C in either 25 or 36°/ooS. At 18°C (Fig. 72) magnesium ion level in 36°/ooS re- mained the same as in 32°C. In other salinities the ion concen- tration increased to hyperosmotic levels. The mean control magnesium concentration in shrimp acclimated and tested in 32°C (Fig. 73) was 5.5 mEq/L, which was lower than the 8.2 mEq/L level of the animals acclimated and tested in 25°C. Among the test salinities, ionic concentration was hyposmotic only in 2°/ooS. The ionic variations were not significantly different from each other in the low-salinity range. Magnesium content in- creased about four times in 25°/ooS; it increased about ten times in 36°/ooS and was still rising by the end of the study. After the first day ionic stabilization started in 25°/ooS. The animals in 25 °C (Fig. 74) controlled the magnesium influx very effectively and better than in 32°C on the first day after transfer. But the concentrations leaped to levels higher than in the previous group from the second day onward. No major changes appeared in the low- salinity range except that the animals in 5°/ooS were hyposmotic. 139 66 62, 58 54 50, 46. 42. _l l^ 38. I 2 34. CO ^ iO. o t 26. 22. 18. 14 . 10. 6. 0 - 0 0 - 0 0 - 0 - 0 - 0 0 - 0 • 0 - 0 - 0 - 0 ■ -//- ACCL 25'C TEST 25"C ..//'■ / * t / CONTROL - i/ I0%o /CONTRi . ^^'^^ l5%o ■7> _i_ -^ 36%, 0 12 4 6 10 16 24 4 ( TIME — hours 72 96 780 740 - 700 660 620 580 540 500 460 Z L57oo ■ 5 /oo Z'Z, 420 V) 380 « 340 300 260 220 180 140 100 60 20 168 Figure 70. Comparison of blood magnesium ion levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 25°C 140 2 ^ Z 66.0 62.0 58.0 54.0 50.0 46.0 42.0 38.0 34.0 30.0 26.0 22.0 18.0 14.0 10. a 6.0 2.0 VA- ACCL 25''C TEST 32»C / <'"'"■— ^ -''' ^z" .^■ .X' y / I I / 367o, ^257o» - *r •H^ ^,1-'' CONTROL 157. 0 12 -//- _i I l_ 780 740 700 660 620 580 540 500 460 2 420 « UJ z 380 <2 340 300 260 220 180 140 100 60 20 10 16 24 48 72 96 TIME— hours 168 Figure 71. Comparison of blood magnesium ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 25°C 141 66. 62. 58. 54. 50. 46. 42. Id 38 E i 34 CO W 30 I 26 22 18 14 10 6 2 -ff- ACCL 25''C TEST le'C /X 36%, 0 12 10 VA- 780 740 700 660 620 580 540 500 460 S. 420 f^ 60 20 16 24 48 TIME -hours 72 96 168 Figure during 72. Comparison of blood magnesium ion levels of Penaeus aztecus adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 25°C 142 66.0 62.0 58.0 54.0 ACCL 32°C TEST 32°C 0 12 10 16 24 48 72 96 TIME -hours Figure 73. Comparison of blood magnesium ion levels of Penaeus aztecus during adaptation to various salinities at 32°C. The control conditions were 15°/ooS and 32°C 143 660 620 580 540 500 460 420 380 2 340 CO UJ 300 z < -//- ACCL SgoC TEST 25»C -.'//' .^'^ .^' I / ,^' 36%, 257o, - 1240 1180 1120 1060 1000 940 880 820 760 700 2 to 640 580 W CONTROU ^ l5%o ■JJM~'^^"--j VA 520 460 400 340 280 220 160 100 40 Z < 0 12 4 6 10 16 TIME— hours 24 48 72 96 168 Figure 74. Comparison of blood magnesium ion levels of Penaeus aztecus during adaptation to various salinities at 25°C. The control conditions were 15°/ooS and 32°C 144 The ionic levels were controlled more effectively in 25 and 36°/ooS at IS^C (Fig. 75) than at 25° or 32°C. The magnesium ion content increased about twice that of the control in 25°/ooS and about four times the control in 36°/ooS. Moreover, in both media new steady states appeared from the fourth day onward. The changes in low salinities were no different from those tested at 25°C. The magnesium ion content in animals acclimated to 18°C (Fig. 76a, b,c) was similar to those acclimated to 32°C. Hyposmotic ion levels were present in 2, 5, and 10°/ooS at 18°C (Fig, 76a); vari- ations in the ion levels between these salinities were not sig- nificant. In 25 and 36°/ooS the ion concentrations were hyper- osmotic. However, these hyperosmotic levels were well below the levels of animals acclimated to 25° and 32°C. At test tempera- tures 25° and 32°C (Figs. 76b and 76c, respectively) magnesium concentration in 36°/ooS dropped below the level of 25°/ooS. The salinity response curves became more widely separated from each other with the increase in test temperature from 18° to 32°C, thus indicating a possible salinity and temperature interaction effect on the magnesium content. Potassium concentration The potassium concentration increased with the external sa- linities but not proportionately. With the exception of 2 and ^(s J2^°/ooS, hyposmotic and hyperosmotic ion levels in the test sa- linities were not maintained consistently when there was a change in water temperature. Shrimp acclimated and tested at 25°C exhibited hyposmotic potassium ion concentrations in 2, 5, and 10°/ooS and hyperosmotic concentrations in 25 and 36°/ooS with respect to the ion levels in 15°/ooS (Fig. 77b). The ions were held at more or less consistent 145 66.0 62.0 58.0 54.0 50.0 46.0 42.0 _l S 38.0 E I 2 34.0 UJ 30.0 z § 26.0 -y/- ACCL 32X TEST I8''C /A' -V/ 56%o ._ 25%o: 5 /oo J 1 I I L. -//- 1240 1180 1120 1060 1000 940 880 820 760 700 2 64 0 ''' 580 O < 520 ^ 460 400 340 280 220 160 100 40 16 24 48 72 96 TIME -hours 168 Figure 75. Comparison of blood magnesium levels of Penaeus aztecus dur- ing adaptation to various salinities at 18°C. The control conditions were 15°/ooS and 32°C 146 22. 18. 14. 10. 6. 2. ACCL IS'C TEST 32^. / ^^i?f.iji»Sii9Ri«iktJir.?.rT.-.rTT; .^^^' /> CONTROL ^^ 36 «>o i5%r' - c :.-.:T:.-.atAfi»A»»',A»»»»jw4>.«'"*~*' 27o< 0 12 10 ' //- 16 TIME -hours 340 300 260 220 180 140 100 60 20 24 48 72 96 168 Figure 76. Comparison of blood magnesium levels of Penaeus aztecus dur- ing adaptation to various salinities at 18°C (section A^of the figure), 25°C (B) , and 32°C (C) . The control conditions were 15°/ooS and 18°C 147 14.0 12.0 10.0 8.0 6.0 4.0 14.0 E 12.0 I 3E 2 10.0 to CO < 8.0 V/- ACCL 25°C TEST I8°C 25%o CONTROL t' c-^ /oo \^\ji^_i_i^^^^^_^^_^,^_^ _I I u -//- O 0. 6.0 4.0 14.0 12.0 10. 0 8.0 6.0 4.0 -//- ACCL ZS-C TEST 25"^ /A- ■ S5%t/A- 15%, 36%. i ■' — "Z^ — r."" T.rjV,'^ *^ •5*' V • 5% CONTROL' "). — 2^ v>- V/^ ACCL asx TEST 32»C 36%, _!_ 0 12 10 ' //- 16 24 TIME-hours 160 140 120 100 80 60 40 160 140 120 ^ 100 to to i5 80 5 a. 60 40 160 140 120 100 80 60 40 48 72 96 168 Figure 77. Comparison of the blood potassium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure) , 25°C (B) , and 32°C (C) . The control conditions were 15°/o°S and 25°C 148 levels in 2, 25, and 36°/ooS throughout the study; but the ion levels increased from ten hours after transfer in 5°/ooS and after the first day in 10°/ooS, both becoming hyperosmotic by the end of the test period. At 32°C test temperature (Fig. 77c) the tendency was to maintain ion concentration in all salinities well above the levels at 25°C. As a result, the ion concentrations became hyper- osmotic to levels in the control animals from the second or third day and continued so thereafter. In 18°C the trend was reversed (Fig. 77a) and the ion levels in the respective salinities were generally lower than in 25° and 32°C. Except in 36°/ooS the ion levels were hyposmotic from the third day onward. The ion concentration pattern of animals acclimated and tested at 32°C (Fig. 78c) was similar to those acclimated and tested in 25°C (Fig. 77b). Hyposmotic and hyperosmotic ion levels in the different salinities remained unchanged. However, when the test temperatures were lowered to 25°C (Fig. 78b), the potassium ion levels decreased gradually in 25 and 36°/ooS and increased in 5, 10, and 15°/ooS. Some other important differences appeared be- tween these two test temperatures. Tlie potassium ion concentra- tion levels were lowered in all salinities. At 25°C the ionic levels were hyperosmotic to the level of the control shrimp in all salinities except 2°/oo. On the other hand, at 18°C the ionic concentrations were hyposmotic except in 36°/ooS. Acclimation to 18°C improved the potassium regulation. The shrimp tested at 18°C (Fig. 79a) maintained hyperosmotic ion levels in 25 and 36°/ooS from the beginning as they also did at 25° and 32°C (Figs. 79b and 79c) in 5, 10, and 15°/ooS. In 2°/ooS the potassium concentration was hyperosmotic after the first day. It should be added that the potassium concentration level of the control shrimp at 18°C was lower than in the other control temperatures, 25° and 32 °C. 149 16 TIME— hours Figure 78. Comparison of the blood potassium ion levels of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B) , and 32°C (C) . The control conditions were 15°/ooS and 32°C 150 14. 12. 10. 8. 6. 4. 14. _l o- 12. LlI E 55 n to ° ■ 2 6. -y/- ACCL I8»C TEST I8°C ^ 27. -//- 200 180 160 140 120 100 80 60 4.0 - ■VA- ACCL IS^C TEST 25''C 25%, '^C^^ _-^-s^> — r ^ — CONTROL B _l L. 14, 12, 10, 8, 6, 4. 200 180 ss 160 140 V) 120 ^ o 100 0. 80 60 ACCL I8°C TEST 32"^ 25%. 36%.' ^: — '''^ x'''5%o 5%, CONTROL ■y/- 200 180 160 140 120 100 80 60 0 12 10 16 24 48 72 96 TIME-hours 168 Figure 79. Comparison of the blood potassium ion levels of Penaeus aztecus during adaptation to various slainities at 18°C (section A of the figure), 25°C (B) , and 32°C (C) . The control conditions were 15°/ooS and 18°C. 151 Osmotic and Ionic Regulation in Relation to the Isosmotic Line In Figures 80 to 84 the isosmotic lines represent the concentra- tion levels of the respective ions in the external test salinities ranging from 2 to 36°/ooS. The figures were drawn with the mean os- motic and ionic concentration values in the different experimental conditions. The mean concentration values were computed from the new steady-state levels. In some conditions new steady -state levels were not established. Mean stabilization values were used in such cases. Osmoregulation The osmoconcentration values for brown shrimp acclimated to 18°, 25°, and 32°C are shown in Fig. 80. Regardless of test temperature, blood salt concentration increased with increasing salinity. How- ever, the osmoconcentration appeared to be more efficient in the range of 5 to 25°/ooS than in other salinities. Within this sa- linity range the blood osmoconcentration was subjected to relatively less change in relation to the test salinities. Outside this range there was either a greater salt efflux or influx. The blood salt concentration was isosmotic at about 25°/ooS level or slightly below. Shrimp acclimated to 25°C showed less osmoregulatory variation in response to temperature changes. In contrast the shrimp accli- mated to 32 °C became more temperature sensitive. The extent of the sensitivity to test temperatures was determined on the basis of separation of the temperature related osmotic curves from each other. Moreover the osmoregulatory efficiency seemed to be im- paired more in dilute salinities than in other shrimp acclimated and tested at 18° or 32°C. These animals experienced a sudden drop in the salt concentration levels in salinities below 10°/ooS. At 25°C a similar drop occurred in 2°/ooS only. 152 "" 1 1 1 1 1 ■ T I I ^^ o o < 1 L __, 1 1 1 1 1 1 1 L. 1 1 I I 1 — 1 — A - r o o !/1 O c 3 o .— I •H 03 +-> > « 03 fn i-H C e oi if> C •H 1-0 o 0) c u <; XI T3 c c O o ctf o o ■ 1 — 1 o t/i x> e C o to O LO 0 o •H CNl -C ■P ■M o •H - JZ 13 o c H C 00 o O — 1 o • 13 W) (U (D q — c c > 03 Q. n3 •H •H °- X 1— ( -t-" CO 1 o O o >- o 0) 0 t- ■^ •H CL,^ s ^ ■l-J t/) o 2 3 o OJ LO _l -M fc; ^ -H < o3 (/) CO U O (D ^4-1 B (/) q X q O »-H i-H nj ^ 03 ■!-> t/5 !h I/) q 1 o M-i C X a O •H 13 03 S +-> t/1 u (t) tj m t+-i M^ O M-i +-I q 13 UJ M 03 (1) 05 O 4-> • f) (U O 3 +-> +-> CO aj q 03 (B (U c (/) ^H 0 o 3 o. u W) p. •rH 1+-I o O O -H 00 ^ +-> rH X (D O o jz: 0) -o H CI. C T3 (/) c3 o (u O • !h CO >— 1 (U o XI C o c ^ •H -H C -M t+H O O t/) o e -H (U t/l 0) t/) M O > c C ui OJ O 03 tH .— 1 .H X +J r- o 4-> c —1 O C <1> C 03 -T3 ^ o3 E C Do 03 c e ^-1 ■H l4-l X .-P ■!-> I/) c -a •H 3 CD (U c o !/) 4J •H <1> CU t/) ^ -M ^1 03 3 O C •— 1 !fi (D (D 03 ^ M-i Cu > o! ^ e m <+H c -H o o c •H < • C 4-) ^ O 03 00 -H f-( . ■!->+->(/> \ o^'-^V. fH\ ^v:^ ^ ^i\. xw\ < *■><..... 1 c t/1 o c u O O o C -H CM o e 4-> K^ •H 3 -H •>H -o -a e o c c 3 •-I O rt •H rt o o o ^ 0) 0 Oj 0) > LO U 4:: -H (N H •!-> -o 0 - 0 00 0 • Cuoo 1— ( 0) Ul r-l X) C 0) •H >H t3 (U -H c X (D OJ •■pox: •H +J (/) C •(-> 0 0 0 C 0 e -H ^ (U VI 0 tJO 0 tfl LO C tn i-l r-l rt -H 0) 0.x; > 4-1 S 0000 1 x ^ >. 0) +-> t/) (- '-' < !-i C (/> -r-l (O OJ 0 1 -13 P.T-1 X C e -t-" -d 0 (U nJ 03 0 •M ^ 0) 0) +-» ^ -0 (-1 tn 0 C fi ca c c 4-> •H 03 C X , 0) 0 +J Ul 6 0 •H 3 C 0 ■M e •H (U !-( If) 4-> 03 OJ 0 C t/) 4-1 3 • C C to (M 0 0 ■— 1 00 -H -H 03 ■i-i -i-i e c f= •H - w ■M o 03 o C o ■H •H LO c ■W t— * o o in e -H U-l a) lA (U o M o > ^ c £ p p H <1^ +-> 03 -H 1 ^ ■P T3 O W C -1 4-J ■!-> 1 O < 03 X O w ^ c T3 o o 03 r—i p^ ■H ■P Si o 03 m p +-" f-H C Ol c o T3 ^ 03 O C tu 03 c B B ■H O X +-) ^ ■P !/) C '+^ ■r-H 3 (U c u i/l TD •H (U O 03 3 a> O C -1 3 OJ !U 03 M-i G. > in <+-( .-H PJ 4-1 C 03 O O B • c P C K5 o 03 < 00 •H U ■p P OJ 03 C • Vl r-H 0) tn D 3 o c w; OO C O •H (D O -H U. ^-1 U P -1/b3LU-wniS3N9VW 158 The magnesium regulation was apparently very efficient in all salinities in animals acclimated at 18°C. Temperature change to 25° or 32°C did not alter the efficiency significantly as in other ac- climation temperatures. Potassium regulation Blood potassium increased with the test salinities (Fig. 84) . The ion regulation appeared very efficient in the range of 5 to 25''/ooS in shrimp acclimated to 25° and 32°C and tested in 18° and 25°C. The ion influx was, however, not effectively checked in 25 and 36°/ooS at 32°C. At 18°C potassium was present in the lowest con- centration of all temperatures regardless of the acclimation tem- perature. The ion concentration increased progressively with test temperatures 25° and 32°C. Animals acclimated to 25°C were less sensitive to test temperature variations than those acclimated to 18° and 32°C. These shrimp exhibited greater variations with tem- perature. Tlie ion concentration was hyperosmotic to the isosmotic line in salinities of 25"/oo and below. Interaction of Salinity and Temperature on Osmotic and Ionic Regulation The effect of temperature change on osmotic and ionic regulation was explained on the basis of the responses observed in shrimp accli- mated and tested at 25°, 32°, and 18°C. The control mean blood os- motic or ionic concentration levels represent the values in 15°/ooS in the respective acclimation temperatures. Some references were already made in the previous topics on the impact of temperature on the regulatory process in brown shrimp. However, the major trends of temperature effect will be reported in this section. Osmoregulation At 25 °C brown shrimp maintained a lowest salt concentration (643 mOsm) in 15°/ooS of all temperatures (Fig. 85). The concentra- tion levels were 657 mOsm at 32°C (Fig. 86) and 674 mOsm at 18°C 159 ■; T r 1 1 r ' I I -■ T I I F « *. • S^>\\ ^ y\ cvj\ "\ x5 o (U u U-1 U C "p 1>] O C -H T3 O +J (U •H 03 p ^ ui B +-- (U in 3 C ■P ,—i ■H (D tn o <; - V£5 O o m a> Ph LO X • rsi +-> (D Ul ,c: C - C H O o O •H 00 .^ P i-H in a. (U . •H Ol DO 0) T3 -73 1 c c c c >- 1- 03 -rH O 03 ^ ^ O O CO z O (U o _J (U -H > o in < ^H -P •H ~^ •—i CO 3 O P o +-> B O LO 03 (/) (D rH o ^1 O Oh f-H O m ui Uh Ph-H C -H 1^ •H t3 m X C c ■P o (/) o • H -H -H a C P a> • H 03 > --I 1— I .-H (D O 03 ^ t^ t^ t-~ IS) ^D iO -* o ^ CM IP 'S> o o o o o o o O CD iX) "d- CM O CO (X) ") U~) iD iT) ID "^ 6 O Q o CD ^ ^ OJ Cn uo in i'^ .:loo im ^11^ s K \ s / /! / CM co 'J- CM to o — UJ ID CM o o o o o o (D o o t« c o a. B !/) O 0) !h U '4-1 x-o ^ +J t/1 03 (D ^ ■!-> 3 oo (u u (D fH o fH (U LO O S r^i E 1/1 t/) "3 O -H C oj 03 e H to C o D < o tn ^^ !-( o 3 • LO O (U ^ O W) C O 03 o c+^ O C o If) U 3 ■!-> 03 U D e u e o o u +-> c • o LO -H 00 ■(-> a) 1—1 !-( 0 3 U ■H C ujso"-AinV"IOI^SO 161 o CO o O o O o o <0 fCO tD \f (M <) (X> <.D ID is>r> IT) lo If) in ■^ rt c CO •H O o to -^ 3 3 SI in 3 (D CO C a. o v> c o -a c c o •iH O t/l I— I (U o t/1 f-l C 4-1 o c &I o t/l o B in T-i 3 < O •H c ■!-> 03 4-> a> M-i 3 O ■!-> 03 C " o to ^1 o e (L> aJSQUJ-AlllVlOk^SO ca &, e (u o X u -P o • -p 00 C o 3 03 00 .-I • H (U 162 (Fig. 87) . The criterion for efficient regulation is the ability of animals to maintain relatively steady osmotic or ionic levels in re- lation to large changes in the test salinities. On this basis, the osmoregulation at 25°C appeared to be more efficient than in 32° or 18°C. In 2, 5, and 10°/ooS the animals tested at 25°C and 32°C reg- ulated the salt levels more effectively than in 18°C. In 25 and 36°/ooS the osmoregulation was relatively more efficient at 25° than in 32°C. In 36°/ooS brown shrimp were less efficient in controlling the influx of salts at 18° and 32°C. At 18°C the animals tended to lose the salts initially to the dilute media. Subsequently, the losses were partially recovered with the result that the final steady- state levels were similar to the levels in 25° and 32°C. Acclimation to 32°C seemed to impair the osmoregulatory ability, particularly in 2 and 36°/ooS (Fig- 86) throughout the test tempera- ture range. The animals tested at 25 °C recovered from the salinity stress in 2°/ooS faster than in 18° or 32°C. At 18°C the shrimp failed to control the salt influx for 24 hours in 36°/ooS. In this salinity the shrimp tested at 25° and 32°C appeared to be better regulators than in 18°C. But the regulatory fluctuations were larger in 32°C than in 25°C. Acclimation to 32°C, however, conferred cer- tain advantages to the shrimp in 5 and 10°/ooS and 32 °C combinations, particularly in 10°/ooS. The salt concentration levels at 18°C were relatively low in most of the dilute media. The shrimp exhibited better regulatory efficiency when accli- mated and tested at 18°C (Fig, 87). This was shown from the fact that the shrimp did not experience the initial rapid salt loss in 2, 5, and 10°/ooS as the shrimp did when acclimated to 25° or 32 °C and tested in 18°C. The osmoconcentration levels in these salini- ties were maintained on par with those in 25°C; but the shrimp ex- perienced severe osmotic problems in 2 and 36°/ooS at 32 °C. In 2°/ooS they failed in controlling the salt efflux effectively. In 163 \ ■ a CX) ID 8\ / / \ ^\ 51 S! ^1 / 11 / I \ OOOQOOQOOO m M t— ( crt U-i t/) o 3 OJ 01 rt c C o 0) •H a. +-) o t/1 I— I c o O ^1 CI, 4-> t/) c (U o ^ o X e ^1 o o u ^-> <+j nj ^ Td M 4-1 (1) t/1 W c O ^H 3 ■!-> rt J-i (I) Oh B C o (/I •H 03 PL, (D e -p o *-> r- o 00 ■(-) •H 03 164 36°/ooS great fluctuations occurred. However, the shrimp tested at 25°C were consistently efficient osmoregulators in the entire sa- linity range. Blood chloride ion The temperature effect on chloride ion regulation was similar to the osmoconcentration in brown shrimp acclimated at 25°C (Fig. 88), 32°C (Fig. 89), and 18°C (Fig. 90). The chloride ion regulatory pat- tern in 2, 5, and 10°/ooS was similar in 25° and 32°C. The ion loss was relatively less in these conditions than in 18°C. Temperature effect was not significant on chloride regulation in 15 and 25°/ooS. In 36°/ooS responses were similar at 25° and 32°C on the first day of transfer. From the third day onward the animals from 32° and 18°C responded alike in 36°/ooS. Acclimation and testing in 32°C (Fig. 89) improved the ability for chloride ion regulation in 36°/ooS over that of shrimp accli- mated to 25°C and tested in 32°C. However, animals acclimated to 32 °C experienced considerable disadvantages in 36°/oo and 2°/ooS when tested at 18°C. Temperature effect was not seen in 10, 15, and 25°/ooS. The shrimp tested in 25°C and 32°C responded quite similarly in all the salinities except in 5°/ooS. The chloride ion regulation improved considerably in animals acclimated and tested at 18°C (Fig. 90). The acclimation also made it possible to maintain nearly the same ion levels in 2, 5, and 10°/ooS as in 25°C, without the initial rapid loss. The ion regu- lation of shrimp acclimated to 18° and tested at 32°C was not im- paired much except in 2°/ooS where there was a rapid loss of chlorides during the initial four hours. In 25 and 36°/ooS the ion regulatory pattern was much the same in both 25° and 32 °C, 165 in •.fO /- / f I 1/ ll - M - /V r- -T r- 1: ; ~r- o' 90 J ,(\J in 1 : 1 " \\' - ^ H 1 . r ''^ 1 •v n . oili f^ii n 1 1 - 1 } i^e q: i^ - •••iki 0 6 Q 0 CD '^r -^ eg 0 m ro ro K) OJ CM CM I ■ 1 1 o. in Is O 00 u) ^ cd § 00 ro CM '" "' "' - — I o o I :' h J -- ^ ~ w-~tD^CM CMCMCMcO~ rOCMCMCMCM n/b3UJ-3aiyo"IH3 V 1; O I ' ijl r 03 (X> CD CD CM CM !2 o CD CM o CM O CD ro ro CM CM CM ^ 8 § o ni I— ( U o O QJ o m o c •H -H C -H o -o •H C •M O 03 U I— ( D '-< tiC O (U f-i U ^-J C C O O O 0) o •H 4h O 13 .— I 1) X +-> a u) o X . ■(-J aj bn ^H C 0 rt — CO H o t/) (U •H ^ a. 03 s o u &, 00 00 u 00 ^\ 0^^" lOl CM 1 h vi ! ' - 1 vi 1 CM ; 1 "J 1 - d .o: 1° 1: 6 6 6 CVJ O 03 ro ro CJ 1 1 \ 1 1 r — I- I 1 i" / 1 / / V, " \ : ^, \ - I \ - 1 ^ \ 1 ^ : ' "^^ \ 1 I \ i 4, J J. ^ • T > > I : I I I j I : I : I ! I I I I I 1 I i I : I : O § CM 8 S po ro ro <>J 5^° CM I i r- I « S'l S CM\ \ \ \ • 1 1 1 \ • - \ ; \ / . \ : 1 "i W \\ *. / \\ _ O Q O "^J- CM O CD CM CM CM — '66666 6 gd CD ^ CM ro CM n/b3uu-3aidOnH3 o rt ^1 o M-i o c o O 13 •H C ■P O oJ O r-H P r-l OJ) O 0) i-l U +J c c o o o •H e O o -T3 ^H •H U-t U O 13 •— I a> o to <+-! •(-> o o u ^ s 3 O W 05 •H C iL o to ^ forOrOCVJOJrOrorO O O O CM Q CD f^ CM O O O O O O O CMQOpl^'^CMOCO ^ '^ to ro ro to CM CO (0 c o •H ■M nj t— ) ^ U o 00 ^ C -H (T) V) C ^ 03 ^ 52 0) ■!-> CO o 1 rt o o 1 t/) lO (C 1 0) i-H •? 03 <+-l C o h- O 03 U U) d --I 't (0 O t/) se of ere te 10 U 5 CD O 1/1 ^ O r-l en B -H ^ •H C ^ 3 5 •M 0) 00 ^ c O 03 c u o O^ 03 E fH O 1/ '1 - /\ / n - r ^^ V 1 . \' - \ K - i\ . - 1 1 1 J 1 J ; "T 1 1 T in s to CO t^ 00 o I LU CD - "^f O O Q O Q O O O 00 U3 rf CO o 00 K) C\J OJ CO CO CO ~ I l^ I I I I V li l/\ l\ I I I I I 1 1 it 6 A 6 6 6 ^'6 A 6 6 A' ' A 6 A A COOOOtD^COcJOOOlD'T (VJciOOcD COCO— COCO~~~ CM^j— — c o •H ■l-J nj I— I f-i o CA) +-> C -H O T3 •H C ■P o 3 --1 CO M O (U U U +-> o c c o o o E e o 3 ,f^ m O '0 --I T3 of ca teste CD t/1 (-1 0) ^ • CD 1 O C O tJ LlI t/l > ■H (U o 1- Compar eratur ID a. E ■—1 4-> ^ CTl CJ O ^1 3 •H a, "l/b3uj-y^jnionvO -T 1 ■ 11 . -o-l T \ \ 0} W ' U' J ~ j _ 1 c (E / H 1 f^ • o ^i • \l w 6 6 6' •.CJ I- n in (M \ I V I I I I I I 1 •A I > o CD ro S / . I "i / • 'O : - ^ CM C\J CD O O Q U) '^ C\J 00 CM CO O Q. O Q Q _ eg ^ CM O > \ \ \ \ Vx S / rO ro cn CM « o CD I Q CO CM (5^ CO rj- CM CM CM CM 6-^ ?3\ &; o in \ \ \ 6 6 6 6 6 6 6 CD -cJ- CM O 00 Cfi '3- CM CM CM CM \ i I I '1 I I '( /■ ]1\ f / z o J /■ \ jj o O CD CM O 00 CM c N D 0) C 0) M-i O ui C o o -o ■H C ■M O 03 O . — I 3 — DO O (D 7> U U (U !-( 3 n/b3iu-;^ni01V0 r "1 r- CO ~l 1 in 1 i 1 : 1 I \ 1 1 // pj . X / \ / 1 / - U i 1 h > > > ' » 1 1 1 1 1 6 o o o o (\j O 00 ?! i / / / I in c\j '> > > ill 1 w \V\ \ \ c q: H u •■■v^ ' u >i*^'-' CM C\J C\J <\J 00 CVJ o o q O Q Q (£1 ^ C\J O 00 to CJ CO CM OvJ ~ ~ si CVi, 1 1 \ ■ \ •• l^ 1 \ I I \ Aj I r I J—. Q O O Q CM O 00 10 , ro rO CM CM CM CM CD CM ^ J2 ^ ui CD CM O <^ cvJ S 1 1 1 1 1 ■^ •. ~| -1 CM \ 1 • 1 \ N . • L 4. i.-l 4. 1 i 1 i a: h- c — J — i^ 1 1 1 o o o o o <^ CM O 00 U3 ^ CM CM CM — — — -1 1 r J- in 6 6 6 6 6 cS ^ cM o 00 CD 'd- CM CM CM — — — o o o o o o o CD ^ CM O CO IX) ^ CM CM CM CM — — ~ CO 3 O ca J-l o 00 t/1 o M (U C o c c o C -H O TJ ■H C i-> o 03 O 1 — ( 3 ^ bO O (U ^ ^1 4-> C (= o o u •H e s o 3 U ■ H <4-l u O -M t/) O -P 0) (U !/) ^-1 5h (1) 3 S O O (/) 0) rt e e •H -H ■P C O 3 r-H W) O 0) fH !h +-> c C o O o H B K o 3 ^H H U-1 t/l o Tl c 03 tfl E d) -|/b3LU-tMniS3N9VlAi O 0) u tn 3 !h 3 t/l O ^ o rt 6 (U -r-l E C •H <: (D . j:: (1> +-1 at) C CH 03 O X o c O (U t/) f-l •H 3 Vh -P 03 03 Ph ;-! e I I I I I I I I . I V \ \ i \ ^-■-...^ -->^ 6 6 6 6 6 6 6 ■•Pi V^^. col ~l / ^ \ "-^ / \ f \ \ \ \ \j' Sfe..S o to ro CM CM CO CM o q o -^^ O Q Q ^^S^S^SSS^^^SS* Q O CM O O O Q CO 3: Q CO CO CM 52 3 O CM O O 1\ / 11 f // • / r o o o o o o O CO CD 'd- CM O < I I I I: I in SP I: I I - / \ I O O O Q O 00 CD ^ CM O O O O O Q O CO CD '^ CM — 1 — ^^ — ' ' — o o o o O 00 CD ^ CD CM 00 ^ CD - CD CM -I o Q O CM O n/b3UJ-^MniS3N9VlAJ o C 13 C 03 in 3 u tu ■p CO O c o C -l-i c o •H +-» M 0) C o T3 C o o u c o o e o e <+^ D ■ H 13 1/1 CD C W DO -.i[ Q CVJ O Q O CO CM loi I J / -1 1 1 1 1 1 1 1 1 1 1 r ?i ( ^ I r I / \ ■S CD ■\ \ I \ \ M 3 O CO Q6c)6Qci)666666ci>66666i» ^i d ^ 1 1 1 . 1 1 1 1 1 1 / / ( / ■ / ;• i 1 1 / 1 ._I L. o CD O CO o q 'J- cM 06660 CM O CD CO 'i- c o ■r-i U ■»-> O nj 00 •—< 1— ( D u -a c C a! •H CO t/1 o 0) p ■I-' nl LO o (/I c o C XI •H C o c o o ■H .-( ■M O ■—I •»-> 3 c 00 o a> o t^ e c o o u •H tH e -T3 3 a> •H ■!-> t/i 1/1 E U M C 3 ■(-> oJ (U e o u o o c o •H f-1 03 U 3 l/b3Uj-iftiniS3N9VW 100 80 60 40 20 120 100 80 60 120 ^ 100 u e 80 i 60 i 120 CO S 10.0 -//- CONTROL szx — //- -/A- _252C 2%oH CONTROL ■■jiffMiiyy^ ***"* 32^. I8»C VA- _1 L 5% oo VA- CONTROL -I8»C O Q_ -//- -//- _1 L. 10% 80 60 120 100 80 160 140 120 10.0 80 CONTROL 32»C 25«C ■""" y-' .^^ :;;: ^ -//- -//- I8*C -I l5%o / / --*'""-Zm. — — • -^ —^ " CONTROL ,J8!£ _1 I I I L. 25°/< otf -VA- -VA- r'A- _25*!£.. I8°C J^ " "CONTROL VA- 367c oo -/A- 0 12 4 6 10 16 24 48 72 96 TIME- hours 168 Figure 97. Comparison of the time course of potassium ion regula- tion in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 25°C increased from a lowest level in 18°C to a highest in 32°C. This pattern was seen throughout the salinity range. Animals tested at 25°C responded like those in 32°C in most of the salinities. No significant deviations were seen from the above pattern in shrimp acclimated to 32°C and tested in 18°, 25°, and 32°C (Fig. 98). The shrimp tested in 18°C maintained a lowest ion concentration in all salinities. The important difference was that in 25°C the potas- sium levels were highest in 5, 10, and 15°/ooS. The shrimp tested in 25° and 32°C responded almost identically in 2 and 36°/ooS. In shrimp acclimated and tested in 18°C (Fig. 99) the potassium levels did not increase from the previous lowest levels. The low potassium levels indicated that by acclimation to 18°C the shrimp derived no advantage with respect to altering the potassium regu- lation. In 32°C the shrimp held the highest ion levels. However, the shrimp acclimated to 18°C became more temperature sensitive. This was indicated by the wide separation of temperature -related response curves from each other except in 36°/ooS. The animals tested in 25°C responded similarly as in 18°C in 2 and 36°/ooS while in other concentrations they reacted more like those tested in 52°C. Effect of Sex on Osmotic and Ionic Regulation The results of t tests comparing mean levels of blood osmolality and ionic concentrations in male and female brown shrimp are given in Tables 2 through 6. Significant differences (P=0.01 or 0.001) were noted in a few isolated cases but there was no consistent pattern. It appeared, therefore, that sex of the shrimp within the size range used in these studies had no effect on ionic or osmotic regu- lation. 178 100 80 60 40 ■y/- CONTRQL -/ f- 2% 100 80 60- 4.0 -y/ — I 1- -y/ 25«C - CONTROL ..//■• •35»e" I8»C J ^ ^^ 1 L. 57oo-l 100 o.U- T^, -f /- 3gy„ IS'C I0%o- 0 12 4 6 10 16 24 48 72 96 TIME -hours 168 Figure 98. Comparison of the time course of potassium ion regula- tion in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 32°C 179 cr E I O a. 6.0- ''t CONTROL 40 57c oo IZO 100- 80- 60 40 _l I I L. .//•• ^"^""SyJTROI " 25»C I8X -//- I0%o- 120 100 80 60 -//- -//- h^-^control" ^'^ , 25*^ -y/- 15% 0 12 4 6 TIME— hours Figure 99. Comparison of the time course of potassium ion regula- tion in Penaeus aztecus in relation to temperature change. Animals were tested from control conditions of 15°/ooS and 18°C O 0 tn !/) !/J t/5 t/) Ul c c c c c c rsi ■^ ^ ^0 CT> CTi CM 00 -^ r^ rvj r^j LO un vO vO t^^ o3 .— I Ln 00 r~- 00 t--- t-o Oi ^ r~- r-j r\i tn LO \D vD r^ 00 to t/i t/) t/l t/l !/l c c c c c c o 00 00 -^ •* r^i ho t-H to LO o Ln LO \D >.0 O l~~- 00 rH (7l (Nl r~- (Nl LO (Ni o '^ un ■— I vO L/5 vD -£) vO I~^ 00 oj rsi CM rj r-j rg ^O t-0 t-O t-O ^0 to (O (/)(/) t/i t/i t/i c c c c c c \£3 ^ t'^ Ln r^ CTi LO t^ "^ LO 00 00 lO LO v£) vD vD r-~ r^ 00 -^ CTi o o ho \£) to Ln cr> o Ln LO >J3 vD vO CO CM LO O LO LO v£> 1— I I— ( (Nl to LO UO LO LO lO l-O rsi CM r4 r\i CM CM c o o O 00 CN CM r\l rsi r-j rsi to to to to to to (/) t/l t/) t/1 I/) tf) c c c c c c ly) to LO \0 rH .— I (M ,— I rsj to en "* LO \0 O vO ^ CO r~~ 1— I o t^ o^ ■— I to •— I to rf 00 to LO \D ^ ^D \0 00 (Nl LO O LO LO vO ,— ( ■— I (Nl to LO lO lO to LO LO CNJ to ■— I O ^ i-H to (Jl 'd- O t-O LO ^O \D ^O 00 c o LO INI un o to to \0 ~^ r-i I— I CNi to u o CM 00 00 00 00 00 00 r— t T-H .-H t— ( I— t t—t • u to -^ CTl to CO ^ o ■* r-- .— I r^ o to LO to >£) O CTl (Nl LO O LO LO \D ,— I .-H (Nl to 00 00 00 00 00 00 03 o • H (4-1 •H c DO O to c t/i O (U ■M N 03 3 (1) 03 C (U o 3 CO D Oi (D -a •H o -a o o c o o (1) UJ X +-> 0) •P to H o —< o 05 --^ CO o +-> Dh t/1 E U (U CDO H H +-< •M t/i ■1-1 lU J X C3- UJ B CD J 03 e u. X CT UJ B 4-J o CO o H ^ 0 ■M Ph W e u CD ID ■M J 03 X O" s UJ o t/1 "5 -v" +-< CL. in B U CD OJO H H c C/l C II 1/) c C o i-H !7i o CM O ^0 \0 1^ .— I to l^ C7i 00 ro I— I \0 00 (7^ i-H vO (N (N CM CM CM CM to to CM LO O LO LO \0 .-H r— I rM to (M O (D <+-! OJ X 0) (1) ix cr w UJ t/) o ■— 1 0 CO o +-> Oh t/1 B U (U a>o H H Ix cr (1) .-I ; ||x a- CO o O^ IX Ol 6 ||x 0) •M Oh c O 03 O o 03 U t/5 t/1 (/)(/)(/) O c c c c c • II CI. CN r^j r-i fo r^ r^ \0 CM o o to ^ ■— I rsi r-j rsi CM tN .— t \0 •— ' O 00 OnI r- r^4 o r-H ro ■* ^H r\i CM rsi rg rg (N Ln O LO LD >o f— I f— I CM hO rsi CM CnI OJ rsi CM to to bO to hO to (/) !/5 (/) t/l (/) t/i C C C C C C Ol to O to CT) '^ CTi vD 00 O 00 vO .— I f— I .-H (N I— ( r\j (Tl rH LO 00 Ol CTi cn r-- 00 cr> CTi vo ^H .-H t— I .— I t-H r\i rsi Lo o i-o Lo vo .— I .— I rg to LO LO LO LTJ LO LO (Ni rg (Nj rsi (Nj r^ (/) t/i ui t/i t/i t/j C C C C C C 00 Ol rH -a- O 00 r^ 00 o to o> to r-< rH r\i (M .— { CM o 10 (/) (/) t/l (/I t/l c c c c c c CO ^ oo •^ Tf Ol \0 Ol 00 00 CN LO .— ( (-H I— ( r— ( (N] Csl tN 00 to \D rg t-- Ol 00 Ol rg ^O rH I — I r— I .-H OJ rg CNJ LO O LO LO ^£> rH 1— ( (Nl to rj rg rvi CM rsi rM to to to to to to t/i t/5 t/i o in w c c c • c c II OJ Ol to to 00 00 O Ol 00 ^ LO I— I Ol r— I t— I OJ Ol vO LO t~~- -^ O l~^ O Ol O ^H l-O ^ Ol .— ( Ol Ol OJ Ol LO O to LO \0 r— I f— I OJ to LO LO LO lO LO LO OJ Ol OJ Ol OJ Ol to (/5 t/l t/5 1/5 t/5 c c c c c c LO CO v^ Ol Ol o vO l~~ 00 Ol ■— I LO f— I .-H T-H .— I Ol 04 c o ■p 03 t/1 t/) (/)(/) l/l t/1 q c c c c c Ol vO CO LO Ol Ol Ol Ol rj Ol LO .— I Ol Ol Ol Ol Ol vO vO vD Ol Ol \0 r^ o Ol Ol to -^ r-l Ol OJ Ol Ol Ol Ol LO O lO LO \o rH ^H OJ to OJ OJ Ol OJ Ol Ol to to to to to to q C C C C C \£) O Ol vO LO LO Ol 00 r-^ 00 LO to t— I I— I 1— I .— I 1— I Ol i-H O O •— I o t-^ Ol 00 r^ Ol 'S- «^ I — I I — I .— I .— I 1 — I Ol Ol LO O LO LO \0 I— I 1—1 Ol to LO LO LO LO LO LO Ol Ol Ol Ol Ol Ol 10 t/l (/) 10 (/) !/) q q c c c c Ol to OJ .— I ^H r^ 0 00 Ol O to LO 01 r— I ,— I Ol Ol Ol r-^vO00LO\O00 CO OOOOvOOlOLO CO LOOlt^tOOlOl -.O Ol Ol to ^H Ol ^H .-H r-H Ol Ol Ol OJ LO O LO LO \0 .— I .— I Ol to 00 CO 00 00 00 00 ^ vO 00 00 r— I LO r-H r-H ^H .— ^ OJ Ol OJ Ol 00 o to o Ol .— I 1— I Ol OJ to u LO OJ oa LO r— t OILOOLOLO^O ^^ OILOOLOLO'O I— li— lOltO C_J .— !■— (OltO o Ol to 000000000000 ooooooooooco I — It ^r— 1< ^1 ^1 — I • . — 1< — I,— tr— li — It— I u c 05 u +-1 o q CO q in V) o -t-> m in 3 o rt C (U o ■H ■M a I— I 3 bO (D Qi --I O ^ l-H H S 3 • H I/l c OS O O c o ■!-> O 4) <^ U X u v: ■p Oh in e c_; 0 QJO H H ■M ■M 0) •M IX CT- nS e X cr +J CO CD H o 0 ■M D. t/) e CJ — < '^ ■^ LO r-O LT) LO vO 00 r— I LO LO ro 00 r~- LO •* LO LO vD 00 00 CM Ln o LD Ln •-D r\i rj c^i rj rg rsi to Ki t^ to ^0 ^O t/l t/i (/I ^^ t/i to C 1= C „■ C C a. -d- r\i ^H hO to vD LT) LO LO LO to r^ \0 "^ O "-H >-H \o LO -ct >0 1^ to PnI rg LO O lo LO \£) —I .—I CM to LO LO LO LO LO LO rj CM r\i r\i (N CM (« to t/) !/)(/) t/i C C C C C C 00 LO LO r^ cnj •d- vD LO r^ o >— I O rM LO o^ o rs) LO \0 vO 00 00 Ol .-H to rsl LO O LO LO \£) 1—1 I— I rg to (M rg ^^l csi (^) rsi to to to to to to t/i to to to to t/5 C C C C C C LO ■— I LO O (N \0 r^ LO 00 00 CTl 00 ^ to \0 rsi 00 •^ LO 00 to Lo t)0 00 00 r~- I— I to rsi LO o LO LO \D .-H rH ^^J to LO LO LO LO LO LO 04 rsi Osi ri (N CM t/1 to t/) t/) t/l t/1 C C C C C C r~- i~-~ Ol vO o^ o^ LO ^ \0 LO 00 "* .—I to LO <-H rg '^ I— * LO ■^ vo r^ LO -^ \£) (N to CM LO O LO LO v£) 1— I I— I CN to (N r^i CM (Nj rj r\i to to to to to to to to to to to (/) c c c c c c to to 00 LO to ^o \0 vD vO vO (^J ■^ 00 f^ 00 •— ( \0 CTi LO LO vO r^ "^ '-H eg LO o LO LO ^D r— I ^H rg to LO LO LO LO LO LO CnI (N og rg (^j (^1 * o if> to to to 1/1 to t/) to t/) to to t/) to to t/1 to (/) o c C C c C c C c c C c C C C c C c n c c • o o o D. H •H •H ■M ■M ■!-> 03 E H rt tl3 E •H LO 1— 1 00 ■ri- o t~^ E •H 00 o ^ .— t rs! CTi o CO o^ Ol •* t-H r— ( (J u vO LO v£> ■^ r^ to u o \D 00 C7^ ^H t— ( (Nl t— t (J o t^ ■^ o ^o f-H 00 r-H f— ( (N to f-H r— 1 n3 rt cd \0 rg \o LO vO O CO r^ CT) CM LO O CTl m ^ Tf CM ^ 'S- og 0 ^ v£) vO v£) r^ rg o r-- vO O^ o to to o o ^£) LO r^ \D O r^ o i-H -^^ t-H rj to -^ f-H rg o 0 LO LO LO f—H .— t 1— ) ^ rg LO O LO LO vO ^^ rg LO O LO LO ^ ^^ rg LO O LO LO vO o r— < ^ rg to 0 r— < (-H rj to U o I-H r—t rg to 00 LO rg ^^ rg to 00 00 00 00 00 00 00 00 00 00 00 oo 00 00 00 00 00 00 aa u ^o X3 H 3 o ■(-> D C +-> 03 1 — I 3 c o m n) ■M O G. 13 O O c o o m X ;ix a1 t= f - E 0 t/1 H 03 CO 0 0 4-> Ph (/) fcl U (1) a>o H H 0) cr UJ e UJ e H CO d) 0)0 m IX ai tu e H C O t/) t/> V) t/) t/) i/l q c c c c c LO 00 00 O --H Ol r~ I^ 00 CTl I— t CT) 00 .— I Csl 00 en CTl Ln 00 00 00 o 00 (N LTJ O LO LO vD .— I .-H (Nl K) rsi CM oj rg rM r^ to to M to to ro to t/l (/) t/l !/) t/l c c c c c c 'S- LO \D O •* O vO P^ r^ 00 CT> CTl to to CTl ■^ 00 LO \0 r-- r~ 00 CTi 00 CTi 00 o 1— 1 a^ t~^ o CT) to LO r~- 00 CO 00 o i~Ni r^j LO o lo lo vo r— I ^H r\i to (Nj rj r\i r\i rsi (Ni to to to to to to t/l t/l 10 t/i t/) t/i C C C C C C 00 O \0 00 CTl to \0 O^ 00 00 CT^ r-H CM .— I to OO O "d- OM O O P^ o >* r^ 00 00 CT^ o c_) LO r\l LO O LO LO ^O ^H T— I rg to 00 00 00 00 00 00 o 4-> 03 t/l t/! (/I !/) t/l I/l c c c c c c to CM ■ct o r^ oj \0 00 CO CTi .— I rsi ^ to r-H ts- o «* \0 t~^ CO CO <^l O) (M LO O LO LO MD ^H I— I (NJ to r\i rsi (Ni CM (N) tN to to to to to to !/l (/) I/) V) t/) t/1 c c c c c c LO LO -^ LO T— I r^ vo 00 cri CTi o ^H vD t^ t^ ■— I 'O vD \£) 00 OO CT^ G^ .— I CM LO O LO LO vO 1— I .— I (N to LO LO LO LO LO LO Csi r^i tN CM rsi (Ni W !/) t/1 t/1 t/1 C C ii' c c c a. o r^ r^ •* ^ o LO \0 ^O I^ t^ o c/) LO \0 CTi CT^ O^ <~-J o o LO LO LO \D r^ o u to u r^4 LO O LO LO ^£) — I ^H (N) to 00 00 00 CO 00 00 03 o <+^ c o c (/I Oxygen Consumption in Time Course of Adaptation Effect of 25°C acclimation on oxygen consumption Respiratory rates in Penaeus aztecus acclimated to 25°C are shown in Figs, 100, 101, and 102 in the time course of adaptation. The horizontal line is the control mean oxygen consumption in 15°/ooS and 25°C. Means are connected by solid lines and the moving averages by broken lines. The shrimp experienced an initial shock effect following their transfer into the test conditions. The sudden change in the oxygen uptake indicated this effect in conjunction with the hyperactive behavior under similar conditions. The immediate response resulted in an overshoot in the oxygen uptake at 25° and 32°C (Figs. 100 and 101, respectively) and in an undershoot at 18°C (Fig. 102), a situa- tion which occurred consistently throughout the salinity range. The shock effect was present for about two hours in 15°/ooS (Fig. 100) and six hours in other salinities. Animals tested in 25°C showed significant variations (P=0.01) in their respiratory rates (Table 7) in all salinities except 15°/ooS which indicated their sensitivity to salinity changes. This was also documented in their behavior. The shock effect increased when shrimp from 25°C were transferred to 32° and 18°C, The extent of the initial shock can be understood from the significant differences existing between the initial respiratory rates and the steady-state levels at 32°C (Table 8) and 18°C (Table 9). Stabilization of respiratory rates commenced at 25°C faster than in 32° and 18°C. At 25°C stabilization started in almost all salini- ties within four hours after transfer and new steady-state levels were established in less than a day. The criterion for a new steady state was the flatness of the metabolic curve, the presence of 186 0. 50 120 144 168 TIME- Figure 100. Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 25°C. The control conditions were 15°/ooS and 25°C. The mean control level represents the mean response of 80 shrimp 0.50 0.40 0.30 0.20 0.10 0.00 0.50 0.40 0.30 0.20 0.10 ■5' 0.00 0.50 0.40 E 0. 30 1 0.20 z o 1- Q. O o 0.10 0.00 0. 50 0.40 0.30 z UJ o > X o 0.20 0.10 0.00 0. 50 0.40 0.30 0.20 0.10 0.00 0.50 0.40 0.30 0.20 0. 10 0.00 Figure 101 course of -ff- 27o. >-^ ?■— VI 3 3 0 12 4 6 10 24 48 72 96 120 144 16f TIME— hours Oxygen consumption rates of Penaeus aztecus in the time salinity adaptation at 32°C. The control conditions were 15°/ooS and 25°C 188 CVJ o I z o I- Q. o u UJ o >- X o 0. 0 . 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 50 40 30 20 10 00 50 40 30 - 80 -/f- 2%, -//- CONTROL -I // I I I ^ 5%, £QNIEQL J •/ L. _l I L I07oo - 30 20 10 00 50 40|- 30 201- 10 CONTROL 00 50 -• i t- _l I L. 15%. CONTROL l_ — „_^ 5 a S *g 257oo- ^MHt--^'^ CONTROL 0. 0. 0. 0. ±=i^ 3d /oo - - 80^—* 1 — I — 1_ 1 i_ -/H CONTROL ^r^^-fs -/f- ?^9 T8 id -^TT^. 10 79 ^8 f7 0 12 10 24 48 TIME — hours 72 96 120 144 16E Figure 102. Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 18°C. The control conditions were 15°/°°S and 25°C ^ v. l/l Ul (/I !/I t/l c c c c c c c CCCCCCCCCCC 71 in wi in (/I c c c c c C 03 ^-, -H C -a o V) O -H 0 4-1 4-) ■M tn Wl U 03 ^H +J w C ^^ T) e O o c o • H 4-) 03 4-1 4-1 c 4-1 CI. t— ( u o e o ^ 3 M LO ^^ in C rj c •r-t M o I— < -a o u B. C o E 03 ^-v. c 03 o r^ ID I/O LO LO M o , C O T3 , — 1 X (LI ■\ C £ o O 0 OJ t« 3 LO H (4^ 4-> ■—1 , s O (U C ca o o c~ c lU e -H J ■H u J= int/ivitnvjirtn'/i cccccccc (/) in VI !/■, tn -yi C C C C C in vi tn i/^ in VI in in CCCCCCCC 0-4 (/j -yj in m IT. in c: c c c C c c - C' c: o o ■!-» t/i u OJ cu +-• t/) -a B c O 03 U o o ^ LO ' — ' r^j ,, — ., CO T3 -d o OJ c o 3 03 "\ C o ■H m to 'M o c o o -■--^ -T3 U 0 c: ^ — ' LO i-H 03 r^ ,— ^ O c dJ ■M o •H ^ T3 +-> rt (1» o H 4-» CD 03 tn E ■fH p , _~t O o ^-J cccccccccc ccccccccc c c c c VI lA '/) 01 y. 01 Ln c c c c c c c c c c c c c -y) ift tn 01 t/1 c c c c c l/l 1/1 t/1 VI I/. 1/1 yj 0". y, 01 VI c c c c c c c 01 VI VI t/i VI 01 VI c c c c c c c 01 VI y. '/i 01 01 VI c c c c c c c y V) V) VI vi l/l VI (fl 01 I/l c c c c c Vi V) VI 01 U o (U -^ o e •H c ) ^ OC rsl O O T CC - -w T r-- cr. ("J *T -^ 191 cccccccccccc ccccccccc 'fi 'j\ ir. ■ nl ,-, C •o o U -H 4-> -P OT O 0 U o OJ ~-^ SO Ln P-CM E •H C fH -H j: in 0) ^ cccccccccc cccccccc cccccccc t/1 1/1 Ul VI (/I 1/1 c c c c c c O 1/J t/. ifi (/I c c c c cccccccccc 0^/ll/l(/l^/ly^vllrt^/l ■ cccccccc o tn in irt in (/) • c c c c c c c c c c O l/l (/I 01 71 • c c: c c O !/i (rt « 192 lA ui tn i/i w (/) c c c c c - c c c: c m C£i ra 4-> O > di c ij T^ to o 01 C •H 4-) ca E 4J c o D. M U -M B O 4-* a OO LO O U) c n ^ c: •H \ — ' o rH T3 o CL C I/O e OJ o c CO o 00 0) C/D m ^ 00 o o a> l;-^ c O LO .— 1 >: (U ^^ ^ o 0) O T3 ca 3 u-) C E- M-l 4-» ^ nj O D M O .— . 01 t-> C O m O c 3 t3 -h ra o 0> 4-* o P-rM 01 E ,J ■H c ^H -H JS in i/lOi/lOVlt/l'/ii/l c ■ c . C c c c i/lOt/lOvlt/lt/lt/l (/I (/I l/v l/l o o ccccccccc t/1 (/I trt i/i cccccccc (/I in tfi 1/1 i/i II c c c c c ui i/i t/) 01 c/1 01 c c c c c c: c c c c c: f/1 I/l t/l !/•. t/l 195 -a o -M in o D 0) ^-J tf> T3 e c o (t3 +-1 fJ u o o Xi ID ^^ (NJ , — ^ (/) -a T3 o o C o 3 m ^ C o • H t/5 LO ■M O T—t c o o ^ -a o o C \~^ LO 03 00 , — , O C ■!-> u o o Xi If) "—^ , — ^ (Nl T) en O -T3 0 -a C ° 3 rt -^ .—1 o L) tn o c o c^ o o U ^-, -a o d u^ nj 00 .—1 0) O C f— 1 ■P o x> ■ H cS x> *-> f- o o t-> S tfl(/llftl/lI/lUlUlUl cccccccc c c c c c c c: 1/1 ;i (/I tr 'Jl C C C C C (/■j (/I ui w t/i l/l (/) l/l 01 i/l c c c c c c (/) trt (/) t/1 (/■. Wl c c c c c c 195 O O O Vi VI ra .H If) c M ■o o 6 o o 3 OO LO X3 m C Cv] '~~^ C •H o ^ TJ C/5 o a C o B CO 0 c to ~~- 0) en OO o 00 O U~i >, c o X OJ ^ -o o 01 0 C 3 LO CO U-l UP — t o a (!) E C ^J ■H -H u tn c c c c c c c OOOVii/ii/it/iui • • • c c c c c c c c c c: c c: 'Jl. I/] O 1/11/11/1 O O O '/I ■ - • c D- a. G, II II — > V, n = 5 196 c c c c C c VI Ul l/l '/> l/l coo C' (/lO(/^ i/i Vl !/)01 tS ^s C •a o -a E C o pj p •M u o 0 J^ LO ^^ (Nl .^ — , CO T3 ■^ o c; C o 3 OJ ~-^ C o •H en ui ■P o ^ c o o ~~^-T3 o o C ^^ LO CT3 <— 1 CTl / — ^ O C 0) +J O i-H •H ^ -a +-1 ni 0 o H 4-1 0) rt 1/1 E •rt CX r-C O O +J (J ^-^ cfl CO 0 o ^H O (D ^~. so o g.- •H fi H -H X O O 01 ''^ ccccctrcc ■ cccccccc O'/i'/^i/l"^'^'''''' Cj fi tfi LO •^^ rg •a c/5 o JD ■rH n! -o «-> f- 0) 'J +J dj oj tn E •rH n. -H o O 4-1 O ^-' « C/0 W wi (/I 'Jl V) (/) (/I O « i/l 01 1/1 1/1 V, c c c c c c O w t/i 1/1 '^ O '/I VI t/j CCCCCCCCC 0(/ivit/ii/)wi(rtcnui ■CCCCCCCC ■ C C C C C C c O 1/1 V5 (/I Ul VI W c c c c c O VI 1/1 Vl (fl C; VI VI O — ' f^l T O C 198 nonsignificant differences between the adjacent means and the state of the experimental animals. The state of the animals was recognized from their activity level and other behavior. The new steady-state levels were usually higher than in 15°/ooS. In 10, 15, and 25°/ooS, where the animals apparently experienced relatively less salinity stress, there was a slight decline in the respiratory rates after the fifth day. This may reflect a possible starvation effect which was higher in 32°C than in 25°C but was not observed at 18°C. Within six hours stabilization started at 32° and 18°C. The shrimp at 32°C reached a steady state from the third day in 10 and 15°/ooS and from the fifth day in 25°/ooS (Fig. 101). After the third day the shrimp were apparently under the influence of hunger. Comparison of the oxygen means between the first and third days showed significant differences in 10 and 15°/ooS and to a lesser extent in 25°/ooS (Table 8). Therefore, the conclusion about the new steady state was inconclusive and was based solely on the flat- ness of the metabolic curve. The starvation effect was not noticed in 2, 5, and 36°/ooS where the animals were under both salinity and temperature stress. There was a high mortality rate in these con- centrations. The possibility of reaching a new steady state in these conditions was doubtful. In 18°C the respiratory rates dropped to very low levels within a few hours after transfer and remained so in all salinities (Fig. 102). After some days the respiratory rates, however, tended to return toward the level in 15°/ooS and 25°C in 10 and 15°/ooS but not in other media. All shrimp in 2°/ooS died within four hours. In 5°/ooS there was also a high mortality. The shrimp at 18°C were obviously less affected in 25 and 36°/ooS. In 18°C steady-state metabolic levels were reached in 5, 10, 15, and 25°/ooS within six hours. 199 Effect of 18°C acclimation on oxygen consumption After seven days of acclimation in 15°/ooS and 18°C brown shrimp were tested in 18° (Fig. 103), 25° (Fig. 104), and 32°C (Fig. 105). The metabolic rates increased gradually with temperatures from 18°C to 25° and 32°C. At 18°C the initial shock effect continued for a maximum of two hours (Fig. 103) as opposed to longer periods in 25° or 32 °C where the effect lasted for several hours. At a test tempera- ture of 32°C, all shrimp were dead in 2°/ooS by four hours. Stabili- zation commenced within two hours at 18°C and within a day in some of the salinities in 25° and 32°C. The flatness of the metabolic curve in 5 and 36°/ooS at 32°C should be considered with caution in view of the high mortality rates. On the basis of visual examination of the metabolic curves, statistical analyses of the respiratory means (Table 10) , and fa- vorable survival rates, it appeared that the shrimp at 18°C attained steady-state metabolic levels in all but 2°/ooS. The animals at 25°C attained such levels in 10, 15, and 25°/ooS within the first day but not in 2, 5, and 36°/ooS where the mortality was also high. Significant differences occurred in the respiration rates in most of the media from the third day onward (Table 11); but the importance of these variations was not clear. In 10 and 15°/ooS they might re- flect starvation effect and in others, a recovery state from the ini- tial shock. The shrimp acclimated to 18°C and tested in 32°C could not establish steady-state metabolic levels within the test period in salinities other than 10 and 15°/ooS. Steady-state level appeared in 25°/ooS on the first day but all the shrimp died after six days. From the statistical comparison the respiratory rates in 5 and 36°/ooS were erratic (Table 12). The extent of stress caused by a sudden temperature change from 18° to 32°C was evident from the high mortality in most of the salinities. The starvation effect was not 200 d" I z o o o X o 0.50 0.40 0. 30 0.20 0.10 0.00 0. 50 0.40 0. 30 0. 20 0. 10 0.00 0. 50 0.40 0.30 0.20 0. 10 0.00 0. 50 0.40 0. 30 0.20 0. 10 0.00 ^ff~^^ CONTROL 10%. ^,._^9_.^>048_ 15%. m ON^K)L~~^ § — ^ 25%. 36%. :Sg:^A-^,8__^9^.^5 ^l^^7 - -//- _i_ 0 1 2 10 24 48 TIME -hours 72 96 120 144 Id Figure 103. Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 18°C. The control condi- tions were 15°/ooS and 18°C. The mean control level represents the mean response of 105 shrimp -//- ■VA 2%, -/ f- 3 CONTROL 0.40 0.30 0.20 0.10 0.00 1 0 50 ^ o^ 0 40 E 0 30 1 0 20 z o 0 10 t- Q. n on ^ 0 50 z o 0 4 0 o 0 30 z UJ o 0 20 > X 0 10 o :^^ 5%, ^—^ 105 CONTROL -■ «■ yA- _i_ I07oo - .8. 1 9 i8_i8 Q 0. 50 0.40 0.30 0.20 0.10 0.00 367o. ^^^^^^-4^^-^. ^ '105 CONTROL -// ^4 -//- _!_ _1_ JL. _l_ _i_ 0 12 10 24 48 TIME -hours 72 96 120 144 16( Figure 104. Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 25°C. The control conditions were 15°/ooS and 18°C ■f/- CONTROL J /f. I I ' I I 1 L. o E I Z) z o o UJ X o 0 12 10 24 48 72 96 120 144 16f TIME-hours Figure 105. Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 32"C. The control conditions were 15°/ooS and 18°C CCCCCCCCCCCC m -o c (/) c U ■a o O C E •H ■M to o ■U c w c^ 1 — 1 U 4-> e o o D CU3 oo Xi to C C •H o 1 — t T3 CO o o. C o e a; o c 03 v^ o 4) to to o 1—4 oo O LO >, c o o X 0) \ -a r-~i o 0) 0 c Xi 3 LO CO rt U-l ■M t- o (1) ^ , OH o c ID *-> o O Ul •H C 3 •a -M (0 u 0) o CJ (U ■(-> 01 •H 4-» Oi t/1 ^H M E •H c« •^ Ch c —■ o oo t/l U J-> •H 3 o ■-^ W 0) e" C J ■^ -H 4) VI 'yi wi vi i/i 01 (/) C C C C C C C t/i i/i t/l w y, V, (/I (/I t/1 O t/) CCCCCCCCC -C I/". Ul Vl t/l '/I t/l 01 ■/, V5 ^ ooooooooo l> II II II II H M N II a.Q.a.D.a-D.D_cD. c c c c c 01 01 (/)(/) in o 1/1 V) O i/i (/) in i/i O O trt i/i t/i • ^M .a- O C ■ 204 l/lf/lVlt/lt/Vtfll/ll/) cccccccc a. a. D- a. o o o o o o ti ti ri II II II a. a. c_ ci. c- a. 1/1 t/i (/I i/i tn (/) 1/1 w t/i ccccccccc tS ^^ c ■o O (D -H v *-> u) a (U (U ■♦-» t/i •a E C o Ctf J-> ■4-' u o o J3 00 ^^ , — ^ r-t T3 CO 0 ■O o 3 C o C rt ^^ ■ H o ■M to LO c o .— < o o o --. T3 \ ' 0 C LO Oj o -— 1 ,-H , — ^ o c -•^ 3 o O CI. .— « e •H C u •H ^ (/I m ^ • (/I Wl (/I n c C C C CJ o t/) ^ ""^ 205 CC -CCCCCCCCC ui « O v; (rt i/i 03 OO o O o J-. ^ (D o r4 01 o-. O i/l W yi 1/1 C C C C C C O -a •r-( u ■M ■M O w Q> 0 yi •M E •a o c +-» nj +J o U c c c c c c c c c c c c c c c c c c c c c c = tn 01 01 (/I c c c c -o -a o 3 c •^^ OJ o o vD c C/0 1^, o o o 0 -a ' — ' ~-~ c o oJ c u-> • — r •-H / — V C CJ o o ^H ■M -H ^ +J C3 -3 O H CJ o ■M tn n! e !X •M O W. O VI « (/■■ VI C C C C c c '/. 'f, V) (A c c c c 206 OOOol(/^u^"*iW^'/l'^ S aj . •^^ T3 'c' ra (D O a) +-> -H u m VI ■(-» n) .-( t/1 C u T3 O 01 c e •H J_l tfl o 4J c +j a 1— 1 (_) 4-> e o o 3 ao 00 J3 Cfl c .— 1 ^ — ' c •H o t-~i t3 CO u d. C o B 03 o c ra ~^ ,_( (U CO CO o ,—1 ao O LO :>, C o 0) X 01 -^ -a r-4 o 0) o C -D 3 LO Oj ClJ LH 4-t ^H H o 0) ,— ^ pa O C 0) •P O o m •H a 3 T3 -M a O (L> O a 01 4-> 1) •H 4-1 Cl3 tn u-l N e •H n) •H C^ C ^ o OC U] U -t-J •H 3 Ci ^-^ cn (1) ca n) CO M-l C d) 0 0 0) U 0 CU C~ a) e c eJ • H -H c c c c c c c c ■ ■ c c c c c c c c c c O U"i 01 '/I y^ ''I . c c c c c O Wl tf^ 01 VI ■ri o o o o o o O O O 3 O Cu a. Cl. a. — o o o c: o : II II II II n D. Cu C Ci. C^ D- Q. Cu D- C_ '/I f ) t/1 W) M X ■ oO ri O O ■ 207 WC-OOCOC !/> VI i/;o wi (0 to O o ^ o o ^ 3 o c c^ E •H c c- c^ Cu c c a. Ci-C_C_CD_C_C_Q.C- a c o o o c; C C C C ■^. c c c o ^ trt ,,^ C XJ o (U •H ■M +-J Ul t> H 4) O 4-1 m nj in 6 •H P. rt O O -l-J U v_/ rt m O 0 !h o (/: c Ij T3 o 0) C E •H 4-1 03 O 4-1 c 4-1 D. l-l U 4-1 B o o 3 00 00 JD m c c •H o rH T3 C/1 u Cu C o E rt o C re ^.^ r-j QJ c/2 C/5 o »-H 00 o u-i >, C o (U X tx QJ E C hJ •H -H ^4 X in (U ^ 1/1 !/l -yi Ul c c c c 01 Ul ul (/I c c c c O Wl O I/) V5 tfl O 1/1 in Vl ui O t/l VI tfi o o a. a. ■ r-j T O O • c/^ — ■ 210 O O i/l "> "/l 1/1 t/1 m (J OJ o +J to ■o 6 c o rt 4-> ■!-> u o o ^ 00 '—^ ^— ^ •-H -o tn 0) T3 0 3 C o C oJ -^ • H o ■M CO LO C O i-H o o o ^. -o * — ' o C LO OJ CM t— 1 .— 1 o c o ♦J O •H J3 T3 -t-l ro ^ i/i vi (/I 01 in i/i i/i (/I tfl in i/i in VI t/i cccccccc c c c c c c in in t/1 tn i/i C C C C C in in t/i irt • in I/) i/i i/i n c c c c DO —• "T 00 "^ ^ O T 00 ri T r- CT^ '~J T O 211 cccccccc Oi/ioivii/lt/i 5/jui(/)wi c c c c c c c c c tfi t/1 1/1 ir, in '71 tn C C C C C C C C TJ o (U • rH +-> •M V) O 0) (U 4J VI T3 E C o aj ■M •M U O o ^ t/l t/1 71 01 t/) c c c c c C/3 Ul Ul 1/1 II crcccccccc VI • • w m in tn (ft CM II c c c c c VI yi 1/1 t/i VI t/i ui C C C C C C C c I/} vO o o hO ■J o ^ — ' \ -a o c rj LO t« i— t .-H 0) o c 1 — 1 ■P o ^ •H rt -a *-! H z o o UJ o X o e-^^^-^T-V^fe-f^-^^-^^ J I I L. 0 12 4 6 10 24 48 72 96 120 144 16f TIME- hours Figure 107. Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 18°C. The control conditions were 15°/ooS and 32°C CM O g H Q. 3 CO o o LlJ X o 0.40 0. 30 0.20 0.10 h 0.00 "j^m^. CONTROL _L- _1_ _1_ J- _JU 0 12 10 4H 72 96 120 144 16S TIME -hours Figure 108. Oxygen consumption rates of Penaeus aztecus in the time course of salinity adaptation at 25°C. The control conditions were 15°/ooS and 32 C Q. a. a. Q- a. CM a. o- Q- Q. i/i t/i v) ^ft '/J '/; i/i o y. v. VI 0) o tfll/lWt/lUll/lVlv; c c c c c OS c o a, E ID X X o c 00 > c B ce o ■M u *-> o O r^j XI Kl -o C>~j C 0 CO 0 ■^^ u^ o o LO o ^^ -a o c in rt 7—i o p. *-> o •i-H -o J-" i/i (/I t/1 01 (/) c c c c c c c c c c c c c c c c c c c c c c ui 01 ui m w c c c c c (/)(/)invi;/ii/iuit/i cccccccc (/I wi 1/1 ui i/i tn o c c c c c c - c c c c c c c c c ■ 00 n o 3 ■ 217 cccccccccccc cccccccc -cc V • tS ^ «s T3 O a) -H 4J 4-> in o a> ID +j in 13 S c o cd ^M ■p u o 0 ^ (NJ '—' t — s ro T3 in a) T3 o 3 C 0 C n) --^ •H o 4-1 CO Lo c O .— 1 o o o \ -a ^ — ' o C LO OJ M rH r-H , — s o c (U ■M O i-H •H J3 -d +J ra (U (J t- +J 0) g m •H fl. rH O O 4-> O v-/ nJ t/3 0 o U o 1) ^ 3 o o CX-H E • rt c ^< -H j: in tft u". t/1 (/I (/I (A !/l CCCCCCCCCC trtt/ii/)(rttO(rttfii/)(/i CCCCCCCCC i/i (/) in v) in i/i i/i (/I cccccccc in in ift wi I/) i/> yj c c c c c c c i/i VI in in in tn C C C C C C in in in 'ji in tn ui in 01 CCCCCCCCC i/iininintni/imin CCCCCCCC tn '>! in in in c c c c c in in in m in in in (n in in c c C C C c in in tn (rt tfi c c c c c C TO ^^ •-^ > J- 218 nJ CO (U o ^- o -M tfl O (U (U 4-> cfi T3 6 C O Oj ■(-» 4-> u o o ^ ,^ — , tN ^-^ T3 ro (D c/:i t3 13 o 3 C o ca ~~-. (J o C CO ^ O o to O o ^^-d o C NO i/l 03 r-H -—1 +-) u o o /D rvl ^-^ ,,-^ ro -o CO (U t3 0 D C o C a! ^^ • H o +-' C/: LO C O ^-* o O o ^-0 o C LO n! rt I-H f— I o c o •1-1 o t— 1 •H XI -O ^-' n) Hi o H +J 0) oj en E ■H CL. •-I O O j-i o --^ c« t/) ^ o U o -^ SO o CU-H E •H C (-. -H J= trj 0) ^ c c c c l/l l/i !/■. '71 !/■) c c c c -^ c- I/; ui Vi '.n 1/1 1/1 :/) o O ' 221 4J . rt ,-^ C T3 O <0 -H 4J -P Ul U 0) 0) •p in •a e c o td 4-> •M U O o J3 I — V rsi ^-^ •o to (U CO -o -a o 3 C 0 nS ~^ o a c cn vO o o to u o ^ T3 o C «^ LO nj f-H .— t 0 O C i-H *-i o XI • H cfl T3 <-) f- 0) O +J D Cd V) E •H O, ^ O O ■(-> o ^-^ nl C/3 (U o U o 1^ L. 3 O o E *-< s: a. c- CL Q, D- c_ a. tx t/lWl(/ll/)VIl/lI/)(/l cccccccc in VI Vl (/I !/i Ul Vt c c c c c c c (/■i tfi lyi t/i yi i/i C C C C C C • Ul i/l i/i vi 01 II c c c c c 222 Ol/lOOO'/lV'Ul c c c c c c c o o o o £X c- a. a. C o o c 01 M X X o T3 C ttJ O ■(-> .H tn W 4-> nH 0) O CO ■M in t_t T3 0) C E ■M 03 O c 4-1 1 — 1 U •U o o an rj Xi C Kl ^-^ w ^-1 ■a c/i Q_^ C o E rt o ca ~^>^ c/o C/) o o Ln c o o -^ -a o r/l • H D 13 +J U IK O +-> rt f) [-] e 03 •H C ^ o o ^ 3 o ^ X OOOOOO IT. o-j (/) UlOi c c c c c c- cL a, c^ Cl- 1- OOOOOO 1/1 (/) t/l "^ c c c c c c D. a. c c- O t/<000 (/^ tni/i (/v C C C C OOOOOOO I/I c II II II II II II II Q. a. cl. a. CI. Cl. Cu c c c c a. G. Q_ c- o o o o II II c c c Cu D- Cl C- in tn 01 t/1 o o o o a. s- a. c- ir-iTtvCOTOorvi.OOrroo .-■. r-l -T 1^ Oi *"■-] ^ -D c rt J-. — > 3 " I- O nl C '"^ 223 • c c c c c c c c c c (^j w I/) A - t/1 (/I m (^ IB 0 <-) H o c c c c c 1/1 in i/i '/) lyi •a o; . 4-> , ^ w C (U O 4-> ■M •o O c f—^ M •(-> T3 o a) T3 ^ 3 C C CO •H w ■M t/1 o c o o o o ^ o c ■M O CC -H • H U c q c c c c ccrccccccc cccccccc t/1 yi i/i '/I VI (/I 01 C C C C C C C to t/l Vl ■/! I/) l/l c c c c c c (/I 1/) (/) Vl l/l c c c: c c ^ m ITi t/i c c c c o o • • >.-. 25%o ^''Y'-rf : ,367oo tr-*^ "" ~" "~ "* »i ■I'.'tw^" 'iiM ...mT CONTROL -•, ••«rjUVoo l5%o 250 Z 200 2 a z o o ISO 100 50 IxJ O X o -//- ACCL ZS'C TEST SZ'C ^, 25 /oo -//- j_ _i I I I- ;50 200 150 100 50 0 12 10 24 48 72 96 120 144 168 TIME -hours Figure 109. Comparison of the oxygen consumption rates of Penaeus aztecus during adaptation to various salinities at 18°C [section A figure) , 25°C (B) , and 32°C (C) . Animals were tested from control ditions of 15°/o°S and 25°C of the con- 227 0.50 0.40 0.30 0.20 - 0.10 0.00 -//- 0. 50 (VI O 0.40 9 0. 30 t- CL w 0.20 z o o 0.10 - > X 0.00 o ^• o.soh 0.40 0. 30 - 0. 20 0. 10 0.00 ACCL SZ-C TEST I8»C f^"NTRni _l L. _// I I I I I I L. 125 100 75 50 V/- ACCL sz-x: TEST 25-0 - 125 5^ CONTROL ZIZ'Z'^'- -^"^ ^'C ^ ^-ZT "^^ >^ _2£ I i_ ■yA- _l L 100 - 50 ACCL SZ'C TEST 32°C /y^^^S^^.-r '^--^^ - 125 - 100 10% 2B7oo 2%. -i_ -L. _1_ -l_ _1_ 0 12 — ' //"— 10 24 48 72 96 120 144 168 TIME -hours - "5 - 50 - 25 g O. to z o o z UJ X o Figure 110. Comparison of the oxygen consumption rates of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B), and 32°C (C) . Animals were tested from control con- ditions of 15°/ooS and 32°C 0. 50 0.40 - 0.30 0.20 0.10 O^ 0.00 0. 50 0.40 -V/- ACCL. I8»C TEST IS'C I0%< ^aKl— = 25%, _i 1 I i_ _1 y^ , 1 L. -I I l_ O 0. 30 I- a. ^ 0.20 z o o 0.10 X 0.00 o -//- ACCL.ISt TEST 25"^ 25%o CONTROL .'-'•^ - ~^x: 36%. io%;---.>..^ ■ -J — •/- 500 400 300 200 100 500 0. 50 ■ 0.40 0. 30 0.20 0.10 - 0.00 /2%, ACCL I8°C - TEST 32°C •//y. .-•-' ^^' ^^^^--^ J, • I0%o 5%o CONTROL -//- -//- _i_ _i_ -L. _1_ 300 JIIO 100 600 400 500 200 0. 3 CO z o o o X o - 100 0 12 4 6 10 24 48 72 96 120 144 168 TIME— hours Figure 111. Comparison of the oxygen consumption rates of Penaeus aztecus during adaptation to various salinities at 18°C (section A of the figure), 25°C (B) , and 32°C (C) . Animals were tested from control conditions of 15°/ooS and 18°C 229 consumption of shrimp tested at 25°C between 15°/ooS and the other media (Table 16); but the respiratory rates in 2, 5, and 36°/ooS did not vary significantly from each other as did the rates between 10°/oo and 25°/ooS, The oxygen consumption increased with the devia- tion of the test salinities from 15°/ooS. The corresponding varia- tions in the blood osmoconcentration would suggest the presence of a possible interaction between the ionic and respiratory rates. However, this relationship was not found consistently in other test conditions. Tlie respiratory rates at 32°C increased to highest levels of all test temperatures (Fig. 109c) . Oxygen consumption rates were ini- tially highest in 15 and 25°/ooS. In 2 and 5°/ooS the rates were low while in 10 and 36°/ooS they were median. Some important differences were noticed in the salinity-related responses between the test tem- peratures 25° and 32°C. Comparison of the respiratory means at 32°C indicated that oxygen consumption occurred at the same rate between 2 and 5°/ooS, 10 and 36°/ooS, and 15 and 25°/ooS. Significant dif- ferences (P=0.01) were found in other comparisons (Table 16). There was a high mortality in 2°/ooS. The low respiratory rates in this concentration may perhaps indicate a state of depression. At 18°C the oxygen consumption rates decreased to the lowest levels of all test temperatures (Fig. 109a). In 2°/ooS the experi- mental shrimp died within four hours. The respiratory rates in 36°/ooS dropped to a low level like those in 15°/ooS. With the ex- ception of these two salinities the respiratory pattern in other salinities was similar to that of animals acclimated to 25°C. Com- parison of the oxygen consumption means between 2 and 5°/ooS, 10 and 25°/ooS, and 15 and 36°/ooS did not show significant variations (Table 16). However, in other salinity combinations the comparisons were significantly different (P=0.01). 230 Table 16 Effect of Salinity Change on the Respiratory Rates of Penaeus aztecus in the Process of Adaptation to Salinity and Temperature The animals were transferred for adaptation from 15°/ooS and 25°C background. Adaptation Test Temperature (°C) Salinities (Voo) Compared 18 25 32 2 vs 5 ns* ns ns 2 vs 10 P=.01 P=.01 P=.01 2 vs 15 P=.01 ^ P=.01 P=.01 2 vs 25 P=.01 P=,01 P=.01 2 vs 36 P=.01 ns P=.01 5 vs 10 P=.01 P=.01 P=.01 5 vs 15 P=.01 P=.01 P=.01 5 vs 25 P=.01 ns P=.01 5 vs 36 P=.01 P=.01 P=.01 10 vs 15 P=.01 P=.01 P=.01 10 vs 25 ns ns P=.01 10 vs 36 P=.01 P=.01 ns 15 vs 25 P=.01 P=.01 ns 15 vs 36 ns P=.01 P=.01 25 vs 36 P=.01 P=.01 P=.01 ''ns: not significant. 231 Effect of 32°C acclimation The oxygen consumption of shrimp acclimated to 32°C dropped sud- denly when tested in 18°C. The low respiratory rates continued throughout the test period (Fig. 110a). No marked variations were observed by visual examination in the respiratory rates due to salinity changes. Comparison of the respiratory means did not show significant changes between 5, 10, 25, and 36°/ooS. Respiratory means in 2 and 15°/ooS exhibited significant differences with the responses in other salinities (Table 17). The shrimp acclimated to 32° and tested in 25 °C maintained a low initial respiratory rate in 15°/ooS and high rates in 2 and 36°/ooS (Fig, 110b). These responses were similar to those of shrimp accli- mated and tested in 25°C under the same salinity conditions. In 36°/ooS the respiratory rates were maintained at the initial high level while in 2 and 5°/ooS the rates dropped during the successive time intervals. Consequently the animals in 36°/ooS showed signifi- cant differences with the rates in other salinities. At the same time the shrimp in 15°/ooS did not show significant changes with those in 2, 5, 10, or 25°/ooS (Table 17). It was not known to what extent the decreased oxygen uptake in 2 and 5°/ooS represented a state of depression. The shrimp acclimated and tested in 32°C used the lowest amount of oxygen in 36°/ooS and a lower amount in 15°/ooS than in the other salinities (Fig. 110c). In these respects the respiratory rates were different from the shrimp acclimated to 25°C and tested at 32°C (Fig. 109c). The respiratory rates in 15°/ooS showed significant differences (P=0.01) with the responses in 25 and 36°/ooS but not in 2 or 10°/ooS. Also the shrimp in 10°/ooS responded like those in 5 and in 25°/ooS (Table 17). In 36°/ooS the oxygen consumption rates differed significantly from the rest of the salinities except in ^ / o oo • 232 Table 17 Effect of Salinity Change on the Respiratory Rates of Penaeus aztecus in the Process of Adaptation to Salinity and Temperature The animals were transferred for adaptation from 15°/ooS and 32°C background. Adaptation Salinities (°/c ■o) Compared 2 vs 5 2 vs 10 2 vs 15 2 vs 25 2 vs 36 5 vs 10 5 vs 15 5 vs 25 5 vs 36 10 vs 15 10 vs 25 10 vs 36 15 vs 25 15 vs 36 25 vs 36 Test Temperature (°C) 18 25 32 p= .01 p= :.01 p= :.01 p= :.01 p= ^.01 ns p= :.01 ns ns P:: :.01 ns ns P = = .01 P = = .01 P= = .01 ns * ns ns P= = .01 P= = .01 ns P= = .01 P= = .01 ns ns P= = .01 ns P: = .01 P= :.01 P= :.01 ns P= :.01 ns ns P= = .01 ns P= = .01 ns ns P= = .01 P= = .01 P= = .01 ns P=.01 P=.01 "ns : not significant. 233 Effect of 18°C acclimation The shrimp acclimated and tested in 18°C (Fig. Ilia) showed some similarities in oxygen consumption with those acclimated to 25°C and tested in 18°C (Fig. 109a), The oxygen uptake in 2°/ooS was high until the shrimp died in a state of hyperactivity. In 5°/ooS the respiratory rates were low despite the fact that the shrimp were not depressed. The respiratory means in 15°/ooS exhibited signifi- cant differences (P=0.01) with other test salinities (Table 18). However, the rates were similar between 10°/ooS and both 25 and 36 /ooS. The initial respiratory rates at 25°C were higher than in sub- sequent intervals in 2 and 36°/ooS and lower in 10 and IS^/ooS (Fig. 111b). The shrimp acclimated and tested at 25°C exhibited a similar pattern in relation to the salinity changes (Fig. 109b). Respiratory rates between 2 and 25°/ooS and between 2 and 36°/ooS did not exhibit significant variations nor did comparisons between 5 and 15°/ooS and between 5 and 36°/ooS (Table 18). The brown shrimp acclimated to 18°C and tested in 32°C consumed a high amount of oxygen in 15°/ooS (Fig. 111c). The shrimp accli- mated to 18°C responded much the same in the test salinities as those acclimated to 25°C and tested at 32°C (Fig. 109c). In 2°/ooS the shrimp were hyperactive until death and used more oxygen. In 5°/ooS the oxygen consumption was lowest. Significant differences (P=0.01) were present in the metabolic rates between 2°/ooS and other salinities (Table 18), Animals in 15°/ooS responded like those in 25 and SS^/ooS as did the shrimp between 5 and 10°/ooS. However, the respiratory responses both in 5 and 10°/ooS were significantly different (P=0.01) from those in 15, 25, and 36°/ooS. Heavy mortali- ties occurred in 2, 5, 25, and 36°/ooS probably because of the large variation between the acclimation (18°C) and test (32°C) temperatures. 234 Table 18 Effect of Salinity Change on the Respiratory Rates of Penaeus aztecus in the Process of Adaptation to Salinity and Temperature The animals were transferred for adaptation from 15°/ooS and 18°C background. Test Temperature (°C) Adapt; ation Salinities cv 0 o) Compared 2 vs 5 2 vs 10 2 vs 15 2 vs 25 2 vs 36 5 vs 10 5 vs 15 5 vs 25 5 vs 36 10 vs 15 10 vs 25 10 vs 36 15 vs 25 15 vs 36 25 vs 36 18 25 32 P=.01 P=.01 P=,01 P=.01 P=.01 P=.01 ns* P=.01 ns P=.01 P=.01 ns ns P=.01 P-.Ol P=.01 ns P=.01 P=.01 P=.01 P=.01 P=.01 P=.01 P=.01 P=.01 ns P=.01 ns P=.01 P=,01 P=.01 P=.01 P=.01 P=.01 P=.01 P=.01 P=.01 P=.01 ns ns P=,01 P=.01 ns ns P=.01 ''ns : not significant. 235 Although the respiratory responses at normal temperature (25°C) were influenced primarily by salinity, the test temperature had exerted a greater effect in altering the response pattern. However, when returned to 25°C, either from IS^C or from 32°C test conditions, the experimental animals exhibited a tendency to resume the original salinity response pattern. Interaction of Salinity and Temperature on Respiration In IS^/ooS the respiratory rates increased with temperature. But it was not known how a simultaneous change in the salinity factor would alter the temperature influence on respiration. This effect was shown by reproducing the salinity-related metabolic response curves of shrimp acclimated at 25°C (Fig. 112], 32°C (Fig. 113), and 18°C (Fig. 114). The oxygen consumption of shrimp acclimated to 25 °C increased in 15°/ooS when tested at 32°C and decreased in 18°C (Fig. 112). The differences were highly significant (P=0.001) between the three test temperatures (Table 19). Such variations occurred consistently in all salinities between 18° and 25°C. However, the situation was different between 25° and 32°C. In salinities other than 15°/oo there was no proportionate increase in the oxygen uptake when tested at 32°C. Nevertheless, the respiratory rates between 25° and 32°C showed significant differences in 10 and 25°/ooS, but not in 2, 5, or 36°/ooS. This was because in extreme test salinities 2, 5, and 56°/ooS the animals failed to increase the oxygen uptake at 32 °C to above the oxygen consumption levels in 25°C. In Fig. 113 the respiratory responses of shrimp acclimated to 32°C were shown. The animals exhibited more or less similar salinity response patterns, except in 5°/ooS, like those acclimated and tested in 25 C, In 5°/ooS the oxygen consumption increased with temperature 236 CVJ o O I- CO o o o >- X o 0. 50 0 . 40 0. 30 0.20 0. 10 0.00 0.50 0.40 0. 30 0.20 0. 10 0.00 0. 50 0.40 0. 30 0.20 0. 10 0.0 -VA- 0.00 2%. /. if .r--.-_ .::.r /> 2 CONTROL /'. __ / CONTROL iU»»t»tamm» \8°C 25*^ 32°C -//- 5%. /' ggT^nr- ^, Z in /^ , .,'/."••• I0%o - - ,/__^ ""•••..52°C I8°C 1 1 1 1 — , — 1 1 1 1 .^ 1 1 1 1 10 24 TiME-hours 120 144 168 Figure 112. Comparison of the time course of oxygen consumption responses in Penaeus aztecus in relation to temperature change. The control conditions were 15°/ooS and 25°C O e I 3 O IjJ o >- X o 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 50 40 30 20 10 00 r/ •• 2%. CONTROL ■ "^- — irr^rrr 1 — 1 — I 1 1 1 /* 1 1 1 32<^ J 1 1 1 50 - 40 30 20 10 00 50 ■ 40 - 30 - 20 - 10 - 00 - 50 40 - 30 - 20 - 10 - 00 50 40 30 20 10 00 50 40 30 20 10 00 -//- 57oo - I8°C :::::::::/ >™^ I07oo -I CONTROL ^ ■^'/. ■■! ■32^- 25°C- .I8°C- -i'/ L. _i_ -//- l57oo -I CONTROL ^■w" " ■ ' "• :■ 32"C- 25°C I8°C' -**- •//• 257oo CONTROL V: -/A- '••32'^ '25X ■-I8»C -v/- -1 I I i_ V ^^ta«^Mrl£S^CVMi1A&*. V. E?fc--Ul-:>^ 367o« CONTROL _l I 1 L. _l_ 0 12 — // ' >— 10 24 48 TIME— hours I8°C 72 96 120 144 16f Figure 113. Comparison of the time course of oxygen consumption responses in Penaeus aztecus in relation to temperature change. The control conditions were 15°/ooS and 32°C O E I 2 O CO o u o >- X o 0. 50 0.40 0. 30 • 0.20 ■ 0. 10 0.00 0. 50 0.40 ■ 0. 30 0.20 0. 10 0.00 0. 50 - 0.40 ■ 0. 30 - 0.20 0. 10 0.00 0. 50 0.40 0. 30 0. 20 0. 10 0.00 0. 50 0. 40 0. 30 0.20 0.10 0.00 •/>• .'y CONTROL ^P^- ?2°C 25"^ -y/- 10%. CONTROL .'A ~" " ■ / ^ ^ ~" ~~ ~" ■"■ ■" — — . '.S — 32°C _25°C — 18^' -//- -/>• 15%, .32*^; ■* — ^ 25°C IfiTf ^^ CONTROL -VA- 25%o - ■■// 32 °C CONTROL _i I I I — I8°C- ^1=^ 3d /oo - d6fJTRCL -L. •32^ ^25T -//- _i_ -L. 0 12 10 24 48 TIME- hours 72 96 120 144 163 Figure 114. Comparison of the time course of oxygen consumption responses in Penaeus aztecus in relation to temperature change. The control conditions were 15°/ooS and 18°C 239 Table 19 Effect of Temperature Change on the Respiratory Rates of Penaeus aztecus in the Process of Adaptation to Salinity and Temperature The animals were transferred for adaptation from 15°/ooS and 25°C background. Test Comparison Level of Salinity Temp rc) vs Temp CQ Signifi- / oo Accl Test Accl Test cance 25 18 25 25 P=.001 2 25 18 25 32 P=.001 25 25 25 32 ns* 25 18 25 25 P=.001 5 25 18 25 32 P=.001 25 25 25 32 ns 25 18 25 25 P=.001 10 25 18 25 32 P=.001 25 25 25 32 P=.001 25 18 25 25 P=.001 15 25 18 25 32 P=.001 25 25 25 32 P=.001 25 18 25 25 P=.001 25 25 18 25 32 P=.001 25 25 25 32 P=.001 25 18 25 25 P=.001 36 25 18 25 32 P=.001 25 25 25 32 ns ''ns: not significant. 240 increase from 25° to 32 °C and a comparison of the means showed a significant difference (Table 20), On the contrary, the shrimp acclimated to 18°C and tested in 5°/ooS (Fig. 114) did not show significant differences in the oxygen consumption levels between 25° and 32°C (Table 21). But in 36°/ooS significant temperature related differences were found between 25° and 32°C. Comparisons in 2°/ooS were not valid due to the high mortality. These findings suggest that the shrimp acclimated to 25°C were apparently less sensitive to temperature increase in very dilute or in more saline media. The shrimp acclimated to 32° or 18°C were more temperature-sensitive in low (5°/oo) or in high (36°/oo) salinities, respectively. Effect of Temperature Background on Adaptation Adaptation to a single factor, i.e. salinity or temperature, was found to occur faster than to two simultaneously changed factors; but in nature brown shrimp are simultaneously exposed to several environmental variables. As such, it is important to know the salinity and temperature combinations to which the shrimp can adapt quickly. The background environmental factors, particu- larly temperature, seemed to influence the salinity and temperature adaptation considerably. In order to understand some of these processes, the metabolic response curves were reproduced from the different adaptation conditions in Figures 115, 116, and 117. Brown shrimp which were adapted to 18° or 32°C were found from the respiratory rates to re-adapt faster to their original control conditions (15°/ooS and 25°C) (Fig. 115). A comparison of the respiratory means between the three groups of shrimp in 15°/ooS showed no significant differences (Table 22). However, in salinities other than control, the rate of adaptation varied 241 Table 20 Effect of Temperature Change on the Respiratory Rates of Penaeus aztecus in the Process of Adaptation to Salinity and Temperature The animals were transferred for adaptation from 15°/ooS and 32°C background. Test Comparison Level of Salinity Temp ("C) ,,, Temp CQ Signifi- / o o Accl Test Accl Test cance 32 18 32 25 ns* 2 32 18 32 32 P=.001 32 25 32 32 ns 32 18 32 25 P=.001 5 32 18 32 32 P=.0Ol 32 25 32 32 P=.001 32 18 32 25 P=.001 10 32 18 32 32 P=.001 32 25 32 32 P=.001 32 18 32 25 P=.001 15 32 18 32 32 P=.001 32 25 32 32 P=.001 32 18 32 25 P=.001 25 32 18 32 32 P=.001 32 25 32 32 P=.001 32 18 32 25 P=.001 36 32 18 32 32 P=.001 32 25 32 32 ns ^ns : not significant. 242 Table 21 Effect of Temperature Change on the Respiratory Rates of Penaeus aztecus in the Process of Adaptation to Salinity and Temperature The animals were transferred for adaptation from 15°/ooS and 18°C background. Test Comparison Level of Salinity Temp ("C) ,,, Temp (^C) Signifi- °/ / o o Accl Test Accl Test cance 18 18 18 25 P=.01 2 18 18 18 32 P=.001 18 25 18 32 P=.001 18 18 18 25 P=.001 5 18 18 18 52 P=.001 18 25 18 32 ns * 18 18 18 25 P=.001 10 18 18 18 32 P=.001 18 25 18 32 P=.001 18 18 18 25 P=.001 15 18 18 18 32 P=.001 18 25 18 32 P=.001 18 18 , 18 25 P=.001 25 18 18 18 32 P=.001 18 25 18 32 P=.001 18 18 18 25 P=.001 36 18 18 18 32 P=.001 18 25 18 32 P=.001 ^ns: not significant. 243 0 1 2 10 24 48 TIME-hours 96 120 144 168 Figure 115. Effect of control temperature on the time course of oxygen consumption rates in Penaeus aztecus during salinity adap- tation at 25°C. Control temperatures are shown along with the corresponding respiratory responses 10 24 TIME -hours 72 96 120 144 168 Figure 116. Effect of control temperature on the time course of oxygen consumption rates in Penaeus aztecus during salinity adap- tation at 32°C. Control temperatures are shown along with the corresponding respiratory responses o E I CL z o o o >- X o 0. 50 0.40 0. 30 0. 20 0. 10 0.00 0. 50 0. 40 0. 30 0.20 0. 10 0.00 0. 50 0.40 0.30 0.20 0. 10 0.00 0. 50 0.40 0. 30 0. 20 0. 10 0.00 0. 50 0.40 0. 30 0. 20 0. 10 0.00 0. 50 0.40 0. 30 ^f 2%o ■ 32°C - . 1 1 1 1 1 \ ll 1 1 1 1 1 1 5°/oo 32 °C .25^^ i8°c — ^'T ~-~~-~~.r... ;,..., ^. L. . . 1 1 1 1 1 1 ^/ I I I 1 1 1 • 1 - I07oo- 32-^: - ■ - -25'cJ\ ~ i_i_, 1 1 1 ., 1 1 1 1 1 1 1 - ff 257oo - 32°C - • •25^ O E I ID O O O >- X o D-.'^O 0.25 0.20 0.15 0 . 1 0 2hr. ' — "*-» ^ ACCL le'C TEST 25°C CONTROL I). 50 - 0.45 - 0.40 0.35 0.30 SALINITY- ppt Figure 118. Comparison of the oxygen consumption rates of Penaeus aztecus during the first 10 hours of adaptation and the new steady-state levels. From the control conditions 15°/ooS and 18°C, the shrimp were transferred for salinity adaptation at 18°C (section A of the figure), 25°C (B) and 32°C (C) I). IS - II. Ill (1.05 r^ CONTROL ACCL 25°C TEST 18°C / t X \l hr // ... \^> 2 hr ,4hr STEADY STATE •^6'hr. O n . 30 z 13 0.25 o o LlI X B ACCL 25°C TEST 25°C ^^' - Ih^. _ — — ^Vt •••->- 10 hr. V . 0.20 0.50 0.45 0.40 0.35 - 0.30 0.25 0.20 STEADY STAT?^-^^!^'"^^::-;^-^'*"'^-^ ::.>'"'-' ,^^ ^ CONTROL 10 15 25 .^6 SALINITY- ppt Figure 119. Comparison of the oxygen consumption rates of Penaeus aztecus during the first 10 hours of adaptation and the new steady-state levels. From the control conditions 15°/ooS and 25°C, the shrimp were transferred for salinity adaptation at 18°C (section A of the figure), 25°C (B) , and 32°C (C) n . V) i'.;s 0.20 0.15 - Ph (/) e u OJ 0X3 H H (U -J (U DO 1/1 (U llx M a. e u oo H c o E o o on u o t/l 1/) tA) 7) II II (X c c c c t-~ CTl r-- vO r^ CM Osl CM rsj to to to tM "d- O 00 VD LO LO (Nl to to to to r^j LO o LO LO \D ■-H 1—1 CM to rsi (Ni rsi (N (Ni CM to to to to to to (/)(/) t/l !/) t/l to c c c c c c O (Nl ^ CM t^ ■^ to CM r— I (M (NJ CN ^ LO (^ rsi CTi r^ r-i (NJ r-H (-Nj rxi rxi On! LO O LO LO vO •—I r-H (M to UO LO LD LO LO LO (~\l (M (Nl (Nl (N) rN t/l l/l 10 (/5 (/) c c c c c rM CTl (NJ ,-H K) ^H (N O i-H i-H >-H .-H C o •H (J o CO C/t) t/l t/l t/l t/l t/l c c c: c c r~- \D o 1^ 00 (N fM to to to \0 VO (Nl t-H 00 (M (NJ to •^ to (Nl LO O LO LO i-H I— t (Nl (Nl (NI (NJ (Nl CN to to to to to to t/l to to (/) C II c c c c a. O^ to to iTi >* .— I (N (N (N t-H (NJ ro (7l (7i (N (Ti LO •^ INJ (N (>a I— I (NI to (NI LO O LO LO \D f-H 1— I (N to LO LO LO LO LO LO (NI (NI (Nl (NJ (NJ (N t/l t/l to to to to c c c c c c VD LO O 00 .-H C?l 1— ( 1-H ^H O i-H O c o ■H 4-> nl E ■H f-H tj O CO to t/l ^^ (/I to t/l C C ii" C C C a. to r-l to rt- CTl i-H to ta- to to to to ■— I O O Tt o ^ to •^ rf to ■* (NI (Nl LO O LO LO \0 r-l I— I CM to (NI (Nl (NJ (NJ CM og to to to to to to to (/I t/l t/l t/l ^-^ C C C C C ii' r~- (5^ to 1-H .— I r-- (N .-H (NI (NJ (Nl (Nl LO CO 00 O (NI LO CM ■— I (Nl CM CM to (NI LO O LO LO \D •-H ^H (Nl to LO LO LO LO LO LO CM CM (N (N CM CM (Nl r-H to t/l c c to 00 t-H O t/l to c c CMOOCMCTltOrM "^ v£)(NltOC7ltO00 ^-^ ^Or-l(7^ (NIO— <0.-H^ O ^CMrtOr-HO o K) |^ r-(^ °^ o lO CM fN ^o OOOOOOOOOOOO COOOOOOOOOOO OOOOOOOOOOOO ^,-H,-HrtrHr^ ^^^^^^ . ^^^^^^ OQ (_j c (30 o c t/l c * ion concentrations (Table 26). The control medium was prepared with synthetic sea salt. Tests were conducted at 18°, 25°, and 32°C. Behavior in control salinity The experimental shrimp were active in 25 °C for about 30 minutes, possibly as a reaction to handling during the transfer to the contain- ers. One shrimp died within the 24 hour observation period. Two shrimp became stressed after three hours and the others resumed nor- mal activity after one hour. The shrimp were hyperactive at 32 °C for almost two hours. One shrimp died due to stress in seven hours; a second one died during the night (ten hours later) after molting. The surviving shrimp became quiescent for the duration of testing following the initial hyperactivity. The shrimp were generally inactive in 18°C. They walked or swam around the tank occasionally for about 30 minutes. Later they were quiescent for the rest of the test period. The behavior in the artificially constituted control medium did not differ from that in the natural seawater in the three test tem- peratures. Effect of deviated sodium Most shrimp survived in 25 °C except for deaths which occurred due to molting in media with 85 and 120% sodium (Na) ion concentrations (Table 26). The shrimp were hyperactive for two to three hours in 95% Na and for one hour in 85% Na before becoming quieter. The animals were inactive in 120% Na throughout the test period. At 18°C all of the shrimp were quiet from the start and remained in- active throughout. Two shrimp developed abdominal cramps in 95% Na and died within 24 hours. More shrimp died at 32°C than in 25° or 18°C. The shrimp were all hyperactive for one to two hours after transfer and later became 257 Table 26 Effect of Deviated Ions on the Mortality Rates of Penaeus aztecus* The shrimp were tested in 15°/ooS with deviated ions from 15°/ooS (normal ionic concentration) and 25 °C background. Ionic Test Temp( sratures Concentration 18" 'c 25°C 32' ^'C Co) D s D S D S Control 0 0 1 0 2 0 Sodium 85% 0 0 2 0 l+(2) 1 95% 2 0 1 0 2 0 120% 0 0 1 0 4 . 2 150% 0 2 C2) 0 3 0 Calcium 5% 1 6 5 5 8 2 10% 1+(1) 3 6 6 6 4 15% 0 1 1 1 7 3 25% 0 0 4 0 4 4 35% 0 0 3 0 5 1 Magnesium 0% 0 0 1 0 1 0 1% 0 1 2 0 1 2 4% 0 0 0 3 0 0 6% (1) 1 1 1 1 0 Potassium 10% 0 2 5 0 5 6 30% 0 2 1 2 3 1 40% 1 2 1 0 4 1 50% 0 0 0 1 5 0 60% 1 2 2 0 8 0 *The percentages represent the actual ion concentrations present in each test medium. Mortalities are shown in terms of actual deaths (D) , shrimp stressed (S) beyond the point of recovery, or deaths due to molting (in parentheses) . Ten shrimp were tested in each condition. 258 quiescent. In 85% Na one shrimp was dead by 1-1/2 hours and two others died after molting. Three shrimp died in 12 hours in 95% Na and in 24 hours in 120% Na. The mortality rate did not indicate any trend associated with the deviated sodium ion. It is likely that the deaths were due to a combined effect of high temperature (32°C) and deviated sodium concentration. Effect of reduced potassium In media of low potassium (K) concentration the brown shrimp exhibited different levels of activity, experienced stress and mor- tality, and developed paralysis and abdominal cramps. At 25 °C the shrimp showed symptoms of stress in the medium with 10% K within one hour after transfer. Five animals died after three hours. Out of these, one was molted and two had become paralytic. The five survivors were quiet. In 30% K the animals felt the stress from six hours but only one shrimp died by 24 hours. The remaining animals were active throughout the observation period. In 40, 50, and 60% K the shrimp were initially hyperactive. Later some experi- enced stress. Three shrimp died in 60% K as opposed to one in 40% and none in 50% K. The incidence of abdominal cramping was high at 18°C. The rate was six in 10% K, highest of all concentrations. The lowest rate was two in 50% K. Usually cramps developed by six hours and continued thereafter; they exhibited no trend in relation to the ionic concen- tration. Mortality was lowest at 18°C, one each in 40% and 60% K media. At 32 "C the mortality rate was higher than in the other tempera- tures. The rate did not show any pattern because the highest number of deaths occurred in 60% K. In 10% K, which was the lowest potassium concentration, only four shrimp died. Lowest mortality was in 30% K. Generally the shrimp were hyperactive in all the test media, possibly 259 due to the temperature raise. Cases of paralysis or cramps were not known. The pattern of activity level was much the same as in the control--a short period of increased activity at 25°C, longer hyper- activity at 32°C, and relative inactivity at 18°C. Effect of reduced calcium Reduced calcium (Ca) levels had the greatest impact on the sur- vival rates of shrimp of all the ions tested. The mortality rate increased roughly in proportion to the reduction of calcium ion. In addition, high temperature increased the death rate (Table 26) . At 25°C the salinity media with 5 and 10% Ca concentration levels proved to be "lethal." The term "lethal" implied that none of the shrimp survived in these concentrations (Table 26) . The salinity media with 15, 25, and 35-o Ca were "critical" for the survival of some of the test shrimp. No deaths occurred in salinities containing calcium levels above 35-0. The lethal calcium concentration range in- creased to 5, 10, and 15% Ca at 32°C. The critical range included 25 and 35% Ca levels. In contrast to this condition, in 18°C none of the reduced calcium levels were lethal for the shrimp. Calcium levels 5, 10, and 15% appeared to be critical for the survival of some of the shrimp only. However, at 18 °C the shrimp developed muscular cramps in the reduced calcium concentrations at the rate of three in 10% Ca and one each in 15 and 25% Ca. Another difference was that the shrimp were more active than those in normal water of 15°/ooS at 18°C. Effect of reduced magnesium The shrimp appeared more active in the reduced magnesium (Mg) concentrations at the three temperatures than those from the control. However, in these media the animals showed signs of stress later. At 25°C the shrimp became hyperactive after 20 hours in salinity with 0% Mg. Two of them were under stress by eight hours in 0% Mg. ^60 Two of them were under stress by eight hours but none were dead by the end of the test period (Table 26) . The times at which stress began in 1, 5, and 6% Mg were erratic, as were the death rates. There was one death in 6% Mg and two in 1% Mg. The latter deaths occurred after molting. At 18°C reduced magnesium had little effect on survival rates within the 24 hour period. A few cases of stressed animals were present in 0, 1, and 6% Mg with only one death in 0% Mg. At 32°C more animals exhibited stress than in 18° or 25°C. Four shrimp died in 0% Mg during the 24 hour period. One animal each in 1% and 6% Mg became stressed, and they were later killed and eaten by the others. The feeding response usually indicates the presence of normal behavior. Effect of Deviated Cation Concentrations in Low Salinities on the Behavior and Survival Brown shrimp were acclimated separately for one week in synthetic seawater of 5 and 10°/ooS concentrations at 25°C temperature. The acclimated animals were tested in the respective control salinities having normal ion concentrations and in salinities with deviated cation concentrations. Their behavior and survival rates were de- termined in each condition (Table 27). In 5°/oo control salinity the shrimp were somewhat more active than the animals tested in either 10 or 15°/ooS at 25°C. Otherwise the general beK-avior was normal in both 5 and 10°/ooS. One shrimp, however, developed cramps in 5°/ooS. Effect of deviated sodium In both salinities 5 and 10°/oo there was no marked effect of decreased or increased sodium concentration on the behavior and 261 Table 27 Effect of Deviated Ions on the Mortality Rates of Penaeus aztecus The shrimp were tested from a background of 25 °C and normal 5°/oo and 10°/ooS media. Ionic Ionic Concentration 5' / o oS Concentration CO 107o oS (%) D S D s Control (2) 0 Control (1) 0 85% Sodium 2 1 120% Sodium (1) 0 15-0 Calcium 1 0 15% Calcium 2 1 25% Calcium - - 25% Calcium 0 0 0% Magnesium 4 1 0% Magnesium 8 0 40% Potassium 2 0 40% Potassium 6 1 50% Potassium 1 1 60% Potassium 1 0 60% Potassium 1 0 ''The percentages represent the actual ion concentrations present in each test medium. Mortalities are shown in terms of actual deaths (D) , shrimp stressed (S) beyond the point of recovery, or deaths due to molting (in parentheses) . Ten shrimp were tested in each condition. 262 survival of shrimp. However, two shrimp died in 5°/ooS after ex- periencing an initial stress in 85% Na concentration. In 10°/ooS one shrimp died in 120% Na after molting. Effect of reduced potassium In 5°/ooS the initial behavior in 40% and 60% K media was similar to the control shrimp. Seven hours later two shrimp in 40% K started experiencing stress and both died in about 23 hours. In 60% K one shrimp died. In 10°/ooS the shrimp developed stress earlier and in greater numbers. As a result of the stress, six shrimp out of ten died in 40% K medium. In 50% K one shrimp died by 11 hours and the others were depressed. In 60% K one shrimp died by 22 hours but it was not stressed until after 11 hours. Effect of reduced calcium The lethal concentration levels of 5% and 10% Ca were not used in these studies. At 5°/ooS containing 15% Ca the shrimp apparently behaved similar to the control shrimp. However, one animal was stressed after 11 hours and died in 22 hours. The shrimp were very quiet in 10°/ooS with 15% Ca concentration. One animal was stressed and one died by 11 hours. By 23 hours the stressed animal died and another became stressed. In 25% Ca the animals were active and appeared normal. Effect of reduced magnesium The shrimp in 5°/ooS with 0% Mg were initially active. After one hour one animal became cramped and died by 11 hours. Later two more shrimp were under stress until they died by 22 and 23 hours. The surviving shrimp were inactive and some of them were undergoing stress by the time the studies ended. In 10°/ooS with 0% Mg half of the shrimp died by 22 hours. Three more died by 25 hours. In the 3% Mg medium survival rates improved. The only shrimp that was dead by 25 hours had shown signs of stress after 23 hours. At that 263 time two more shrimp were stressed. The remaining shrimp were quiet but occasionally swam around. Effect of Acclimation to Media with Deviated Ions on Tolerance in Extreme Salinity and Temperature Brown shrimp were acclimated at 25°C to 5 and 10°/ooS in which the normal concentration of cations was changed as described in the material and methods. It was found that large numbers of brown shrimp did not survive in the deviated media over a long period. Mortalities in the acclimation process increased from 50 to 100% in certain com- binations. Very few shrimp survived acclimation to 10°/ooS with 40% K and 6% Mg. In 5°/ooS with 40% K concentration few animals survived. Therefore, the availability of acclimated animals became a limiting factor in performing the tests. Tlie surviving shrimp were tested in 2.5 and 42.5°/ooS at 18° and 32°C combinations. Survival of shrimp in these tests was very low (Table 28). Shrimp tranferred from any of the acclimation salin- ities to 42.5°/ooS did not survive at either temperature. The shrimp died sooner at 32°C than at 18°C. At 32°C the shrimp were hyper- active for 10 to 30 minutes. Some showed signs of stress, lying on their sides and beating the pleopods feebly. Others were depressed. Some survived for 12 to 18 hours but most of the shrimp died within one to five hours. At 18 °C the shrimp were quiet from the beginning and became depressed (or stressed) within two hours. Deaths occurred between five and 20 hours. The surviving three animals after 24 hours were severely stressed and could not have lived much longer. Almost all shrimp acclimated to 10°/ooS were dead in 2.5°/ooS at both temperatures. Only one shrimp from the 15% Ca medium sur- vived at 32°C. The animal was, however, inactive. Some shrimp acclimated to deviated ion media in 5°/ooS survived in 2.5°/ooS, 264 but they were all inactive or depressed (Table 28), None showed nor- mal behavior. Combinations of extreme salinity and temperature test conditions caused the shrimp to undergo some unusual contortions. Normally, in the resting posture the pleopods extend forward with the exopodites angled down. But in 2.5°/ooS some shrimp rested with the exopodites curled forward, while in 42,5°/ooS the exopodites were angled back- ward with the tips curled up. This was noticed in animals acclimated to 5 and 10°/ooS. Abdominal cramps were seen in both test salinity and temperature combinations. Some shrimp were also cramped in the control salinities (5 or 10°/oo) at 18° but not at 32°C. In 5 or 10°/ooS most of the shrimp survived but they were in- active. There was a higher mortality at 32" than in 18°C, Oxygen Consumption in Deviated Ion Media The respiratory rates of brown shrimp were determined in rela- tion to deviated ionic ratios of calcium, magnesium, and potassium in the seawater. Within the concentration levels used in our studies, sodium had no apparent adverse effect on the behavior or survival of shrimp during a 24 hour period. Therefore, sodium was excluded from these studies. Oxygen consumption in synthetic seawater Oxygen consumption rates were determined at test temperature 18°, 25°, and 32°C by directly transferring brown shrimp from 15°/ooS and 25°C, The control test medium (15°/ooS) was prepared with syn- thetic sea salt and contained the normal ion ratios. The purpose of these experiments was to determine whether the respiratory rates ex- hibited significant differences from the rates in natural salt water. 265 Table 28 Effects of Acclimation of Penaeus aztecus to Salinities of 5 and 10°/oo with Deviated Ionic Ratios and Testing in Normal but Extreme Salinities 2.5 and 42.5°/oo* Accl Ionic Concentration Test Temp Test Sail nities (Voo) Sal 2 .5 Control 4 2.5 (Voo) (%) (°C) x* * D S T D S T D S 5 150% Na 18 10 4 2 10 0 1 10 9 1 32 8 4 1 8 1 2 8 8 - 5 15% Ca 18 7 6 0 7 2 1 6 5 1 32 8 4 0 8 2 1 8 8 - 5 6% Mg 18 4 2 2 4 0 1 4 3 1 32 8 5 0 7 3 1 7 7 - 5 50% K 18 _ _ _ _ _ _ _ - _ 32 6 6 - 6 9 0 6 6 - 10 120% Na 18 4 2 2 4 0 0 4 4 _ 32 8 8 - 8 5 1 8 8 - 10 15% Ca 18 2 2 _ 2 1 1 2 2 _ 32 8 7 0 8 4 0 8 8 ~ *The percentages represent the actual ion concentrations present in each test medium. Mortalities are shown in terms of actual deaths (D) , shrimp stressed (S) beyond the point of recovery, or deaths due to molting (in parentheses) . **T refers to total number of shrimp tested. 266 The zero hour reading in Fig. 121 represents the metabolic rates of brown shrimp in natural sea water of 15°/ooS and 25°C. The control animals (those tested at 25°C, 15°/ooS) maintained a steady metabolic state at a level slightly lower than 0.2 mlO_/L/g (Fig. 121). The new steady-state level was established in about six hours after an initial increase in the respiratory rate. This ini- tial increase might have occurred in response to the introduction of synthetic seawater into the respiratory chamber replacing the original salt water control. The initial increase in the oxygen uptake at 32 °C was greater than the increase in 25°C. Also, the steady-state metabolic level at 32°C, established at about six hours, was higher than in 25°C. At 18 °C there was a gradual drop in the oxygen consumption during the immediate response phase from the control level 0.19 mlO„/L/g. After four hours, the new respiratory steady state was established at 18°C well below the control level. During the stabilization process uniformly high individual metabolic variations occurred between one and four hours through- out the test temperature range (Ref . standard deviation values) . Although the tests were made in 15°/ooS synthetic seawater, sta- bilization and new steady-state levels occurred much the same as in natural seawater of the same concentration. Survival of the test animals was low at 32°C. Oxygen consumption in reduced calcium The effect of 25% Ca on the respiratory rates of shrimp was studied at 18°, 25°, and 32°C (Fig. 122). The respiratory pattern in 25% Ca concentration varied from the control shrimp in two respects. The immediate responses at 25° and 267 0 . 50 0. 40 0 . 30 - 0 20 0 10 -? 0 00 _l ^ c\ O 0 S(l E 1 1 7 0 40 O \- Q. n ^0 s. 3 to 2 0 20 O o 0 10 z UJ 1) 00 o >- X o 0 50 _l L J I I I L. 0.4 0 0.30 0. 20 0 . 10 0.00 -8 J I I I L 0 1 3 4 10 TIME-hours ACCL 25°C TEST jex ACCL as'x: TEST 25°C ACCL 25X TEST SZ*^ ^2 Figure 121. Oxygen consumption rates of Penaeus aztecus in 15°/ooS synthetic seawater of normal ionic composition at 18°, 25°, and 32°C. The control conditions were 15°/ooS normal seawater and 25°C 268 0.50 0.40 - 0. 30 - 0. 20 0. 10 0. 00 I E 0.50- I ^ 0.40 0. 30 0. 20 0. 10 -1 I 1 L Q. CO o >- X o 0.00 - 80i J 1 I L. 0 1 10 TIME— hours ACCL SS'C TEST I8X ACCL 25°C TEST 25°C 4 - Figure 122. Oxygen consumption rates of Penaeus aztecus in 15°/ooS with 25 percent calcium ion concentration 269 32°C continued for three hours instead of one hour as in control. At 18°C there was no change in the oxygen uptake from control (Fig. 121). Secondly, the survival rate was considerably lower at 25°C; at 32°C there was a total mortality within the first day. Survival rate was not affected at 18°C. At 32°C oxygen consumption in 25% Ca was higher than the consumption in the control medium at the same temperature. At 18°C the shrimp did not exhibit any marked changes in 25% Ca from the control animals also tested in IS^C, either in deviation of the stabilization period or in the steady metabolic level. The steady-state level at 25°C was similar to that in the control con- ditions (Fig. 121) , but the stabilization process took longer. Mor- tality rate increased progressively with the temperature rise from 18°C. Eff-ect of total elimination of magnesium Oxygen consumption rates were determined at 18°, 25°, and 32°C in 15°/ooS synthetic salt water with 0% Mg (Fig. 123). The respira- tory rates during the immediate regulation were higher in 18° and 25 °C than in control shrimp tested at the same temperature. But the rates at 32°C were similar to the rates in the control medium at 32°C. Stabilization of the metabolic rates was faster at 18° than in 25°C in the same test conditions. The new steady metabolic level at 18°C was slightly higher than in control media 15°/ooS and 18°C (Fig. 121). The high oxygen consumption rate in 18°C was in synchrony with the reported increased activity in the behavioral ob- servations in the corresponding test conditions. Removal of magne- sium affected the survival rate more adversely at 32°C than in 18° or 25°C. The shrimp survived in 32 °C for less than ten hours. During 270 ^ CM O 0. 00 I I I I I 1_ ■^ 0.50- 0. 40 g I- Z3 to z o o UJ o 0. 00 4^ I I I I L 0 12 3 4 10 TIME -hours ACCL 25°C TEST I8°C ACCL 25°C TEST 25X ACCL 25°C 0. 50 r TEST sa*^ - 0. 40 T " - 0. 30 10.-. I/T II I :4 - 0.2 0 "SOcV^ i|0 ^ I 0.10 1 1 1 1 1 1 1 ~ 24 Figure 123. Oxygen consumption rates of Penaeus aztecus in 15°/ooS with 0 percent magnesium 271 this period the respiratory rate of these animals was similar to the shrimp tested in the control medium at 32°C. Steady metabolic level at 25°C was also similar to the level in the control shrimp (15°/ooS and 25°C, Fig. 121). Effect of reduced potassium on oxygen consumption Respiratory rates were determined at 18°, 25°, and 32 °C in 15°/ooS media with 30% K concentration (Fig, 124). At 18° and 25°C the immediate responses and stabilization pro- cesses in 30% K solution took the same amount of time and followed the same pattern as in the 0% Mg tests. In both test temperatures the steady metabolic levels were maintained at the same levels as in the control conditions (15°/ooS and 18° and 25°C, respectively; Fig. 121). The steady-state level in 32°C was at a slightly higher level than in the control (15°/ooS and 32°C, Fig. 121). The survival rates at 18°, 25°, and 32°C were similar to the control test condi- tions. Metabolic rates in relation to temperature Temperature effect on the metabolic rates is shown comparatively in media with 25% Ca, 0% Mg, and 30% K concentrations (Fig. 125). In all these test conditions, the respiratory rates increased with a temperature rise from 18° to 32°C. In 25% Ca and 30% K con- centrations the oxygen consumption levels at 32°C were higher than in control shrimp tested in 32°C. The respiratory rates in 0% Mg did not increase at the same rate as in the other test conditions. The temperature effect on the oxygen consumption was not significantly different in 18° and 25 °C from the control shrimp tested at the same temperatures. 272 .p> O I O »- Q- =3 O) 2 O o UJ o >- X o 0.50- 0. 40 0 . 30 0 . 20 0. 10 0. 00 0. 50 0. 40 0. 30 0 . 20 0.10 0.0 0 _i I I ' J L 0 12 3 4 8 10 TIME— hours ACCL 25°C TEST I8*C ^8 - ACCL 25X TEST 25°C -H8 - ACCL25°C TEST 32'C- 24 Figure 124. Oxygen consumption rates of Penaeus aztecus in 15°/ooS with 30 percent potassium concentration 273 0 . 10 I) . .so - CONTROL 0.20 25°C - 1 1 1 1 1 1 \e°c 0.10 1 1 C7I 0.40- 0 12 3 4 1 0 TIME-hours 2 4 Figure 125. Effect of temperature change on the oxygen consumption rates of Penaeus aztecus in deviated ion media 274 Metabolic rates in relation to ionic changes Metabolic rates of shrimp tested in the different deviated ion media are compared by test temperature in Fig. 126. The oxygen con- sumption levels of shrimp tested in 0% Mg at 25°C were similar to the control level (15°/ooS and 25°C). The respiratory levels in media with 25-6 Ca and 30% K were alike at 25°C; but these levels were higher than in the control. The metabolic rates in 25% Ca were consistently high in all test temperatures; but the oxygen consumption in 0% Mg and 40% K exhibited an opposite trend in relation to temperature changes. For instance, the oxygen consumption level in 0% Mg medium was higher than in 40% K at 18°C. This trend was reversed at 32°C with the result that the oxygen consumption in 40% K was higher than in 0% Mg. The shrimp in 25% Ca and 0% Mg experienced a total mortality in 32 °C within ten hours . 275 CM O O I- Q. CO z o o liJ o >- X o 0.4(1 0.31) ''.20 0.10 0.40 0 . 30 0.20 0.10 0.40 0.50 0.20 0.10 ACCL 25°C TEST IS^C ^•^-i^fiS^ CONTROL ^307d< ACCL 25°C TEST 25»C A / \ 'f/ •• V *" S. 25%Cq. ^v^coTrT;Ro" - - -;r 07oMg. -1 1 1 L. ACCL 25X TEST 32"^ 39% K CONTROL •♦07oMg -I 1 I I L 0 12 3 4 () 10 TIME -hours Figure 126. Comparison of the effects of deviated ionic ratios on oxygen consumption rates of Penaeus aztecus at 18°, 25°, and 32°C 276 IV: DISCUSSION Time Course of Salinity Adaptation The results demonstrate that salinity adaptation in brown shrimp depends upon the magnitude as well as the direction of salinity or temperature change. The data also show that the metabolic rates, determined on the basis of oxygen uptake rates, change in accordance with the external salinity or temperature conditions. Alterations in the metabolic rates are usually associated with changes in osmotic and ionic concentrations of body fluids. Changes in the functional responses indicate that the adaptation to salinity or temperature occurs through immediate regulation, stabilization, and steady-state phases. Blood chloride ion regulation generally parallels the osmoreg- ulation. Minor ionic deviations apparently do not affect brown shrimp adversely, but major changes in calcium and potassium seem to create severe physiological problems and threaten their survival. Salinity adaptation in brown shrimp is apparently related mainly to their background on the one hand and to the deviation in test sa- linity and temperature conditions on the other hand. The simplest case of salinity adaptation was observed in animals acclimated and tested at 25°C in a salinity range of 2 to 36°/oo. In these test salinities, maximum initial changes in the respiratory rates oc- curred within two hours. This was followed by another few hours of stabilization. New metabolic steady-state levels were finally reached in less than a day, which indicate the completion of sa- linity adaptation. In the same test conditions the initial osmotic and ionic changes occurred within two hours in 5, 10, 15 (control), and 25°/ooS and in six hours in 2 and 36°/ooS. Both osmotic stabili- zation and steady-state levels were obtained in less than a day. 277 However, in the process of salinity adaptation there was a relatively high mortality in 2 and 36°/ooS, probably due to either salinity stress or the process of molting. In this case where a single test variable, i.e. salinity, was used the salinity adaptation occurred within a day in the animals that survived. Also the mor- tality rate was generally low. Besides there was good synchrony between the steady respiratory responses and blood osmotic or ionic regulation. The correlation between these two responses was dis- turbed in experiments where two variables, i.e. salinity and tempera- ture, were applied simultaneously. In animals acclimated to 25 °C and tested in 32 °C the salinity related respiratory sequence was changed from 25°C test conditions. Oxygen uptake levels were higher than in 25°C. Animals were hyper- active from 1/2 hour in 15°/ooS to four hours in 2°/ooS; in 25 and 36°/ooS they appeared normal. Mortality was higher than in 25°C. The decreasing oxygen consumption levels in 10, 15 Ccontrol) , and 25°/ooS after two or three days suggest the possible impact of starvation. New steady-state levels appeared faster in osmotic or ionic regulation than in the metabolic rates. At 32°C test conditions the increased activity level apparently had influenced the respira- tory rates and delayed the new steady-state metabolic levels. There were no indications, however, that the behavioral responses could directly influence the osmotic and ionic regulation as much as they did the metabolic rates. The discrepancy between the two physiological responses was even more pronounced in shrimp acclimated to 25°C and tested in 18°C. Steady metabolic levels were obtained within six hours in all but 2 and 36°/ooS. There was a total mortality in 2°/ooS within four hours. Steady-state levels were not seen in ionic or osmotic regu- lation until three or four days although the initial changes in 15 and 25°/ooS were completed in two hours and in other salinities in 278 six hours. However, salinity adaptation seemed to be complete in 5 to 25°/ooS within the one week period. While judging the adaptive state in 2 and 36°/ooS the mortality rates should not be ignored. The high mortalities indicate that adaptation to these extreme sa- linities is difficult in a direct transfer from 15°/ooS and 25°C. The adaptation process was further complicated by a simultaneous temperature change. Adaptation to these extreme salinities should be made easier by a gradual change of the salinity and temperature factors. Despite the gradual change, when salinity and temperature factors are involved simultaneously, the salinity adaptation ob- viously becomes slower than in normal temperature conditions. In shrimp acclimated and tested in 18°C salinity adaptation seemed to occur fast within the 10 to 36°/ooS range. Within two days there was a total mortality of shrimp used in metabolic studies in 2°/ooS and a high mortality in 5°/ooS. In other salinities there were no mortalities other than those in molting. With the exception of 2°/ooS, steady-state levels were achieved in metabolic and os- motic or ionic regulation in all salinities within six hours. In 2 and 5°/ooS although major osmotic changes were completed within six hours, steady-state levels were not seen until the fourth day. By acclimation to 18°C the survival and rate of adaptation were improved at 18°C test temperature in 10, 15, 25, and 36°/ooS but not in 2 or 5°/ooS. Low-salinity and low temperature combinations impaired the rate of salinity adaptation in brown shrimp. Similar findings were made in our previous studies (Venkataramiah et al. 1974). It also fits previous field observations showing that penaeid shrimp leave the cool, low-salinity bays in winter (Gunter 1950). The shrimp acclimated to 18°C and used in the respiratory studies at 25°C experienced a high mortality in 2 and 5°/ooS. Stabilization in the metabolic rates and steady-state levels occurred in 10, 15, and 25°/ooS within the first day and in 2, 279 5, and 36°/ooS after three days. Conversely, steady-state osmotic and chloride ion levels were attained within the first day in all salinities except in 5°/ooS. In 18°C test conditions the metabolic rates reached stable levels sooner than the osmotic and chloride levels. The tempera- ture increase was shown to influence the respiratory rates more than the osmotic or ionic regulation. Naturally this would result in a lack of harmony between the two responses in the process of adaptation. The mortality was very high in shrimp acclimated to 18° and tested in 32°C. The shrimp used for oxygen studies died in 2°/ooS within four hours. In six days there was a similar mortality in 25°/ooS. Survival was generally poor in 5 and 36°/ooS. However, in 10 and 15°/ooS new steady-state levels appeared both in metabolic and salt regulation within the first day. In the animals that sur- vived in 36°/ooS steady metabolic and chloride levels appeared after four days. In 25°/ooS the deaths occurred much later after reaching a steady-state metabolic level on the first day. In 2 and 36°/ooS the animals failed to reach steady-state levels. Sudden temperature change from 25° to 18° or 32°C obviously reduced the range of sa- linity adaptation from 5 to 25°/ooS. The shrimp acclimated to 32 °C experienced a heavy mortality in 2°/ooS in the three test temperatures. At 18°C the animals used for the oxygen studies survived in 2°/ooS for six hours only. In the other salinities death rate was moderate to high. One possible ex- planation for a higher mortality rate at 32°C than in test tempera- tures 18° or 25°C was the starvation effect. Starvation was shown to lower the respiratory rates; but its effects on the salt regula- tion on a short-term basis were not known. Newell (1973) observed that starvation may reduce the scope for activity in the intertidal 280 invertebrates. He considered this as an important adaptation for using the metabolic reserves during periods of stress. There were discrepancies observed between the mortality rates of brown shrimp in the oxygen studies and those in the osmotic and ionic regulation studies. There are two possible reasons for this outcome. In the metabolic studies, fewer shrimp (11 or less) were used in each test condition than in the osmoregulation and ionic regulation where up to 200 were used. Also in the oxygen studies, the shrimp were confined to narrow respiratory chambers with just a small amount of sand in the bottom. In the other studies shrimp were able to swim about or bury freely which probably contributed to their better survival rates in unfavorable test conditions. Irrespective of test temperature the shrimp acclimated to 32°C attained new steady-state levels in the metabolic, osmotic, and chloride regulation in 10 and 15°/ooS within the first day. At 32 °C test temperature steady-state metabolic levels appeared in 5°/ooS on the first day but not in 25°/ooS. In 25°/ooS there was a gradual drop in the oxygen consumption. However in 25°/ooS steady-state osmotic and chloride levels did appear. Between the first and fourth day large osmotic fluctuations occurred in 2, 5, and 36°/ooS and slowed the rate of adaptation process. Chloride ion reached steady state on the first day in 10, 15, 25, and 36°/ooS and after two days in 2 and 5°/ooS. In shrimp acclimated to 32°C and tested at 25°C steady metabolic rates appeared on the first day in 10, 15, 25, and 36°/ooS and on the third or fourth day in 2 and 5°/ooS. Steady-state levels in osmotic and chloride regulation in 2 and 5°/ooS appeared on the fourth day more or less at the same time as in the metabolic rates. The osmotic and chloride ion regulation in 36°/ooS was still in the process of stabilization by the end of one week. 281 At 18°C the surviving shrimp in 2°/ooS failed to control the salt loss. In view of the large osmotic fluctuations in 2 and 36°/ooS the adaptation was considered incomplete. In 10, 15, 25, and to some ex- tent in 5°/ooS, steady-state levels in metabolic and salt regulation were reached within the first day. Our results indicated that the large juvenile brown shrimp can be more readily adapted to 2 to 36°/ooS range by a direct transfer at normal temperature 25°C than at 18° or 32°C. With a temperature change from 25°C to 18" or 32°C the range of salinity adaptation de- creased. At these temperatures, adaptation was possible to a nar- rower range of 5 to 25°/ooS and more favorably to 10 and 25°/ooS without heavy mortalities. Between 18° and 32°C salinity adaptation proceeded more favorably at 18°C with fewer deaths. Temperature Influence on Osmotic and Chloride Regulation Although it was suggested above that at 25°C salinity adapta- tion was possible to a range of 2 to 36°/ooS, the osmotic and chlo- ride concentration levels in Figs. 101 and 102, respectively, indi- cated that such adaptation became more effective to a 5 to 25°/oo salinity range. In other salinities osmotic or ionic regulation was less stable. Also the animals acclimated to 25°C withstood better the temperature changes (18° or 32°C). The osmotic and chloride regulatory pattern of shrimp acclimated to 18°C and tested in 18°, 25°, and 32°C appeared much the same as in 25°C acclimated shrimp. The only difference was that the low tempera- ture acclimated animals were temperature-sensitive. The sensitivity was determined from the compensatory responses noticed in the regula- tory pattern toward the temperature change. This would result in the wide separation of the response curves from each other. The shrimp acclimated to 32°C were even more temperature-sensitive than others. 282 In these animals the salt concentration levels decreased in tempera- tures other than 32°C. Furthermore, the shrimp lost large amounts of salts in salinities below 10°/ooS even in 32°C which was their acclimation temperature. The chloride regulation in shrimp acclimated to 25°C was less affected by temperature change within a salinity range of 5 to 25°/oo. Nor was this influence noticed in a 10 to 25°/ooS range in animals acclimated to 32 °C. The low temperature acclimated shrimp were, however, more temperature-sensitive in the chloride regulation as were the 32°C acclimated animals in osmotic regulation. Although chloride regulation usually paralleled the osmoconcentration, variations occurred between the two responses depending upon test temperature. Temperature Influence on the Steady-State Levels Conclusions made on salinity adaptation at 18°C exclusively on the basis of respiratory rates may be misleading. At IS^C the res- piratory rates dropped to very low levels within a few hours and re- mained throughout. At 18°C these levels usually designated as the steady-state levels appeared faster than in 25 °C in most of the sa- linities. On the basis of these levels it is hard to conceive the idea of faster salinity adaptation in 18°C than in 25°C. Moreover, the appearance of steady metabolic and osmotic levels in 18°C at different intervals provides conflicting evidence on the state of salinity adaptation. Bulnheim (1974) studied the respiratory metabolism in the isopod Idotea balthica from the Baltic Sea. The salinity in this area aver- ages 15°/oo. The laboratory control salinity was 10°/oo. Habitat temperature was not reported; but the isopods reproduced at 15°C temperature in the laboratory which indicates its proximity to the ambient conditions. The isopods transferred from 15 °C achieved 283 steady metabolic rates in 5°C within two hours and showed no further fluctuations in the succeeding 24 hour period. IVhen the test tempera- tures were reversed from 5° to 15°C the new metabolic steady-state levels were reached in about 15 hours followed by prolonged fluctua- tions. It is not known why the isopods took five times longer to acclimate to 15°C than to 5°C. The respiratory pattern in brown shrimp at 18° and 25 °C approximate these responses; but we are not sure whether the sudden drop in the respiratory rates at 18°C can be termed a steady-state level. At 18°C the state of salinity adaptation determined on the basis of osmotic and chloride regulation therefore appeared to be more reliable than the exclusive respiratory responses. The meta- bolic rate can be taken as a reliable criterion when the responses are in harmony with the osmotic or chloride ion regulation under identical test conditions. Behavioral studies of shrimp at 18°C suggested that adjustment to low temperature was a slower process than was indicated from the respiratory levels. Salinity and Temperature Requirements in Relation to Size In our previous studies shrimp acclimated to 21°C responded more adversely to temperature changes than those acclimated to 31°C (Ven- kataramiah et al. 1974). Conversely in the current experiments the animals acclimated to 18°C exhibited better survival and faster sa- linity acclimation than those acclimated to 32°C. The shrimp accli- mated to 32°C experienced heavy mortality. The rate of salinity adaptation was relatively slow. The discrepancy in the temperature relationship of shrimp in the two studies may be due to size vari- ations of the experimental animals. Juveniles of 70 mm average length were used previously. The mean length of shrimp in these studies was 95 mm. In the life cycle of brown shrimp, some impor- tant physiological variations are likely to occur within these two sizes. 284 This possibility was shown in the isosmocity of blood osmotic and chloride levels with the external salinities. In the 70 mm juveniles blood chloride concentration was isosmotic at about 17°/ooS level (Venkataramiah et al. 1974). In the present experiments the chlorides were isosmotic at 20-22°/ooS level and the osmotic concen- tration at 22-25°/ooS level. The chloride isosmotic level agrees favorably with the isosmotic level of 700 to 800 mOsm (about 24 to 28°/ooS) in Penaeus aztecus reported by McFarland and Lee [1963). The small variations in the isosmotic levels between these two studies can be attributed to the larger sized shrimp (>100 mm) of McFarland and Lee which were collected from a higher salinity (690 mOsm or about 23.7°/ooS). Other studies from our laboratory have shown that salinity tolerance is also a function of size in brown shrimp. While fol- lowing the development of salinity tolerance in postlarval brown shrimp Biesiot (1975) observed that the tolerance range expanded gradually from the time the postlarvae were 6 mm long (10 days old). From 19 days of age (9-10 mm length) the postlarvae grew faster in 18°/ooS than in either 25 or 32°/ooS. In earlier studies salinity tolerance of postlarvae (20 mm) and juveniles (size 1: 21 to 45 mm range; size 2: 50 to 75 mm range) was studied by Venkatara- miah et al. (1974). It was found that postlarvae survived longer than juveniles in the extremely high salinity 59.5°/oo. In low salinities 0.34 and 3.4°/oo juveniles (size 1) survived longer than the others. The salinity tolerance expanded gradually and reached a widest range in late postlarvae. From this stage onward the tolerance range decreased gradually as they became older. Chew (1975) carried bat comparative respiratory studies with two size groups of shrimp of 60 and 100 mm mean length by direct transfer and by acclimation to a series of salinities. The respiratory rates showed significant differences between these groups in re- sponse to salinity changes. The responses of larger shrimp (100 mm) 285 were more favorable toward the high salinities (36 and 45°/ooS) than the smaller ones (60 mm) and vice versa. O'Driscoll (1975 unpub- lished data) found a narrower salinity tolerance range in adults of 140 mm mean length than in the adults of 110 mm mean length. The evidence indicates that wide salinity tolerance range is important for the survival of the species when they are young; it becomes narrower in later life. In young shrimp this ability pro- motes a wider dispersal in the estuaries and bayous. In adults that are about to emigrate offshore, tolerance to low salinities has no obvious survival value. The shrimp (95 mm) in our present experiments were about to emigrate to the open sea. As such, the size factor has another im- plication concerning the temperature requirements. Offshore tem- peratures are normally lower than the summer and fall temperatures in coastal waters. It was shown in the earlier part of the discus- sion that the shrimp acclimated to 18°C were faster in salinity adaptation than those acclimated to 32°C if not at 25°C. In view of the impending offshore emigration it is likely that the shrimp are undergoing a sort of physiological preparation to meet the low temperature and high-salinity conditions. Such preparation may occur on a seasonal cyclic basis in brown shrimp. This possibility was shown below on the basis of rates from an earlier study by Venkataramiah et al. (1974). Prior to the experiments the juvenile brown shrimp (70 mm) were acclimated to 8.5, 17.0, 25.5, and 34.0°/ooS and 21°, 26°, and 31 °C combinations for more than three weeks. Then they were tested in the same conditions as their acclimation. The respira- tory rates were lower in low salinity (8.5 and 17.0°/oo) and warm temperature (26° and 31°C) and high salinity (25.5 and 34.0°/oo) and low temperature (21 °C) combinations than in others. The low 286 respiratory rates indicate reduced motor activity in the respective combinations. These combinations associated with low respiratory rates approximate the seasonal habitat conditions of brown shrimp. Another important observation was made in the present behavioral studies. The survival rates were generally high in 25 and 36°/ooS regardless of temperature as opposed to 18°C and 2 and 5°/ooS combi- nations. However, when the transfer was made directly from 15°/oo to 36°/ooS the salinity adaptation was either slow or did not occur at both 18° and 32°C. In nature such an abrupt change from 15 to 36°/ooS is unnatural. In the course of offshore emigration the shrimp are exposed to high salinities in stepwise increments. In laboratory experiments the shrimp did not exhibit great variations in 25°/oo from 15°/ooS either in osmotic or in chloride regulation regardless of test temperatures. The shrimp responded as if they were more favorably inclined toward a higher salinity than the 70 mm long shrimp. The optimal salinity range of the smaller juveniles seemed to exist in a concentration range of 8.5 and 17.0°/ooS. Significant differences were also observed in other physiological responses be- tween low and high salinities (Venkataramiah et al . 1974). Osmoregulation and Energy Relations Brown shrimp acclimated and tested at 25°C exhibited hyperosmotic regulation in 2, 5, and 10°/ooS and hyposmotic regulation in 25 and 36°/ooS with respect to the test salinities. The magnitude of the osmotic or chloride changes was roughly in proportion to the devia- tion of test salinities from 15°/ooS. Corresponding increases in the oxygen uptake were observed in the respective test conditions. The re- sponse pattern suggested a positive interaction between the respiratory rates on the one hand and the osmotic and chloride regulation on the other. In Pachygrapsus crassipes. Gross (1957) observed a similar in- crease in the oxygen consumption in salinities other than the control. 287 He explained the increased oxygen consumption on the basis of higher motor activity in other salinities where they attempted to escape the salinity stress. Lofts (1956) and Rao (1958) attempted to cor- relate the oxygen consumption in prawns from marine and brackish water habitats with their osmotic gradients in their respective sa- linity media. Lofts found that when tested in a fresh water to 65°/ooS range the marine population of Palaemonetes varians con- sumed the lowest amount of oxygen in 26°/ooS which was close to their natural habitat. The brackish water population had the lowest respiratory rates in 6°/ooS. In the Indian prawn Metapenaeus mono- ceros, Rao (1958) observed a similar respiratory pattern between the marine and brackish water populations. From a more recent study Kutty et al. (1971) reported that the prawn Penaeus indicus accli- mated and tested in a 5 to 60°/ooS range exhibited the lowest oxygen uptake in 10-15°/ooS. These findings suggest that the energy spent for osmotic regulation can be measured from the amount of oxygen consumed. Also the results imply that the higher the osmotic gra- dient between the blood salt concentration and the external salin- ities, the more energy prawns would require for regulation. The results of the present study showed a good correlation at 25 °C between the metabolic and osmotic responses, thereby tending to confirm the findings of the above authors. When the test temperatures were altered from normal to 18° or 32°C the above correlation did not exist any more. Under the changed temperature conditions the osmotic and chloride regulation pattern remained intact as in 25 °C with a few quantitative changes; but the salinity- related respiratory pattern was altered from the original. At 32 °C the oxygen consumption was higher in IS^/ooS than in other media. In 2 and 5°/ooS the consumption was lowest. The respiratory rates no longer showed any relation to the osmotic gra- dient in the respective salinities. In animals acclimated to 25° and tested in 18°C the respiratory rates were well below the normal level. In some of the salinities the rates maintained no correlation with the osmotic or ionic gra- dients. For instance the oxygen consumption in 36°/ooS was at the same level as in 15°/ooS. In 2°/ooS the consumption was similar to 10°/ooS. However, in 5, 10, and 15°/ooS some kind of correlation was sustained between the two responses as in 25°C. The salinity-related respiratory sequence of the shrimp accli- mated to 18°C and tested in 25° or in 32°C more or less resembled the pattern in animals acclimated at 25°C and tested in the same tempera- tures. Nevertheless, in the test temperature of 18°C minor variations appeared in the respiratory rates between the rest of the salinities, except in 2 and 5°/ooS. At the same time, the osmotic and chloride regulatory pattern did not change from the original. Consequently there was no consistent correlation between the osmotic regulation and the respiratory rates. In animals acclimated to 32° and tested at 18°, 25°, and 32°C the oxygen consumption rates showed no relation to the osmotic or chloride levels in the respective salinities. In animals acclimated and tested in 25°C the metabolic responses were influenced mainly by the salinity changes. But in 18° and 32°C, as a result of temperature influence the correlation between the oxy- gen consumption and osmotic or chloride gradients disappeared. Potts and Parry (1964) observed that changes in metabolic rates are, in most cases, much too large to attribute to energy expenditure for ionic and osmotic regulation alone. They stated that increased metabolic rates caused by salinity change were not confined to the tissues involved in osmotic work. Therefore we are not certain that the respiratory rates in shrimp reflect the energy spent to maintain the osmotic or chloride gradients. 289 The effect of salinity variations on the metabolic rates of marine and brackish water invertebrates have been grouped by Kinne (1971) under four headings: 1. Increase in subnormal salinities and/or decrease in supra- normal salinities; 2. Increase in subnormal and supranormal salinities; 3. Decrease in subnormal and supranormal salinities; 4. Remain essentially unaffected. Types 1 and 2 represent, according to Kinne, largely the meta- bolic rates in euryhaline invertebrates; type 3 represents the stenohaline forms; and type 4, the holeuryhaline (or extremely euryhaline) forms. In classifying these types Kinne evidently did not consider the tempera- ture effect. When both temperature and salinity factors are involved simultaneously, types 1, 2, and 3 metabolic responses are observed in the same species of brown shrimp. Metabolic Compensation to Temperature Change At 32°C the respiratory rates did not increase above the levels in 25°C in 2, 5, and 36°/ooS. On the contrary, in 15°/ooS the oxygen uptake increased in proportion to the temperature rise from 18°C. The temperature effect on respiration was consistent between 18° and 25°C in all test salinities; but the test temperature effect of 32°C disappeared progressively as the test salinities were in- creased or decreased from 15°/ooS. Consequently, variations in the respiratory rates became nonsignificant between 25° and 32°C in 2, 5, and 36°/ooS. This might reflect a possible failure to increase the oxygen consumption at 32°C beyond the levels in 25°C. Since the failure occurred at 32°C, naturally one might suspect the likelihood of a starvation effect. Earlier in the results, the decline in res- piratory rates at 25° and 32°C in 10, 15, and 25°/ooS were attributed to a possible starvation effect; but no such effect was seen in 2, 5, or 36°/ooS. It appeared as though in extreme salinities the shrimp did 290 not exhibit the normal appetite. Therefore, starvation was an unlikely factor for the low oxygen uptake at 32°C in 2, 5, and 36°/ooS. On the other hand, this appears to be a case where the shrimp failed to re- spond to temperature rise. Animals acclimated to 18° and 32°C and tested in 25° and 32 °C conditions, exhibited more or less the same trend. Regulation of Other Cations The results have shown that magnesium regulation was consistently hyposmotic and calcium regulation hyperosmotic to the test salinities. Magnesium concentration changed relatively less in salinities below the control as did the calcium and potassium. In 25 and 36°/ooS magnesium increased considerably in animals acclimated to 25°C and tested in 18°, 25°, and 32°C. Similar increases were also found in shrimp acclimated to 32°C and tested in 25° and 32°C. The ion in- crease was much less in animals acclimated to 18°C and tested at 18°, 25°, and 32°C as well as in those acclimated and tested at 32° and 18°C, respectively. In 25 and 36°/ooS calcium increased uniformly regardless of acclimation or test temperatures. Animals acclimated to 25° and 32°C exhibited slightly higher ion concentrations at 32°C than in other test temperatures. Potassium was hyperosmotic below the 25°/ooS level at 18° and 25°C, without regard to the acclimation temperature. At 32°C test temperature the ion was hyperosmotic to the entire test salinity range in shrimp other than those acclimated to 18°C. In Penaeus aztecus and P^. setiferus, McFarland and Lee (1963) observed muscle calcium and magnesium at fairly constant levels in brackish water and seawater; but potassium increased with salinity. 291 In mantis shrimp Squilla empusa the serum magnesium, calcium, and potassium decreased with decreasing salinity (Lee and McFarland 1962) , Below 60% seawater the calcium level remained constant, at a concentra- tion which appeared similar to the blood calcium level in our studies. Concentrations of blood calcium, magnesium, and potassium were measured in crabs Hemigrapsus nudus and H. oregonensis in eight sa- linities (6 to 175% seawater) and three temperatures (5°, 15°, and 25°C) (Dehnel and Carefoot 1965; Dehnel 1966, 1967). In 6 to 75% seawater the blood potassium and calcium were considerably hypertonic to the external salinities. In 100 to 175% seawater the ion concen- trations approached isotonicity. The potassium regulation is in agreement with our findings in P^. aztecus; but calcium regulation paralleled the isosmotic line in 25 to 36°/ooS range. The shrimp acclimated at 18°C exhibited isosmocity between 32 to 36°/ooS level at 18° and 32°C. Blood magnesium in the crabs was hypotonic in all salinities above 12% seawater. In H. nudus and H. oregonensis test temperatures exerted no in- fluence on the regulation of blood potassium and calcium. High tem- perature impaired the magnesium regulation. There is contrary evidence in brown shrimp that temperature variations influence the calcium, po- tassium, and magnesium regulation. Potassium concentration levels in shrimp increased with test temperature regardless of acclimation tem- perature. Temperature was shown to influence the regulation of cal- cium and magnesium. Evaluation of the physiological implications of these ionic changes is beyond the scope of these experiments. However, changes in the ionic ratios in the test salinities are shown to influence the behavior, survival, and metabolic rates in brown shrimp. High or low sodium levels used in these studies evident- ly have no effect on the behavior and survival. Removal of magnesium from the salinity medium at 18°C increased the activity level above 292 that in 15°/ooS at 18°C. Normally the shrimp were quiet in 18°C with good survival rates in the test salinities; but the survival de- creased with a temperature rise and there was a total mortality at 32°C in low salinities. The calcium ion has a greater impact on survival than the other ions. At 25°C mortality increased progressively as the calcium levels decreased below 35%. In 5 and 10% calcium there was a total mortality. In 15% calcium there was also total mortality at 32°C. In 25 and 35% calcium levels more animals died at 32° than in 25°C. None of the test calcium levels were lethal at 18°C. In 18°C no deaths occurred in calcium levels above 15%. However, at this temperature abdominal cramps developed in salinity media with 10, 15, and 25% calcium levels. Schwenke (1958) had shown in red algae that among the calcium, magnesium, and potassium ions absence of calcium caused the highest degree of cell damage. Damage increased with increasing exposure time. Species of red algae showed variations in tolerance to the absence of calcium. Lack of calcium resulted in rapid loss of potassium in Porphyra species. The presence of calcium, potassium (and probably also magnesium) is required for normal functioning of the cellular processes, including ion transport (Eppley and Cyrus 1960). It was shown that mortality in shrimp increased in reduced ion concentrations with temperature rise and reached a maximum level at 32°C. Calcium is known to exert a stabilizing effect on protein structures and metabolic processes in estuarine invertebrates re- sulting in an overall increase in tolerance, especially to high temperatures. The high mortality which occurred in brown shrimp at 32°C in low calcium media may be attributed to this condition. Schlieper and Kowalski (1956) observed that additional amounts of calcium and magnesium increased the thermal stability of tissues 293 of Mytilus edulis, while additional potassium decreased it. At reduced ionic concentration levels, nearly 40% of the test animals survived in 40% potassium while in 0% magnesium and 25% calcium levels none survived beyond ten hours at 32°C. Besides improving the thermal tolerance, calcium content plays an important role in the permeability as was shown in the estuarine turbellarian Gunda ulvae. These invertebrates can tolerate temporary exposure to fresh water only in the presence of sufficient calcium. In fresh and low saline waters they suffer from extensive water uptake and salt loss unless both media have a supranormal calcium content (Pantin 1931a, b; Weil and Pantin 1931). The high mortality in low potassium media was probably due to the combined influence of the ionic deficiency and high tem- perature than by either of them singly. Mortality was highest of all at 32°C. Abdominal cramps developed mainly in 18°C. Although the incidence of cramping did not follow any trend that was related to the potassium levels, the highest number of cases was found in the lowest level of 10%. It was shown from the behavioral studies that abdominal cramps also occurred at 18°C mainly in 2 and 5°/ooS. In these salinities the potassium levels were generally lower than in 10, 15, 25, and 36°/ooS regardless of acclimation temperature. All this evidence points to the fact that the abdominal cramps are likely to occur mostly in combinations of low temperature and low potassium levels. Literature on abdominal cramps in brown shrimp is absent to the best of our knowledge. From the oxygen consumption studies, metabolic rates were shown to be influenced primarily by temperature changes and to a lesser extent by reduced levels of calcium, magnesium, and potassium. Also temperature changes altered the respiratory rates induced by the test solutions. The respiratory rates in 25% calcium were consis- tently higher in 18°, 25°, and 32°C; but the rates in 30% potas- sium and 0% magnesium media exhibited opposite trends to each other 294 in response to temperature change. In 0% magnesium the oxygen up- take decreased with temperature increase and in 30% potassium it increased. The high oxygen uptake in 0% magnesium at 18°C corre- sponded with the relatively high activity. In 30% potassium there was a good correlation between the increased metabolic rates and the progressively increasing potassium levels with temperature. The conclusions derived on the effects of deviated ions on the physiological responses on a 24 hour basis may not be similar to those derived from long-term experiments. The high mortality which occurred in the process of acclimation to 5 and 10°/ooS with 40% potassium and 25% calcium ion concentrations indicated more serious consequences of long-term exposure than the short-term effects. Also, very little is known about the sublethal effects that might occur in shrimp due to ionic deviations. The osmotic regulation and metabolic studies provided no evi- dence in favor of sex effects on the functional responses in brown shrimp. However, this conclusion leaves us in the dark regarding the disproportionate distribution of the sexes in natural conditions. Our own field collection during several years had shown that usually females outnumber males in the catches in local bayous and estuaries. Euryhaline invertebrates are known to exhibit greater osmo- regulatory capacity in the lower part of their normal temperature range than in the upper (Kinne 1971). The degree of osmotic in- dependence in these animals also tends to increase with decreasing tolerable temperature. The partial accuracy of these conclusions is shown in the high survival rates of brown shrimp at 18°C. The survival was best of all the test temperatures at 18°C as long as the shrimp were held in the control salinity (15°/oo) or in its vicinity. Also the survival was highest at 18°C in salinity media with reduced calcium, potassium, and magnesium levels; but the 295 degree of osmotic independence was limited at 18°C to a narrower sa- linity range than in 25°C. At 25°C the salinity adaptation was faster than in the other temperatures; the rate of overall mortality due to salinity change was lower. The range of salinities to which the shrimp can be adapted by direct transfer was wider than in 18° or 32°C. This is possible in Penaeus aztecus because of its geo- graphic distribution which is confined to tropical and subtropical waters. 296 V: SUMMARY The time course of salinity adaptation is determined in Penaeus aztecus by studying their behavioral and physiological responses. These responses include survival and metabolic rates, blood osmotic and ionic regulation of chloride, magnesium, calcium, and potassium. Experimental shrimp of 95 mm mean length were acclimated for about a week to 18°, 25°, and 32°C in a control salinity of 15°/oo. The acclimated shrimp were then transferred separately to 2, 5, 10, 15, 25, and 36°/ooS for salinity adaptation at 18°, 25°, and 32°C. At 25°C the activity level and other behavioral responses were influenced mainly by salinity changes from the control salinity. At other temperatures (18° and 32°C) the interaction of salinity and temperature apparently determines these responses. Three phases--immediate responses, stabilization, and new steady- state levels--were recognized in the salinity adaptation process. The duration of each phase varied with the test condition. The attainment of steady states of metabolic and osmotic or chloride levels indicated the completion of adaptation to the respective con- ditions. Salinity adaptation was faster in 25°C than in 18° or 32°C. It occurred in a wider range from 2 to 36°/ooS within a week. The range of adaptation decreased from 5 to 25°/ooS at 18°C, and from 10 to 25°/ooS at 32°C. In these salinity ranges mortality was low during adaptation. Outside of these salinity ranges adaptation was slower. Adaptation to some conditions was not possible by direct transfer. Prior acclimation of shrimp to 18° or 32 °C accelerated the rate of adaptation in the respective test temperatures. Also the range 297 of salinity adaptation increased; but the temperature change from 25°C to 18° or 32°C was not beneficial in any respect. Between 18° and 32 °C, the low temperature was more favorable for faster adapta- tion with higher survival rates. Temperature modified the behavior in shrimp considerably. Be- havior apparently altered the respiratory rates more than it did the osmoregulation. The variations in behavior influenced the metabolic responses differently and distorted the usual synchrony between the metabolic and osmotic responses. Therefore, conclusions made on sa- linity adaptation in temperatures other than normal, particularly in 18°C, might be misleading. Under such conditions presumably the steady-state osmotic levels would provide a more reliable evaluation on the state of adaptation. The osmotic and chloride regulation was hyposmotic in salinities above and hyperosmotic in salinities below 15°/oo. This was more or less a consistent pattern throughout the test temperatures. At 25°C the respiratory rates varied in accordance with the osmotic gradient in the test conditions and thus exhibited a positive interaction between the osmotic and metabolic responses. This inter- action was nevertheless absent or confined to fewer salinities in 18° and 32° than in 25°C. The inconsistent interaction between the two physiological responses obviously did not endorse the hypothesis that oxygen consumption reflected the energy expenditure involved in osmotic regulation. Salinity and temperature requirements were shown to be size re- lated in brown shrimp. As the shrimp grew to adulthood they favored salinities above 10°/oo and temperatures below 25°C. In contrast, the smaller juveniles (70 mm) of our previous study preferred salini- ties below 17°/oo and temperatures 26° or slightly higher but not 298 lower temperatures. The preference for high salinity and low tempera- ture combinations may indicate a physiological preparation of the ju- veniles for offshore emigration. On this basis a possibility for the existence of seasonal salinity-temperature rhythms was suggested. Magnesium, calcium, and potassium ions comprised a minor portion of the blood ions compared with chlorides. The concentration of these ions increased with external salinity. Temperature change influenced the regulation of each ion differently. Potassium exhibited a more consistent temperature-related pattern than others. The behavior and survival of shrimp were not significantly changed by minor changes in the composition of sodium, magnesium, calcium, or potassium; but major changes in calcium and potassium affected the shrimp more ad- versely than other ions. Below 35% of the normal calcium concentration the animals started dying. The death rate increased with reduction in calcium levels. In 5 and 10% calcium all test shrimp died. Also the death rate increased with temperature. A similar trend was noticed in solutions with low magnesium and to a lesser extent with low potassium. In media with low potassium levels there was a high incidence of abdominal cramps at 18 "C. At 25° and 32 °C the incidence was none or rare. Tlie cramping was observed in the behavioral studies mainly in 2 and 5°/ooS media at 18 °C. It was inferred, therefore, that low potassium levels and low temperatures were likely combinations in which abdominal cramps occur. 299 VI: EPILOGUE The information presented here confirms certain ideas concerning life history previously discovered by studies in the field. In one aspect it gives a physiological background and an explanation of some ecological phenomena, more specifically the goings and comings, sea- sonal cycles, size and growth, and distribution over an environment with variable salinity and temperature limits, and the seasonal pat- terns and cycles that are preeminent and dominant in life histories. These patterns are not always regular and they undergo perturba- tions that lead to variations close to or even beyond tolerable limits of temperature and salinity at a given time and place. In part this report defines some of these limits and variations. When the tolerable environmental limits are reached or trans- gressed the result is catastrophe. These sometimes occur under natural conditions at the margins of the sea, and are usually un- controllable because they are large-scale phenomena connected with weather, climate, etc. The same results sometimes can be obtained locally, and slowly because of the works of man. These works are modifiable and in part their impact on the environment can be seen, if we have the proper understanding. This report adds to the under- standing of the basic environmental factors, temperature and salinity, on shrimp. Brown shrimp were selected for study in part because of their great abundance and commercial importance, which similarly en- hances them as ecologically important representatives of estuarine species. Possibly the most worthwhile generalization of this report is that it reveals that a slow gradualism in change of basic environ- mental factors is best for the organisms and permits their adaptation in many ways; therefore, from the standpoint of biological management and fisheries this principle should always be held in mind in the planning of works with hydraulic, hydrological , and hydrographic im- pacts upon the estuaries and the margins of the sea. 300 During the past few years, including the flood year of 1973, the shrimp catch statistics of the north central Gulf of Mexico, the area of greatest production in North America, have reflected the influence of an above average supply of fresh water. The total hydrographic data may be available for a broad analysis of this general question, although it had not been made, but at least with the present data we are in a much better position to give some ex- planation of how populations of this abundant fishery species, the brown shrimp, are influenced by salinity and temperature changes. We may even see the hope of an approach to a predictive situation of some reliability, which in a sense is a major goal of fisheries and wildlife management, as set forth in the broader view by John Stuart Mill (1848), who said, "Of all truths relating to phenomena, the most valuable to us are those which relate to their order of succession. On a knowledge of these is founded every reasonable anticipation of future facts, and whatever power we possess of in- fluencing these facts to our advantage." The above remarks concern large general or regional phenomena; but there is another approach to the effects of temperature and salinity changes and that concerns the more local changes of river basins. Some studies have been made by the Louisiana Department of Wildlife and Fisheries (White 1975) in individual estuarine drainage basins . It would seem, for instance, that an examination of the Tennessee- Tombigbee project as a local work, in the light of some of the above re- sults, may yield information of value for operation of this project within the General Design Memorandum. After all it sits on the fourth largest drainage basin of North America and Mobile Bay is a productive fishery area. In fact we may suggest, in the light of these results that any large project within the shrimp productive area of the southeast coast of the United States might be examined with some profit in the planning stage. 301 VII: ACKNOWLEDGMENTS The authors wish to express a deep sense of gratitude to Mr. Frank A. Herrmann, Jr. , Assistant Chief, Hydraulics Labora- tory, Waterways Experiment Station, Vicksburg, Mississippi. Mr. Herrmann has been monitoring these investigations on penaeid shrimp continuously from 1970. In this capacity he happened to be intimately associated with us. Mr. Herrmann's personal in- terest in these studies and handling of the problems with under- standing are greatly appreciated. We are indebted to Dr. Harold D. Howse, Director, Gulf Coast Research Laboratory, for his active concern and help during these investigations. Our gratitude is due to Mr. Robert P. Ochsner, Administrative Officer, Mrs. Lee Rasor, Financial Secretary and Mrs. Eleanor Wasmer, Purchasing Officer for their services during this project. The authors also acknowledge with thanks the technical assis- tance received from Mr. Dennis L. Chew and the help of Mrs. Mary Ann Macias, laboratory artist, Mrs. Ann McCaslin and Mrs. Sharon Christmas of the Physiology Section in the preparation of the manuscript. Mr. J. Y. Christmas, Fisheries Research and Manage- ment Section, allowed us to use the computer from his section and Mr. David Boyes, Data Processing Section, assisted in analyz- ing part of the data. We appreciate the help received from both of them. 302 VIII: LITERATURE CITED Aldrich, David V., Carl E. Wood and Kenneth N. Baxter. 1968. An ecological interpretation of low temperature responses in Penaeus aztecus and P. setiferus postlarvae. Bulletin of Marine Science of the Gulf and Caribbean. 18(1): 61-71. Anderson, W. W. , J. E. King and M. J. Lindner. 1949. Early stages in the life history of the common marine shrimp, Penaeus seti- ferus (Linnaeus). Biological Bulletin (Woods Hole). 96: 168- 172. Biesiot, Patricia. 1975. Salinity tolerance of postlarval brown shrimp Penaeus aztecus in relation to age and acclimation sa- linity. M.S. Thesis. Bowling Green State University, Bowling Green, Ohio. 63 pp. Birshtein, J. A. and G. M. Beliaev. 1946. The action of the water of Balkash Lake on the Volga-Caspian invertebrates. (Russ.; Engl, summary). Zoologicheskii Zhurnal . 25: 225-236. Bulnheim, H. P. 1974. Respiratory metabolism of Idotea balthica (Crustacea, Isopoda) in relation to environmental variables, acclimation processes and moulting. Helgolander wissenschaft- liche Meeresuntersuchungen. 26: 464-480. Burkenroad, Martin D. 1934. The Penaeidae of Louisiana with a dis- cussion of their world relationships. Bulletin of the American Museum of Natural History. 68(2) : 61-143. Burkenroad, Martin D. 1939. Further observations of Penaeidae of the northern Gulf of Mexico. Bulletin of the Bingham Oceano- graphic Collection. Yale University. 6: 1-62. Burkenroad, Martin D. 1949. Occurrence and life histories of com- mercial shrimp. Science. 110(2869): 688-689. Chew, Dennis L. 1975. Studies on the effects of variations in sa- linity, size, and sex on the respiratory rates of brown shrimp, Penaeus aztecus Ives 1891. M.S. Thesis. University of Southern Mississippi, Hattiesburg. 83 pp. Clarke, F. W. 1924. The data of geochemistry. Bulletin of the U. S. Geologic Survey. 770: 1-841. Cronin, L. E. 1967, The role of man in estuarine processes. In George H. Lauff (Ed.) Estuaries. Publ. No. 83. American Asso- ciation for the Advancement of Science. Washington, D.C. pp. 667-689. 303 Dehnel, P. A. 1962. Aspects of osmoregulation in two species oY~ intertidal crabs. Biological Bulletin (Woods Hole). 122: 208-227. Dehnel, P. A. 1966. Chloride regulation in the crab, Hemigrapsus nudus . Physiological Zoology. 39: 259-265. Dehnel, P. A. 1967. Osmotic and ionic regulation in estuarine crabs, In George H. Lauff (Ed.) Estuaries. Publ. No. 83. American Association for the Advancement of Science. Washington, D.C. pp. 541-547. Dehnel, P. A. and T. H. Carefoot. 1965. Ion regulation in two species of intertidal crabs. Comparative Biochemistry and Physiology. 15: 377-397. Duval, M. 1925. Recherches physico-chimiques et physiologiques sur le milieu interieur des animaux aquatiques. Modifications sous 1' influence du milieu exterieur. Annales de I'Institut oceanographique. Monaco. 2: 233-403. Eppley, R. M. and B. S. Cyrus. 1960. Cation regulation and survival of the red alga Porphyra perforata in diluted and concentrated sea water. Biological Bulletin (Woods Hole). 118: 55-65. Gordon, Malcolm S. 1972. Animal Physiology: Principles and Adapta- tions^. The Macmillan Co"! U.Y'. 592 pp. Gross, W. J. 1957. An analysis of response to osmotic stress in selected decapod Crustacea. Biological Bulletin (Woods Hole). 112: 43-62. Gross, W. J. 1963a. Acclimation to hypersaline water in a crab. Comparative Biochemistry and Physiology. 9: 181-188. Gross, W. J. 1963b. Cation and water balance in crabs showing the terrestrial habit. Physiological Zoology. 36: 312-324. Gunter, Gordon. 1950. Seasonal population changes and distributions as related to salinity, of certain invertebrates of the Texas coast, including the commercial shrimp. Publications of the In- stitute of Marine Science, University of Texas. 1(2): 7-51. Gunter, Gordon. 1962. Shrimp landings and production of the state of Texas for the period 1956-1959, with a comparison with other Gulf States. Publications of the Institute of Marine Science, University of Texas. 8: 216-226. 304 Gunter, Gordon. 1967. Some relationships of estuaries to the fisheries of the Gulf of Mexico. In George H. Lauff (Ed.). Estuaries. Publ. No. 83. American Association for the Ad- vancement of Science. Washington, D.C. pp. 621-638. Gunter, Gordon, J. Y. Christmas, and R. Killebrew. 1964. Some relations of salinity to population distributions of motile estuarine organisms, with special reference to Penaeid shrimp. Ecology. 45(1): 181-185. Gunter, G. and H. H. Hildebrand. 1954. The relation of total rain- fall of the state and catch of the marine shrimp (Penaeus seti- ferus) in Texas waters. Bulletin of Marine Science of the Gulf and Caribbean. 4(2): 95-105. Kahler, H. H. 1970. Uber den Einfluss der Adaptationstemperatur und des Salzgehaltes auf die Hitze- and Gefrierresistenz von Enchy- traeus albidus (Oligochaeta) . Marine Biology. 5: 315-324. Karpevich, A. F. 1958. Uberlebensdauer, fortpftanzung und Atmung von Mesomysis kowalevskyi (Paramysis lacustris kowalevskyi Czern.) in Brackwasser der UdSSR. (Russ.; Engl, summary). Zoologicheskii Zhurnal. 37: 1121-1135. Kinne, 0. 1971. Salinity. In Otto Kinne (Ed.) Marine Ecology, Vol. 1, Part 2. Wiley-Interscience. N.Y. pp. 683-1033. Kirsch, M. 1956. Ionic regulation of some of the major components in river-diluted sea water in Bute and Knight inlets, British Columbia. Journal of the Fisheries Research Board of Canada. 13: 273-289. Kutty, M. N., G. Murugapoopathy, and T. S. Krishnan. 1971. Influence of salinity and temperature on the oxygen consumption in young juveniles of the Indian prawn Penaeus indicus. Marine Biology. 11: 125-131. Lee, B. D. and W. N. McFarland. 1962. Osmotic and ionic concentra- tions in the mantis shrimp Squilla empusa Say. Publications of the Institute of Marine Science, University of Texas. 8: 126- 142. Lindner, M. J. and W. W. Anderson. 1956. Growth, migration, spawning and size distributions of shrimp, Penaeus setiferus. Fishery Bulletin, U.S. Fish and Wildlife Service. 56(106): 555-645. Lobza, P. G. 1945. Salt composition of the Kara-Sea waters and its variations by influence of fluvial waters (Russ.) In Dok 1 ady Juvileinoi Sessii Arcticeskogo Naucno-Issledovatelskogo Insti- tuta, 1920-1945. Izd, Glavsevmorputi, Moscow. 305 Lofts, B. 1956. The effects of salinity changes on the respiratory- rate of the prawn Palaemonetes varians (Leach) . Journal of Ex- perimental Biology. 33: 730-736. Longley-Cook, L. H. 1970. Statistical Problems. Barnes § Noble Books, New York. 297 pp. McCoy, Edward G. and James T. Brown. 1967. Preliminary investiga- tions of migration and movement of North Carolina commercial penaeid shrimps. Proceedings of the Annual Conference, South- eastern Association of Game and Fish Commissioners. 21: 277- 295. McFarland, W. N. and B. D. Lee. 1963. Osmotic and ionic concentra- tions of penaeidean shrimps of the Texas Coast. Bulletin of Marine Science of the Gulf and Caribbean. 13: 391-417. Mill, John. 1848. Principles of political economy. 2 Vols., Boston. Newell, Richard D. 1973. Factors affecting the respiration of inter- tidal invertebrates. American Zoologist. 13: 513-528. O'Driscoll, Philip. 1975. Salinity tolerance of the adult brown shrimp Penaeus aztecus. Gulf Coast Research Laboratory, Ocean Springs, MS. Unpublished. Pantin, C. F. A. 1931a. The adaptation of Gunda ulvae to salinity. 1. The environment. Journal of Experimental Biology. 8: 63-72. Pantin, C. F. A. 1931b. The adaptation of Gunda ulvae to salinity. 3. The electrolyte exchange. Journal of Experimental Biology. 8: 82-94. Pearse, A. S. and G. Gunter. 1957. Salinity. In J. W. Hedgpeth (Ed.) Treatise on Marine Ecology and Paleoecology, Vol. I. Memoirs of the Geological Society of America. 67: 129-157. Pearson, John C. 1939. The early life histories of some American Penaeidae, chiefly the commercial shrimp, Penaeus setiferus (Linnaeus). Bulletin of the U.S. Bureau of Fisheries. 49(30): 1-73. Perry, Harriet M. , J. Ronald Herring, Thomas Van Devender and James R. Warren. 1974. Fisheries assessment and monitoring, annual re- port. CFRD project 2-215-R, segment 1. Gulf Coast Research Laboratory, Ocean Springs, MS. Unpublished data. Potts, W. T. W. and G. Parry. 1964. Osmotic and Ionic Regulation in Animals. Pergamon Press, Oxford. 506 Rao, K. P. 1958. Oxygen consumption as a function of size and sa- linity in Metapenaeus monoceros Fab. from marine and brackish- water environments. Journal of Experimental Biology. 35: SOT- SIS. Schlieper, C. and R. Kowalski. 1956. Uber den Einfluss des Mediums auf die thermische und osmotische Resistenz des Kiemengewebes der Miesmuschel Mytilus edulis L. Kieler Meeresforschungen. 12: 37-45. Schwenke, H. 1958. Uber einige zellphysiologische Faktoren der Hypo- tonieresistenz mariner Rotalgen. Kieler Meeresforschungen. 14: 130-150. Sokal, Robert R. and F. James Rohlf. 1969. Biometry. The Principles and Practice of Statistics in Biological Research. W. H. Freeman and Company, San Francisco. 776 pp. Spaulding, M. H. 1908. Preliminary report on the life history and habits of the "lake shrimp" (Penaeus setiferus) . Bulletin of the Gulf Biologic Station. 11: 1-29. Standard Methods for the Examination of Water and Wastewater. 1965. American Public Health Association, Inc. N.Y^ pp. 406-410. Sverdrup, H. U. , Martin W. Johnson and Richard H. Fleming. 1942. The Oceans. Their Physics, Chemistry, and General Biology. Prentice-Hall, Inc. , New Jersey. 1087 pp^ U.S. Army Corps of Engineers. 1971. Effects of engineering activi- ties on coastal ecology. Report to the Office of the Chief of Engineers. 48 pp. Venkataramiah, A., G, J. Lakshmi, and G. Gunter. 1974. Studies on the effects of salinity and temperature on the commercial shrimp, Penaeus aztecus Ives, with special regard to survival limits, growth, oxygen consumption and ionic regulation. U.S. Army Engi- neers Waterways Experiment Station, Vicksburg, Miss., Contract Report H-74-2, 134 pp. Vinetskaya, N. I. 1959. Salinity of the North Caspian waters. (Russ.) Trudy vsesoyuznogo Nauchno-Issledovatel ' Skogo In- stituta Morskogo Rybnogo Khozyaistva I Okeanografii. 38: 36-51. Viosca, Percy, Jr. 1920. Report of the biologist. Fourth Biennial Report, Louisiana Department of Conservation, 1918-1920, pp. 120-130. New Orleans. 307 Weil, E. and Pantin, C. F. A. 1931. The adaptation of Gunda ulvae to salinity. 2. The water exchange. Journal of Experimental Biology. 8: 73-81. Wengert, Marvin W. , Jr. 1972. Dynamics of the brown shrimp, Penaeus aztecus Ives 1891, in the estuarine area of Marsh Island, Louis- iana in 1971. M.S. Thesis. Louisiana State University. Baton Rouge, La. 94 pp. Weymouth, F. W. , M. J. Lindner and W. W. Anderson. 1933. Preliminary report on the life history of the common shrimp Penaeus setiferus (Linn.). Bulletin of the U.S. Bureau of Fisheries. 48(14): 1-26. White, Charles J. 1975. Effects of 1973 river flood waters on brown shrimp in Louisiana estuaries. Louisiana Wildlife and Fisheries Commission. Technical Bulletin No. 16. 24 pp. 308 APPENDIX A Definition of Terms Acclimation An ecological phenomenon comprising adjustments of organisms to alterations in the intensity patterns of variables in their environ- ment. The success of this phenomenon results in a relative increase in their capacity to survive, reproduce, or compete with other species. The terms adaptation and nongenetic adaptation are synonyms for acclimation. Adaptations often consist of a variety of adjustments of both functions and structures. They may be genetically determined (genetic adaptation) or environmentally induced (nongenetic adaptation). Brackish water A mixohaline water of between 0.5°/oo and 30°/ooS (Venice System) usually found in restricted coastal regions such as estuaries or salt marshes and in larger landlocked seas such as the Baltic, the Caspian and the Ural Sea. Estuaries Bodies of water where seawater is measurably diluted with fresh water from land drainage. Euryhaline Animals and plants which are tolerant of a wide range of salini- ties are euryhaline; as opposed to this, those that are restricted to a narrow range of salinity, usually to full strength seawater or fresh water, are called stenohaline. Hyperosmotic A solution that is more concentrated than the comparison solution. Al Hyposmotic A solution that is more dilute than the comparison solution. Ionic regulation The capacity of organisms to regulate specific ion concentrations in their body fluids by a selective process at the cell surface. Ionic regulation is a general and primitive capacity at both cellular and organismic levels. Isosmotic One solution is said to be isosmotic with another if the two are equal in osmotic concentration. Although many authors use the term isotonic as synonymous with isosmotic, the terms are not identical. Tonicity is defined in terms of the response of cells immersed in a solution. An isotonic solution is generally also isosmotic, but this is not necessarily so. Metabolic rates The terms oxygen consumption rates, respiratory rates, and meta- bolic rates are used synonymously. These rates are measured in a variety of units. The units in most common use in static determina- tions are volume of oxygen converted to standard pressure and tempera- ture consumed by an animal per unit time (cc of 0_,/hr; liters of 0^/ day) . In flow through respirometry, the oxygen consumption rate was expressed in ml 0-/L/g. Nongenetic adaptations Involve gradual adjustments of individuals wihtin their genetic limits which are directly induced by the environment and not passed on as such to the next generation; as opposed to the above, genetic adaptations involve changes in the genotype and are the result of speciation and evolution. A2 With respect to both genetic and nongenetic adaptations to tem- perature, one may differentiate between resistance adaptations (varia- tions in tolerance to extreme salinity or temperature) and capacity adaptations (variations in performance within tolerated ranges) . These two types of adaptations are closely related and may occur simultaneously in one and the same individual. Osmoregulators Organisms that can maintain internal concentrations different from that of the concentration in the outside medium and can with- stand wide environmental changes. On the other hand osmoconformers cannot regulate in a medium that is more dilute or more concentrated than their internal concentration with the result that their body fluids reach a concentration approximately equal to that of the sur- rounding water. Osmotic concentration The total effective concentration of all solutes present in a solution is osmoconcentration. It is often expressed in osmoles, i.e., the total num.ber of moles of solute per liter of solvent. The units mOsm used in this report are the number of millimoles of sol- ute per kilogram of solvent. Salinity "The total amount of solid material in grams obtained in one kilo- gram of seawater, when all the carbonate has been converted to oxide, the bromide and iodine replaced by chlorine, and all the organic mat- ter completely oxidized." (Sverdrop, Johnson and Fleming 1942). Serum The liquid which remains after allowing either whole blood or plasma to clot. A3 Standard metabolism Ideally the standard metabolism ratio should be an animal's metab- olism under the simplest and least physiologically demanding condi- tions. For animals other than mammals and birds the minimum metab- olism of fasting individuals at a given temperature is referred to as the standard metabolic rate. In the case of mammals and birds these rates of fasting adult animals under no thermal stress are usually referred to as the basal metabolic rate (Gordon 1972) . Weight specific metabolic rate For purposes of comparing organisms of different sizes, it is often convenient to divide the 0_ consumed per hour by the weight of the animal being measured (Volume 0^=ml 0^/g/hr) . This value is also known as the weight-relative metabolic rate. A4 APPENDIX B Tables I. through IX. Tables X. through XVIII. Tables XIX. through XXVII. Tables XXVIII. through XXXVI, Tables XXXVII. through XLV. Tables XLVI . through LIV. Tables LV. through LVII. Mean blood osmotic concentration + S. E. Mean blood chloride concentration + S. E. Mean blood potassium concentration j|^ S. E. Mean blood calcium concentration +_ S. E. Mean blood magnesium concentration ;|^ S. E, Mean oxygen consumption ;^ S. E. Mean oxygen consumption J^ S. E. in media having variation in cation concentration. Bl Table I. Mean blood osmotic concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 25°C Sampling Interval Test S; ilinit y - °/ 0 o (hour) 2 5 10 15 25 36 0 643+ 2.7 643+ 2.7 64 3+ 2.7 -- 643+ 2. .7 643+ 2.7 1 596^ 7.2 617 + 5.7 652 + 6.1 654 + 9.6 680+ 7, ,7 735j^ 8.8 2 578+ 7.8 63U 4.0 650+ 3.5 654+: L3.2 673+ 6. ,5 791+ 7.4 4 531+ 8.3 621 + 6.5 634 + 5.7 644+: 11.8 680+ 8, .2 846+14.7 6 524+ 9.1 631 + 6.4 649 + 4.9 646+_ 8.5 690+ 5, ,5 873+10.4 10 524+14.1 607 + 4.7 651 + 5.9 647+ 3.2 690 + 4, .1 860+24.9 16 528+ 2.2 609 + 9.6 630 + 2.8 632 + 5.4 680 + 8. ,0 836+15.0 24 523+ 8.2 61 u: LO.O 627 + 5.2 654 + 7.3 691 + ] L5, ,1 863+ 5.2 48 547+ 9.5 594 + 9.9 620 + 4.8 637+ 4.2 703+ 1. ,6 839+ 7.0 72 508+10.3 619 + 7.7 611 + 4.4 635 + 5.9 690+ 5, .2 840+ 8.5 96 508+12.7 606+ 4.3 600 + 4.4 636+ 6.0 704 + 3. .1 858+13.8 168 490+10.3 583 + 2.9 602+ 5.0 632 + 5.0 709+ 3. .0 850+11.1 Avg 551+ 6.6 617 + 2.7 631 + 2.6 643+ 2.0 683+ 3, .0 803+10.2 B2 Table II. Mean blood osmotic concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 32°C Sampling Interval Test Salini ty - Voo (hour) 2 5 10 15 25 36 0 643+ 2.0 643+ 2.0 643+ 2.0 643+ 2.0 643+ 2.0 643+ 2. ,0 1 578+ 3.4 627+ 7.4 660+ 8.1 671 + 3.7 688-^ 9.0 767+ 6. .0 2 555 + 8.6 636+ 8.8 664+ 9.4 665+^ 3.0 708+ 7.0 803+12, .4 4 527+ 6.8 641 + 6.2 659+ 6.4 661 + 7.6 699 + 6.3 854+ 9, ,3 6 514 + 8.7 638+ 4.0 651+ 7.0 653 + 7.1 712 + 4.4 849+11, ,2 10 533 + 7.8 615 + 9.5 636+11.3 670j^ 9.2 712 + 5.6 877+_ 6, ,9 16 516+: LI. 9 585 + 3.4 639+ 3.9 664 + 4.4 705 + 4.6 884+12, .2 24 525 + 8.9 599 + 5.6 634+ 3.2 654 + 4.7 716j^ 4.8 870+15, ,6 48 50 8+ : 11.1 596+ 4.9 636+^ 6.2 635+_ 6.0 714 + 3.7 861+ 9, .5 72 487+: 13.0 587+ 4.4 622+ 5.2 638+ 2.6 718+ 2.6 903+ 1, .8 96 5 19+: 10.7 612 + 9.1 621+ 5.2 654+ 1.8 726 + 2.5 909+ 8, ,8 168 507+ 7.4 602 + 8.6 622+ 3.2 648+ 4.5 745 + 2.1 883+ 9 .6 Avg 545j^ 6.6 617-*^ 3.1 640+ 2.3 654+ 2.0 701 + 3.7 821+11, .8 B3 Table III. Mean blood osmotic concentration +_ S. E. in P_. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15°/oo Test Temperature 18°C Sampling Test Salinity - °/oo Interval (hour) 2 5 10 15 25 36 0 643+ 2.7 643+ 2.7 643+ 2.7 643+ T .7 643+ 2.7 643+ 2.7 1 534+26.6 604+ 8.2 618+ 8.2 b4l+_ 4, ,2 667+^ 5.9 716+ 4.8 2 551+13.1 576+ 7.8 622+ 4.2 627+_ 6. .8 679 + 4.3 740+ 3.1 4 497+22.5 556+ 6.8 616j|^ 5.2 625 + 7, .3 686+ 4.5 790+ 5.4 6 491+ 4.9 552+ 9.7 609+11.4 626 + 5. .6 686 + 2.6 820+ 4.0 10 488+12.8 533+12.8 614+ 7.3 64U 8. ,1 683+^ 1.7 867+ 8.7 16 483+ 5.8 544+ 8.1 540+14.3 6424^ 7. ,3 685 + 4.8 888+19.1 24 455+ 2.9 507+ 8.1 580+11.7 623 + 6, ,4 698 + 4.1 895^14.3 48 485+10.6 528+_25.3 575+13.1 639+ 8. ,1 702 + 1.7 878+ 5.5 72 505+ 9.6 542+12.4 610+16.4 641 + 3, .0 705+ 2.2 877-t^ 8.2 96 533+ 9.1 553+10.8 597+17.4 650 + 6, 2 709 + 2.3 881+ 2.7 168 552+23.5 567+_12.6 642+ 7.1 641 + 5, .9 719 + 3.5 905+ 6.5 Avg 535;^ 9.0 565+ 5.8 611+ 4.4 63 7j^ 1. .9 685 + 3.0 8104^11.7 B4 Table IV. Mean blood osmotic concentration + S. E, in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 32°C 32°C Control Salinity 15°/oo Sampling Interval Test Salini ty - °/oo (hour) 2 5 10 15 25 36 0 657+ 2.5 657^ 2.5 657+ 2.5 -- 657+ 2.5 657+ 2.5 1 543+21.0 598+12.3 624+10.6 650+ 5.0 702 + 6.7 769+ 5.9 2 516+23.6 574+11.0 627+10.2 660+ 2.9 716 + ] LI. 7 822+15.6 4 472+ 2.5 529+22.3 613+ 9.7 661+ 5.4 705 + 4.0 838+ 6.0 6 461+ 8.5 532+21.3 606+ 4.0 632+ 7.3 719+ 1.7 861+13.4 10 462+11.7 532+12.9 613+ 4.7 645+ 4.7 708+ 6.7 850+ 7.8 16 449+30.0 545^^19.8 603+10.0 642+ 7.6 718+ 4.3 851+ 5.9 24 486+ 6.8 609+ 4.8 638+ 4.2 658+11.9 712 + 5.6 830+15.2 48 494+13.0 552+ 6.2 667+ 5.9 657+ 7.5 722 + 2.2 842+ 4.9 72 520+ 7.4 550+16.1 625+ 7.5 662+10.6 732 + 7.8 851+ 3.7 96 486+28.8 499+11.8 637+ 4.5 682+ 4.8 725 + 8.5 882+ 4.3 168 460+10.7 550+22.1 645+ 4.6 684+ 1.4 738+ 3.1 887+17.3 Avg 525+11.3 573+ 7.7 633+ 3.3 657+ 2.5 708+ 3.5 809+10.2 B5 Table V. Mean blood osmotic concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32 = 'C Control . Salinity 15''/oo Test Temperature 25^ 'C Sampling Interval Test S; ilini ty - °/ f O O (hour) 2 5 10 15 25 36 0 657+ 2.5 657+ 2.5 657+ 2.5 657+ 2.5 657 + 2.5 657+ 2.5 1 545+ 9.0 582 + 8.7 617 + 1.4 665+ 8.8 714 + : LI. 4 732+10.6 2 490+35.5 574 + 8.8 590 + 6.1 656+ 5.7 7 16 + : L9.3 765+ 5.0 4 481+10.2 555 + 8.0 572 + 6.2 643+ 3.9 692 + 3.7 812^12.2 6 454+17.5 540+ 3.3 566 + 5.1 654 + 9.1 697 + 5.9 826+13.0 10 468+19.3 554 + ; LI. 9 569 + 5.8 645+ 9.7 687+ 2.2 808+14.3 16 462+ 5.3 550 + ; L0.9 567 + 8.5 618+ 6.4 713 + 7.2 834+27.3 24 435+15.0 556+ 7.7 585 + 7.8 645+ 3.4 703 + 5.4 843+15.0 48 503+25.5 529 + 9.8 597 + 7.3 639 + 9.4 705 + 5.5 793+18.0 72 523+28.5 564+^ 7.3 589 + 7.5 624 + 4.5 706+ 7.3 826+11.2 96 543+17.5 590+: L5.5 602h^ 5.6 635 + 5.5 697 + 5.0 865+ 6.5 168 550+ 4.5 573 + 3.3 616+ 5.3 661 + 9.0 689+; L2.8 934+17.3 Avg 533+11.9 578+ 5.3 600+ 3.9 646 + 2.5 691 + 3.7 786+11.1 B6 Table VI. Mean blood osmotic concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32°C Control Salinity 15°/oo Test Temperature 18°C Sampling Interval Test Salinit y - °/oo (hour) 2 5 10 15 25 36 0 657+ 2.5 657+ 2. .5 657+ 2.5 657+ 2.5 657+ 2. .5 657+ 2.5 1 547+10.8 624+11. ,1 598+15.5 634+ 3.5 685 + 6. 2 744+ 7.9 2 516+ 2.5 594+16. ,7 580+ 1.7 630^^ 3.7 680 + 6. .2 748+18.4 4 478+15.6 540+32. .1 573+ 7.5 629+13.2 692 + 5. .3 815+20.3 6 454+_24.5 518+14, ^ 2 569+16.8 6384^ 7.0 683+ 5. .5 875+ 7.0 10 456+18.0 561+20. .0 591+^21.8 620+ 6.7 670 + 3. .3 890+22.5 16 458+18.0 _- 587+18.8 614+10.0 676 + ~i .9 924+25.8 24 478+ 0.0 463+31. ,0 566+11.5 622+ 4.6 679+ 5. .0 1035+79.4 48 -- 518+ 3. .0 583+16.4 625+ 3.3 695 + 8. .4 893+36.8 72 -. 529+31. ,5 562+16.0 612+14.7 689+ 5. .2 942+ 8.8 96 -- 569+ 6, .5 576+30.3 603+ 8.7 664 + 5. .6 893+10.2 168 564+ 0.0 556+ 6, .5 575+19.0 630+ 6.1 65 7+; L7. ^ 2 821^^ 6,3 Avg 554+15.4 581+10, _ 2 594+ 5.8 630^^ 3.0 675 + 2 .5 828+16.6 B7 Table VII. Mean blood osmotic concentration +_ S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Ss ilinity 15°/oo Test Temperature 18°C Sampling T n t p rv a 1 Test Salini ty - Voo (hour) 2 5 10 15 25 36 0 674+^ 2.1 674+ 2.1 674+ 2.1 -_ 674 + 2. ,1 674+ 2. 1 1 592-t^ll.O 633+20.4 644+ 7.7 673+12.6 693+] L2. ,6 757+ 6. ,0 2 574+ 5.1 572+ 9.8 650+17.0 659+13.2 710+ 6. ,8 773+13. ,1 4 541+ 4.8 587+10.6 662+ 7.4 663+ 7.0 710 + 4. ,0 832+ 8, .2 6 513+13.2 563+32.4 654+^ 8.2 678+11.0 713+ 8. ,7 820+11, .6 10 494+ 8.0 558+17.4 635+13.0 679+ 2.3 708+ 9. ,3 869+17. .8 16 517+27.0 572+16.4 650+1 1 . 5 674+ 3.7 712 + 6, ,4 880+10. ,1 24 504+22.8 542+ 9.5 632+ 2.3 684+ 5.6 726+ 5, ,0 848+ 3. .4 48 521+12.1 595+ 4.9 628+12.2 667+ 9.9 717+ 3, .8 851+ 9, .5 72 523+32.4 590+23.8 656+ 3.5 684+ 2.8 723+ 2, .8 857+10, ,5 96 513+14.4 589+16.9 632+ 8.4 678+10.6 720+ 3, ,8 856+ 8, .8 168 566+ 7.1 581+11.8 599+ 6.2 669+ 3.9 735 + 6. .0 836+ 5, .5 Avg 56U 9.2 599+ 6.9 647+ 3.4 674+ 2.1 707+ 3, .0 803+10, .1 B8 Table VIII. Mean blood osmotic concentration j|^ S. E. in £. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 18°C 25°C Control Salinity 15°/oo Sampling Test Salini ty - °/oo T n t" f^ TA/ ?i 1 (hour) 2 5 10 15 25 36 0 674+ 2. 1 674+ 2.1 674+ 2.1 674+ 2.1 674+ 2.1 674+ 2. 1 1 620+14. 1 636+ 8.0 667+ 3.9 664+ 9.0 676+ 7.6 7494^ 9 . 7 2 594+ 9. , 3 615+11.1 650+ 8.5 686+ 9.3 695+ 8.2 782+ 5. 9 4 562+11. ,3 608+ 2.7 669+ 5.4 684+ 6.3 694 + 7.4 814+13. 1 6 548+17. ,1 598+ 8.2 653+ 8.5 670+ 4.8 682+ 2.0 793+ 6. ,7 10 543+19, ,7 611+17.5 663+ 4.8 662+ 5.6 689+ 6.3 791^^14. ,2 16 521+ 8, .5 544+ 9.4 634+10.0 656+ 4.7 682 + 8.4 810+ 6. .9 24 527+12. .1 465+23.1 632+10.9 648+ 4.9 652+: 22.2 813+13. ^ 2 48 525+24, .4 520+ 7.6 623+ 4.2 634+ 2.8 7 16 + : 33.0 808+ 9, .5 72 533+ 9, .8 519+10.6 597+13.2 639+ 7.9 667+ 14.3 812+ 6, .8 96 500+17, .6 571+12.1 595+ 7.3 650+ 4.6 714 + 17.9 812+ 7, .7 168 522+ 8, .2 579;^10.0 628+ 1.1 650+ 3.4 704 + 4.4 824+ 8, .3 Avg 571+ 8, .7 589^^ 8.3 643+_ 3.8 660+_ 2.4 686 + 3.2 776+ 7 .3 B9 Table IX. Mean blood osmotic concentration +_ S. E. in P_. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15 /oo Test Temperature 32°C Sampling Test Salinity - °/oo Interval (hour) 2 5 10 15 25 36 0 674+2.1 674+2.1 674+2.1 674-1^2.1 674^^2.1 674+2.1 1 584+23.5 612+7.3 675+23.3 683+7.8 714+2.4 789+6.8 2 529+7.9 592-1^5.5 682+8.1 678+10.3 723+7.1 816-1^9.2 4 51U 3.9 579 + 26.5 637+^24.6 693+7.0 714jt^3.8 825-4^8.6 6 501-1^2.8 569-1^14.8 654+7.3 676+^6.1 727-|^9.9 844-t^2.8 10 -- -- 570-1^19.1 630+9.4 695+^11.6 695+16.9 799+7.2 16 -- -- 578ji^l3.9 634^^ 7.4 664^^14.0 718+^6.0 814 + 12.5 24 465+0.0 58U2.7 636h^8.0 676+4.2 734-1^6.0 790-t^l2.2 48 527-1^0.0 577+8.7 634^^5.9 669-1^6.3 751^^6.4 814-1^4.3 72 453+0.0 598-1^7.4 643ji^4.9 642^^18.7 75U6.3 830-t^l 1 . 1 96 566-1^0.0 623+14.1 647-t^l5.2 669+11.8 736+^2.1 853+3.9 168 570+ 0.0 614+ 7.4 638+ 6.5 689+ 3.3 746+ 6.6 934+12.0 Avg 578+13.9 608+5.9 652+3.7 675+2.8 719+3.7 797+9.8 BIO Table X. Mean blood chloride concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 25°C 25°C Control Salinity 15°/oo Sampling Interval Test Salinit y - Voo (hour] 2 5 10 15 25 36 0 292+1.1 292+1.1 292+1 . 1 -- 292+_l.l 292+ 1.1 1 255+1.6 267+6.7 288+^3.3 304+_4 . 2 309+1 . 6 346+ 3.8 2 241+7.1 272+3.3 287+4.8 294+2.4 300+9.4 355+ 6.6 4 230+^5.4 275+2.9 278+6.1 287+4.3 304+5 . 7 369+13.0 6 213+9.1 282+4.4 290+3.6 288+5.5 311+3.3 400+ 6.5 10 219+^5.6 267+3.4 289+4.5 290+2 . 1 312+5.3 402+16.7 16 226+7.6 264+3.7 276+5.3 286+2.5 306+4 . 3 380+14.1 24 221+3.5 268+4.7 274+7.1 293+7.0 311+5.2 388+ 6.1 48 223+4.3 254+3.7 277+5.0 293+2.8 329+1.6 386+13.5 72 213+4.6 265+5.2 273jt^3.7 287+1.8 310+4.9 381+ 8.0 96 207+6.6 257+3.1 270+3.0 291+^3.7 326+5.1 360+10.4 168 198+2.5 258+3.6 273+4.4 294+1.9 323+2.2 375+ 7.2 Avg 234+^3.9 270+1.8 28U1.5 292-1^1.1 310jf^l.8 362-|^ 5.0 Bll Table XI. Mean blood chloride concentration j|^ S. E. in £. aztecus during the time course of salinity-temperature adaptation. .o Control Temperature 25°C Control Salinity 15 /oo Test Temperature 32°C Sampling Test Salinity - ° 1 / o o (hour) 2 5 10 15 25 36 0 292 + 1.1 292 + 1.1 292+ 1.1 292 + 1. ,1 292+ 1.1 292+ 1.1 1 251 + 3.8 273+ 4.3 296+ 3.5 306+ 2. ,7 313 + 4.2 350+ 3.3 2 234 + 5.6 265 + 7.0 307+ 5.0 291 + 9. ,0 318 + 5.4 357+ 6.2 4 209 + 3.3 273 + 4.1 286+ 6.6 294 + 2, .7 301 + 4.3 390+ 5.5 6 198+ 6.2 270 + 5.1 290+ 4.0 288+ 5. ,7 316 + 5.0 394+10.9 10 228+ 3.8 259 + 7.5 276 + 7.6 293+ 4, ,1 312 + 7.0 412+ 7.5 16 200+: L7.5 258+ 4.0 287+ 3.1 299+ 5, ,9 315 + 3.9 389+14.5 24 228+ 1.9 260 + 3.7 269+ 5.0 279 + 2 ^ ^ 2 308+ 6.2 389+14.0 48 215 + 5.9 257+ 4.2 2754^ 3.7 279+ 6, .6 317 + 6.7 3854^ 3.6 72 209+ 6.9 257 + 3.4 273 + 3.0 282 + 6, _ 2 335 + 3.8 420+ 2.8 96 228+ 6.9 265 + 7.0 274 + 2.0 284j^ 2, .2 330 + 5.1 430+ 4.9 168 205+: 10. 4 259 + 4.8 285 + 1.8 294 + 3, . 3 332 + 2.1 393+10.0 Avg 231^ 4.1 268+ 1.9 285 + 1.6 290+ 1 .5 313 + 2.1 373+ 5.8 B12 Table XII. Mean blood chloride concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 18°C Sampling Test Salinity - °/oo Interval (hour) 2 5 10 15 25 36 0 292-1^1.1 292+1.1 292-1^1.1 292+1.1 292+^1.1 292-|^l.l 1 232+^15. 0 280-t^4.0 274+5.0 292-)^ 1 . 8 302+_3.6 338-1^4.3 2 232+^10.0 267^^3.5 280-1^4.3 285h^2.0 311+_2.9 3444^5.8 4 198h^11.3 2604^5.2 264-1^6.7 288h^6.0 306j*^7.3 375+3.7 6 200+8.3 250-t^4.6 26U4.9 284-1^7.6 300^^6.7 389h^8.3 10 186^^7.6 243+5 7 269-f^ 5 . 1 297+^2.6 308+3.9 407-|^8.5 16 190jt^ 9.0 2474^2.5 246+14.1 297'4^4.2 306^^5.0 410j|^9.1 24 184-1^6.7 234-f^6.7 2704^8.1 285^^5.6 323-+ 2.9 424-1^6.7 48 2094^5.0 244+15.1 274+6.3 292-)^ 4.2 324+3.5 424^^8.0 72 216-|^ 7.2 242+ 3.2 295-i^ 7.1 292+ 6.0 333+ 2.0 422+_ 7.4 96 232-(^6.4 251+^5.3 292-1^9.9 294-1^4.2 330j^ 4 . 3 406ji^5.3 168 2464^8.2 252+6.3 290+3.3 282+' 6.3 339jt^3.4 429+3.9 Avg 228+ 5.4 258+ 2.7 278+ 2.3 290+ 1.3 308+ 5.0 381+ 6.1 B13 Table XIII. Mean blood chloride concentration + S. E. in P^. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32°C Control Salinity 15 /oo Test Temperature 32 C Sampling Test Salini ty - °/oo Interval (hour) 2 5 10 15 25 36 0 283+ 1.4 283+ 1.4 283+ 1.4 -- 283+ 1.4 283+ 1.4 1 233+14.1 249+ 4.7 266 + 6.6 279+ 3.0 329 + 2.7 334+ 3.7 2 192+ 7.6 237+ 9.7 270+ 9.2 279+ 3.8 324 + 7.6 381+ 6.0 4 185+ 8.8 204+15.8 262 + 4.5 291+ 3.3 318+ 2.8 377+ 7.9 6 172+ 6.2 216+15.6 269 + 6.4 273+ 5.3 305 + 2.6 372+14.7 10 193+ 5.6 220+10.9 254 + 5.2 285+ 4.7 304 + 5.4 376+ 9.5 16 200+15.5 232+10.6 254 + 2.3 286+ 2.4 308+ 3.7 3744^ 6.9 24 227^ 6.9 255+ 7.2 264 + 5.1 295+ 1.8 323+ 3.3 364+ 6.3 48 218+ 9.7 231+ 7.4 263 + 7.5 282-t^ 5.1 329 + 2.6 390+ 6.4 72 228+ 8.2 235+ 8.7 257 + 3.5 276+ 5.7 332 + 4.9 386+ 6.7 96 213+10.6 209+ 3.9 275 + 6.7 277+10.9 330 + 2.7 394+ 5.2 168 206+ 4.7 228+ 7.8 254 + 8.9 286+ 7.6 329 + 6.9 392+12.0 Avg 222+ 5.6 240+ 4.1 266+ 2.0 283+ 1.4 315 + 2.3 359+ 5.3 B14 Table XIV. Mean blood chloride concentration j|^ S. E. in P^. aztecus during the time course of salinty-temperature adaptation. Control Temperature 32°C Control Salinity 15 /oo Test Temperature 25°C Sampling Interval Test S; alinity - °, / o o (hour) ~> 5 10 15 25 36 0 283+ 1.4 283+ 1.4 2S3+_ 1.4 283 + 1.4 283+ 1.4 283+ 1.4 1 252+ 2.8 256 + 3.6 267+ 2.0 285+ 5.7 338+ 1.0 335+10.6 2 206+14.9 247+ 4.7 260+ 3.6 285 + 2.3 318+ 3.2 369+ 4.0 4 201+ 8.0 250+ 4.2 248+ 5.8 285+ 3.7 318+10.5 401+ 6.2 6 195+11.5 244 + 6.4 250+ 6.2 292 + 5.1 328+ 3.4 386+10.3 10 204+ 8.4 249 + 3.2 247+ 7.8 284 + 3.1 314+ 0.7 372+10.7 16 209+ 3.5 255 + 1.9 256+ 3.6 272 + 6.1 344+ 8.0 387+18.5 24 193+14.5 248 + 4.4 274 + 2.4 279 + 4.3 338+ 8.3 400+ 7.4 48 232+23.5 223+ 3.7 257 + 9.9 284 + 4.6 316+16.3 366+ 8.0 72 225+16.5 247 + 8.6 261 + 4.0 275 + 5.9 318+ 6.4 483+86.1 96 254+14.5 252 + 6.1 273+ 3.0 284 + 5.4 325+16.5 405+10.6 168 249+ 8.9 253 + 1.8 283+ 2.6 294 + 5.0 319+ 8.3 409+35.1 Avg 253+ 5.4 254 + 2.2 265 + 1.9 283+ 1.3 316+ 3.5 369+ 9.7 B15 Table XV. Mean blood chloride concentration +_ S. E. in P_. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 18°C 32°c Control Salinity 15 /oo Sampling Test Salini ty - 7 o o Interval (hour) 2 5 10 15 25 36 0 283+ 1.4 283 + 1.4 283+ 1.4 283+ 1.4 283+ 1.4 283+ 1.4 1 253+ 6.3 281 + 6.4 269+ 3.9 277+ 5.6 333+10.2 333+ 4.4 2 220+ 6.2 248+ 7.8 259j^ 2.1 295+_ 8.6 318+ 4.8 356+10.5 4 194+11.4 244 + 8.2 259+ 5.2 292^ 5.1 314+10.0 384+ 7.5 6 175+ 9.0 211 + 8.7 252+ 9.0 287+ 4.0 299+ 7.7 416+ 5.3 10 202+ 2.5 234 + : 15.6 273+10.4 270+ 9.9 307+ 4.0 420+10.7 16 144+37.5 -- -- 273+ 5.1 280+ 8.8 312+ 9.5 445+12.8 24 190+ 0.0 205 + : 15. 0 265+ 2.4 280 + 2.1 309+ 7.9 454+11.6 48 __ 225 + : 10. 5 276+ 7.2 290+ 5.9 315+ 5.2 427+16.1 72 -- 224 + : 14.0 266+ 6.4 278+ 8.5 320+ 7.3 445+ 4.5 96 -- 254 + 9.5 268+12.8 277+ 5.4 301j|^ 3.6 459+10.9 168 128+ 0.0 259 + 2.0 263jt^ 5.7 294 + 4.6 368+58.8 421+ 7.5 Avg 230+51.1 252 + 4.8 269+ 2.0 283+ 1.7 311+ 5.0 389+ 8.8 B16 Table XVI. Mean blood chloride concentration +^ S. E. in P_. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 18°C 18°C Control Salinity 15°/oo Sampling Interval (hour) Test Salinity - °/oo 10 15 25 36 0 298+ 1.8 298+ 1.8 298+ 1.8 -- -- 298+ 1.8 298+ 1.8 1 256j|^4,2 268-1^5.6 274-1^5.6 280+9.8 293^^16.4 324+6.8 2 253+5.3 246+7.0 285^^5.3 291+3.4 314+7.6 34U9.2 4 237+^7.8 240+9.0 285+5.4 299^^5.8 308j|^6.0 371-|^15.1 6 222-1^1.5 228-1^12.9 274-1^8.0 303+6.9 309+8.6 3524^3.7 10 193+10.8 222 + 18.0 268-1^6.3 301+8.3 310+4.5 415+8.1 16 222+10.3 241+8.9 279+9.8 294j^3.1 313+8.3 404+4.8 24 218-t^ 5.3 2294^9.3 279+9.6 303+8.0 305+6.2 362^^30.0 48 242-1^5.0 252+2.2 281+4.7 296+7.8 322+3.2 392 + 13.1 72 235+14.2 249+10.3 272+3.8 300+5.1 304+8.8 368+3.0 96 232+4.3 248+13.1 270+5.0 301+5.1 322+5.0 383+2.8 168 253+7.7 247-)^ 4.4 2644^6.0 315+4.0 314jt^9.4 378-|^6.2 Avg 246+ 4.5 254+ 4.0 280+2.1 298+1.8 308+2.2 357+^5.7 B17 Table XVII. Mean blood chloride concentration +_ S. E. in P^. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15 /oo Test Temperature 25°C Sampling Test Salinity - °/oo Interval — — — ^ (hour) 2 5 10 15 25 36 0 298+ 1.8 298-t^ 1.8 298+ 1.8 298+ 1.8 298+ 1.8 298+ 1.8 1 270+8.3 275+2.3 297+3.1 292+_3.4 322+5.8 358+5.5 2 260+5.8 276+3.6 294+3.2 297+5.5 310+7.0 372+8.4 4 243+6.2 260+5.9 297+5.6 301+8.0 323+3.9 387+10.5 6 230h^18.0 253+9.8 274h^6.0 293j1^4.4 323h^ 4.9 358+^10.8 10 217^^ 3.8 253+9.8 292+4.9 295+1.4 324+3.4 348+11.9 16 226+8.5 232+12.4 276+5.2 292+5.6 325+4.0 374+8.6 24 236+7.3 204+10.5 283+4.9 297+0.7 321+9.0 370+22.9 48 236+13.4 230+ 7.0 28U 2.7 278+ 1.4 316+^ 5.2 346+33.0 72 237+9.6 216+5.8 266+8.8 281+5.7 308+15.0 356+8.4 96 221+6.1 228+12.6 267-t^9.6 291+^2.4 334+4.7 379+6.8 168 231+9.3 247+3.8 287+3.0 292+5.3 329+3.4 377+5.9 Avg 249+ 4.3 252+ 4.1 286+ 2.0 293+1.4 317+2.0 353+4.9 B18 Table XVIII. Mean blood chloride concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15 /oo Test Temperature 32°C Sampling Test Salini ty - °/oo (hour] 2 5 10 15 25 36 0 298+ 1.8 298+ 1.8 298+ 1.8 298+ 1.8 298+ 1. ,8 298+ 1.8 1 253+ 8.4 274+ 7.4 284+ 8.0 306+ 8.7 306+ 2. .8 354+ 7.5 2 205 + ; L0.5 250+ 4.0 317+11.7 316+ 9.5 308+10. .7 363+ 8.6 4 175 + 4.1 247+18.8 280+10.1 303+10.9 319+ 9, ,4 361^^11.2 6 173 + 6.1 242+10.9 291+11.4 285+ 7.0 329+ 3, .8 387jt^ 3.5 10 -- -- 236+ 7.5 276+ ".9 305+ 8.8 304+ 7. T 341+13.0 16 -- -- 260+ 6.9 283+11.4 277+10.1 315+ 3, ,9 357+10.2 24 219 + 0.0 254+ 5.7 288+ 6.6 293+ 5.9 315+ 6, ,9 344+ 6.6 48 242 + 0.0 252+ 9.7 298+ 1 .9 298+ 9.8 309+12. . 3 354+ 8.6 72 210+ 0.0 279+ 6.4 3024^ 4.1 291+11.2 325+ 3, .0 370+ 6.9 96 262+ 0.0 285+11.0 298+ 9.6 311+ 3.7 328+ 2, .3 350+ 9.7 168 272 + 0.0 281+ 6.6 295+ 7.8 309+ 5.8 332+ 7, .6 423+ 7.4 Avg 241 + 9.2 269+ 3.5 293+ 2.4 299+ 2.3 314+ 2. _ 2 351+ 4.9 B19 Table XIX. Mean blood potassium concentration +_ S. E. in P^. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 25°C Sampling Test Salini .ty - °/oo Interval (hour) 2 5 10 15 25 36 0 8.8+0.2 8.8+0.2 8.8+0.2 -- 8.8+0.2 8.8+0.2 1 5.1+0.5 6.2+0.3 7.6+0.2 6.6+0.3 8.6+0.2 10.2+0.4 2 5.7+0.4 6.8+0.4 7.6+0.2 8.7+0.7 9.4+0.3 10.2+0.4 4 4.9+0.4 7.3+0.5 8.5+0.5 -- 9.0+0.2 10.9+0.9 6 6.5+1.0 7.4+0.3 8.2+0.2 8.9+0.7 9.2+0.4 11.2+0.3 10 6.2+0.2 8.4+0.2 8.7+0.1 8.6+0.7 10.2+0.2 12.5+0.9 16 6.3+O.S 8.6+0.4 8.0+0.4 9.7+0.3 10.6+0.4 11.3+0.4 24 7.8+0.3 8.2+0.3 7.5+0.4 8.4+0.2 10.0+0.2 11.4+0.2 48 7.2+0.2 9.4+0.6 8.4+0.7 7.8+0.4 10.3+0.4 11.3+0.5 72 6.6+0.3 10.3+0.3 9.1+0.4 9.2+0.4 10.2+0.6 11.2+0.5 96 7.2+0.5 9.1+0.5 8.3+0.5 9.1+0.4 10.8+0.2 11.4+0.2 168 7.7+0.5 10.4+0.3 10.4+0.3 10.8+0.3 11.2+0.2 11.8+0.4 Avg 6 . 8+0 . 2 8.4+0.2 8.4+0.1 8.8+0.2 9.8+0.1 10.8+0.2 B20 Table XX. Mean blood potassium concentration +_ S. E. in P^. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 32°C Sampling Interval (hour) Test Salini .ty - 7oo 2 5 10 15 25 36 0 8.8+0.2 8.8+0.2 8.8+0.2 8.8+0.2 8.8+0.2 8 . 8+^0 . 2 1 7.7+0.4 7.4+0.3 7.1+0.2 7.4+0.2 9 . 2+^0 . 3 10.3+0.5 2 7.1+0.3 8.0+0.3 7.5+0.3 6.7+0.3 9.7+0.2 11.9+0.5 4 7.0+0.3 8.7+0.4 7.1+0.4 7.2+0.4 9.8+0.6 12.0+0.4 6 6.8+0.4 8.6+0.2 7.7+0.1 8.5+0.6 10.1+0.5 10.7jt^0.4 10 7.1+0.4 8.3+0.3 9.4+0.2 8.8+0.7 10.0+0.3 13.7+1.5 16 7.9+0.4 8.3+0.6 9.6+0.2 10.2+0.2 10.5+0.4 13.0+0.8 24 7.8+0.4 9.0+0.2 8.2+0.3 8.2+0.7 10.2+0.4 11.0+1.0 48 8.1+0.3 9.5+0.3 9.7+0.1 9.4+0.3 10.9+0.8 12.7+0.3 72 8.2+0.2 9.1+0.2 9.2+0.5 9.7+0.4 11.4+0.5 12.9+0.5 96 9.8+0.2 11.1+0.3 8.9+0.3 9.4+0.2 12.1+0.4 12.8+0.5 168 8.3+0.4 9.7+0.4 9.2+0.3 10.1+0.3 11.8+0.2 13.6+0.3 Avg 8.1+0.1 8.8+0.1 8.6+0.1 8.7+0.2 10.2+0.2 11.6+0.3 B21 Table XXI. Mean blood potassium concentration +_ S. E. in P_. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 18°C Sampling Interval (hour) Test Salini ty - Voo 2 5 10 15 25 36 0 8.8+0.2 8.8+0.2 8.8+0.2 8.8+0.2 8.8+0.2 8.8+0.2 1 4.4+0.2 7.7+0.3 8.0+0.2 7.6+0.3 8.7+0.5 9.2+0.4 2 4.2+0.1 6.5+0.2 7.4+0.3 7.9+0.3 8.3+0.4 9.1+0.3 4 4.4+0.2 7.4+0.1 7.3+0.4 8.1+0.2 8.8+0.3 9.9+0.4 6 2.8+0.9 7.5+0.2 7.6+0.2 7.9+0.2 8.4+0.3 10.8+0.2 10 4.1+0.2 7.8+0.3 7.7+0.4 8.1+0.5 8.6+0.4 11.2+0.6 16 4.9+0.3 7.3+0.2 8.4+0.6 8.8+0.5 8.6+0.3 11.5+0.5 24 4.6+0.2 7.6+0.5 8 . 1+^0 . 2 8.1+0.5 9.0+0.1 11.4+0.4 48 5.1+0.1 7.1+0.6 8.3+0.3 8.2+0.4 9.0+0.2 11.0+0.4 72 6.1+0.3 7.1+0.1 8.7+0.4 8.4+0.2 8.7+0.2 10.9+0.2 96 6.8+0.8 7.2+0.2 8.7+0.4 8.9+0.2 8 . 8+^0 . 2 10.7+0.5 168 6 . 2+^0 . 6 8.4+0.2 9.8+0.2 8.3+0.2 9.5+0.2 11.9+0.4 Avg 5.6+0.5 7.6+0.1 8.7+0.1 8.3+0.1 8.7+0.1 10.4+^0.2 B22 Table XXII. Mean blood potassium concentration j|^ S. E. in £. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32°C Control Salinity 15 /oo Test Temperature 32°C Sampling Tn f PTva 1 Test Salini .ty - /oo (hour) 2 5 10 15 25 36 0 8.5+0.1 8.5+0.1 8.5+_0.1 -- -- 8.5+0,1 8.5+0.1 1 6.1+0.2 7.1+0,4 7.5+0.6 8.0+0.5 11,0+0.5 11.3+0.2 2 5.5+0.3 6.8+0.4 7.3+^0.6 8.6+0.4 11.3+0.2 12.8+0,6 4 5.0+0.5 6.5+0.4 7.6+0.4 6.7+0.4 11.9+0.3 12,9+0.3 6 5.7 + 0.4 6.8+0.7 7.6+0.5 8.6+0.1 11.9+0.3 13.5+0.5 10 6.1+0.5 7.4+0.3 8.2+0.1 7.8+0.5 12.3+0.5 12.1+0.6 16 5.7+0.2 7.5+0.8 8.8+0.4 9.4+0,3 12.2+0.3 12.8+0.3 24 7.2+0.3 8.2+0.4 8.6+0.1 8.0+0.4 12.4+0.3 10.7+0.5 48 7.6+0.3 8.0+0.2 8.7+0.2 8.3+0.6 11.6+0.3 9,2+0,5 72 7.5+0.4 8.0+0.3 8.4+0.4 8.4+0.1 11.7+0,4 13,1+0,4 96 7.6+0.4 7.0+0.2 8.1+0.5 8.1+0.3 1 1 , 8+^0 , 4 13,2+^0,7 168 7.1+0.4 8.7+0.2 8.9+0.4 9.3+0.2 12,2+0.3 13,1+0,4 Avg 6.9+0.2 7.7+0.1 8.2+0.1 8.5+0.1 11.3+^0.2 ll,7+_0,3 B23 Table XXIII. Mean blood potassium concentration ^ S. E. in £. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 32°C 25°C Control Salinity 15 /oo Sampling Interval Test Salinity - °/oo (hour) 2 5 10 15 25 36 0 8.5+0.1 8.5+^0.1 8.5+0.1 8.5+0.1 8.5+0.1 8.5+0.1 1 5.8+0.8 7.4+0.6 7.6+0.5 7.3+0.4 7.9+0.1 10.1+0.4 2 6.7+0.8 9.6+1.0 7.7+0.1 7.4+0.2 9.3+0.9 11.6+0.6 4 6.5+0.5 8.0+0.2 7.9+0.2 8.3+0.4 10.4+0.4 11.8+0.1 6 7.1+0.4 7.5+0.2 8.5+0.5 9.2+0.2 10.2+^0.6 12.6+0.3 10 7.7+0.7 8.3+0.4 9.1+0.2 8.8+0.3 10.3+0.4 12.3+0.6 16 5.0+0.6 9.3+0.2 9.4+0.2 10.6+0.5 10.5+0.5 11.1+^0.4 24 6.0+0.5 8 . 6+^0 . 5 9.7+0.5 9.6+0.8 8.6+0.7 12.4+0.7 48 6.8+0.4 8.4+0.7 10.2+0.6 9.8+0.8 9 . 4 + 0 .4 11.7+0.0 72 6.6+0.8 9.2+^0.5 9.8+0.4 11.1+0.4 10.5+0.4 11.1+0.7 96 7.1+0.5 9.9+0.3 9.8+0.2 9.5+0.5 10.5+0.9 11.3+0.7 168 7.6+0.2 9.5+0.1 9.7+0.4 10.5+0.3 11.1+0.4 12.3+1.4 Avg 7.0+0.2 8.6+0.2 8.9+0.1 9.2+0.2 9.5+0.2 11.0+0.2 B24 Table XXIV. Mean blood potassium concentration +_ S. E. in P_. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32°C Control Salinity 15 /oo Test Temperature 18°C Sampling Interval Test Salini ■ty - Voo [hour) 2 5 10 15 25 36 0 8.5+0.1 8.5+0.1 8 . 5 +_0 . 1 8.5+0.1 8.5+0.1 8.5+0.1 1 5.2+0.5 6.4+0.5 5 . 9+^0 . 1 6.7+0.6 7.2+0.2 8.4+0.5 2 6.5 + 1.3 5.6 + 0.4 5.8+0.4 6.5+0.3 7.1+0.6 7.8+0.6 4 5.2+0.4 6.1+0.6 5.6+0.7 7.5+0.1 7.6+0.4 9.8+0.7 6 4.4+0.4 5.6+0.5 6.1+0.5 7.5+0.3 7.2+0.3 10. 9+0. S 10 4.2+0.2 5.9 + 1.1 6.2+0.3 7.5+0.5 7.2+0.3 10.7+0.7 16 4.9 + 0.5 -- -- 7.0+0.4 7.2+0.3 7.5+0.4 10.7+0.6 24 6.0+0.0 5.5 + 1.2 6.5+0.4 7.8+0.4 8.3+0.3 10.1+1.2 48 __ __ 4.9+0.0 7.1+0.3 7.6+0.3 9.4+0.2 11.0+0.4 72 -- -- 5.1+0.1 6.3+0.4 7 . 5 +_0 . 4 8.6+0.1 10.8+0.2 96 -- -- 5.6+0.0 6.6+0.8 6.3+0.4 7.4+0.4 11.0+0.2 168 7.0+0.0 8 . 1+^0 . 1 7.1+0.3 7.8+0.2 7.7+0.4 9.2+0.3 Avg 6.5+ 0.3 6.6+0.2 6 . 8 +_0 . 2 7.5+0.1 7.9+0.1 9.7+0.2 B25 Table XXV. Mean blood potassium concentration +_ S. E. in P_. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15 /oo Test Temperature 18°C Sampling Test Salini ty - °/oo Interval (hour) 2 5 10 15 25 36 0 6.3+0.1 6.3+0.1 6.3+0.1 __ 6 . 3+0. 1 6.3+0.1 1 5.6+0.4 5.0+0.3 5.4+0.1 5 . 8+0. 3 6.4+0.2 7.1+0.5 2 5.6+0.2 4 . 5+0- 8 5.2+0.2 5 . 7+0. 2 5.7+0.5 7.7+0.3 4 6.5+1.2 4.7+0.3 5 . 0+0. 1 6.1+0.3 7.1+0.3 8.5+0.1 6 4.4+0.3 4.6+0.3 5.0+0.1 6.0+0.4 6.8+0.2 7 . 8+0. 1 10 5.5+0.2 5 . 4+0. 2 5.5+0.3 6.6+0.4 6.5+0.1 9.4+0.7 16 4.2+0.3 4.6+0.2 4.9+0.2 6.1 + 0.3 7.0+0.5 8.3+0.7 24 4.7+0.4 5.2+0.1 5.6+0.2 6.1^0.1 7.0+0.2 8.5+0.1 48 6.8+0.6 6.7+0.1 5.8+0.2 6.7+0.4 7.7+0.2 8.1+0.6 72 6.8+0.5 6.3+0.3 6.5 + 0.2 6.0+0.3 8.2+0.5 8.1 + 0.3 96 6.7+0.2 6.1 + 0.5 6.5+0.2 7.1 + 0.1 8.3+0.5 8.3-1^0. 2 168 7.1 + 0.3 6.3+0. 3 7.4+0.4 7.1+0.2 10.5+2.3 9.6+0.5 Avg 5.9+0.2 5.6+0.1 5.8+0.1 6.3+0.1 7.2+0.2 7.9+0.2 B26 Table XXVI. Mean blood potassium concentration + S. E. in P^. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15 /oo Test Temperature 25°C Sampling Interval Test Salini -ty - °/oo (hour) 2 5 10 15 25 36 0 6.3+0.1 6.3+0. 1 6.3+0.1 6.3+0.1 6.3+0.1 6.3+0.1 1 5.7+0.2 5.4+0.5 6.6+0.5 7.6+0.2 9.0+0.3 7.5+0.4 2 6.0+0.4 7.0+0.2 6.7+0.3 6.9+0.7 9.0+0.2 8.1+0.4 4 5.4+0.2 6.6+0.0 6.6+0.5 7.3+0.4 9.7+0.5 9.0+0.4 6 5.8+0.6 7.0+0.6 7.5+0.7 7.5+0.4 9.8+0.2 8.9+0.6 10 5.4+O.S 7.2+0.1 7.6+0.2 7.5+0.3 9.7+0.5 8.4+0.4 16 5.9+0.2 6.7+1.0 7.2+0.3 7.7+0.7 9.1+0.7 8.9+0.4 24 6.7+0.6 6.7+0.4 7.4+0.3 7.4+0.2 8.4+0.3 9.1+0.9 48 6.6+0.4 8.4+0.3 7.9+4.0 8.2+0.4 10.3+0-7 8.3+0.2 72 6.7+0.2 9.8+0.7 8.6+0.5 8.8+0.7 8.2+1.3 8.5+0.3 96 7.5+0.5 8.0+0.6 9.6+1.3 9.2+0.3 9.1+0.7 8.7+0.6 168 8.2+0.5 8.0+0.5 10.5+0.5 11.5+0.2 12.1+0.5 10.3+0.1 Avg 6.3+0.1 7.1+0.2 7.5+0.2 7.9+0.2 9.0+0.2 8.2+0.2 B27 Table XXVII. Mean blood potassium concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. .o Control Temperature 18°C Control Salinity 15 /oo Test Temperature 32°C Sampling Interval Test Salinity - °/oo (hour) 2 5 10 15 25 36 0 6.3+0.1 6 . 3+^0 . 1 6.3+0.1 6.3+0.1 6.3+0.1 6.3+0.1 1 5.8+0.4 6.9+0.3 7.0+0.2 8.2+0.2 8.9+0.1 7.6+0.5 2 5.6 + 0.6 6.9+0.3 6.7+0.7 8.6+0.2 10.3+0.3 7.5+0.4 4 6.8+0.6 7.3+0.6 6.5+0.2 8.1+0.6 10.8+0.3 8.0+0.2 6 4 . 4+^0 . 2 6.9+1.3 7.9+0.1 7.4+0.7 10.9+0.4 8.9+0.5 10 __ -_ 7.2+0.1 7.8+0.3 8.8+0.3 9.9+0.2 8.7+0.3 16 -- -- 7.6+0.4 8.4+0.2 8 . 5+0 . 3 10.4+0.2 9.7+0.1 24 6.4+0.0 7.3+0.1 8.5+0.3 9.1+0.3 10.9+0.2 9.6+0.3 48 7.1+0.0 9.1+0.4 9.4+0.2 9.2+0.6 12.0+0.3 9.9+0.2 72 8.8+0.0 9.4+0.3 9.8+0.2 9.6+0.4 11.7+0.5 10.8+0.3 96 8.3+0.0 10.2+0.6 10.4+0.5 10.2+0.2 12.2+0.2 9.9+0.3 168 13.7+0.0 8.6+0.4 9.9+0.2 9.7+0.2 11.6+0.1 11.6+0.2 Avg 6.4+0.5 7.6+0.2 8.0+0.2 8.3+0.2 10.1+0.3 8.7+0.2 B28 Table XXVIII. Mean blood calcium concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15°/oo Test Temperature 25°C Sampling Interval Test Salini ty - 7oo (hour] 2 5 10 15 25 36 0 19.7+0.3 19.7+0.3 19.7+0.3 -- 19.7+0.3 19.7+0.3 1 20.9+0.8 21.3+0.3 20.6+0.8 20.7+0.6 18.1+0.5 17.4+1.2 2 19.2+0.9 21.6+0.4 21.2+0.5 21.9+0.4 19.6+0.6 21.3+0.2 4 19.6+1.1 20.4+0.9 20.9+0.4 __ 19.9+0.2 24.1+1.0 6 17.6+0.8 21.1+0.8 20.5+0.3 21.4+1.1 22.5+1.0 26.1+0.8 10 18.4+1.0 19.7+0.6 20.3+1.2 20 .1 + 1.7 22.0+1.1 27.1+1.6 16 17.4+1.6 20.0+0.8 20.2+1.0 20.3+0.6 20.7+1.3 23.7^^3.3 24 18.3+0.8 20.5+1.0 18.4+1.3 21.1+0.4 23.2+0.7 28.1+0.7 48 18.5+1.9 20.0+1.5 19.0+1.6 17.3+1.0 23.1+0.9 27.6+1.0 72 16.1+0.8 22.7+1.1 18.2+1.0 19.4+1.1 20.6+0.8 26.7+1.4 96 14.5+1.7 20.4+1.1 17.6+1.4 18.0+0.9 21.9+0.7 27.8+0.7 168 16.8+0.9 19.6+0.5 18.7+1.0 18.7+1.1 20.7+0.7 25.4+0.8 Avg 18.3+^0.3 20.5+_0.2 19.5+0.3 19.7+0.3 20.9-t^0.3 24.2+0.5 B29 Table XXIX. Mean blood calcium concentration +_ S. E. in P_. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 32°C Sampling Interval Test Salinit y - Voo (hour) 2 5 10 15 25 36 0 19.7+_0.5 19.7+0.3 19.7+0.3 19.7+0.3 19.7+0.3 19.7+0.3 1 22.6+0.5 20.9+1.2 21.4+0.5 19.1+0.6 18.6+0.7 21.4+1.0 2 23.5+1.4 21.5+0.3 21.5+0.4 20.4+0.4 20.0+0.5 23.2+0.5 4 19.3+0.1 20.9+0.7 19.7+0.8 18.2+1.0 21.4+1.0 21.8+0.8 6 17.8+0.3 19.9+0.3 19.9+0.5 20.3+1.3 20.0+1.1 25.3+0.9 10 17.4+0.7 19.7+0.6 18.6+1.0 17.9+1.7 21.9+0.8 27.1+1.2 16 16.2+1.6 14.6+0.9 18.3+1.1 19.3+0.9 24.5+^0.9 29.6+1.1 24 17.0+0.3 20.1+0.3 18.4+1.1 17.5+1.7 26 .5+1.0 22.8+1.6 48 14.0+0.8 17.7+1.0 19.3+0.7 17.U1.5 22.0+1.4 28.1+1.0 72 15.5+1.0 19.2+1.0 16.7+1.4 18.5+1.2 26.7+0.9 26.9jf^2.0 96 15.5+0.4 20.5+0.7 16.8+0.5 20.7+_1.2 24.6+1.5 30.6+2.5 168 14.0+1.0 18.6+0.9 16.7+0.5 19.2+0.7 25.3+0.8 29.1+0.6 Avg 17.5+0.4 19.4+0.3 19.0+0.3 19.1+0.3 22.3+0.4 25.0+0.6 B30 Table XXX. Mean blood calcium concentration +_ S. E. in £. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 18°C Sampling Interval Test Salini ty - Voo (hour) 2 5 10 15 25 36 0 19.7+_0.3 19.7+^0.3 19.7+0.3 19.7+0.3 19.7+0.3 19.7+0.3 1 19.5+0.9 18.2+0. 2 19.6+0.6 18.9+0.7 20.3+0.6 20.4+0.6 2 21.0+0.2 16.5+0.6 18.7+0.4 -- 20.9+0.7 21.3+0.3 4 18.2+1.0 17.4+0.6 18.3+0.6 18.8+0.7 20.4+0.6 20.8+0.5 6 -- 18.0+0.6 18.9+0.8 18.8+0.5 21.7+0.8 23.3+1.5 10 16.2+0.6 17.1+0.6 18.4+0.4 18.7+0.6 19.7+2.0 25.3+0.5 16 17.7+0.7 17.8+0.9 16.0+0.9 20.4+0.9 22.6+0.5 28.0+1.1 24 14.9+0.6 17.9+1.2 17.1+0.9 17.3+1.3 22.2+0.7 29.3+0.4 48 15.6+0.7 15.1+1.4 17.7+0.5 17.4+_0.9 23.6+0.9 28.6+0.5 72 16.2+0.5 16.9+0.6 1 7 . 8+^0 . 7 18.0+0.7 21.4+0.7 25.6+0.4 96 17.0+0.8 16.7+0.8 18.3+0.7 20.1+1.2 21.2+1.0 28.2+0.6 168 17.2+0.7 17.5+^0.9 20.3+0.7 16.7+1.0 20.9+0.8 26.6+1.1 Avg 17.3+0.4 17.6+0.2 18.6+0.2 18.7+0.3 21.1+0.3 24.4+0.5 B31 Table XXXI. Mean blood calcium concentration ^^ S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32°C Control Salinity 15 /oo Test Temperature 32°C Sampling Test Salinity - °/oo Interval (hour) 2 5 10 15 25 36 0 22.8+0.3 22.8+0.3 22.8+0.3 -- 22.8+0.3 22.8+0.3 1 21.5+1.6 24 . 0+^0 . 9 22.7+0.5 23.0+0.7 20.6+0.8 21.7+0.7 2 21.4+0.7 22.2+0.5 22.0+0.8 24.4+0.7 20.9+0.4 21.3+1.7 4 15.0+1.1 19.5+0.5 22.0+0.2 23.9+0.3 21.3+0.6 23.2+1.1 6 17.5+0.3 19.7+1.1 20.6+0.5 23.5+0.6 19.8+1.4 25.8+0.7 10 19.3+0.7 20.5+0.5 21.7+0.9 19.64^1.4 22.3+0.7 24.4+2.0 16 19.3+1.5 19.9+0.8 21.8+1.4 22.6+0.9 21.6+1.4 25.6+0.7 24 17.9+2.0 21.6+0.5 21.5+0.5 21.5+1.1 25.1+0.8 22.4+2.0 48 17.0+0.5 19.8+0.6 23.0+0.4 21.7+0.5 23.2+0.8 24.4+1.0 72 18.5+0.3 19.3+0.7 23.2+2.1 22.2+1.0 25.6+1.3 27.4+1.2 96 16.2+0.8 19.7+0.6 22.7+2.1 22.4+0.7 29.5+4.1 30.4+1.6 168 14.3+0.4 23.7+1,5 27.1+1.0 25.8+0.6 23.7+1.2 29 . 5+_2 . 2 Avg 19.1+0.5 21.3+0.3 22.6+0.3 22.8+0.3 22.9+0.4 24.8+0.5 B32 Table XXXII. Mean blood calcium concentration +_ S. E. in P. aztecus during the time course of salinity - temperature adaptation. Control Temperature 32°C Control Salinity 15 /oo Test Temperature 25°C Sampling Test Salinity - °/^^ Interval (hour) 2 5 10 15 25 36 0 22.8+0.3 22.8+0.3 22.8+0.3 22.8+0.3 22.8+0.3 22.8+0.3 1 18.6+2.5 17.7+0.8 18.4+0.6 19.8+1.0 10.1+0.4 18.1+0.9 2 23.5+_2.6 18.5+1.8 18.1+0.6 19.5+_0.7 10.9+1.2 20.6+1.2 4 19.6+0.4 17.9+1.1 16.8+0.9 19.0+^0.5 1 1 . 7 +_0 . 1 21.5+0.9 6 20.8+1.2 17.5+1.3 17.1+1.3 20.0+0.4 13.0+0.4 23.7+^0.9 10 19.4+1.8 17.3+0.7 17.2+0.3 19.9+1.2 14.6+0.9 25.8+_0.8 16 14.6+1.7 16.7+1.1 17.4+0.7 17.4+1.2 16.4+1.1 26.5+0.4 24 16.2+0.4 18.3+0.8 16.5^^1.3 20.3+0.7 14.1+0.9 25.6+1.5 48 18.3+0.3 16.1+0.5 16.4+0.4 19.1+0.8 13.6j!^2.3 25.5j|^1.7 72 18.5+0.5 17.5-1^1.0 17.5+^0.9 15.8+0.7 18.2+0.5 22.5+1.4 96 19.7+0.0 19.8+1.2 16.4+0.5 18.0+1.8 19.1+0.7 24.9+1.6 168 20.2+0.4 19.9+0.4 18.6+0.5 18.9+1.1 22.6+1.6 34 .7+4.2 Avg 20.1+0.5 18.7+0.4 18.2+^0.3 19.5+_0.3 1 7 . 0 +_0 . 7 23.9+_0.6 B33 Table XXXIII. Mean blood calcium concentration h^ S. E. in P^. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32°C Control Salinity 15°/oo Test Temperature 18°C Sampling jes^ Salinity - °/oo Interval (hour) 2 5 10 15 25 36 0 22.8+0.3 22.8+0.3 22.8+0.3 22.8+0.3 22.8+0.3 22.8+0.3 1 21.7+1.3 24.9+2.9 21.3+1.9 19.3+0.7 24.5+0.4 22.3+1.6 2 26.5+3.4 22.0+_2.3 18.4+1.0 18.5+1.7 22.5+0.5 21.0+0.5 4 25.3+1.2 20.1+1.7 16.4+2.2 19.7+0.9 24 .6+1.2 25.2jf^l.5 6 16.4+1.2 16.9+4.5 19.1+0.9 22.1+1.0 24.8+1.5 26.7+0.7 10 17.6j|^1.5 19.0+1.6 20.6+1.3 22.1+1.8 23.7+2.0 30 .0+1.7 16 18.4+0.8 -- 19.6+1.5 20.1+1.2 24 .4+1.9 31.5+1.5 24 24.6+0.0 18.2+1.8 19.5+1.0 21.4+0.6 26.1+1.6 35.1+1.8 48 -_ 17.2+^0.3 19.5+1.3 19.3+0.6 24.0+1.6 32 .2+2.4 72 -- 16.2+0.2 17.0+^0.9 19.9+0.8 22.2+0.4 30.6+0.9 96 -- 17.9+0.0 17.5+1.5 18.5+^0.3 20.8+0.1 28.6+0.8 168 16,8+0.0 17.7+0.3 19.7+1.4 20,9+1.3 20.4+1.4 28.8+2,7 Avg 22.3+0.7 20.4+0.7 19.7+0.4 20.7+0.3 23.3+0.4 27.2+0.7 B34 Table XXXIV. Mean blood calcium concentration + S. E, in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 18°C 18°C Control Salinity 15°/oo Sampling Interval Test Salini ty - °/oo (hour) 2 5 10 15 25 36 0 23.4+0 .2 23.4^0. .2 23.4+0.2 -- 23.4+0.2 23.4+0. .2 1 21.4+1, .0 20.9+1. .3 20.5+^0.4 22.3+0.7 19.7+0.5 20.6+0. .2 2 19.2^1, .0 16.0+2, .8 21.0+0.3 22.6+1.1 18.3+2.1 19.7+0. .4 4 17.1+0, 2 18.0+1. .1 20.1+0.8 23.5+0.9 20.8+0.2 21.3+_1. ,0 6 16.7+0, .6 18.3+1. ,4 20.0+0.9 22.6+0.1 20.3+0.3 21.5+0 .3 10 15.0+0, .4 19.7+0. ,7 20.1+1.0 23.5+0.4 20.5+_0.7 23.2+0 .8 16 15.3+0, .5 19.0+0. ,6 18.5+1.4 23.3+0.6 21.5+0.5 23.8+0 .8 24 14.8-K), .8 19.7+1. , 2 19.1+0.7 23.2+0.7 22.0+0.2 25.3+0 .4 48 16.8+0, .8 21.6+1. 2 18.5+1.0 23.5+0.6 22.3+0.4 23.3+0 .5 72 16.6+1, .9 20 . 4+0 . ,7 20.8+0.4 23.8+0.2 22.6+0.5 25.4+0 .7 96 16.0+0, .4 19.5+p. .6 20.6+0.5 24 .8+0.4 21.7+0.5 25.1+0 .5 168 18.0-K), .8 19.4+1. .0 20.2+0.7 24.2+0.2 27.4+5.0 25.5+0 .5 Avg 18.3+0 , .5 20.1+p. .4 20.65) .3 23.4-K) .2 21.9-K).5 23.2+0 .3 B35 Table XXXV. Mean blood calcium concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15 /oo Test Temperature 25°C Sampling Interval Test Salini ty - Voo (hour) 2 5 10 15 25 36 0 23.4+0. ,2 23.4+0. ,2 23.4+0.2 23.4+0.2 23.4+0. .2 23.4+0. ,2 1 22.4+2, ,0 17.4+1. .4 21.4+0.6 20.3+^0.8 17.2+1. ,1 20.9+0. ,5 2 22.7+0. ,8 19.9+1. ,1 19.7+0.7 18.4+1.3 16.6+0. ,9 23.0+0. .2 4 21.5+0. .3 17.9+1. ,5 17.8+1.7 20.2+0.9 17.5+0. ,7 26.5+0. .5 5 19.0+1. .4 19.1+0. .8 18.5+0.8 21.1+0.6 18.6+0. 2 27.7+0. ,7 10 19.2-1^1. . 3 18.2+1. .0 18.8-t^l.l 20.9+0.5 19.6+1. ,2 26.5+_0. ,9 16 18.4+0. ,3 16.9+1. .1 17.7+0.4 20.7+_0.3 19.6+0. ,2 26.9+0. .8 24 20.5+0. .8 13.4+1. .3 19.0+_0.6 17.8+0.4 19.9+0. .4 29.3+1. .0 48 19.1+1. .0 16.6+1. 2 18.2+0.6 20.3+0.7 23.8+2, ,0 26.7+0, .3 72 18.9+0. .7 16.5+0. ,9 15.0+1.1 20.6+0.8 26.1+1, .1 29 . 3+0 , 2 96 17.8+0. ,6 17.1+0. .7 16.3+1.9 21.4+0.6 20.5+1, .4 28.2+1, .1 168 19.2+0. .6 15.8+1, .0 18.9+^0.6 18.8+_0.6 20.9+1, .1 31.1+0, .5 Avg 20.6+0. .3 18.2+0, .5 19.2+^0.4 20.6+0.3 20.3+0, ,4 26.1+0, .4 B36 Table XXXVI. Mean blood calcium concentration j|;_ S. E. in P^. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15 /oo Test Temperature 32°C Sampling Interval (hour) Test Salini ty -7oo 1 5 10 15 25 36 0 23.4+0.2 23.4+0. 7 23.4+0.2 23.4+0.2 23.4+0.2 23.4+0.2 1 20 .8+1.9 24.0+0. ,7 21.1+0.9 19.7+0.6 20.7+1.7 19.8+0.9 2 17.0+1.4 24.3+^0. ,1 17.3+1.6 19.8+0.2 21.6+0.7 22.4+0.4 4 14.6+0.6 22.6+1, ,7 18.0+0.9 21.0+1.0 21.5+0.5 23.1+0.3 6 14.3+0.7 23.5+0, 2 22.3+1.1 22.3+0.5 24 . 0^1 . 2 24.3+0.6 10 -- 24.0+0, ,9 19.6+0.8 22.4+0.2 21.8+1.3 23.8+0.2 16 -- 21.7+1, .1 21.3+0.4 21.7+0.4 22.9+0.6 24.7+0.5 24 15.6+0.0 22.1+0. .3 21.8+1.1 21.8+0.4 24 .7+0.8 22.4+0.4 48 14.2+0.0 21.8+0. ,5 20.8+0.6 21.7+0.9 27.8+0.6 25.5+1.1 72 15.5+0.0 21.0+0. .6 19.4+0.5 20.0+1.0 25.8+0.6 28.0+0.6 96 17.2+^0.0 22.2+1, . 3 20 . O-t^O . 6 18.4+0.7 23.3+1.0 29 .1+0.7 168 24.6+0.0 20.3+0, .7 19.4+0.7 20.2+0.5 25.6+0.9 24.9+1.7 Avg 19.1+0.8 22.7+0, .2 20.7+0.3 21.3+0.2 23.7+0.3 24.2+0.4 B37 Table XXXVII. Mean blood magnesium concentration j^ S. E. in £. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 25°C Sampling Test Salini ty - Voo Interval (hour) 2 5 10 15 25 36 0 8.2+0.5 8 . 2+^0 . 3 8.2+0.3 -- 8.2+0.3 8.2+0.3 1 7.5+0.5 10.2+0.5 10.1+0.3 9.4+0.3 11.2+0.4 15.6+0.3 2 5.1+0.3 5.4+1.0 8.5+0.2 6.3+0.4 12.8+0.6 17.7+0.5 4 4.1+0.3 6.3+1.0 9.2+1.1 -- 16.5+0.9 21.0+1.9 6 3.4+0.1 4.5+0.7 8.5+0.2 7.2+1.7 17.4+3.2 22.0+1.6 10 2.9+0.1 3.9+0.2 8.4+0.5 8.9+0.2 23.6+2.9 37.5+^5.3 16 5.2+0.4 5.5+1.2 7 . 9+^0 . 2 8.0+1.0 -- 20.5+4.0 24 2 . 7 +_0 . 5 5 . 9 +_1 . 0 8.5+0.4 8.2+1.1 23.6+1.9 50.7+3.7 48 2.2+0.5 3.4+0.2 8 . 8+^0 . 3 7.9+0.9 24.0+2.0 70.0+7.1 72 2.3+0.3 3.8+0.5 8.8+0.2 8.1+1.4 23 .7+2.7 61.7+7.5 96 1.9+0.5 3.8+0.2 8.1+0.5 8 . 3j^l . 1 17.9+2.2 44 .8+9.2 168 1.6+0.2 3.7+0.1 8.7+0.2 9.0+1.0 17.8+1.9 40.0+8.2 Avg 4.1+0.3 5.5+0.5 8.6+0.1 8.2+0.3 18.7+0.9 38.1+3.0 B38 Table XXXVIII. Mean blood magnesium concentration ^S. E. in P^. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 32°C 5°C Control Salinity 15 /oo Sampling Test Salinity - °/oc Interval — (hour) 2 5 10 15 25 36 0 8.2 + 0.3 8.2-|_0.5 8.2 + 0.5 8,2+0.3 8.2+0.3 8.2 + 0.3 1 14.7+1.1 9.9+0.7 8.1 + 1.1 11.4 + 1.2 12.8+0.6 17.9+^1.2 2 9.8+^0.7 6.6+^0.6 6.5 + 0.6 9.4+^0.9 13.6+^1.1 24.5 + 3.0 4 3.6+0.6 4.7 + 0.1 5.6j+1.4 7.1+0.5 16.2 + 1.0 27.5ji^3.8 6 2.8+^0.2 5.2+0.3 6.6 + 0.8 8.3+0.6 16.1ji^l.8 32.7 + 1.3 10 5.1+0.6 6.3+0.6 6.3+^0.2 9.6+0.9 18.3 + 1.4 30.2 + 2.5 16 4.6 + 0.6 6.2+0.1 6.9+0.4 9.1+^0.5 27.6+^4.8 50.1+4.9 24 3.6 + 0.3 6.0+0.5 6.3+0.6 7.9 + 0.9 25.7 + 2.8 50.0+^4.3 48 3.0+^0.7 6.0+0.6 6.4+_0.6 8.6+^0.3 19.6+2.8 52.6+3.8 72 2.8+1.1 5.1+0.8 5.4+1.0 7.8+0.6 24.8+2.2 48.4+6.7 96 3.1 + 0.2 6.2+_0.5 4.9+^0.7 8.7+0.9 14.3+1.2 42.0+^9.4 168 3.7+0.8 7.2+0.2 4.1+0.4 5.2+1.0 15.4+1.8 61.2+2.6 Avg 5.4+0.5 6.6+^0.2 6.3+^0.2 8.4_+0.3 16.8+0.9 34.6 + 2.3 B39 Table XXXIX. Mean blood magnesium concentration ^ S. E. in £. aztecus during the time course of salinity-temperature adaptation. Control Temperature 25°C Control Salinity 15 /oo Test Temperature 18°C Sampling Test Salinity - °/oo Interval (hour) 2 5 10 15 25 36 0 8.2+0.3 8.2+0.3 8.2+0.3 8.2+0.3 8.2+0.3 8.2+0.3 1 9.1+0.3 9.4+0.6 11.5+0.7 12.3+0.4 13.1+1.3 13.2+0.6 2 9.9 + 0.4 8.3+0.2 12.2+0.5 18.9+0.9 13.0+0.9 14.8+0.5 4 9.7+0.5 8.5+0.4 10.4+0.2 9.8+1.0 13.4+0.8 18.5+1.9 6 7.0+0.9 9 . 3+^0 . 6 10.1+0.7 10.7+0.8 16.2+1.7 18.5+1.0 10 7.6+0.2 8.9+0.5 10.0+0.5 11,9+0.8 18.4+2.1 18.5+1.1 16 6,9 + O.S 5.9+0.5 9.3+0.8 10.U0.6 26.3+1.5 42.4+4.0 24 6.7 + 0.1 8.2+1.3 10.2+0.8 14.7+2.3 27.4+2.6 33.9+3.9 48 6.0+0.1 5.8+0.4 8.7+0.3 10.4+0.5 35.9+^2.4 54.2+5.4 72 5.1+0.2 4.7+0.2 8.7+0.2 9.4+0.4 31.2+3.1 50 .7+2.1 96 4.8+0.2 4 . 3 4^0 . 2 8.6+0.3 9.2+0.4 27.3+5.1 57.4+8.4 168 4.8+0.3 6.0+0.6 7.0+0.7 8.3+0.4 20.84^2.5 45.2+3.2 Avg 7.2+0.3 7.4+0.3 9.4+0.2 10.3+0.3 19.7+1.2 29 .3+2.3 B40 Table XL. Mean blood magnesium concentration j|^ S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32°C Control Salinity 15 /oo Test Temperature 32°C Sampling Interval Test Salini -ty - 7oo (hour) 5 10 15 25 36 0 5.5+0.2 5.5+^0.2 5.5+0.2 __ 5.5+0.2 5.5+0.2 1 6.8+0.3 7.2+0.3 7,4+0.4 4.2+0.5 12.8+1.0 14.2+0.7 2 6.2+0.5 6.0+0.2 6.4+0.4 4.6+0.2 14.4+1.3 21.2+0.8 4 5.6+0.7 5.9+0.4 6.0+1.0 4.8+0.4 16.9+1.1 23.0+1.4 6 3 . 7+^0 . 6 6.1+0.3 6.0+0.2 4.0+0.1 21.0+1.6 28.0+1.3 10 2.8+0.1 5.9+0.2 7.5+0.2 5.1+1.0 24 .9+1.2 37.0+6.7 16 5.3+0.4 6.1+0.2 6.9+0.1 6.0+0.4 31.5+4.0 37.5+2.8 24 5.4+^0.7 6.1+0.3 7.1+0.1 4.9+0.1 25.2+3.3 -- 48 3.1+0.3 6.2+0.1 7.5+0.3 6.2+0.4 22.9+2.8 -- 72 3.6+0.8 6.3+0.2 7.7+0.2 6.7+0.2 18.2+0.9 47.640.1 96 4.7+0.1 6.3+0.1 7.8+0.2 6 . UO . 6 16.6+_2.1 63.2+ 8.9 168 4.0+0.3 6 . 1+^0 . 3 8.0jt^0.6 7.3+0.1 16.7+2.0 51.142.3 Avg 4.8+0.2 6.1+0.1 6.8+0.2 5.5+0.2 17.7+1.1 28.7+ 2.7 B41 Table XLI. Mean blood magnesium concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32°C Control Salinity 15 /oo Test Temperature 25°C Sampling Test Salinity - °/oo Interval (hour) 2 5 10 15 25 36 0 5.5+0.2 5.5+^0.2 5.5+0.2 5 . 5 +_0 . 2 5.5^^ 0.2 5.5jt_0.2 1 7.2+1.5 7.U0.5 8.3+^0.4 8.7+_0.7 16.1ji^0.9 12.2+0.3 2 10.7+^0.9 7.1 + 0.7 6.7+^0.6 5.5+^0.7 17.4ji^ 0.8 16.4+0.8 4 6.5+^1.2 6.2 + 0.3 6.U0.3 5.3+^0.5 23.6jt^ 3.6 19.3-+1.6 6 8.3+^0.4 5.3+^0.3 7.1+^0.4 5.4+^0.4 20. U 2.8 16.3+1.0 10 6.8+^0.4 5.8+^0.3 6.3+0.5 5.1+_0.7 25.3j|^4.7 14.2_+0.9 16 2.9+^0.4 6.2+^0.5 7.1 + 0.3 8.7+_0.4 21.8-t^3.0 16.7+2.1 24 4.0+0.3 4.6+^0.4 6.3+^0.8 6.3^^1.2 20.6h^ 1.7 22.0+^1.8 48 3.6+0.0 4.2+^0.2 6.3+0.3 8.1+0.8 26.4+2.6 19.2-t^3.8 72 3.4+^0.2 4.1+_0.7 7.1+_0.9 7.6+0.6 42.4 + 10.7 46.5+^1.7 96 3.5+0.0 5.6+0.3 6.6+^0.3 8.5+^0.5 31.5-1^4.3 48.3+4.9 168 3.7+^0.1 7.8-1^2.8 6.7+^0.5 5.3+^0.5 20.4j^2.5 82.1 + 7.8 Avg 5.8+0.4 5.8+0.2 6.6+0.2 6.6+0.2 19.0+ 1.6 21.2+2.5 B42 Table XLII. Mean blood magnesium concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32°C Control Salinity 15°/oo Test Temperature 18°C Sampling Test Salinity - °/oo Interval (hour) 2 5 10 15 25 36 0 5.5+0.2 5 . 5 +_0 . 2 5.5+0.2 5.5+0.2 5.5+0.2 5.5+0.2 1 7.0+0.6 6.7+0.9 8.0+0.5 6.4+0.5 8.5+0.5 11.8+0.1 2 7.6+1.7 4 . 9+^0 . 3 7.8+0.7 7.4+0.8 10.7+0.9 15.2+1.3 4 8.2+0.9 7.3+0.5 6 . 6j^l . 3 8.5+0.6 14.3+1.0 19.3+2.0 6 5.2+0.2 6.5+1.4 9.2+0.6 7 . 6 4^0 . 5 12.2+0.9 18.7+1.9 10 6.7+0.3 5 . 3+^0 . 6 8.1+0.6 8.0+0.7 11.0+0.9 22.4+1.8 16 4 . 9j*^l . 1 -- -- 6.5+0.1 6.3+0.7 10.9+1.2 20.3+1.1 24 6.1+_0.0 4 . 7+^0 . 3 6.4+0.7 6.7+0.1 10.7+1.1 24.5+3.6 48 -- -- 4.0+0.2 6.7+0.6 5.7+0.5 9.3+0.6 28.0+4.3 72 -- -- 3 . 5^0 . 7 5.7+0.8 6 . 1+^0 . 5 12.4+1.7 22.3+2.8 96 -- -- 2 . 9+^0 . 6 6.0+0.4 4.8+0.6 13.4+1.8 21.6+3.1 168 1.3+0.0 2.9+0.0 6.3+0.7 6.9+0.5 7.1+1.0 13.3+2.4 Avg 6.2+0.4 5.3+0.3 6.7+0.2 6.5+0.2 9.9+0.5 16.9^^1.1 B43 Table XLIII. Mean blood magnesium concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15°/oo Test Temperature 18°C Sampling Test Salini ty - °/oo Interval (hour) 2 5 10 15 25 36 0 6.5+0.1 6.5+0.1 6.5+0.1 -- 6.5+0.1 6.5+0.1 1 11.2+1.0 8.3+0.6 7.8+0.5 6.4+0.2 6.7+0.2 8.4+0.5 2 8.5+0.6 7.4+1.4 7.4+0.5 6.7ji^0.4 6.9+0.5 9.8+0.4 4 7.6+^0.1 6 . 2+^0 . 4 7.2+0.4 6.4+0.3 6.8+0.3 9.4+0.4 6 6.3+0.5 6.5+0.5 7.0+0:6 7.2+0.8 7.7+0.6 9 . 3+0. 7 10 6.6+0.4 6 . 0+^0 . 4 7.5+0.1 6.7+0.2 7.9 + 0.5 13.6+1.9 16 6.2+0.2 5.0+0.1 6.2-1^0.6 6.1+0.2 7.3+0.1 12.8+1.4 24 4.4+0.7 5.6+0.7 6.0+0.3 6.6+0.4 7.2+0.4 13.3+0.9 48 6.1 + 0.1 5.2+0.5 6.2+_0.1 6.3+0.1 7.4+0.4 17.4+1.8 72 4.7+0.6 4.4+0.1 5.7+0.2 6.2+0.4 7.2+0.3 18.0+2.6 96 5.4+0.4 4.2+0.4 5.9+0.4 6.1+0.1 7.5+0.4 15.0+1.0 168 5.2+0.1 4.9+0.2 6.7+0.2 6.4+0.5 9.2+1.9 11.1+0.6 Avg 6.5+0.2 5.9+0.2 6.7+0.1 6.5^0.1 7.2+0.2 11.3+0.6 B44 Table XLIV. Mean blood magnesium concentration + S. E. in P^. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15°/oo Test Temperature 25°C Sampling Test Salini ty - °/oo (hour) 2 5 10 15 25 36 0 6.5+0.1 6.5+0.1 6.5+0.1 6.5+0.1 6.5+0.1 6.5+0.1 1 8.0+0.7 6.0+0.6 8.0+2.2 5.8+0.4 10.4+1.5 8.7+0.9 2 9.0+0.9 6.1+1.0 6 . 6+^1 . 6 4.0+0.7 10.1+0.6 1 1 . 0;^1 . 2 4 7.0+1.7 4.6+0.8 4 . 9-^1 . 4 4.0+0.5 12.2+0.7 11.2+1.6 6 7.7+2.4 5.5+0.5 7.2+1.1 5.1+0.5 16.1+1.5 23.3+^6.5 10 3.6+0.5 3.2+0.3 4 . 4+^0 . 6 4.7+0.8 13.6+1.7 22.0+6.4 16 3.4+0.1 2 . 6+^0 . 3 4.4+0.3 5.4+0.7 10.9+1.2 17.7+3.0 24 4.8+0.2 4.3+0.4 4 . UO . 8 6.6+0.9 11.8+2.4 18.3+5.4 48 3.7+0.3 5.1+1.4 4.9+0.3 7.8+0.6 14.0^^1.9 22.3+3.0 72 4.4+0.3 4.5+1.3 6.6+0.5 7.7+1.6 14.9+4.9 36.5+4.6 96 4.6+0.1 5.3+1.5 5.8+1.1 9.8+1.3 1 1 . 8 4^1 . 1 46.2+9.0 168 4.2+0.5 2.8+0.5 5 . 7+^0 . 7 8.6+0.7 17.2+2.0 18.3+6.1 Avg 5 . 7+^0 . 3 4 . 9+^0 . 3 5.8+0.3 6.4+0.3 11.9+0.6 18.0+1.8 B45 Table XLV. Mean blood magnesium concentration + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15°/oo Test Temperature 32°C Sampling Tnterva 1 Test Salini ty - °/oo (hour) 2 5 10 15 25 36 0 6.5+0.1 6 . 5+^0 . 1 6.5+0.1 6.5+0.1 6.5+0.1 6.5+0.1 1 6.9+0.8 7.7+0.4 6.8+0.3 6.9+0.8 10.0+0.8 6.1+0.3 2 6.6+1.1 5.6+0.6 4.7+0.9 6.9+0.5 13.3+1.3 6.2+0.2 4 2.5+0.1 4.6+0.2 4.4+0.3 5.5+0.4 19.1+1.0 7.4+0.4 6 2.2+0.2 4.8+0.4 4.9+0.6 6.3+0.8 22.3+3.2 8.0+0.4 10 -- 5.4+0.6 4.8+0.7 5.3+0.4 12.8+_1.1 7.5+0.5 16 -- 5.2+0.3 4.3+0.4 6.2+0.2 20.9+1.3 9.2+0.8 24 3.2+0.0 4.9+0.1 5.3+0.2 7.0+0.3 19.6+2.1 8 . 2+^0 . 8 48 2.3+0.0 6.0+0.1 5.2+0.4 7.1+0.7 23.6+2.4 12.1+4.5 72 3.2+0.0 5.3+0.1 5.6+0.5 7.8+0.8 21.9+1.6 17.2+3.2 96 2.7+0.0 5.5+0.2 6.3+0.4 6.4+0.6 20.7+1.1 9.5+0.8 168 3.3+0.0 5.8+0.1 5.1+0.1 8.0+0.4 17.4+1.7 14.0+2.4 Avg 5.0+0.4 5.7+0.1 5.5+0.2 6.6+0.2 16.4+0.9 9.0+0.6 B46 Table XLVI. Mean oxygen consumption ;|^ S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 25°C 25°C Control Salinity 15°/oo Sampling Test Salini ■ ty - Voo T n 1" p T\f a 1 ■LJII...CX vCl-.i. (hour) 7 5 10 15 25 36 0 .19+.01 .19+. 01 .19+.01 .19+. 01 .19+. 01 ,19+. 01 1 .35+. 04 .34+. 03 .24+. 04 .19+. 02 .28+. 02 ,37+, 04 2 .38+. 04 .32+. 03 .25+. 03 .21+.02 .27+. 02 , 36+ , 04 4 .32+. 02 -- -- .26+, 03 .23+. 03 .21+, 02 , 35+ . 04 6 .31+.02 .33+. 03 .24+, 03 .21+. 03 ,24+. 02 ,31+. 04 10 .31+.02 .27+, 02 .19+. 02 ,18+. 01 ,27+. 02 .31+,02 24 .22+. 01 ,23+. 02 .24+. 03 .19+. 02 ,23+, 03 ,31+. 03 48 .26+. 06 .26+. 05 .18+, 02 __ -_ ,28+. 03 ,28+, 06 72 .23+. 02 ,23+, 05 .23+, 02 ,18+, 02 .25+, 03 ,26+. 02 96 .24+_.03 ,25+. 04 .24+. 02 .16+.02 .25+. 03 ,28+. 05 120 .22_+.04 ,24+, 04 .23+. 03 .17+, 02 ,22+. 03 , 25+ , 04 144 .22+. 04 .23+,07 .16+. 01 __ __ ,24+, 03 , 26+ , 04 168 .21+. 04 -- -- .11+.02 .14+. 02 ,17+. 04 .24+. 02 Avg .29+. 01 ,27+. 01 ,22+, 01 ,19+. 01 ,24+, 01 .32+. 01 B4 7 Table XLVII. Mean oxygen consumption + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 25°C 32°C Control Salinity 15°/oo Sampling T n t p TV a 1 Test Salini ty - °/oo (hour) 2 5 10 15 25 36 0 .19+.01 .19+. 01 .19+. 01 .19+.01 ,19+,01 ,19+, 01 1 .39+. 02 .31+.01 .34+. 03 .38+. 04 .42+, 03 ,42+, 03 2 .29+. 02 .28+. 03 .37+. 02 .45+. 02 ,36+, 03 ,38+, 04 4 ,32+. 02 .23+. 02 -- .48+,02 ,43+, 01 ,34+, 03 6 .28+. 02 .23+. 03 .37+. 03 ,45+, 03 ,43+, 02 ,32+. 02 10 .28+. 02 .20+. 03 .34+. 03 ,44+. 03 .43+, 03 .32+, 03 24 .20+ -- .33+. 04 .35+.03 ,42+.02 .39+, 02 ,33+, 03 48 .20+.01 .29+. 02 .40+. 02 ,37+,03 ,38+, 02 ,31+,03 72 -_ __ .30+, 04 .33+. 04 ,31+. 04 ,36+, 02 ,28+. 03 96 .17+. 02 .27+. 02 .24+. 02 .25+. 03 ,37+, 04 .28+. 02 120 .19+.01 .26jt^.02 .15+.01 .26+. 07 ,31+, 02 .31+, 04 144 .17+ -- .29+. 02 .23+.03 .28+. 03 ,30+, 02 .26+, 02. 168 .16+ -- -- -- .16jt^.01 .20+. 01 ,29+, 03 ,22+, 05 Avg .27+. 01 .26+. 01 .32+^.01 .37+. 01 ,38h^.01 ,32+. 01 B48 Table XLVIII. Mean oxygen consumption ^S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 25''C 18°C Control Salinity 15°/oo Sampling Interval (hour) Test Salinity - °/o, 10 15 24 36 0 .19-1^.01 .19+. 01 .19+. 01 .19+. 01 .19+. 01 .19+. 01 1 . 20+ . 02 .22+.03 .14+. 02 .17+. 02 .10+. 02 .10+. 02 2 .07+. 01 .22+. 02 .10+. 01 .14+. 02 .10+. 02 .09+. 01 4 .08+. 01 .15+.01 .08+. 01 .09 +.01 .1U.02 .09+. 02 6 __ __ .12+. 02 .10+.02 .07+. 01 .10+. 02 .07+. 02 10 -- .15+.02 .09+. 02 .04+. 00 .ll+.Ol .08+. 01 24 -- -- .16+. 01 .08+. 02 .06+. 01 .14+. 01 .07+. 01 48 __ __ .16^^.03 .12+.01 .07+. 01 .13+. 01 .08+. 02 72 -- -- .13ji^.02 .ll+.Ol .08+. 01 .14+.02 .07+. 02 96 __ __ .13+.02 .10+. 01 .08+. 02 .12+. 02 .08+. 02 120 -- -- .11+. 02 .11+.02 .08+.01 .11+.02 .08+. 01 144 -- -- .10+. 02 .12+.01 .08+. 01 .10+. 01 .08+. 01 168 __ __ -_ __ -- -- .08+. 01 -- -- .08+. 02 Avg .16+. 02 .18+. 01 .12+.01 .09+. 00 .12+. 00 .08+. 00 B49 Table XLIX, Mean oxygen consumption + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 32°C 32°C Control Salinity 15°/oo Sampling Interval (hour) Test Salini ty -°/oo T 5 10 15 25 36 0 .34+. 01 .34 +.01 .54+. 01 .34+. 01 .34+. 01 .34+. 01 1 .46+. 04 .42+. 03 .45+. 01 .33+. 04 .50+. 03 .32+. 02 2 .36+. 02 .38+. 04 .39+. 06 .35+. 03 .45+.02 .30+. 02 4 .40+. 02 .42+. 03 .4U.05 .35-1^.04 .44+. 03 .31+.02 6 .32+. 02 .39+. 04 .37+. 04 .36+. 04 .41+.03 .33+. 02 10 .32+. 07 .39+^.02 .38+. 04 .34+. 04 .46+. 02 .29+. 03 24 .32+. 03 .42+. 01 .36+. 03 .36+. 03 .42+. 04 .36+. 04 48 .30+. 04 .40+. 01 .35+. 03 .35+. 02 .36+. 02 .32+. 02 72 .24+. 02 .37+. 02 .35+. 01 .29+. 02 .37+. 04 .23+. 03 96 .26+. 05 .35+. 04 .29-1^.03 .33-1^.03 .31+. 02 -- -- 120 .21+. 05 .36+. 06 .28+. 02 .34-^.05 .28+. 02 .23+. 04 144 .19+. 04 .29+. 01 .27+. 01 .30+. 05 .25+.01 .19+.02 168 .16+. 01 __ .28+.01 .32-^.06 .24+. 02 -- -- Avg .32+.01 .40+. 02 .37-1^.01 .34 +.01 .39+. 01 .30^^.01 B50 Table L. Mean oxygen consumption + S. E. in P^. aztecus during the time course of salinity-temperature adaptation, Control Temperature 32' 'C Control Salinity 15 /oo Test Temperature 25' 'C Sampling Test Salinity ° 1 loo Interval [hour) 2 5 10 15 25 36 0 .34+. 01 .34+. 01 .34+. 01 .34jt^.01 .34+. 01 .34+. 01 1 .28+. 02 .28+. 04 .32+. 05 .29+. 02 .24+. 01 .28+. 03 2 .29^.02 .27^.03 .27+. 04 .28+. 02 .22+. 02 .30+. 03 4 .31+. 02 .22_+.02 .25j|^.02 .21+. 03 .24+. 02 .30+. 03 6 .28+. 02 .14+. 02 .28+. 04 .20+. 02 .20+. 01 .29+. 04 10 .25+.02 .13+. 01 .25+.03 .18+. 04 .21+. 02 .30+. 04 24 .27+. 02 .16+. 02 .24+. 03 .13+. 03 .21+.02 .28+.02 48 .17+. 03 .21+.01 .25+. 02 .20+. 01 -- -- .27+. 06 72 .12+. 02 .11+. 01 .22+. 02 .11+. 01 -- -- .26+. 02 96 .17+.02 .13+. 01 .23+.03 .10+. 01 .15+.01 .SOh^.OI 120 .15+. 02 .12+. 02 .20+. 02 .14+. 02 .20+. 04 .29+. 02 144 .16+. 01 .10+. 01 .21+. 02 .15+. 02 -- -- .27+. 01 168 .18+. 02 .12+.01 .21+. 02 .144^.01 .20+. 05 -- Avg .25-t^.Ol .18^^.01 .25j^.01 .2U.01 .22+. 01 .29+. 01 B51 Table LI. Mean oxygen consumption + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 32' 'C Control Salinity 15 /oo Test Temperature 18' ^C Sampling Interval Test Salini ty - °/oo (hour) -> L. 5 10 15 25 . 36 0 .34+. 01 .54+. 01 .34+. 01 .34+. 01 .54^01 .34+. 01 1 .26+. 02 .20+. 04 .07+. 03 .09+. 0 2 .09+. 02 .13+. 02 2 .30+. 02 .15+.03 .14+. 02 .07+. 01 .12 .02 .10+. 02 4 .16+. 02 .12+. 02 .14+. 01 .09+. 01 .11^.02 .10+. 01 6 .07+.01 .12j^.01 .08+. 01 .09+. 01 .12+.01 .09+. 01 10 -- -- .10+. 01 .12+.01 .09+. 01 .14J^.01 .13+.01 24 -- .08+. 01 .09ji^.01 .lOji^.Ol ■ lA^.Ql .13+. 02 48 -, .09+. 02 .10+. 01 .10+. 02 .14^.01 .13+. 02 72 -- -- -- -- .10+. 01 .08-1^.01 — .11+. 01 96 _- __ .10-^.02 .13+. 00 .09+. 01 .12:^.01 .11+. 03 120 -- -- .09+. 01 .08-^.01 .06+. 0 2 .10+. 01 .11+. 02 144 -- .11+. 02 .06+. 01 .07+. 01 .09+.02 .06+. 01 168 -- __ .06+. 01 .10+_.01 .07+. 01 .0 8_.01 .06+^.00 Avg .22+. 02 .12j^.01 .12+. 01 .08+. 00 . 1 2+ . 0 1 .11+. 00 B52 Table LII. Mean oxygen consumption + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature Test Temperature 18°C 18°C Control Salinity 15°/oo Sampling Interval (hour) Test Salinity - °/oo 10 15 25 36 0 .10 + .00 .10 + .00 .10 4^.00 .10 ■^.00 10 _^.00 .10 + .00 1 .35 jf^.04 .13 + .02 .17 + .03 .12 + .01 17 + .02 .14 + .03 2 .27 + .03 .09 +^.01 . 16 + .02 .13 + .01 18 + .02 .13 + .01 4 .19 + .02 .09 + .01 .16 + .02 .12 + .01 13 + .01 -- -- 6 .16 + .02 .07 + .01 .15 + .03 -- -- 12 + .02 .11 + .01 10 .19 + .02 .05 + .00 .12 + .01 .10 + .01 10 + .01 .12 + .01 24 .18 + .02 .07 + .01 .11 + .02 .09 + .01 13 + .01 .09 + .01 48 .16 + .04 .08 j^.Ol .11 1-01 .10 + .02 11 + .01 .10 + .01 72 -- -- .08 + .01 .12 + .02 .10 + .01 13 + .01 .10 + .01 96 -- -- .04 + .00 .10 + .01 .10 + .01 11 H^.02 .10 + .01 120 -- -- .08 + .01 .09 + .01 .12 + .01 12 + .01 .12 + .01 144 -- -- .07 + .00 .08 + .01 .11 + .01 12 + .01 .08 + .01 168 -- -- .07 + .01 .09 + .(U .07 + .01 12 + .01 .12 + .01 Avg .22 + .01 .08 + .00 .12 + .01 .10 ■t^.OO 13 + .00 .12 + .00 B53 Table LIII. Mean oxygen consumption jj^ S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18' ^C Control Salinity 15''/oo Test Temperature 25' = C Sampling Interval Test Salinity / o o (hour) 2 5 10 15 25 36 0 .10+. 00 .10+. 00 .10+. 00 .10+. 00 .10+. 00 .10+. 00 1 . 36+ . 04 .21+. 02 .22+. 03 .27+. 02 .22+. 02 .32+. 01 2 .32+. 02 .25jt^.02 .17+. 02 .28+. 02 .32+. 02 .30+. 01 4 .27+. 02 .29+. 02 .19+. 01 .23+.01 .24+. 01 .30+. 02 6 .25+^.03 .28+. 03 .22+. 02 .22+. 02 .28+. 02 .27+. 01 10 .24+. 02 .30jt^.03 .17+. 01 .24+. 01 .32+. 02 .25+. 02 24 .23+. 03 .31+. 02 .19+. 02 .23+. 02 .27+. 04 .26+. 01 48 .23+. 03 .16+. 02 .14+. 01 .24+. 03 .28+. 03 .21+. 02 72 .16+. 03 .16+. 01 .14+. 01 .20+. 03 .30+. 04 .17+. 02 96 .19+. 03 .16+. 02 .13+. 02 .22+. 02 .27+. 03 .18+. 02 120 .19+. 01 .12+. 01 .14+. 01 .15+. 03 .23+. 02 .16+. 01 144 -- -- -- -- .11+. 01 .09+. 01 .21+. 02 .14+. 01 168 -- __ -- -- .08+. 01 . llj^.03 .21+. 03 .14+. 01 Avg .27+. 01 .23+. 01 .17+. 01 .21+.01 .27+. 01 .25+. 01 B54 Table LIV. Mean oxygen consumption + S. E. in P. aztecus during the time course of salinity-temperature adaptation. Control Temperature 18°C Control Salinity 15 /oo Test Temperature 32°C Sampling Test Salinity - °/oo Interval (hour) 2 5 10 15 25 36 0 .10+, .00 .10+. 00 .10+. 00 .10+. 00 .10+. 00 .10+. 00 1 .48+, .05 .32+_.01 .2,5^.02 .42+. 02 .31+. 01 .36+. 03 2 .49+, .14 .24+. 01 .31-^.03 .39+. 02 .35+. 02 .37+. 02 4 — — .26+. 02 .32+^.02 .38+. 03 .34+. 02 .37+. 02 6 -- -- .31+. 03 .32+. 03 .39+. 03 .39+. 02 .34+. 02 10 -- -- .31+.02 .30+. 02 -- -- .37+. 01 .35+. 02 24 -- -- -- -- .30+. 02 -- -- -- .34^^.05 48 -- -- .34+. 03 .26+. 02 .31+. 04 .39+. 03 .34+. 04 72 — -- .32+. 01 .26+. 02 .29+. 03 .39+. 04 .34+. 02 96 -- -- .22+. 02 .26jf^.03 .28+. 03 -- .03 .28+. 02 120 -- — .21+. 02 .27+. 05 .32+. 02 .41+. 08 .28+. 03 144 -- -- .21+. 03 ,25+. 03 .36+^.05 .33+ -- .26+. 02 168 -- -- -- -- .24+. 03 -- -- -- .28^.02 Avg .47 + .04 .26+. 01 .29+. 01 .37+. 01 .37^^.01 .34+. 01 B55 Table LV. Effect of variations in the cation concentration on the oxygen consumption of P. aztecus. The concentrations of the variable cations are represented in percentages. Control Salinity 15' /oo Cont: rol Temperature 25°C Test Salinity 15°/oo Test Temperature 25°C Sampling Interval Control 25% Ca 0% Mg 30% K (hour) 0 .19+. 01 .19+. 01 .19+. 01 .19+. 01 1 .25+. 04 .25+. 05 .35+. 03 .25+. 02 2 .23+. 04 .28+. 04 .26+. 03 .23+. 02 3 .22+. 02 .29+^.03 .22+. 02 .22+. 03 4 .22+. 02 .26+. 02 .18+. 02 .22+. 02 6 .15+. 02 .27+. 02 .16+. 01 .15+. 01 10 .17+^.02 .21+. 02 .17+.01 .17+. 02 24 .19+. 04 .21+. 03 .20+. 02 .19+. 02 B56 Table LVI . Effect of variations in the cation concentration on the oxygen consumption of P. aztecus. The concentrations of the variable cations are represented in percentages. Control Salinity Test Salinity 15°/oo 15°/oo Control Temperature 25°C Test Temperature 32°C Sampling Interval Control 25°o Ca 0% Mg 30% K (hour) 0 .19+. 01 .19+. 01 .19+. 01 .19+. 01 1 .32+. 03 .33+. 04 .24+. 01 .33+. 03 2 .30+. 02 .39+. 06 .33-t^.03 .38+. 04 3 .26+. 04 .37+. 06 .33+. 03 .33+. 07 4 .26+. 04 .37+.05 .21+.01 .32+. 05 6 .24+. 01 .34+. 03 .24+. 03 .31+. 04 10 .25^.03 .38+. 06 _- _- .33+. 05 24 .27+. 02 -- -- -- .28+. 01 B57 Table LVII. Effect of variations in the cation concentration on the oxygen consumption of P_. aztecus. The concentrations of the variable cations are represented in percentages. Control Salini Lty is' /oo Control Temperature 25°C Test Salinity is' /oo Test Temperature 18°C Sampling Interval (hour) Control 25''b Ca 0% Mg 30% K 0 .19+.01 .19+. 01 .19+. 01 .19+. 01 1 .18+. 03 .24+. 02 .30+. 03 .28+. 03 2 .16+. 02 .22+.01 .21+.02 .22+. 02 3 .13+. 02 .18+. 01 .134^.01 .17+. 01 4 .13+. 02 .17+. 02 .15+. 02 .12+. 01 6 .15+^.02 .16+. 01 .16+. 02 .13+. 01 10 .13+. 02 -_ __ .12+.01 .13+^.01 24 .11+. 02 .11+. 01 .15+. 01 .11+. 02 B58 In accordance with letter from DAEN-RDC, DAEN-ASI dated 22 July 1977, Subject: Facsimile Catalog Cards for Laboratory Technical Publications, a facsimile catalog card in Library of Congress MARC format is reproduced below. Venkataramiah, A Studies on the time course of salinity and temperature adaptation in the commercial brown shrimp Penaeus aztecus ives / by A. Venkataramiah . . . ^et al j , Gulf Coast Research Laboratory, Ocean Springs, Mississippi. Vicksburg, Miss. : U. S. Waterways Experiment Station, 1977. 308, A, 58 p. : ill. ; 27 cm. (Contract report - U. S. Army Engineer Waterways Experiment Station ; H-77-1) Prepared for Office, Chief of Engineers., U. S. Army, Washington, D. C, under Contract No. DACW 39-73-C-0115. Bibliography: p. 303-308. 1. Aquatic ecosystem. 2. Crustacea. 3. Environmental effects. A. Salinity effects. 5. Shrimps. 6. Temperature effects. I. Bieslot, Patricia, joint author. II. Gunter, Gordon, joint author. III. LakshDii, C. J., joint author. IV. Valleau, John D. , joint author. V. Ocean Springs, Miss. Gulf Coast Research Laboratory. VI. United States. Army. Corps of Engineers. VII. Series: United States. Waterways Experiment Station, Vicksburg, Miss. Contract report ; H-77-1. TA7.W3Ac no..H-77-l V f ^^