CHANGES m gwm: mus mums campasmw 0? mac BASS {Ammmmgs RU?E$TR[§) AS PHYSEQLQGECAL CRITERIA C}? STRESgFUL CONMTEONS That: fos- i‘fm Degree of: DH. D. MICHIGAN STATE UNIVERSETY Gerald Ray Bouck 1966 LIBRARY I Michigan Stew University ,1 + This is to certify that the thesis entitled CHANGES IN BLOOD AND MUSCLE COMPOSITION OF ROCK BASS (Ambloplites Rupestris) AS PHYSIOLOGICAL CRITERIA OF STRESSFUL CONDITIONS presented by GERALD RAY BOUCK has been accepted towards fulfillment of the requirements for Ph.D degree in Fisheries & Wildlife msngxQ $4M, Major professor Date “LUV/l ‘j [0] E G (If I 0-169 W3 >3 ABSTRACT CHANGES IN BLOOD AND MUSCLE COMPOSITION OF ROCK BASS (AMBLOPLITES RUPESTRIS) AS PHYSIOLOGICAL CRITERIA OF STRESSFUL CONDITIONS by Gerald Ray Bouck Studies of the physiological responses of rock bass (AmbIOplites rupestris) to sublethal conditions are pre- sented. These include increased hematocrits, hypo— proteinemia, and changes in the composition of the plasma, myogen, and erythrocyte proteins. The activity (and probably content) of certain cellular enzymes was increased in the plasma and indicates that stressful conditions allow or cause certain cells to leak proteins into the blood. Cellular leakage may account for the observed changes in both plasma and myogen protein composition. The composition of myogen and plasma protein appears to be under the control of a homeostatic mechanism(s), and Changes in myogen composition occur more rapidly than the Changes in the composition of blood protein. In both the myOgen and plasma protein, the response to various stresses was a general increase in the amount of low or intermediate mobility proteins. However, these reSponses must be viewed Gerald Ray Bouck as being the observable results of both stressful and ameliorating conditions. Consequently, the changes in pro— tein composition should be followed throughout the course of the stress period and during a subsequent recovery period. These studies demonstrate that sublethal stress con- ditions alter the protein composition of pollution sensitive fish, indicating that presently acceptable standards of water quality are not acceptable. Hence electrophoretic analyses of proteins can provide physiological criteria that would be useful in establishing water quality standards, as well as being useful in detecting stressful conditions in an aquatic ecosystem. CHANGES IN BLOOD AND MUSCLE COMPOSITION OF ROCK BASS (AMBLOPLITES RUPESTRIS) AS PHYSIOLOGICAL CRITERIA OF STRESSFUL CONDITIONS BY Gerald Ray Bouck A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Fisheries and Wildlife 1966 ACKNOWLEDGMENTS I wish to extend my sincere thanks to Dr. Robert C. Ball for his guidance, kind assistance, and stimulation throughout my graduate program. My appreciation is also extended to Dr. Paul O. Fromm, Dr. Gordon Guyer and Dr. Peter Tack for their con- tinual efforts on my behalf. I also wish to acknowledge the invaluable assistance of my fellow graduate students, especially Mr. Kenneth Linton, Mr. Robin Vannote, Mr. Jack WOOd, and Mr. Darrel King. I am also grateful for financial assistance from the Division of water Supply and Pollution Control of the Public Health Service, U.S. Department of Health, Education and Welfare. The early phases were supported by Research Grant WP500369, followed by a Predoctoral Fellowship (Fl-WP—lS, 782), and was completed under Training Grant lTl—WP—lO9. ii TABLE OF CONTENTS INTRODUCTION . . . . LITERATURE SURVEY METHODS Controlled Environment Artificial streams Chemical analyses of water Estimation of dissolved oxygen pressure Collection and Care of Specimens Pretreatment of Glassware Procedures for Testing Blood and Tissue Parameters Collection and preparation of blood samples Collection and preparation of tissue samples Determination of total cell volume in whole blood (hematocrit) of hemoglobin concentration Determination Determination of total protein concentration Assays for Enzyme Activity Determination of lactic dehydrogenase activity Determination of transaminase activity Determination of succinic dehydrogenase activity ElectrOphoretic Methods Separation and evaluation of protein fractions Separation and location of isozyme fractions Statistical Methods ELECTROPHORETIC CHARACTERISTICS OF ROCK BASS PROTEINS . . . . . . . . . . . . . . . . . Results General characteristics of tissue proteins Plasma protein fractions iii Page l2 12 12 19 20 21 21 22 22 23 26 26 27 27 27 29 29 31 31 33 34 36 36 36 37 Muscle myogen fractions Hemolyzate fractions EFFECTS OF LOW OXYGEN ON FEEDING ROCK BASS Introduction and Objectives Experimental Conditions Results Hematocrits, hemoglobin, and total plasma protein concentrations Distribution of protein in the plasma fractions Concentrations of p fractions Distribution of extrac myogen fractions Distribution of extracted hemolyzate fractions Changes in feeding behavior Increment of body weight and length Relationship of body weight and length to plasma fractions Other observations rotein in the plasma ted protein in protein in EFFECTS OF LOW OXYGEN ON FASTING ROCK BASS Introduction and Objectives Experimental Conditions Results Hematocrits, hemoglobin, and total plasma protein concentration Distribution of protein in the plasma fractions Concentrations 0 fractions Distribution of extracted protein in myogen fractions Distribution of extracted protein in hemolyzate fractions Other observations f protein in the plasma EFFECTS OF DIVALENT NICKEL ON ROCK BASS Introduction and ObjeCtives Experimental Conditions Results lobin, and total plasma Hematocrits, hemog protein concentration iv Page 42 42 45 45 45 46 46 49 51 53 55 55 58 58 59 61 61 61 64 64 64 67 69 71 71 74 74 75 76 76 Distribution of protein in the plasma fractions Concentrations of protein in plasma fractions Distribution of extracted protein in myogen fractions Distribution of extracted protein in hemolyzate fractions Liver succinic dehydrogenase activity Other observations COMBINED EFFECTS OF DIURNAL LOW OXYGEN, FASTING, HIGH CARBON DIOXIDE, AND DIVALENT NICKEL ON ROCK BASS . . . . . . . . . . . . . . . . . . . . . Introduction and Objectives Experimental Conditions Results Hematocrits, protein Distribution of protein in the plasma fractions Concentrations of protein in the plasma fractions Distribution of extracted protein in myogen fractions Liver succinic dehydrogenase activity Plasma lactic dehydrogenase activity Plasma transaminase activity Paper electrOphoretic mobility of plasma LDH Otherrobservations hemoglobin, and total plasma DISCUSSION Nbrmal Values and Responses to Stress Hematocrits Hemoglobin concentration Total plasma protein concentration Distribution of protein in the plasma fractions Comparison of native and laboratory fish ' Normal values and responses to stress Concentrations of protein in plasma fractions Distribution of extracted protein in myogen fractions Page 78 78 81 83 83 85 86 86 87 88 88 89 93 95 98 98 100 100 101 102 102 102 105 107 108 108 110 113 118 Distribution of extracted protein in hemolyzate fractions Liver succinc dehydrOgenase activity Plasma enzyme activity Evidence for Homeostasis in the Rock Bass Causes of Change in Blood and Tissue Protein Antibody production Cellular leakage of proteins Effects of hormones Other causes Prognosis for ElectrOphoretic Studies SUMMARY LITERATURE CITED vi Page 122 123 125 126 138 138 139 143 143 144 148 150 Table LIST OF TABLES Summary of water quality data during the ex— posure of fed rock bass (Ambloplites rupestris) to low oxygen conditions Summary of the influence of low oxygen con— ditions on hematocrits, hemoglobin concen— trations, and plasma protein concentrations of fed rock bass (Ambloplites rupestris) Summary of the influence of low oxygen con— ditions on the distribution of protein in the plasma fractions of fed rock bass (Ambloplites rupestris) Summary of the influence of low oxygen conditions on the circulating concentrations of protein in the plasma of fed rock bass (Ambloplites rupestris) . . Summary of the influence of low oxygen con— ditions on the distribution of extracted protein in the myogen fractions of fed rock bass (Ambloplites rupestris) Summary of the influence of low oxygen con— ditions on the distribution of extracted protein in the hemolyzate fractions of fed rock bass (Ambloplites rupestris) Summary of water quality data during the ex- posure of fasting rock bass (Ambloplites rupestris) to low oxygen conditions Summary of the influence of low oxygen con— ditions on hematocrits, hemoglobin concen- trations, and plasma protein concentrations of fasting rock bass (Ambloplites rupestris) vii Page 47 48 50 52 54 56 63 65 Table 10. 11. 12. 13. 14. 15. 16. 17. Summary of the influence of low oxygen con— ditions on the distribution of protein in the plasma fractions of fasting rock bass (Ambloplites rupestris) Summary of the influence of low oxygen con- ditions on the circulating concentrations of protein in the plasma fractions of fasting rock bass (AmbIOplites rupestris) Summary of the influence of low oxygen con- ditions on the distribution of extracted protein in the myogen fractions of fasting rock bass (Ambloplites rupestris) Summary of the influence of low oxygen con- ditions on the distributions of extracted protein in the hemolyzate fractions of fasting rock bass (Ambloplites rupestris) Summary of the influence of divalent nickel on hematocrits, hemoglobin concentrations in fed rock bass (Ambloplites rupestris) Summary of the influence of divalent nickel on the distribution of protein in the plasma fractions of fed rock bass (Amblgplites rupestris) . . . . . . . . . . Summary of the influence of divalent nickel on the circulating concentrations of protein in the plasma fractions of fed rock bass (Ambloplites rupestris) . . . . . . Summary of the influence of divalent niCkel on the distribution of extracted protein in the myogen fractions of fed rock bass (Ambloplities rupestris) Summary of the influence of divalent nickel on the distribution of extracted protein in the hemolyZate fractions of fed rock bass (Ambloplites rupestris) . . . . . . . viii Page 66 68 70 72 77 79 80 82 84 Table Page 18. Summary of the influence of the combined ef- fects of low oxygen, fasting, high carbon dioxide, and divalent nickel on hematocrits, hemoglobin concentrations, and total plasma protein concentrations of rock bass (Ambloplites rupestris) . . . . . . . . . . . 9O 19. Summary of the influence of the combined ef- fects of low oxygen, fasting, high carbon dioxide, and divalent nickel on the distri- bution of protein in the plasma fractions of rock bass (Ambloplites rupestris) . . . . . 91 20. Summary of the influence of the combined ef— fects of low oxygen, fasting, high carbon dioxide, and divalent nickel on the circu- lating concentrations of protein in the plasma of rock bass (Ambloplites rupestris) . 94 21. Summary of the influence of the combined ef— fects of low oxygen, fasting, high carbon dioxide, and divalent nickel on the distri— bution of extracted protein in myogen fractions of rock bass (Ambloplites rupestris) . . . . . . . . . . . . . . . . . . 97 22. Summary of the influence of the combined ef— fects of low oxygen, fasting, high carbon dioxide, and divalent nickel on enzyme activities in rock bass (Ambloplites rupestris) . . . . . . . . . . . . . . . . . . 99 23. Summary of the observed changes in hemato— crites, hemOglobin concentrations, and plasma protein concentrations in rock bass (Ambloplites rupestris) . . . . . . . . . . . 103 24. Summary of the observed changes in the distri— bution of protein in the plasma fractions of rock bass (Ambloplites rupestris) . . . . . . 109 25. Summary of the observed changes in the circu- lating concentrations of protein in the plasma fractions of rock bass (Ambloplites rupestris) . . . . . . . . . . . . . . . . . . 114 ix Table 26. Summary of the observed changes in the distri— bution of extracted protein in the myogen fractions of rock bass (Ambloplites rupestris) 27. Summary of the observed changes in the distri— bution of extracted protein in the hemoly— zate fractions of rock bass (Amblgplites rupestris) 28. Comparison of lactic dehydrogenase activity in tissues of the rock bass (AmblOplites rupestris) . . . . . . . . . Page 121 124 141 LIST OF FIGURES Schematic drawing of artificial stream "A” as used in the study of the influence of low oxygen pressure Schematic drawing of artificial stream "B" as used in the study of the influence of divalent nickel and low oxygen Drawing of the rock bass (AmbIOplites rupestris) showing the location of the heart and the location of muscle used for electrOphoretic analysis Comparative paper electrophoretic mobility of the fastest migrating proteins in the tissues and blood of the rock bass (Ambloplites rupestris) Protein fractions of rock bass (AmbIOplites rupestris) as separated by paper electrophoresis . . . . . . An electropherogram of plasma proteins from rock bass (Ambloplites rupestris) Progressive changes in the hematocrits, hemoqlobin concentrations, and plasma protein concentrations before, during stress and during recovery from stress in rock bass (AmbIOplites rupestris) Progressive changes in the distribution of protein in the plasma fractions during, before, and during recovery from stress in rock bass'LémblOplites rupestris) Progressive changes in the circulating concentrations of protein in the plasma fractions before, during stress, and during recovery from stress in rock bass (Ambloplites rupestris) . . xi Page 14 17 25 39 41 44 128 130 132 Figure 10. Progressive changes in the distribution of protein in the plasma fractions before, during stress, and during recovery from stress in the rock bass (Ambloplites rupestris) . . . . . . 11. Progressive changes in the activities of enzymes before, during stress, and during recovery from stress in the rock bass (Ambloplites rupestris) 12. ElectrOpherogram and muscle myogen fractions of crayfish (Orconectes immunis) xii Page 134 136 146 INTRODUCT ION Changes in the general composition of an aquatic eco— system usually proceed at a slow rate, yet low levels of en— richment or low levels of toxicants can alter the composition of aquatic communities by changing the quality of their environment. When the water quality has become changed sufficiently, the sensitive, less adaptive organisms are sub- jected to increasingly stressfull conditions which in turn evoke physiological responses. If adaptation can not be achieved, these organisms will be eliminated from that habitat. These physiological stress responses suggest a means of early detection of a deteriorating habitat, as well as a deteriorating physiological condition. However, before aquatic organisms can be used, their responses to stress must be recognized, described, and tested for both the physiological and ecological significance. This thesis pre- sents data substantiating the presence of stress responses in aquatic organisms, as judged by the responses of the rock bass (Ambloplites rupestris). 1In this thesis, "stress" denotes the "condition(s) resulting from any environmental change that disturbs the normal functioning of an animal to such an extent that its chances for survival are reduced" (Fromm, 1962). Investigations of the toxicity of pollution have been limited mainly to acute—toxicity bioassays and to analyses of aquatic animal populations. Data from such investigations have been widely accepted as legal evidence of pollutional damage. Unfortunately, these approaches are most applicable when pollution has already caused serious damage. These methods usually do not permit predictions concerning the ef- fects of sublethal conditions. However there is a growing awareness that the result of slow, insidious changes in the environment can be just as detrimental as acutely toxic con- ditions. Consequently, new methods are needed which will de— tect adverse conditions before biOIOgical catastrophy occurs. Several approaches have been used for stress measure— ment but the methods are not generally suitable for investi- gations that require large numbers of specimens. However, the results have shown some of the subtle but adverse ef- fects of sublethal pollution on fish. Among these effects are the thickening of the respiratory epithelium by de— tergents (Lemke and Mount, 1963) and by ammonia (Burrows, 1964); necrosis of the gut tract and inhibited active trans— port of glucose across it (Fromm and Schiffman, 1958; Stokes, 1963); necrosis in the liver and kidney by DDT (King, 1962); decreased hepatic glycogen and ribonucleic acid caused by pulp mill wastes (Fujiya, 1961a) and the death of sac fry when the female fish accumulated high levels of pesticides (Burdick, et al., 1964)- Other approaches are worthy of mention, mainly be— cause they are rapid and easy to perform. Hematocrit de— terminations have been used successfully by Schiffman and Fromm (1959) who point out that these values rise during stressful conditions. The latter unfortunately seems to in— clude the capture of the specimen. Serological studies of aquatic species have become possible with the relatively re- cent development of micro—analytical technics. Among the serological methods now available, electrOphoresis is use— ful for detecting changes in the protein composition of plasma or serum. The importance of proteins to life has been recog— nized by Karlson (1963) in his statement that "all of the basic functions of life depend upon specific proteins." Both the amount and importance of protein to animal life are indicated by White, Handler, and Smith (1959) who estimate that approximately 79% of the dry weight of animal tissue is protein. Thus all of the body's proteins probably perform important functions that developed as that species evolved and these functions appear to be necessary for survival of that species. The body functions performed by these proteins are not isolated events. Rather, these functions are highly interdependent and the relationship exists in the form of a dynamic equilibrium. Thus it is difficult to imagine that an influence on one body function will not also influence other functions of the body. Therefore, one can postulate that stresses which alter the physiological function of tissues must also alter their protein composition to some extent. Changes in the protein composition (or function) do not necessarily imply that the proteins must be attacked directly. For example, hypoxia does not attack proteins directly, but it can result in decreased oxidative phosphory— lation and less usable energy (Conn and Stumpf, 1963). If the energy supply is diminished sufficiently, vital processes of cellular maintenance will be adversely affected, i.e., protein biosynthetic activity, etc. These in turn can result in the leakage of proteins across the cell membrane (Hess, 1963), or may activate intracellular peptidases (Giese, 1962), or still other adverse actions may result. Hormones can also influence the gross protein compo- sition of tissues. It is possible that hypoxic stress activates the hypothalamic-hypOphyseal axis in fish as it does in man (Van Liere and Stickney, 1963) causing the eventual release of glucocorticoids from the adrenal cells. Whether these hormones cause enzyme induction in fish as it does in rats (Knox and Auerbach, 1955; Goldstein, Stella, and Knox, 1962; Caffery, Wichard and Irvin, 1964) is not known. However, Robertson-§t_a1. (1963) and Cordier and Barnoud (1957) have shown that several of these hormones can cause profound changes in fish tissues, which appears to be related to gluconeogenesis. Since all of the previous factors may be operating during stressful periods, it is probable that both the blood and the tissue proteins will be affected by stressful events. Any change in the protein composition of an organism would reflect an alteration in some body function(s). Such a change would be inimical to the general well-being of an organism because (1) this is a change from a homeostatic condition that developed through evolution, (2) even if the change represents an adaptation rather than a malfunction, it requires energy that otherwise might have been used for growth or other biological functions, and (3) adaptations to one stress situation often limits the ability of an organism to resist other adverse conditions. That the plasma or serum protein composition can be altered by stress is widely documented, but the significance of such a change has not been entirely resolved. Bier (1959) believes that the changes are related to the physiological condition of the test organism. Dunn and Pearce (1961) con- curred with Bier and suggested that the general increase in low-mobility proteins in human serum represents a non- specific response to stress; they term this a stress pattern. Fujiya (1961a) and Bouck and Ball (1965) have reported that changes similar to those in stressed humans also occur in the blood of stressed fish. This change was a general decrease in the relative amounts of high—mobility protein and an in- crease in the relative amounts of low-mobility protein. The problems of deciding the significance in such a change can be appreciated when one understands how proteins are separated by electrophoresis. Each protein is made from a RNA template that is different for every protein type, and therefore imparts specific physio—chemical properties to each type of protein. Among the latter is included a particular net surface charge (electrical). Hence a particular protein will migrate with a given mobility when dissolved in an ap- propriate medium and placed in an electrical field. But its own mobility is often similar to other proteins due to the combined effects of molecular weight and surface charge. Al— though there are several thousand different proteins in an organism, paper electrOphoresis will usually separate about six fractions of proteins. Other forms of electrophoresis can separate more fractions, but no electrOphoretic method will separate proteins on the basis of their physiological or biochemical function. An investigation was conducted of the stress re— sponses of fish, and specifically, the investigation con- cerned the changes in protein composition of plasma, muscle, and erythrocytes. For the purposes of this study, a sig- nificant change in the protein composition of the plasma, muscle myogens, and hemolyzates was established as a cri— terion of stress and considered as a change in the well—being of the organism. The specific objectives of these studies were, (1) to determine if stressful conditions produce significant and recognizable changes in the protein composition of erythro— cytes, muscle myogens and the plasma proteins, (2) to de— termine if such changes constitute a generalized response to stressful conditions, and, (3) to determine if cellular leakage of proteins, as judged by plasma enzyme levels, are involved in these changes. Colateral studies were also con- ducted on hematocrit values (total cell volume of whole blood), hemoglobin concentrations, plasma protein concen— trations, and the activity of certain enzymes in the plasma and liver. These objectives were tested by subjecting specimens to different experimental conditions in four separate experi- ments. The experimental conditions consisted of diurnal low oxygen with food offered, diurnal low oxygen without food offered, exposure to divalent nickel, and exposure to di- valent nickel concurrent with low oxygen, high carbon dioxide levels, and fasting. In each experiment controls were used for comparison with the treated specimens. The rock bass was selected for these studies because it has a wide geo- graphical distribution, is empirically classified as pol- lution sensitive, and its myogen proteins are readily separated by paper electrOphoresis. L ITERATURE S URVEY The blood proteins of fish have not been studied ex- tensively except in connection with taxonomic classification. A literature survey revealed fewer than 100 publications concerned with the electrophoretic analysis of fish proteins, and of these only twelve papers dealt with the influence of stressful conditions. None of these papers was concerned with changes in the protein composition of tissue proteins. The two most salient features of the stress studies can be summarized as follows: (1) changes in the blood proteins of fish were always associated with stressful con— ditions such as disease, fasting, toxicity, and thermal stress; (2) induced changes represent a general increase in the low or mid—mobility proteins and in part correspond to the stress pattern of Dunn and Pearce (1961). Lysak and Wojcik (1960) fed diets containing various amounts of protein to one and two year old carp. Low protein diets were associated with reduced amounts of both high and low mobility protein (in serum), but the greatest reduction occurred in the high mobility group. Fujiya (1961b) found similar results in fasting carp, but he did not present quantitative data. Sorvachev's (1957) data agree with the results of these investigators. Neuhold and Sigler (1960) studied the effects of fluoride intoxication on carp. Specimens which displayed symptoms of muscular tetany had significantly different amounts of protein in their serum fractions. The mobility of these fractions was not stated, but from my experience with this species, I assume that the increase was in the lower mobility proteins. Fujiya (1961b) also studied the ef— fects of various toxicants, and although his data were not quantified, the electropherograms indicate an increase in the low and mid—mobility proteins. Flemming (1958) found that carp with infectious drOpsy had increased amounts of low mobility protein in their serum. The effects of photOperiod and ambient temperature were studied by Meisner and Hickman (1962) using rainbow trout. High temperatures (160C) were associated with in— creased amounts of low mobility protein, but the photOperiods that were tested had no apparent effect. Post (1963) followed the immune response of rainbow trout to Aeromonas hydrophila and found that the amount of low mobility protein in the serum decreased as the antibody titer increased. Other studies have been conducted on the rainbow trout by Bouck and Ball (1966) who investigated the effects of various capture methods on the plasma protein fractions. The amounts of proteins in the fractions varied according to the method of capture and the results indicate that 1E.XIXQ coagulation was involved in the observed changes. The 10 magnitude of the induced differences was small, but sta— tistically significant. Sindermann and Mairs (1961) studied serum from pre— and post spawning alewives (Alosa pseudoharenqus). These authors indicated that no significant differences were present inthe serum protein fractions, except after two months of fasting. Then the high—mobility protein was "drastically reduced." Bouck and Ball (1965) investigated the influence of hypoxic stress on three species of warm water fishes. The proportional amounts of low mobility protein were signifi- cantly increased in bluegills (Lepomis macrochirus) and largemouth bass (Micropterus salmoides), but not in yellow bullheads (Ictalurus natalis). This pattern of change con— forms to their empirically determined pollution tolerance. Papermaster, Condie and Good (1962) studied the immune response of hagfish (species not given) to T2 phage infections by immuno-electrophoresis and although their data are not directly applicable here, the precipitin lines indi— cate an increase in the amounts of low mobility protein. Fungal disease (Ichthygsporidium hoferi) in sea herring (no Species given) produced a "drastic slump in the lead anodal peak" (fastest migrating fraction) according to Sindermann and Mairs (1958). They also note that "such pro- nounced electrOphoretic changes have not been accompanied by demonstrable antibody response." Their data could be 11 interpreted to mean that there was a leakage of cellular protein into the blood plasma. Hunn (1964) has reported that while kidney disease in brook trout (Salvelinus fontinalis) did increase the level of the low mobility plasma protein, the fastest mi- grating fraction completely disappeared. In this case, the increase in low mobility protein was a relative increase rather than an absolute increase in concentration. METHODS The experiment was designed to test the influence of stressful conditions on the protein composition of plasma, myogen, and hemolyzates, as well as hematocrits, hemoglobin concentrations, and total plasma protein concentrations. To accomplish these objectives, it was necessary to use arti— ficial streams as a means of controlling environmental con- ditions. Methods for analyzing blood and tissue parameters were selected with regard to accuracy and usefulness in testing small sample volumes. Controlled Environment Artificial streams Two artificial stream systems were used in these studies. The first and smaller stream (Stream A) was used to study the influence of low oxygen pressure and has been described by Bouck and Ball (1965). This stream is shown in Figure l and is described as follows. The control and experi- mental sections consisted of paired acrylic plastic boxes, 30 cm deep, 60 cm wide, and 150 cm long. A regeneration reservoir was included and consisted of plastic coated ply— wood 16 inches deep, 30 inches wide, and 96 inches long. 12 13 . __.., _ __ __ .mCHzmup map Eoum copuHEo mmB cEsHoo mafimmmmop woo .oHSmmmHm comwxo 30H wo codenawca mnu mo Sodom esp ca poms mm :d: Emouum HMHUHMfluHm mo WCHBme oapmamnom .H munmflm 14 1 ml: uwHCHnHKP. BOHHT Hm>0 ] l 2 ‘— F“\ [ll _/ o 0 Au 0 o 0 o NO’}| 1A AlllleucwEuHmmEoo Goapmuod & ? 5%.,i AT. mend IH lli. 41V coflpuwm HNpcoEauwmxm mumauso unmaq Ilfl coapomm Houucoo .w a amwemmHzo UOn-IDZZ i ? mumauso sense 15 This was arranged to form waterfalls, and equipped with aeration by compressed air, a refrigeration coil, and a bronze sump pump (Sears Roebuck Co.). The latter re- circulated the water through the reservoir and supplied water to the streams. Before the water entered the control or the experimental sections, it passed into individual constant head columns four inches inside diameter and four feet high, filled with 0.5 inch raschig rings. Nitrogen gas was bubbled through the columns to strip the water of dis— solved oxygen to the required level before it entered the experimental section. Only the water from the control section was recycled. Lost water was replenished via a float-valve in the regeneration reservoir. Artificial stream "B" is a modification of that described by Kevern and Ball (1965) and was used to test the influence of a heavy metal pollutant (nickel) both with and without low oxygen conditions. This stream is shown in Figure 2 and consisted of paired aluminum shells 10 inches deep, 14 inches wide, and 16 feet long lined with a sheet of polyethylene. A sump pump supplied water to two constant— head columns from a 12,000 gallon concrete reservoir. These columns in turn supplied the control and experimental sections of the stream. The latter section also was equipped with a solution metering pump and mixing box at the front of the stream. The effluent from the metering pump contained sufficient nickelous chloride to bring the experimental 16 .mCHBmHU onu Eouw UoDDHEo ma cofluoom Honucoo map mo Ham .comhxo 30H paw Hoxofic pcoam>flp mo mucosHMCH may mo xpsum map CH poms mm :m: Emonum HMHUHMHDHM mo mCHSMHC oaumfiwnom .m madman 17 cflmuo o6 cofluowm Hmucmfiwuwmxm pmauso AI. Nom mcflxflz coHusHom sexuaz . h 1?. 2.110. mesa mcfluoumz casaou mcflmmmmwnuv 9 upmmm BGMpmcoo ICE? oBOHm H0>0 QEdm poHcH 18 section to the desired concentration of nickel. Oxygen was removed from the experimental water in the same manner that was described for artificial stream "A." Only the water from the control stream was recycled and the lost water was replaced via a float valve in the reservoir. Flow rates in both stream systems were measured by collecting the effluent of a given stream for one minute periods and relating the volume to manometers on the inlets. Valves provided rate adjustment and the flow in both stream systems was held to approximately 10 liters per minute. After the water entered the streams, its velocity was re— duced rapidly and the fish appeared to be in a resting con— dition at all times. Both stream systems were illuminated by fluorescent light. Meisner and Hickman (1962) have shown that photo— period did not have a significant effect on plasma protein fractions, hence photOperiod was not regulated in any of the experiments. Water temperatures for the experiments were main- tained by two methods. Higher temperatures were maintained by adding the desired amount of hot water to the cold water line, before they passed through an activated carbon filter. While passing through this filter, the water temperature was brought to the desired level, and both the hypochlorite and dissolved iron were removed from the water. The temperature of the cold water line was very uniform and its use, after 19 passing through the de—chlorination process, constituted the second method of temperature control. Water for these experiments was moderately hard tap— water derived from several deep wells located on the campus of Michigan State University. Chemical analyses of water All chemical analyses of water were performed in ac- cordance with the procedures set forth in ”Standard Methods for the Examination of Water and Wastewater" (1960). Dis— solved oxygen concentrations were determined using the Alsterberg (azide) modification of the Winkler method. Alkalinity was determined by titrating the sample with N/SO sulfuric acid, first to pH 8.3 and then to pH 4.6; these pH values correspond to the equivalence points of phenophthalein and methyl orange respectively. The pH and bicarbonate alkalinity were used to esti— mate the free carbon dioxide concentration by nomographic de— termination according to Theroux, Eldridge, and Mallmann (1943). Total hardness was determined by the EDTA titration method. Free and combined available chlorine were determined by the Orthotolidine method and total iron was determined by the Phenanthroline method. Ammonia content was determined by direct nesslerization. Dissolved iron, ammonia and chlorine concentrations were consistently low and therefore were considered to be unimportant to the experiments. 20 Estimation of dissolved oxygen pressure The content of gases dissolved in water can be ex- pressed in several ways, but physiologists usually choose to express it in terms of its pressure. For it is this pressure that provides the driving force, hence causes the oxygen to arrive at and load the hemoglobin. Conversely, limnologists usually choose to express oxygen values in terms of the mass or volume of oxygen per liter of water. Fry (1957) suggests that "the density of the oxygen supply is more frequently the limiting aspect of this substance in water than is the pressure aspect." However, oxygen consumption is a function of oxygen pressure over a wide range of temperatures ac- cording to Fry and Hart (1948). Because no single measure of the oxygen content of water is accepted by all, the oxygen pressure was estimated in addition to its corresponding percentage of saturation and mass per liter of water. Barometric pressures were supplied by the U.S. Weather Bureau Substation located at Capitol City Airport, Lansing, Michigan. They believe that these readings would be nearly identical with the pressure in this laboratory. Oxygen saturation values for a given temperature and pressure were derived from Welch (1948). The resulting values represent the pressure in the laboratory and not those at sea level. 21 Collection and Care of Specimens Rock bass were collected from the Red Cedar River using a 230 volt direct current shocker. Immediately after being netted, they were placed in a tub of water and soon after this, they were placed in live—boxes in the river. Later they were transported to the laboratory in an insulated transporting unit filled with river water and aerated by agi— tation. At the laboratory, the fish were transferred to aged tap-water of approximately the same temperature and left undisturbed for one day. After the first day in the laboratory, the specimens were dipped into a strong fungicidal solution of methylene blue (about 10 ppm) and then held in a bactericidal solution of tetracycline (about 0.5 ppm Polyotic, American Cyanamid Co.) for two to three hours. The following day, they were treated with 1:4,000 formalin to remove external parasites. Specimens were not used in experiments unless they appeared to be in excellent condition. Pretreatment of Glassware All hypodermic needles, syringes, and other glass- ware contacting the blood or tissue samples of the Specimens were coated with a biologically inert silicone film (Sili— clad, Clay-Adams Co.). After being used, glassware was washed in detergent (Haemosol, Meinecke Co.), rinsed in tap- water, cleaned with chromic acid, re-washed with detergent, 22 rinsed several times in double distilled water and re-coated with silicone. Procedures for Testing Blood and Tissues Parameters / Collection and preparation of blood samples Each fish was netted carefully and anesthetized in l:80,000 Tricane Methylsulfonate (MS—222, Sandoz Co.). Blood was collected by indirect cardiac puncture (see Figure 2) by probing into the heart through the isthmus. One ml tuberculin syringes (charged with 0.05 ml of 10% trisodium citrate) with one inch, number 22 hypodermic needles were used for this purpose. The blood was drawn rapidly, mixed with the citrate and the total volume was recorded. Each parameter was corrected for the volume of the citrate. After the hypodermic needle was removed, micro-hematocrit tubes were charged, blood films made, and the remaining blood was emptied into a polypropylene centrifuge tube. From the latter, samples were taken for the determination of hemoglobin. The remaining whole blood was chilled in an ice bath, centrifuged at 1500 X G, and then the supernatant plasma was separated from the blood cells. Hemolyzates were prepared from the sedimented blood cells as follows: The remaining plasma was removed from the cells by washing them twice in ten volumes of 0.85% NaCl. 23 Each washing consisted of suspending the cells in the saline and then reconcentrating them by centrifugation. After draw- ing off the saline, the cells were osmotically ruptured by adding an amount of distilled water equal to twice the volume of the packed cells. This process caused the cells to burst and thereby release their cytOplasmic protein into the distilled water. The cell membranes and nuclei were then removed from suspension by centrifuging. This process produces a clear, ruby red supernate which is called a hemolyzate. Collection and preparation of tissue samples Immediately after the blood was safe from coagulation, the ventral aorta was cut and this removed as much blood as possible from the tissues. At this point, the weight and length of the specimen was recorded before tissues were ex- cised. Muscle tissue was removed from the left side of each specimen as shown in Figure 3, skinned, and sealed in a small polyethylene bag with an identification tag. Im- mediately thereafter, these were placed in a freezer and re— mained there until processed for electrophoretic analysis. Preparation of muscle for electrophoretic analysis consisted of freezing and thawing the sample three times to rupture cell membranes, and then grinding the sample with twice its volume of distilled water, in an ice-cooled, glass 24 .mflmwamcm OHumHonmouuomHm How poms oHomDE Mo coaumooH on» pom Home: may mo coflumuoa 03p mCHBOLm Amfluummmsu mouHHQOHQE¢V mmmn xoou osu mo mCHBMHQ .m ousmflm 26 mortar. Grinding is much easier if the specimen is cut into small pieces while still frozen. This method is a slightly modified version of the Potter-Elvehjem method (1936). After grinding, the resulting slurry was re-chilled and centrifuged for 10 minutes at 1500 X G. Determination of total cell volume in whole blood (hematocrit) The total cell volume in whole blood (hematocrit) was determined using heparin coated microhematocrit tubes. Each tube was filled to approximately 85% of its height with citrated whole blood, plugged with clay and centrifuged 15 minutes at approximately 1500 X G. The total height of the blood column and the height of the packed cells (including the milky layer) were measured using a millimeter scale and a magnifying glass. The ratio of the height of the packed cell column to the total height of the blood column was con- verted to packed cell volume / 100 ml of whole blood and ex— pressed as ml/lOO ml. Determination of hemoglobin concentration The acid hematin method as described by Schiffman and Fromm (1959) was used to estimate the hemoglobin concen— tration. A 20 microliter sample of citrated whole blood was added to 10 ml of 0.1 N HCl, mixed rapidly, and allowed to react for 1 hour. Then the sample was centrifuged 10 minutes 27 at approximately 1500 X G to remove nuclei and cell mem- branes. The absorbency of the resulting solution was measured in a spectrophotometer set at 500 mu. Readings were converted to grams of hemoglobin per 100 m1 of whole blood by the manufacturer's calibration curve. Determination of total protein concentration The Biuret method was used to determine the concen— tration of protein in the plasma and tissue supernates. This was adapted for small sample volumes as follows: To SO’pl of sample 1.5 m1 of 0.85% NaCl were added and mixed thoroughly, and to this was added 1.5 ml of Biuret reagent and mixed thoroughly. The reagents were then allowed to re- act at least 15 minutes, but not longer than 30 minutes be— fore the absorbancy was measured in a spectrophotometer set at 550 mu. These readings were converted via a standard curve to the equivalent grams of bovine serum albumin per 100 m1 (3X crystalized albumin, Nutritional Biochemical Corp.). Assays for Enzyme Activity Determination of lactic dehydrogenase activity The general method of Cabaud and Wroblewski (1958) was used with modification for small sample volumes. The 28 essential features of this method are that in the presence of lactate dehydrogenase (LDH) and coenzyme, pyruvate will be converted to lactate. Since pyruvate is an alpha-keto acid, it will react with 2,4—dinitropheny1 hydrazine and in alkaline medium it will form the brown hydrazone which is a quantitative measure of the remaining pyruvate. Therefore, it provides a quantitative measure of the activity of lactate dehydrogenase. The procedures of this assay were as follows. To 50 pl of sample, 0.1 m1 of 0.1% nicotinamide-adenine— dinucleotide (NADH 99% pure, Nutritional Biochemical Corp.) were added and mixed thoroughly. Then 1 ml of phosphate buffered pyruvate (ZOO/ug/ml) was added, mixed thoroughly and allowed to react for exactly 30 minutes in a water bath. Immediately after the incubation 1 ml of 2,4-dinitr0phenyl hydrazine (200dpg/ml) was added, and allowed to react for exactly 20 minutes before adding 10 ml of 0.4 N. NaOH. The latter formed the 2,4 dinitrOphenyl hydrazone of pyruvate. The final reaction was allowed to proceed for 20—30 minutes before the absorbancy readings were made in a spectrophoto— meter set at 550 mu. Readings were converted to the equiva- lent micrograms of pyruvate transformed per ml of plasma per 30 minutes. 29 Determination of transaminase activity In this study, investigation of transaminase activity was limited to that of Glutamic-Oxalacetic Transaminase (GOT) and that of Glutamic-Pyruvic Transaminase (GPT). Both of these enzymes catalyze the transfer of alpha-amino groups from one amino acid to an alpha-keto acid, thereby changing that amino acid to a keto acid and vice versa. The sub- strates for each enzyme are given by their names, i.e., glutamic-oxalacetic and glutamic-pyruvic. Since both of these enzymes are concerned with alpha-keto acids, the same general analytical procedure can be used here as was used for lactate dehydroqenase. The activity of these enzymes was measured using the reagents and methods supplied by the Dade Reagent Co., Miami, Florida. This method is essentially that of Reitman and Frankel (1957), but modified by the manufacturer to stabilize the reagents. The nature of this modification was not avail- able from the manufacturer. Readings were converted to arbitrary units supplied by the manufacturer. Determination of succinic dehydrogenase activity Succinic dehydrogenase is an enzyme of the mito- chondria as well as an enzyme of Kreb's tricarboxylic acid cycle and catalyzes the conversion of succinic acid to fumaric acid. In this assay, the hydrogen and electrous 3O removed from succinic acid reduces triphenyl tetrazolium chloride to its red, aliphatic analOg. The production of reduced tetrazolium is not entirely due to the activity of succinic dehydrogenase, as other dehydrogenases can be ex- pected to contribute to this reaction. However, succinate would be the principle substrate. Thus, this assay presents only a generalized measure of metabolic activity in the re- spective tissue. The method of Fujiya (1960) was used, but modified to improve its application to liver from rock bass. The incubation medium consisted of 2.0 ml of 1.0 M. phosphate buffer at pH 7.8, 0.5 ml of 0.4 M. succinate, 0.5 ml of a solution containing 6.5 mg phenozine methosulfate per 12 ml, 1.0 ml of 0.5% triphenyl tetrazolium chloride and 1 m1 of liver supernate. The latter was prepared by blending one volume of liver with nine volumes of distilled water in an ice cooled glass mortar. After it was centrifuged at ap- proximately 1500 X G for five minutes, the protein concen- tration of the supernate was determined and later was re— lated to enzyme activity. ‘After mixing the above components, the reaction was allowed to proceed 2.5 hours at 250C. Then, one ml of glacial acetic acid and 2 ml of 1% trichloracetic acid were added to end the reaction and to denature the protein. After centrifuging, the colorless supernate was discarded and the sedimented tetrazolium-protein pellet was dried overnight at 31 500C. Then the pellet was crushed to a powder and extracted with acetone to remove the tetrazolium. The absorbancy of the tetrazolium solution was measured in a spectrOphotometer at 490 my and these readings were converted to activity units. One unit of activity equaled a micromole of formazan produced per gram of soluble liver protein per 2.5 hours. ElectrOphoretic Methods Separation and evaluation of protein fractions The Beckman Model R Paper Electrophoresis System with Analytrol Scanner was the principle electrophoretic method used in this study. Separations of protein were con— ducted on S & S # 2095A paper strips. Approximately 20Jul samples were applied to the strips which were moistened previously with buffer, and then the samples were electro- phoresed at approximately 16°C with an applied voltage of 200 volts. Plasma proteins were separated using Smith's (1960) vernol buffer (pH 8.6, u = 0.065), but Tris-EDTA- Borate (Tris) buffer was required for good separation of tissue proteins. Tris buffer consisted of 6.050 g of 2- amino-Z-(hydroxymethyl)-l,3-propanediol, 0.780 g disodium ethylenediaminetetraacetate, and 0.460 g boric acid per 500 ml of water. Vernol buffer was always used to fill the buffer compartments of the electrophoresis cell: tris buffer was used only on the paper strips, thereby forming a discon— tinuous buffer system. 32 The samples were electrophoresed 12 hours after which the strips were suspended in an oven at 1100C for 30 minutes to denature and afix the protein fractions in their respective positions. A 15 minute methanol bath removed the buffer from the strips following which they were stained 30 minutes in 0.1% methanolic bromphenol blue. Three subse— quent baths of six minutes each in 5% acetic acid removed the free dye and the remaining free amino acids. The strips were dried again at 1100C for 30 minutes and then exposed to ammonia vapor for 15 minutes in a sealed chamber. This pro— duced maximum blue color development of the protein—bound bromOphenol blue. Immediately after this, the strips were scanned using the Analytrol Scanner (500 mu filters) and the B—5 balancing cam. Scanning produced an absorbancy curve of the bromphenol blue in each protein fraction which in turn was proportional to the amount of protein in each fraction. The scanner also measured the area under each curve by inte- gration, and recorded it on the electrOpherogram (see Figures 5—6). Plasma protein fractions were expressed and evaluated both as the grams of protein in a fraction per 100 grams of sample protein, and as its concentration per 100 m1 of plasma. This was done using the following formulae: . . area under the curve [: Grams of protein in f a given fraction X 100 gramE] (l) a fraction / 100 grams = .1 , of sample protein total area under all the curves in that sample 33 Grams of protein in grams of protein total concen— (2) a fraction / 100 ml = in that fraction tration of of plasma / 100 grams of X protein / 100 ml sample protein of plasma Myogen and hemolyzate fractions were estimated using formula (1) and are expressed only in terms of the grams of protein in a fraction per 100 grams of extracted protein. Paper electrophoresis as a quantitative technic has been investigated by Henry, Golub and Sobel (1957), by Abdel-Wahab and Laurence (1955) and by Bouck (1963). The procedures used in this thesis conform to the limitations set forth in their recommendations. Using the previously listed standardized conditions for electrophoresis, the location of each fraction was con- sistent in regard to its source, i.e., plasma, myogen, and hemolyzate. In a few instances gross variations occurred, but these were traced to deviations from standardized con- ditions for electrOphoresis. Whenever this occurred, the af- fected paper strips were discarded and other portions of the samples were analyzed. The most usual causes of variation were drafty conditions around the cell, increased ambient temperature, and poor contact between the paper strips and the cell. Separation and location of isozyme fractions Several electrOphoretically different forms of LDH can be distinguished in vertebrate species and certain of 34 these forms of LDH are characteristic of skeletal muscle. These so called "isozymes" can be located after paper electrophoresis by the method of Markert and Ursprung (1962). And when the plasma LDH activity is elevated, the isozymes indicate the tissue which contributed it. The general scheme is similar to the scheme for succinc dehydrogenase activity. As a result of lactic de- hydrogenase activity, a colored tetrazolium is formed on the strip and by this color production, the sites of the iso— zymes are located. This general analytical scheme has been applied widely following electrophoresis in gel media, but does not work well with paper electrophoresis. Here the freshly electrophoresed paper strips were laid on a filter paper soaked with the incubation medium and then sandwiched between two glass plates. Incubation proceeded in the dark until the bands were formed. The incubation medium consisted of 1.0 ml (0.5 M.) hydrazine, 6.0 ml of 0.5 M lactic acid, 20 mg NAD, 0.4 mg phenazine methosulfate, 25 mg Nitrobluetetrazolium chloride, and 15 ml of 0.2 M tris-HCl buffer adjusted to pH 7.4. Statistical Methods All specimens in all of the experiments were assigned to treatments by the use of random numbers. In all cases, whenever the probability of obtaining a given result was equal to or less than 10%, it was listed as being 35 statistically significant. Student's "t" test for small sample sizes was used to compare the means of control and experimental lots for a given experiment. Whenever more than two means were involved, one-way analysis of variance was used to test the results. The results were analyzed without regard to the sex of the specimens because sex could not be determined before dissection, hence not before assignment to treatment. Plasma differences related to sex of fish have been noted only in regard to the associated lipids (Vanstone and Chung— Wai Ho, 1961; Bouck, 1963). Coefficients of variation were also determined for each parameter and provide a measure of the difference among the individuals in a given experimental lot. Furthermore, coefficients of variation are free of units such as feet, yards, etc., hence variability can be compared among different parameters. ELECTROPHORETIC CHARACTERISTICS OF ROCK BASS PROTEINS As a prerequisite to studying the effects of stress on the composition of rock bass protein, it was necessary to establish the general electrophoretic characteristics of plasma proteins in relation to the water soluble proteins of various tissues. The objectives were to establish the rela— tive mobilities of tissue proteins and determine whether these tissue proteins were suitable for subsequent electro— phoretic studies. True mobilities pg; sg were not estimated because the migration of proteins during paper electro— phoresis is not a true function of time (Bier, 1959). Pro— tein fractions were numbered with numerals proceeding from the least mobile fraction to the most mobile fraction. Results General characteristics of tissue proteins Water soluble proteins in liver, gill, stomach, heart, kidney, midgut, highgut, spleen, brain, erythrocytes and muscle were investigated. In most of the tissues, the proteins did not resolve into well defined fractions unless tris buffer was used. The mobilities of the tissue proteins 36 37 were very low in vernol buffer, but tris buffer increased this. However, the comparison of mobilities was made using vernol buffer because this buffer was always used in the separation of plasma proteins. The mobilities of the fastest migrating plasma fraction and tissue fractions are compared in Figure 4. NOne of the tissues had a protein fraction which migrated as fast as plasma fraction IV, and most of the tissue protein did not migrate beyond plasma fraction III. Thus if cellular proteins leaked into the blood, they would remain in plasma fraction I through III. Plasma protein fractions At least six fractions of protein were separated in plasma using vernol buffer (Figure 5). Tris buffer decreased the number of fractions and was not used in subsequent studies of plasma. Fraction III consists of two fractions which did not separate well enough to justify treating them as separate entities. Additional time for migration in— creased the distance between the fractions (from center of fraction to center of fraction) and tended to resolve ad— ditional fractions. However, the shortest possible migration time was preferred because it was associated with less diffusion of the protein fractions and less variability in the resulting values. The standardized conditions for electrOphoretic analyses of plasma proteins are listed in the methods section. Figure 4. 38 Comparative paper electrOphoretic mobility of the fastest migrating proteins in the tissues and blood of the rock bass (Ambloplites rupestris). Samples were migrated concurrently using vernol buffer at pH 8.6. The position of slower migrating fractions is not indi— cated in this illustration. 39 PLASMA FRACTION VI ——1.- LIVER ’ GILLS g5 STOMACH 3 HEART ‘ , KIDNEY es MID & HIND GUT _ V BRAIN #_ r MUSCLE A ' SPLEEN g‘ ERYTHROCYTE l l l l l l l 16 20 3o 40 55 60 7o 80 90 100 PERCENTAGE or TOTAL DISTANCE MIGRATED IN COMPARISON TO PLASMA FRACTION IV Figure 5. 40 Protein fractions of rock bass (Ambloplites rupestris) as separated by paper electrophoresis. A. Plasma buffer B. Myogen buffer C. Myogen buffer protein separated using vernol at pH 8.6. proteins separated using tris at pH 9.1. proteins separated using tris at pH 9.1. Samples were dialized 24 hours and electrophoresed longer than standard conditions. Hemolyzate proteins separated using tris buffer at pH 9.1. Cathode 41 Application Point ‘ _ .9. . . o. .u b . \v . O .4 D. d; .. ‘nv. «0 e s.v...o.rln‘0n1¢x .cittr OJ n ‘01 O c. . o' n 4 an r a . 1. a C a. . . .. 1.: . . .V.‘. . V . o.&. .,n\ . o 1‘ t. . . D. t. 1.. . v *5“. ffluwa. lam-$80.54 {I I IR."..lr .. .0 .. C ”unruly... 0...: I... (.off.£ .. .N s . .‘ n...... 7 v 42 Muscle myogen fractions Muscle myogens did not separate well except in tris buffer. Standard migration conditions produced four fractions (Figure 5B). Longer migrations produced as many as eight fractions and this was aided by dialysis of the samples (Figure 5C). However, preliminary investigations established that variability of the level of each fraction was directly related to the number of fractions that were separated, hence the myogens were analyzed as if they con- sisted of only four fractions. Hemolyzate fractions Hemolyzates did not separate well except in tris buffer. At least two fractions were separated (Figure 5D) and both of these had very low mobilities in comparison to myogens and plasma proteins. At least two electro—chemically different forms of hemoglobin were present and these appear to comprise the bulk of the protein in hemolyzates. 43 Figure 6. An electrOpherogram of plasma proteins from rock bass (AmblOplites rupestris). 44 <8 4U 3.. 35.5.2... 0 no :2: 53 2.5 2:; z: NVNG—n.flvn buxm .556 cu 5:: :3 : _. E: EFFECTS OF LOW OXYGEN ON FEEDING ROCK BASS Introduction and Objectives One of the most common characteristics of polluted water is low dissolved oxygen. Although mildly enriched waters may contain normal amounts of oxygen during the day, the respiration of the aquatic community at night often leads to very low levels of oxygen. This diurnal low oxygen condition has been suspected of contributing to the decline of sport fish populations (Bouck and Ball, 1965). Therefore an experiment was designed to test if diurnal low oxygen conditions significantly altered the protein composition of plasma, muscle, and erythrocytes, as well as changing the levels of hematrocrits, hemoglobin, total plasma protein concentration, feeding activity, and growth. Experimental Conditions Specimens for this study were collected during November 1962, and were held in the laboratory until used. Many fish refused to feed on fish-food pellets and died, but the survivors appeared to become well acclimatized to laboratory conditions. After the fish were assigned to treatments, the diet was supplemented weekly with crayfish 45 46 and minnows, but otherwise consisted of fish food pellets offered daily ad libitum. On May 8, 1963, rock bass were marked by fin clipping and then were randomly assigned to artificial stream "A" (nine fish to each section). The weight and standard length of each specimen was determined on May 8, May 18, and again at the end of the experiment on June 26 (experimental lot) and June 27 (control lot). The experimental conditions were instituted on June 15, and consisted of reducing the dissolved oxygen concen— tration of the experimental section to approximately 3 ppm for eight hours each day for nine consecutive days. This was equivalent to a reduction in oxygen pressure from 127 mm Hg to 52 mm Hg, or a reduction from 86% to 35% saturation. Table 1 presents a summary of the water quality data. The experimental conditions ended at 5 pm, June 23. Blood samples were drawn from the experimental lot on June 24, and from the control lot on June 25. Results Hematocrit, hemoglobin and total plasma protein concentration The average volume of blood cells, and the average concentrations of hemoglobin and total plasma protein are summarized in Table 2. Each of these parameters were sig- nificantly increased in fish that were exposed to diurnal o.m mam om.m me as om.m m.mm -m.m Iowa :mm.n nae mm oo.m -H.Hm momma a ham oo.w mm mm ma.m m.mm m M cofluowm HMDCOEHHOQMM 0.5 mam om.m mma mm oa.m m.mm m -w.m -mmm -om.» -eHH -ms om.e -H.Hm m 2mm e ham em.n has om no.5 m.mm m Ema moomv mm mm EE ca opaxowp 5mm musmmoum .umm 8 8mm 00 connmo mpHCHmead mm cemmxo oo>H0mmHQ .maoa coauomm Houucoo .mcoHuHccoo commxo 30H on Amauummmnn mouaafldaflfimv wmma ROOM pom mo wusmomxw mg» meausp dump wuflamnv neumS mo mumafinm .H magma Table 2. 48 Summary of the influence of low oxygen conditions on hematocrits, hemoglobin concentrations, and plasma protein concentrations of fed rock bass (Ambloplites rupestris). Control: Hematocrit (m1 of cells/ Hemoglobin Plasma Protein 100 ml of blood) (g/100 ml) (g/100 m1) 2 26.3 4.52 2.80 SX 1.86 0.23 0.03 C.V. 16.5 16.2 10.6 N 6 10 9 Exposed to 3 ppm oxygen for eight hours each day for nine days: i 31.8* 4.74** 2 92** SX 2.02 0.10 0.03 C.V. 20.6 5.7 21.9 N 9 7 9 *Significantly different by "t" test. **Significantly different by "f” test. 49 low oxygen. Both the plasma protein and hemoglobin concen— trations were increased approximately 4%, but the total volume of blood cells was increased approximately 20%. The coefficients of variation show that the plasma protein concentrations and hematocrits were more variable in the fish exposed to low oxygen. On the other hand, hypoxia was associated with lower variability among the hemoglobin concentrations. This suggests that the mechanism(s) re- sponsible for regulating these blood parameters were af— fected by exposure to low oxygen conditions. Distribution of protein in the plasma fractions The average weight of protein in each fraction per 100 grams of plasma protein are summarized in Table 3. As compared to the control lot, fish exposed to low oxygen had 22% less protein in fraction I. Fractions II and III were increased approximately 3% and 41% respectively, but fractions IV, V, and VI were decreased approximately 13%, 8% and 35% respectively. Only the changes in fractions I, III, and VI were statistically significant. The weight of protein in fractions III was con- sistently more variable than the values of the other fractions, but not with respect to low oxygen conditions. However, fractions I and VI were considerably more variable among hypoxic fish than in the control fish, indicating that 50 .ucmuweeae msucmuaeacmam.. mcofluomnm mammam m.mm m.e~ N.Hm o.mm m.oa m.am .>.o om.o oe.a on.a mR.H mm.o mm.o xw ..~e.m ma.eH mH.Hm ;.nm.mm em.mm em.e M “An N zv cmmmxo 30H Op @mmomxm m.wm w.ma v.mN m.hm o.ea m.Hm .>.0 x mn.o mm.o mo.m nm.m mo.a No.0 m mm.m mn.ma ma.vm mm.om ©~.mm Hm.w m Acamuoum mo mEmum ooa \ mamumv H> > >H HHH HH H Am u zv Houucou .Amflnumomnu mouflamwanfimv mmmp xuou com mo mcoauomum mammam map as camuoum mo coflusnfluumac on“ so mcoHuHUcoo cwmmxo 30H mo mucoUHMCH may mo mwmeesm .m wanes 51 the fish did not respond uniformly to conditions of low oxygen. In general, the distributions of protein in the plasma fractions were more variable than the values for hematocrits, hemoglobin, and total plasma protein concentration. Concentrations of protein in plasma fractions The average concentrations of protein per 100 m1 of plasma in each of the fractions are summarized in Table 4. These concentrations show the same general trends of change that were found above in the distributions of protein per 100 grams of plasma protein. The concentrations of fractions I and VI were decreased significantly, and fraction III was increased significantly following exposure to low oxygen conditions. Fractions II, IV and V were not significantly different between the control and the experimental lot. The pattern of change does not conform completely to the stress pattern of Dunn and Pearce (1961), but does show a general reduction of the high mobility proteins (fractions IV, V, and VI) and a general increase in the total amount of low mobility proteins (fractions I, II, and III). The average amount of fraction I was decreased, but its average total loss was compensated by a large increase in the average amount of fraction III. The coefficients of variation show that the concen— trations of protein in the fractions was generally more 52 .ucmswueae ssscmoaeacmam.. .poummum SandeAMHcmflme e.se o.es m.em s.mm m.mm m.sm .>.o X as.em oo.am mv.nm mm.sma sm.om os.em m . . . . . . x ..o ems H mom a mmm .o mam o eme .m ems . An N 2v "commxo 30H on comomxm H.om o.mH o.em o.ee m.ea m.mm .>.6 N so.om om.mm am.mm mm.mm mo.om Hp.~m m m.mmm «.mme m.sme m.msm m.eoe H.mmm m AmemHm mo HE OOH \ mEv S > >H HHH HH H 8 n E Houueoo mc0auomum aamuoum mammHm .AmfluumoQSM mmuflHQoHnfidv mmmn Moon pom mo mEmMHQ map CH GHmuOHQ m0 mCOHpmuu Icmocou meapmasouao may no mcoapaccoo commxo 30H mo mucosamca on» mo SHMEEsm .e manna 53 variable in the fish that were exposed to low oxygen. The circulating concentrations of fraction III had the highest coefficients of variability and this was even higher than was observed above in the distribution of protein per 100 grams of plasma protein. Higher variability should be ex— pected because the circulating concentrations reflect the result of the variables in both the distributions of protein and in the total plasma protein concentrations. Distribution of extracted protein in myogen fractions The average distribution of extracted protein in the myogen fractions is summarized in Table 5. As compared to the control values, the fish that were exposed to low oxygen had significantly lower amounts of fraction I and signifi- cantly higher amounts of fractions II and III. Fraction V was decreased, but not significantly in the hypoxic fish. In relative terms, fraction I decreased 17%, fraction II in- creased ll%, fraction III increased 27%, and fraction IV de— creased 14% from the levels found in the control lot. Thus the pattern of response in the myogens to low oxygen was similar to the pattern of response in the plasma protein fractions. The coefficients of variation for the distribution of myogen proteins were generally lower than was observed in the plasma protein fractions. Variability of the myogen 54 Table 5. Summary of the influence of low oxygen conditions on the distribution of extracted protein in the myogen fractions of fed rock bass (Ambloplites rupestris). Control: (N = 8) Myogen Fractions I II III IV (grams / 100 grams of extracted protein) R 37.81 34.99 15.08 11.90 SX 1.571 1.077 0.954 1.520 C.V. 11.7 8.7 17.9 36.1 Exposed to low oxygen: (N = 8) R 31.87* 38 92* 19 12* 10.28 sX 2.207 1.368 1.847 0.889 c.v. 19.7 9.9 24.4 23.9 *Significantly different. 55 values was generally lower in the control fish than in the experimental fish, indicating that low oxygen influenced the uniformity of the myogen response. But more importantly, the coefficients of variation indicate that the composition of muscle myogen is controlled more closely than is the compo- sition of the plasma proteins. Distribution of extracted protein in hemolyzate fractions Significant changes also occurred in the protein composition of erythrocytes (Table 6). The same pattern of change that was observed in the plasma and myogen proteins was also observed in the hemolyzate fractions. Fraction I was increased approximately 7% and the average amount of fraction II was decreased approximately 7%. Both of these changes were statistically significant. Variability was generally low in the hemolyzate fractions of control fish and this was slightly higher in the experimental lot. However, the variability of the values for the hemolyzate fractions was similar to the varia- bility in the values found in the hematocrits and total plasma protein concentrations. Changes in feeding behavior The study permitted observations of feeding patterns in relation to low oxygen conditions. In general, the 56 Table 6. Summary of the influence of low oxygen conditions on the distribution of extracted protein in the hemolyzate fractions of fed rock bass (Ambloplites rupestris). Control (N = 8) I Hemolyzate Fractions II (grams / 100 grams of extracted protein) X 39.15 60.85 S 1.95 1.95 x C.V. 14.1 8.5 Exposed to low oxygen (N = 8) x 42.22* 58.12* sX 2.57 3.576 C.V. 15.6 18.0 *Significantly different. 57 control lot was offered food only once each day and they al— ways fed readily. Food was offered to the experimental fish at various times during the low-oxygen phase to test their inclination to feed during hypoxia. During the first six daily periods of low oxygen, the hypoxic fish ignored food and generally continued to ignore food for about three hours after normal oxygen levels were restored. After the three hour recovery period, the experimental lot readily consumed food. During the sixth hour of hypoxia on the seventh day, two fish in the experimental lot each consumed a single fish food pellet. To further explore their reaction to food, small crayfish were then added to both the control and the experimental sections of the artificial stream. The cray— fish in the control section were consumed rapidly, but only two crayfish were eaten in the experimental section and they were regurgitated later that day. The remaining crayfish in the experimental section were not eaten until after the oxygen level returned to normal. During the eighth and ninth days of the experimental conditions, the hypoxic fish continued to fast during the periods of low oxygen. Thus the only feeding activity during the periods of low oxygen was on day seven and this lack of feeding activity (and regurgitation) indicates that the fish did not acclimate to hypoxia during the period of the experiment. 58 Increment of body weight and length The study also included general observations on changes in body weight and length during the experimental conditions. During the initial acclimation period (May 8— 18) the combined total weight of the control lot did not change, but the experimental lot gained weight (0.7 grams per fish). Between May 18 and June 26, of which low oxygen prevailed during the latter nine days, the control lot gained an average of 10.2 grams per fish. The experimental lot gained only 3.5 grams per fish during this period, possibly reflecting the adverse effects of low oxygen. The average standard length of the fish did not change either in the control or the experimental lot during the experiment. Relationship of body weight and length to plasma fractions At the end of the experiment, the average total weight and average standard length was 63.8 grams and 10.5 centimeters for the control lot, and 71.5 grams and 11.9 centimeters for the experimental lot. The differences in weight and length between the control and experimental fish suggest that the observed differences in the plasma fractions may have been related to each other. Therefore, correlation analyses were conducted to determine the intensity of the relationship between the protein values and the weight, 59 length, and condition coefficients of the specimens. The only two significant correlations were found between the relationships of plasma fraction V and VI (—0.82 and -0.62) to weight. Other correlation coefficients indicated that there was no significant relationship between plasma protein fractions and the weights, standard lengths, or condition coefficients. Other observations Other effects of low oxygen were noted among the experimental fish and these included a loss of body color, a reduction of activity, and higher ventilation rates. Mucus strands were seen occasionally in the water of the ex- perimental section and appeared to be casts of the gut lumen. The fish used in these experiments represent only a small proportion of the fish that were collected during the previous November and held for use in this experiment. As stated earlier, most of these did not acclimate to the con- ditions of the laboratory and these died. Approximately 15% of the original collection acclimated to laboratory con- ditions and these appeared to be in excellent condition. Hence, when one views their response to hypoxic conditions, one must bear in mind that the magnitude of their response is probably different from that of a "normal" pOpulation of rock bass in the river. 60 It is the opinion of the author that the responses in this experiment are minimal to those among rock bass in the river. This View is supported by the following reasons. The collection and acclimation process favor the selection of hardier individuals. Both electroshocking and the prophylactic treatments are stressful as well as potentially lethal. Having survived these treatments, the fish appeared to remain free of diseases and external parasites that were common to rock bass in the river. Finally, the experiment attempted to encompass only one variable, i.e., controlled low levels of oxygen. Such a situation would be unlikely in polluted waters where one would expect to find additional adverse influences such as detergents, ammonia and perhaps other toxic materials. EFFECTS OF LOW OXYGEN ON FASTING ROCK BASS Introduction and Objectives The previous experiment was designed to test the in— fluence of a single variable, i.e., low oxygen conditions. However, during the course of the experiment, hypoxia also induced diurnal fasting and was associated with the regurgi- tation of ingested food. These results suggested that the observed differences in the composition of plasma, myogens and hemolyzate proteins were due to nutritional differences rather than hypoxia per ge. Therefore it was necessary to determine if conditions of low oxygen would significantly alter the values of the plasma, myogen, and hemolyzate pro— teins, as well as the levels of the hematocrits, hemoqlobin, and total plasma protein. Experimental Conditions Specimens for this study were collected on June 27, 1963, and were given the prophylactic treatments described earlier. On July 2, 1962, the fish were randomly assigned to the control and the experimental sections of artificial stream "A." Acclimation to the stream continued for one week, and low oxygen conditions were instituted on July 9th 61 62 and were concluded at the end of July 17th. Blood was drawn from the experimental specimens on July 18th and this was done to the control specimens on July 19th. None of the fish were fed at any time after they were captured in the river, hence they fasted approximately 21 days before their blood was drawn and tested. The experimental conditions consisted of reducing the oxygen concentration in the experimental section from a mean of 7.98 ppm to a mean of 3.13 ppm for eight hours each day for nine consecutive days. This was approximately equivalent to reducing the oxygen pressure from 132 mm Hg to 50 mm Hg, or reducing the oxygen saturation from 98% to 34%. Other water quality data were collected during the degassing phase and are summarized in Table 7. The water quality was similar to that of the previous experiment although the mean tempera— ture was slightly lower (20.16OC compared to 22.800C) and the pH was somewhat higher (8.57 as compared to 8.00). The average size of the specimens used here was similar to the average size used in the previous experiment. The average weight of control fish was 63.8 grams and their average standard length was 11.4 centimeters, while the average weight of the experimental fish was 71.1 grams and their average standard length was 11.8 centimeters. The mean weights and length were not significantly different be- tween the control and the experimental lot, but the variances were significantly different. However, this does not appear 63 O.N VOm . . I . s.m om em m m H am 0 o [mom Iv.m Imv uHm Im.m um.wa mmcmm m.a mam sm.m m.om em ma.m ea.o~ cams .eoauomm HmnameHnmmxm o.m mom s.m mms em m.m H.Hm no.0 loom ue.m Inca umn um.o nm.ma mmcmm m.a mam sm.m 8.Hma mm mm.s ma.o~ cams Ema moomo mm weaxoac and mm as. .umm x and 00 -connmo suacaamxaa mm cmmsxo ew>a08man .QEwe ”coauoom Honugou .mcoauaccoo cmmmxo 30H ou Awauumomsu mouaamoHnE.U X gm.a mv.N om.H hH.H v©.N hh.a m em.h amm.ma amm.©a ©¢.ma rem.mm 80H.¢H M An N CV Uflxommm mcaummm v.mm m.ma ¢.mH m.ma m.eH c.0e .>.U x ©N.H mm.o mv.a em.o om.a No.0 m H>.w mm.om o¢.vm mm.ma hm.mm Ho.o M “camponm mo wEmum OOH \ wEmumv Ah u cv Honucou mcaummm H> > >H HHH HH H mcofluomum mannam .Amauumomsu mouHHQoHQE.O x mv.¢m Om.mm OH.NN O.mm mO.nOH H.mm m m.mma am.Oem aam.mae e.¢mm I.O.Om:. ¥N.mem N Am u zv oaxomhm OCHUmmm O.hm 0.0N H.Hm O.mm m.OH m.Om .>.U x mm.mm O.Vm O>.mv m.mm Ha.ev O.ma m H.¢Om O.mwv H.hnm O.Hom O.mmm m.nma M AmEmmHm mo HE OOH \ OEO Ah u zv Honucoo Ocapmmm H> > >H HHH HH H mCOHnomnm mEmmHm .Awflnumomsn mouflaomwna.0 X me.o «8.0 mm.o mm.o mn.o ma.a m .mo.ne Hm.4e Hm.am oo.am mm.mm ms.m m Am u Zv mmmc o>am How N Hz 8mm m on cowomxm m.om 8.8H H.Ha 8.0m m.me m.em .>.0 K mm.o mo.a mm.o mm.e ma.a 8H.H m 4m.s mo.oe mm.mm 44.4m sm.mm sm.m m Acaeuonm mo mEmnO OOH \ memnmv Am u zv maonpcou ewe H> > >H HHH HH H mcoauumnm .Amenumemmw mouflammanadv mmmn Moon pom mo mcoanumnm MEmMHQ emu Ce caouonm mo coausnwnumflp 05p :0 meoflc pcmHm>H© mo oOGeSHMCH wnu mo hamafism .va oanB 80 .uconmmwflp handmOHMHcmflma 0.0m m.mm 4.0m 0.0m 4.0m 0.mm .>.0 X 00.mm 80.mm s4.mm mm.em 40.04 sm.me m .4.mem .m.0sm 40.444 .m.m04 .H.mm4 m.mme m Aw u 2v mwmc o>aw How N+flz 5mm m 0» Ummomxm m.me 0.0a 8.0a m.em 0.44 0.4m .>.0 X 40.44 m0.mm 08.0w mm.0m 44.0 0e.0m m m.nee 0.0mm 0.04m m.0mm e.m00 0.0Hm m “manned no 48 00a \ 08v Am u zv meouucoo ewe H> > >H HHH HH H mcofluomnm .Amwupmwmdn mopeaQOaneav mmmp xuon com mo wCOHuomnm mammam on» as camuonm mo mCOAmeu Icwocoo OcflumHsonao emu co meoac uceam>ac mo monogamca ego mo mumEEDm .ma magma 81 The average amounts of fractions II through V were signifi- cantly decreased in nickel exposed fish. Fraction VI was significantly increased but fraction I was decreased in nickel exposed fish. When the changes from the control value are expressed as percentages, fraction I decreased 14%, fraction II decreased 23%, fraction III decreased 22%, fraction IV decreased 24%, fraction V decreased 28% and fraction VI increased 20%. These changes indicate that nickel toxicity induced a generalized reduction of the concentrations of fractions I-V, and produced specific changes in fractions I and VI. The coefficients of variation are listed in Table 15 for the concentrations of each plasma fraction. Except for fraction I, exposure to nickel toxicity was associated with a general increase in the variability within the concen— trations of each fraction. Distribution of extracted . protein in the myogen fractions The average amounts of protein in the myogen fractions per 100 grams of extracted protein are summarized in Table 16. Fraction III was significantly increased in the nickel exposed fish, but otherwise their fractions were not altered significantly. When the changes from the control values are expressed as percentages, fraction I decreased 8%. fraction II increased 2%, fraction III increased 8%, and fraction IV decreased 14%. Thus the general pattern of 82 Table 16. Summary of the influence of divalent nickel on the distribution of extracted protein in the myogen fractions of fed rock bass (Ambloplities rupestris). Fractions I II III IV Fed Controls (N = 7) (grams / 100 ml of extracted protein) 2 22.08 44.83 28.03 6.62 SX 3.02 1.94 0.89 0.56 C.V. 36.1 10.6 9.0 22.5 Exposed to 3 ppm Ni 2 for five days (N = 8) R 20.18 45.87 28.22* 5.72 sX 1.70 1.02 1.33 0.60 c.v. 23.9 8.03 10.8 30.0 *Significantly greater. 83 change was an increase in the mid—mobility fractions and de- creases in both the low and the highest mobility fractions. The coefficients of variation for the distribution of protein in the myogen fractions are listed in Table 16. Ex- posure to nickel toxicity was associated with generally lower coefficients of variation in fractions I and II, but the coefficients were higher in fractions III and VI. Distribution of extracted protein in the hemolyzate fractions Other differences in cellular protein composition are reflected by the average distribution of protein in the hemolyzate fractions (Table 17). Fish that were exposed to nickel toxicity had significantly higher amounts of fraction I and significantly lower amounts of fraction II per 100 grams of protein. When expressed in terms of percentages, fraction I increased approximately 22% and fraction II de- creased approximately 9% after exposure to nickel toxicity. The coefficients of variation for the distribution of protein in the myogen fractions are listed in Table 17. Ex- posure to nickel toxicity increased the variability both in fraction I and in fraction II. Liver succinic dehydrogenase activity Nickel toxicity also influenced the average succinic dehydrogenase activity of rock bass liver. Control specimens 84 Table 17. Summary of the influence of divalent nickel on the distribution of extracted protein in the hemolyzate fractions of fed rock bass (Ambloplites rupestris). Fractions I II Fed Controls (N = 8) (grams / 100 grams of ex— tracted protein) 2 22.15 71.55 S 1.52 1.52 x C.V. 15.2 6.0 Exposed five days to 3 ppm Ni 2 (N = 8) 2 34.34* 85.54* sX 2.70 2.70 c.v. 22.2 11.7 *Significantly different. 85 averaged 68.0 units of activity per gram of soluble liver protein, but nickel exposed fish had 135.1 units of activity per gram of soluble liver protein. This represents nearly a 100% increase in enzyme activity over that of the control lot. Coefficients of variation for succinic dehydrogenase activity were rather high. The coefficient for the control lot was 30, but this was 47 in the nickel exposed lot. This increase indicates that nickel toxicity affected the mechan- ism(s) which control succinic dehydrogenase activity in the liver. Other observations After five days of exposure to 3 ppm of divalent nickel, the rock bass had hyperemic vessels in their fins, indicating localized irritation and inflammation. No mortality occurred during this exposure period, and hyperemia was the only overt indication of stressful conditions. (Ventilation and swimming activity were not observed.) COMBINED EFFECTS OF DIURNAL LOW OXYGEN, FASTING, HIGH CARBON DIOXIDE, AND DIVALENT NICKEL ON ROCK BASS Introduction and Objectives The previous experiments were concerned with the in— fluence of one or two stressful variables, yet a much more complex situation would be expected in polluted waters. For example, the Red Cedar River had the following character- istics at approximately 10 am on June 11, 1963: 19 C, 2.3 ppm oxygen, pH 6.95, 121 ppm alkalinity and approximately 30 ppm carbon dioxide (Van Buren Road). This water was not tested for other toxic components, but pesticides, metallic ions, and other adverse factors were believed to be present. Certainly the river's condition was more complex than any of the previous experimental conditions. An experiment was designed to test the combined in- fluences of several potentially stressful factors. The ex- periment was designed to measure physiological responses im- mediately after a three day exposure period and then again after an additional two days had been allowed for recovery. The objectives of this experimental design were to determine the extent of certain physiological responses to multiple 86 87 stresses, to determine if cellular leakage of enzymes oc- curred during the period of stress, and to determine if these physiological parameters would return to their pre- stress levels after a two day recovery period. Physiological tests included the determination of the composition of plasma and myogen protein, the determin— ation of the levels of hematocrits, hemoglobin, and total plasma protein concentrations, and the determination of the activity of plasma transaminase, lactic dehydrogenase and liver succinic dehydrogenase. Additional studies included the determination of the electrophoretic mobility of plasma lactic dehydrogenase in the plasma of control and stressed fish. Experimental Conditions The specimens for this study were collected on August 30, 1963, and were given the previously described prophylactic treatments. These fish were randomly assigned to the control and the experimental sections of artificial stream "B" on September 1. The experimental conditions were begun on September 5 and consisted of adding sufficient nickelous chloride and hydrochloric acid to bring the ex— perimental water to approximately 3 ppm divalent nickel at an average pH of 6.8. Low oxygen conditions consisted of re— ducing the oxygen concentration in the experimental section from approximately 9 ppm to an average of 6.6 ppm for eight 88 hours each day for three days. No food was offered to the fish throughout the duration of the experiment, hence they fasted approximately ten days. After 72 hours of exposure to the experimental stress conditions, blood and tissue samples were collected from seven fish in the experimental lot. The control fish were sampled on the following day, and the remaining experimental fish were sampled approximately 48 hours after the stressful conditions had ended (hereafter called the recovery lot). The weights of the control, experimental, and re- covery lots averaged 100.0, 74.1, and 70.0 grams respective- ly, and these are significantly heterogeneous by one—way analysis of variance. Conversely, the standard lengths of these groups were not significantly heterogeneous and they averaged 13.43, 12.34, and 12.58 centimeters respectively. As discussed before, the differences in weight and length do not seem to be biologically important here. Results Hematocrit, hemoglobin and plasma protein concentration The data for the hematocrits, hemOglobin, and total plasma protein concentrations are summarized in Table 18. Both the average hematocrit and the average hemoglobin concentrations were significantly heterogeneous between the control, the experimental and the recovery lots. Changes in 89 the total plasma protein concentrations were not statistical— ly significant. After three days of exposure to the experimental con- ditions, the average hematocrit value had increased 31% over the control values. Hemoglobin concentrations had increased only 2% and the average total plasma protein concentration had fallen to 10% below their control values. Two days after the experimental stress period had ended, the average hematocrit value was only 17% above the control values. However, the hemoglobin concentration had risen to approximately 37% above their control values. The average plasma protein concentration had returned to "normal" after two days of recovery. The coefficients of variation for the hematocrits, hemoglobin and total plasma protein concentrations are listed in Table 18. Both the hematocrits and hemoglobin values were less variable immediately after the stress period, but variability increased above the control values following two days of recovery. On the other hand, the plasma protein concentrations were more variable immediately after the stress period and decreased somewhat after the two day recovery period. Distribution of protein in the plasma fractions The average distribution of protein in the fractions per 100 grams of plasma protein are summarized in Table 19. 90 Table 18. Summary of the influence of the combined effects of low oxygen, fasting, high carbon dioxide, and divalent nickel on hematocrits, hemoglobin concen— trations, and total plasma protein concentrations of rock bass (Ambloplites rupestris). Hematocrit (ml of cells / Hemoglobin Plasma Protein Fasting 100 m1 of blood) (G/lOOml) (G/lOO ml) Control _ * r X 30.04 5.28 2.56 SX 2.95 1.54 0.05 C.V. 24.0 22.6 4.5 N 6 6 6 Exposed 72 hours to multiple stresses - * * X 39.3 5.36 2.31 SX 3.19 0.02 0.12 C.V. 21.0 8.0 13.6 N 7 6 7 Exposed 72 hours to multiple stresses and allowed to recover for 48 hours a 35.2* 7.25* 2.55 sX 5.37 0.84 0.12 c.v. 30.5 23.2 9.7 N 4 4 4 *Significantly heterogeneous by one—way analysis of variance. 91 .ooc04nm> mo m4m>4mcm mmzloco mp msoocwmonouoc haucmoamacmflm . . .<. . .0 m.mm 0.04 4.mm .>.U 0.0m O O4 0 . . x N4.4 Om.4 mm.4 N4.N m4 v no N m . . . mm.mm mm.w N aha.m aOO ma «.00 ON Nw MH .4. a I i 44 u 5 meson m4 nm>ooon 04 0030440 One .0050: me now mommmnpm 04440458 on cemomxm 110.40 0.40 0.04 0.40 0.0 0.04 .>.o x 4m.O O4.4 mm.4 40.4 O0.0 N0.0 m 400.0 44m.¢4 aO0.0m mO.m4 4mm.mm 844.m M An N zv "mnsos we now mommmnuw 04444458 on Ummomxm 0.04 0.m4 m.O m.¢ 0.0 m.v .>.U 04.0 40.4 00.0 44.0 00.0 08.0 xm *ON.O aom.oa amm.mm Om.m4 smv.ON 4m4.h M Acflmuonm mo madam OOH \ mEmnmv 40 u zv Houucou _ mcHummm H> > >H HHH HH H mcoapomnm .Awflnummmdn moufifimoanfi4© 0cm .wUHXOHU conumo amen .Ocflunmm .cmm>xo 304 m0 muomwmw cmCHQEoo esp mo OUCQDHMCH may mo humEEdm .OH magma 92 Except for fraction III, the weight of protein in the plasma fractions were significantly heterogeneous among the control, stressed, and recovery lots. The average weight of fraction I increased 12% after the stress period, but declined during the recovery period and was only 10% above the control value. Fraction II decreased 3% during the stress period. However its average amount after two days of recovery was 34% above the control mean. Fraction III increased during the stress period to 11% above the control mean and then declined during the re- covery period to only 2% above the control mean. The average weight of fraction IV was 10% below the control mean following the stress period and after two days of recovery its average weight was only 7% below the control value. Fraction V was decreased to 12% below the control mean, and this continued to decline during the recovery period to 20% below the control mean. Fraction VI was increased 56% immediately after the stress period and then declined to 18% below the average amount in the control lot. The coefficients of variatiOn for the distribution of protein in plasma fractions per 100 grams of protein are listed in Table 19. The control values had very low coef— ficients of variation in fractions I, II, and III. After 93 the stress period, variability rose and was still higher following the recovery period. For fractions IV, and V, variability rose during the stress period, but fell during the recovery period. Variability in fraction VI rose during the stress period and continued to rise during the recovery period. Variability may have been increased by the small sample size in the recovery lot, but their coefficients of variation probably indicate the trend of variability during recovery. Concentrations of protein in the plasma fractions The average concentrations of protein in the plasma fractions are summarized in Table 20. Except for fraction III, the concentrations of protein in the plasma fractions are significantly heterogeneous among the control, stressed, and recovery lots. The average concentration of fraction I increased 4% during the stress period. After the recovery period, its average amount rose to 11% greater than the control value. Fraction II declined to 12% below the control value during the stress period and then rose to 33% above the control value following the two day recovery period. Fraction III's values were similar but slightly higher than its control value. After the stress period, the average amount of fraction III was 3% higher than the control 94 .oocm4pm> mo m4mx4mcm >0310co >4 0500:00040405 044cmo4macm4m . . . 4 0.00 0.04 0.04 0.00 0.04 0.04 .>.0 40.00 40.00 00 00 04.04 00.00 00.00 x0 40 404 40.000 40.040 40.040 40.400 40.000 m Aeuzv 0450: 04 now 0040>0004 050 0450: m0 400 00000000 04m4u45E Op Ommomxm 0.04 0.04 0.00 0.40 0.44 0.00 .>.0 00.04 00.04 40.40 44.44 04.00 04.04 xm 40.000 44.400 40.440 40.400 40.000 40.004 W 40 n zv 0450: N0 40% 00000440 04444456 00 Ummomxm O.m4 0.04 0.0 0.44 0.0 O.NN .>.U 44.04 40.40 40.00 40.04 00.04 40.04 40 40.004 40.004 40.000 4N.Ovm 4N.m00 4N.0m4 N 4050044 00 4E OO4 \ OEV 40 u 20 4onucoo OG4wmmm H> > >4 444 44 H chHuomnm (4‘ .4044000Q54 000444044540 mmmn Moon 00 050044 034 C4 C4muonm mo 0:044044cw0coo 02400450440 030 no 40404: uco4m>40 0cm .004x040 connmu nm4£ .OC4pwmm .cmmwxo 304 40 0000000 00244800 0:» mo 00:054MC4 map 00 mHMEE5m .ON @4409 95 value and following the recovery period, its average amount was only 2% above the control value. Following the stress period, fraction IV declined to 12%.below the average amount in the control lot. After the recovery period, this rose slightly and was only 5% below the average amount in the control lot. Fraction V was approximately 20% below the average amount in the control lot, both after the stress period and after the recovery period. The concentration of fraction VI did not decline im— mediately after exposure to stressful conditions and this constitutes an exception to the stress pattern. The average amount of this fraction was 14% higher following the stress period than in the control lot. After two days of recovery, the average amount of this fraction then fell to 20% below the average amount in the control lot. At this time, its values conformed to the stress pattern. The coefficients of variation for the concentrations of protein in the plasma fractions are listed in Table 20. Following the stress period, the coefficients of variation were increased in all of the fractions, and in general, this was even higher after two days of recovery. bistribution of extracted protein in myogen fractions The average distribution of protein in the myogen fractions per 100 grams of extracted protein are summarized 96 in Table 21. The values for each fraction were signifi- cantly heterogeneous between the control, stressed, and the recovery lot. Stress generally reduced the amounts of the lowest and highest mobility fractions and increased the amounts of the mid—mobility proteins. The average amount of fraction I was decreased to 19% below the control value. Following two days of recovery, the amount of fraction I had increased slightly and was only 13% below the control value. Fraction II was increased 14% following the stress period. After the recovery period, fraction II had de— creased slightly and was only 9% higher than the average amount of this fraction in the control lot. Fraction III was 8% higher after the stress period than it was in the control lot. Following two days of re- covery, it decreased to 4% below the control value. Fraction IV was 20% lower in the stressed lot than in the control lot. After two days of recovery, fraction IV had declined to a point only slightly above the control value. Coefficients of variation for the distribution of protein per 100 grams of extracted protein are listed in Table 21. Variability decreased following the period of stress, except in fraction III where it increased. Follow— ing two days of recovery, the coefficients of variation for each of the fractions were higher than was the case for the 97 Table 21. Summary of the influence of the combined effects of low oxygen, fasting, high carbon dioxide, and divalent nickel on the distribution of extracted protein in myogen fractions of rock bass (Ambloplites rupestris). Fractions I II III IV Fasting control (N = 6) (grams / 100 grams of extracted protein) 2 26.98* 40.11* 20.81* 12.08* SX 1.94 2.52 1.66 1.68 C.V. 14.4 14.1 15.9 31.1 Exposed to multiple stresses for 72 hours (N = 7) 2 21.91* 46.02* 22.38* 9.68* SX 1.10 1.84 2.23 0.78 c.v. 12.3 10.6 24.4 21.2 Exposed to multiple stresses for 72 hours and recovered for 48 hours (N = 4) 2 23.37* 44.05* 19.96* 12 59* 3X 3.34 6.54 0.95 3.75 c.v. 21.8 22.7 23.0 45.4 *Significantly heterogeneous by one—way analysis of variance. 98 values in the control or for the stressed lot. The high variability among the fractions of the recovery lot may be related to the small number of specimens in it. Liver succinic dehydrogenase activity The succinic dehydrogenase (SDH) activity of liver homogenates were significantly heteroqeneous among the control, stressed, and recovery lots and these values are summarized in Table 22. After three days of exposure to the experimental conditions, the average SDH activity per gram of soluble liver protein had increased 42% over the control mean. Following two days of recovery, SDH activity was 55% higher than in the control lot. Coefficients of variation for SDH activity are also listed in Table 22. Variability decreased following the stress period and decreased even further after the recovery period. Plasma lactic dehydrogenase activity The plasma lactic dehydrogenase (LDH) activity in the control and the experimental lot were significantly different and are summarized in Table 22. Plasma LDH activity was in- creased 43% after three days of exposure to the stressful conditions. 99 .0050440 . If . > 00 04044050 4031050 44 050050004000: 44050040450004 b V . Z 0 00 0.00 0.04 .>.0 40 4 00.0 00.04 40 400.0 00.0 111 .11 4 0 404.004 4 04500 04 400 0040>0004 050 0450: N0 404 00000400 04440455 00 00004xm 0 0 0 z ®.OH PM.N¢H O.MH 0.0N .>.U 04 4 04.4 00.404 00.0 40 440.04 400.0 40.000.4 400.004 4 0450: 00 400 00000400 04440455 00 00004xm 0 0 4 4 z 0.00 0.00 0.0 0.00 .>.0 00.0 04.0 0.40 0.04 40 400.4 400.04 40.004.4 400.00 m 00400 404>4000 0045: 404>4000 4o40500 000545005048 000545005048 00455 404>4000 00455 404>4000 04>5444 04000040xo 0005000404500 0005000404000 104500540 104500540 040004 04540050 050044 00040 40>44 .4044000454 MW044444nafl9 0000 #004 54 00404>4000 054050 50 404045 05040>40 050 .004x040 500400 504: 40540000 450m4x0 304 00 0000000 00544500 000 00 005054454 0:0 00 4405550 .NN 0400B 100 The coefficients of variation for LDH activity are listed in Table 22. Stressful conditions increase the vari- ability of the LDH values. Plasma transaminase activity The average plasma activity of glutamic—oxalacetic (GOT) and of glutamic-pyruvic (GPT) transaminase are sum- marized in Table 22. The activities of both of these en- zymes were significantly heterogeneous between the control, stressed, and recovery lots. following the three day stress period, GOT activity was 81% below the control values, but following the two day recovery period, GOT activity re— bounded to 98% above the control values. The average activity of GPT in the plasma of control fish was generally low, but in the plasma of stressed fish it was over 1,000% greater than in the control lot. Following the two day re- covery period, plasma GPT had fallen to only 270% above the average value in the control lot. The coefficients of variation for GPT and GOT are listed in Table 22. Stressful conditions increased the variability of the GOT activity levels, but decreased the variability of the GPT levels. Paper electrophoretic mobility of plasma LDH The electrophoretic mobility of isozymes of lactic dehydrogenase in the plasma of stressed fish was typical of 101 the LDH isozymes in muscle and liver. The mobility of plasma LDH was very low and distinct from the high mobility LDH isozymes found in cardiac and nerve tissue. Other observations Initially the experimental design called for oxygen levels approaching 3 ppm, but a higher level was necessary. Whenever the oxygen concentration fell below 4 ppm, the fish in water of low pH, 3 ppm nickel, and high carbon dioxide level became very distressed and lost their equilibrium. Of these factors, the high carbon dioxide pressure (brought about by the reduction in pH) was probably the cause of the acute loss of equilibrium. During the periods of low oxygen, the experimental lot ventilated rapidly, lost their body color, and displayed very little swimming activity. DISCUSSION The basic hypothesis of this thesis has been that stressful conditions elicit physiological responses that are reflected by differences in the composition of plasma, myogen, and hemolyzate proteins, as well as by other physio— logical parameters. That these parameters change in re— sponse to stress has been proven repeatedly in the previous experiments. However, further information can be extracted from the data and this section will be devoted to describing normal1 values for the parameters and their responses to stress. Other t0pics will include the evidence for homeo— statis in these parameters, the possible causes of the ob— served responses, and the prognosis of electrOphoretic studies in connection with stream ecology. Normal Values and Responses to Stress Hematocrits Normal hematocrit values for rock bass appear to be very close to 26 ml / 100 ml of whole blood (Table 23). Ac- climation temperatures between 18°C and 230C did not change lNormal implies the usual value that one would ex— pect to find in experimentally untreated fish when they are maintained under laboratory conditions. 102 103 .0000 050 40 05005 040 00540> 44m mm.N mN.h N.mm WHSOE mV HON H0>OU®H 00 0030440 050 00000400 055 0500 40.0 00.0 0.00 04005 00 4O0 0440045 4054540 050 404045 050404045 544 m 00 00004x0 055 40500 00.0 00.0 0.00 .0004 00 0405 .0540000 00.4 NN.0 0.mm 0400 m 404 404045 050404045 544 m 00 00004x0 055 0500 m0.m 00.m 0.00 .0004 00 0405 400.4 mm.m m0.m 0.0m 04x0445 4054540 055 .0500 00.0 44.0 0.00 .0000 00 0405 .0400 40 0540000 N0.N 00.0 0.4m 04x0445 4054540 055 40500 om.m mm.0 m.0m .00mm 00 0405 «004 445 QO4\0V 445 OO4\0V 400045 40 45 004 0504040500 405552 5400044 5450400504 \ 04400 40 450 4005054404xm 05054404xm 050044 0440000504 .4044000454 000444445545 0005 4004 54 05040040500500 5400044 050044 050 405040040500500 5450400505 400440000505 54 0005050 00>40050 050 40 4405550 .mm 0450B 104 this value, but short exposure to stressful conditions in— creased the hematocrits to nearly 40 ml / 100 ml. Whether or not the hematocrits could reach a higher mean value was not ascertained, but it is doubtful that they would go much higher. The increase in the hematocrits seems to be pro- portional to the stress load. For example, hypoxia increased the mean hematocrit, but an even greater increase occurred when the fish were subjected to fasting with hypoxia. The biological significance of increased hematocrits is somewhat dependent on whether or not the increase was due to polycytemia or osmotic swelling. The hemoglobin concen— trations (Table 23) rose whenever the hematocrits increased, indicating an increase in erythrocyte concentrations. Furthermore, the oval, inflated erythrocytes of fish are not as capable of swelling as are the partially inflated, bi— concave erythrocytes of mammals. Imbibition of water by fish erythrocytes rapidly leads to hemolysis, and this would rapidly decrease the hematocrit value. Thus, the increased hematocrits probably indicate a true polycytemia. During polycytemia, the concentration of blood cells is increased, thereby increasing the friction between suc— cessive layers of blood. Increased friction in turn in— creases the viscosity of the blood, and it increases the re— sistance of blood to flow. But the relationship between hematocrits and blood viscosity is not linear and high 105 hematocrits increase the blood viscosity tremendously (Guyton, 1961). Since highly viscous blood is more diffi- cult to move, high hematocrits probably increase the work load of the heart. The effects of high hematocrits on blood flow in the vessels are indicated by Poiseuille's law (as modified by Guyton, 1961): blood ) £:vesself _ ressur diameteQ . (1) blood flow — blood vessel (C, viscosity) length Assuming that the length of the blood vessel would remain a constant). constant, and that the blood flow would remain unchanged, then an increase in blood viscosity (hematocrit) would re— quire an increase in blood pressure, vessel diameter or both. This formula also suggests that the interaction of these factors could limit the maximum hematocrit value by reaching an equilibrium between the several terms of the equation. Hemoglobin concentration The normal concentration of hemoglobin in rock bass blood appears to be related to the acclimation temperature (Table 23). According to Prosser and Brown (1961) hemo— globin binds less oxygen at higher temperatures, albeit that oxygen consumption would be elevated. This may be related 106 to the higher hemoglobin concentrations in fed fish held at higher temperatures. The hemoglobin concentration also appears to be re~ lated to the nutritional state of rock bass. High hemo- globin concentrations were found in fish that had fasted for short periods (10 days), but low hemoglobin concentrations were found in fish that had fasted for longer periods (21 days). Rock bass that were either fed or had fasted for short periods significantly increased their hemoglobin concentration as well as altered their hemolyzate compo— sition in response to stress. However, rock bass that had fasted 21 days significantly increased their hemoglobin concentration in reSponse to stress, but the composition of their hemolyzate was not changed significantly, presumably because of unsatisfied nutritional requirements. Perhaps the increased hemoglobin concentrations of blood from stressed fish only indicates that higher blood pressures had removed the sequestered cells from the various organs such as liver, spleen, etc. However, the change in hemolyzate composition indicates hemOpoiesis (Marks,_e£_§l., 1959). Except for the fish that had fasted 21 days, varia- bility was decreased in response to stressful conditions. Variability in the former group did not change sufficiently to indicate a trend. 107 Total plasma protein concentrations Normal total plasma protein concentrations were also dependent on acclimation temperatures, nutrition, and stress (Table 23). The plasma protein concentration was slightly lower in fed fish at lower acclimation temperatures. Long term fasting (21 days) reduced the total plasma protein concentration, but short term fasting (10 days) did not seem to affect this. Stresses generally decreased the total plasma protein concentration, but this was not the case among fed-hypoxic fish. The fourth experiment gave evidence that recovery from hypOproteinemia can be achieved in less than two days. The onset of hypOproteinemia appears to be very rapid during stressful conditions. During the early periods of hypOproteinemia, the plasma protein composition is changed little, indicating a general dilution of the blood by isosmotic fluids. This reduction in total plasma protein concentration probably reduces the colloid osmotic pressure of the plasma, and would likely alter the dynamics of fluid exchange at the capillaries (Guyton, 1961). Stressful conditions also influenced the variability of the plasma protein concentrations. In general, the coef- ficients of variation were nearly twice as high in stressed fish as in the control fish. 108 Distribution of protein in the plasma fractions Comparison of native and laboratory fish. Laboratory conditions do not present the experimental organisms with all the benefits and all the disadvantages found in a natural environment. As a result of living in the laboratory, the specimens may have had abnormal levels of protein in their plasma fractions, thereby decreasing the usefulness of the experimental data. Therefore the degree of similarity between the plasma protein composition of wild and laboratory fish would indicate the usefulness of the data derived from fish held in the laboratory. Table 24 presents a comparison of the average amounts of protein in the plasma fractions of wild1 and laboratory maintained rock bass. Both the wild and the lab- oratory fish had similar amounts of protein in their plasma fractions. Fraction I was slightly lower and fraction VI was slightly higher in native fish than in the laboratory fish. Otherwise the means of each fraction are extremely similar. The similarity between the laboratory fish and the native fish is remarkable and perhaps coincidental. However, it nevertheless indicates that laboratory life had little ef— fect on the composition of the plasma proteins in these fish. lCollected June 27, 1962, by electroshocking. Blood was drawn approximately 1 hour after capture. 109 ill/ii . . 0.4m 0.m4 0.0m .0 .0450 N m 0 M4 00 404 40>0004 00 0030M :40 050 00000400 055 .0500 . . 4 0.0N 0.04 ©.mm .0 .04505 0 0 m 0 44 400 404045 000404045 544 m 00 00004x0 055 .0500 0.0 0.04 0.04 0.04 0.00 .4 .0004 00 0405 .0540000 0 1144 0.04 0.40 0.4N 0.44 .04 .0400 .illlilli 0 m 404 404045 050404045 5440 00 0000440 055 .0500 0.4 0.04 0.00 0.40 0.00 .0 .0004 00 0405 .004 m mlw 0.04 0.04 0.04 0.00 .04 .0440445 4054540 055 .0500 . . 0.0m m.m4 0.0N .0 . .UOON n 0 m om 00 0405 .0400 40 0540004 0 0.0 0.04 4.40 0.04 0.44 .0 .04x0545 4054040 0.55 .0500 .11 0.0 0.04 0.00 0.00 0.00 .0 .0000 00 0405 .004 4 . . .00 0.00 4.00 .0 0450400 40040 0 04 N 04 0 45 4 0044500 .0040050 10400040 .5044 0>400z 45400044 40 05040 004 \ 05040 00040>0v 4> > >4 444 44 4 0504040500 405552 400505440444 05054404xm 050400044 050044 .4044000454 00044404554005o 050 40 4405550 .0m 0450B 110 A possible explanation for the lower amounts of fraction I in the native fish has been offered by Bouck and Ball (1966). Fibrinogen is located in fraction I, both in the case of rainbow trout and rock bass, and electroshocking may remove or reduce its amount in the blood plasma. Possibly this difficulty could be overcome by using serum for all such analyses, rather than plasma (which has fibringen). Another possible solution may be to analyze muscle myogens instead of the serum or plasma proteins. In either case, it now appears possible to conduct a survey of sublethal stress in a river by collecting fish at various points and then analyzing their protein patterns for stress reactions. Normal values and responses to stress. Fraction I constituted approximately 9% of the total plasma protein in fed fish and this appears to be the normal amount in rock bass. Both short and long term fasting decreased this amount as did hypoxia. However nickel toxicity, and multiple stresses increased the amount of fraction I, if not in comparison to the normal value, then in comparison to the amount in the respective control lot. Thus the amount of fraction I was usually changed by stressful conditions, but the direction of the change was difficult to predict. Stress also affected the coefficients of variation, usually increasing the variability in the amounts of fraction I. lll NOrmal amounts of fraction II were usually about 23% of the total plasma protein (Table 24). Hypoxia, lower ambient, temperatures, and nickel toxicity had relatively little effect on the amounts of fraction II. In these four experiments fraction II was changed only when the stress was either fasting, or was associated with fasting, i.e., fast- ing with hypoxia, or fasting with nickel toxicity, low oxygen, and high carbon dioxide levels. Apparently fraction II's level is strongly related to nutritional requirements and when these are not satisfied, stressful conditions then cause additional changes. Fasting was also associated with higher coefficients of variation for the amounts of fraction II. When fasting was not included in the experimental condition, stresses then decreased the coefficients of variations. Fraction III was changed more easily by stresses than were the two previous fractions (Table 24). The normal amount of fraction III was approximately 21% of the total plasma protein, and this amount was usually increased by stress. But when long term fasting was involved, the amount of this fraction fell. As with fraction II, the level of fraction III was related to nutritional requirements, but only after long term fasting. Stressful conditions consistently increased the coef— ficients of variations for the levels of fraction III, ex- cept when long term fasting was involved. Under the latter 112 conditions, stresses decreased the variability in fraction III. Fraction IV appears to be relatively stable and did not change greatly in response to stress. The normal amount of fraction IV appears to be approximately 24% of the total plasma protein. The level of fraction Iv was consistently decreased by stressful conditions, and the decrease was greater when the level of stress was higher. Among fasting fish, stressful conditions increased the coefficients of variation for the amounts of fraction IV, but stresses decreased variability when the fish were fed. The normal amount of fraction V appears to be ap— proximately 16% of the total plasma protein (Table 24). Fish that were fasted for 21 days had slightly higher amounts of fraction V. Stresses caused a consistent reduction of the average amount of fraction V and this reduction appears to be directly related to the level of stress. Stresses tended to increase the coefficients of vari— ation for the levels of fraction V, but this was not always the case. Variability was very high in fasting hypoxic fish, but moderate in hypoxic fish. It appears that the degree of variability in fraction V is related to the level of stress. The normal amount of fraction VI appears to be very close to 8% of the total plasma protein. Hypoxia generally decreased the average amount of this fraction, but stressful conditions associated with nickel toxicity always increased 113 the amount of fraction V. Perhaps the latter case is re- lated to the high levels of fraction V which were found in native fish. (Nickel salts are present in the river.) Stressful conditions always increased the coef— ficients of variation for fraction VI, and the increased variability was generally higher at higher levels of stress. A review of the experimental data demonstrates that several factors can influence the distribution of protein in the plasma fractions. Nutritional considerations have strong influences on the resulting values. But possibly the most important factor is the stress load which is the product of the level of stress and the duration of exposure to it. Thus the effects of long term exposure to low concentrations of toxicants may well be comparable to the effects resulting from short exposure to high concentrations of toxicants. The interplay of these factors, and whether or not a re— covery period was allowed may account for much of the vari- ability which has been observed in this type of data (re— viewed by Booke, 1964). Concentrations of protein in the plasma fractions -Average values for the concentrations of protein in each plasma fraction are presented in Table 25. Each fraction is thus independent of the concentration of the other fractions and the actual change (if any) is apparent. The resulting values are more variable than when analyzed on 114 004 Omm 044 000 000 040 .04005 00 404 40>00 I04 00 0030440 050 00000400 055 .0500 000 000 000 000 000 000 .0440545 4054540 5043 04505 04 404 404045 050404045 500 m 00 0000040 055 .0500 004 040 004 000 040 004 .0004 00 0405 .0540000 0 04m 00m 000 000 000 004 .0400 m 404 404045 050404045 500 m 00 0000040 055 .0500 004 000 000 000 000 004 .0004 00 0405 .000 m 004 000 040 000 004 000 .0440045 4054040 000 .0500 000 000 000 000 000 004 .0000 00 0405 .0400 40 0540000 0 004 000 000 000 000 004 .0440045 4054040 000 .0500 000 000 000 000 040 000 .0000 00 0405 .000 4 4050040 40 45 004 \ 05 00040>0v 4> > >4 444 44 4 0504040500 405502 400505440040 0505440040 050400040 .4044000054 000440045500 0005 4004 40 050400044 050040 050 54 5400040 40 05040040500500 05400450440 050 54 0005050 00>4005o 050 40 0405550 .mm 04509 115 the basis of the amounts of protein in a fraction per 100 grams of plasma protein. The higher variability of the concentrations of each fraction are probably related to the hypoproteinemic effect that was described earlier. As a re— sult it is difficult to describe normal concentrations for for each of the fractions. The average concentration of fraction I was 240 mg / 100 ml of plasma, in fed fish held at 230C. In most of the experiments, the mean concentration of this fraction was very close to 200 mg / 100 ml of plasma. Whether this fraction increases or decreases in response to stress seems to depend on the severity of the stress. Mild stresses such as fasting, hypoxia and five days of exposure to 3 ppm of nickel all reduced the amount of fraction I. However, a more severe stress such as long term fasting increased the amounts of this fraction. Stress also influenced the coefficients of varia— bility for the concentrations of fraction I, but the trend was not consistent and did not indicate a pattern. The average concentration of fraction II was 610 mg / 100 ml of plasma in fed fish held at 2300. (Table 25). Fasting for 21 days had little influence on the level of fraction II, but fasting at 180C (10 days) resulted in slightly higher amounts of this fraction. Hypoxia and hypoxia with fasting both increased the concentration of fraction II over the level in their respective control lots. 116 Lower ambient temperatures (18 C) and nickel toxicity (3»ppm for 5 days) both decreased the concentration of this fraction. A similar decrease was found after fish had been exposed to multiple stresses in experiment four, but after two days of recovery the level of fraction II had increased to a level considerably above the control value. The coefficients of variation show that the experi- mental stressful conditions increased the variability of the concentration of protein in fraction II. The average concentration of fraction III was 580 mg / 100 ml of plasma in fed fish held at 23 C. Stressful con— ditions always decreased the level of this fraction in re- lation to the control values. Fasting greatly decreased the level of fraction III and this was decreased even further by additional stresses, i.e., fasting with hypoxia. However, hypoxia caused an increase in the concentration of fraction III in fed fish. Thus the response of fraction III to other stresses would be difficult to predict. Stressful conditions always increased the coef— ficients of variation for fraction III. However, the in— creased variability induced by stress was rapidly reduced after a two day recovery period. The average concentration of fraction IV was 660 mg / 100 ml of plasma in fed fish held at 23 C. Stressful con— ditions always reduced the concentration of this fraction be- low the control values, but not necessarily below the above 117 listed value. For example fasting fish held at 18 C had higher concentrations of this fraction than were observed in any other case. After a reduction of fraction IV by stress, two additional days of recovery brought its level nearly to that of the control value. Stressful conditions were associated with lower coef- ficients of variation at higher temperatures and higher coef- ficients of variation at lower temperatures. In the latter case, two days of recovery decreased the variability markedly. Fraction V had an average concentration of 430 mg / 100 ml of plasma in fed fish held at 23 C and this was con— siderably lower in fed fish held at 18 C. Fasting elevated the level of this fraction, but otherwise stressful con— ditions resulted in a reduction of its protein concentration. Two days of recovery did not return the level of fraction V to that of the control level. The coefficients of variation for fraction V were af— fected by stressful conditions, but the direction of change was not consistent enough to indicate a trend. The average amount of fraction VI was 230 mg / 100 ml in fed fish held at 23 C, and lower than this in all other cases. Hypoxia and fasting both reduced the level of this fraction under that of their respective control values, but stresses associated with nickel toxicity increased 118 fraction VI. After two days of recovery, the level of fraction VI approached that of the control lot. The coefficients of variation for fraction VI were always higher in the stressed lot than in the control lot. Two days of recovery did not return the differences among the experimental specimens, to the level of the control lot. Distribution of extracted protein in myogen fractions Although the myogen supernate may not be such a complex mixture as found in various other cells, the myogen extract is still a very complex mixture. Centrifuging re— moved the cell membranes, contractile protein, nuclei, and probably the nucleoprotein, but the supernate contained a small amount of subcellular organelles and the myogens. How— ever, subcellular organelles comprised only a very small portion of the resulting supernate and they were located only in fraction I. Because of the complexity of the myo— gens, any significant change in their composition is indica- tive of extensive biochemical changes within the muscle cells. The average concentration of myogen fraction I was 37.8 grams / 100 grams of extracted protein in fed fish held at 23 C, but at 18 C the average level was only 22.1 grams / 100 grams of extracted protein. This seems to be an ex— tremely large change in response to only a 5 C difference in 119 acclimation temperature. Since the former was fed mainly on fish food pellets, and the latter group was fed mainly on minnows and crayfish, the differences in the levels of fraction I may be related to nutritional considerations. Each of the stressful conditions decreased the levels of fraction one, except hypoxia concurrent with fast- ing. Under the latter conditions, the average amount of fraction I increased instead of decreasing as before. After two days allowed for recovery (after three days of multiple stress) the levels of fraction I appeared to be returning to normal. The coefficients of variation for the levels of fraction I were usually decreased by stressful conditions. .The average amount of myogen fraction II was 35.0 grams / 100 grams of extracted protein in fed fish held at 22°C. This level was increased in fasting fish and in as— sociation with lower acclimation temperatures. The level of fraction II was always increased by stressful conditions in each of the experiments. After two days of recovery, the level of fraction II appeared to be returning to its previous level. Coefficients of variation for the amounts of fraction II were increased in reSponse to hypoxia and hypoxia with fasting, and decreased 'whenever nickel toxicity was in- volved. Differences in acclimation temperatures (and diet) may have been important influences in these changes. 120 The average amount of fraction III in fed fish held at 23 C was 15.1 grams / 100 grams of extracted protein (Table 26). Hypoxia increased this amount, as did long fast— ing, lower acclimation temperatures, and short term fasting. Each of the stressful conditions changed the level of fraction III in relation to the control level, but the direction of change was not uniform. In fed fish, stresses increased the level of this fraction, but stresses decreased the level of fraction III in fasting fish. Fish that were stressed and allowed to recover for two days had levels similar to their control values. The coefficients of variation for the levels of fraction III were always increased by stressful conditions, but after the two day recovery period, variability was lower than in the stressed lot. The average amount of fraction IV in fed fish held at 23 C was 11.9 grams / 100 grams of extracted protein. Fasting had little effect on this level, but lower accli- mation temperatures (and perhaps diet) greatly decreased this fraction. The stressful conditions tested in the pre— vious experiments always decreased the level of fraction IV in relation to the control values. And these same stressful conditions also decreased the variability in the levels of fraction IV. But two days of recovery returned the levels of fraction IV to the level of the control lot. o.¢¢ v.mm .wuson mv How um>oo low on pw3oHHm pom pmmwmuum pun .mEmm v.0 ¢.mm o.©v m.am .MHSOQ Nb How maxomhn HMCHDHU Ucm meoflc msononc Ema m on pmmomxm pun .mem H.NH m.om H.o¢ o.mm .UomH um pawn .mCHummm v m.m N.mm m.mv m.om .msme m>flu Hon meUHc msononc Ema m on ommomxm non nmem 0.0 o.mm m.¢e H.mm .oomH um mama .omm m H.0H m.mm o.Hv N.©N .mflxommc Hmcusao pun .mEmm 0.0H m.mm m.o¢ o.Hm .uoom pm cam: .msmw Hm mcflummm m m.OH H.mH m.mm s.Hm .mnxons: Hmcsuflo pan .memm m.HH H.ma o.mm m.wm .UOmN um paws «pom a Acamuoum omuomuuxw mo mEmum OOH \ memnm mmmnm>mv >H HHH HH H mcoHUHocou HwQEdz Hmucweflummxm ucwefluomxm mcowuomum ammo»: .Amfluummasu mmuHHQOHQEummno wcu mo >RMEESm .om magma 122 Stressful conditions induced changes in the levels of the myogen fractions, but the direction of change was not always consistent. Fractions I and III sometimes increased in response to stress, but other stresses caused these two fractions to decrease. However, the response of fractions II and IV was consistent; fraction IV was always decreased by stress, and fraction II was always increased by stress. The discovery that myogen fractions are influenced by stressful conditions is important for several reasons. Blood is always difficult to obtain from stressed fish. But muscle samples are easy to obtain and can be obtained in larger quantity than blood. As a result, smaller fish can be used and they can be used in larger quantities (large samples sizes) than is usually done in studies of blood proteins. Furthermore, the responses of fractions II and IV constitute a general response to stressful conditions, and these responses could be useful in detecting threshold levels of stress caused by various pollutants. Distribution of extracted protein in hemolyzate fractions The average amount of protein in hemolyzate fraction I was 39.2 grams / 100 grams of protein in fed fish held at 23 C. Long term fasting decreased this level, as did lower acclimation temperatures (possibly diet). In each of the ex- periments the average amount of fraction I was increased by Table 27. 123 Summary of the observed changes in the distri- bution of extracted protein in the hemolyzate fractions of rock bass (AmblOplites rupestris). Experiment Experimental Conditions Hemolyzate Fractions I II (average grams / 100 grams of extracted proteins) Fed, held at 23cc. Same, but dirunal hypoxia. 39 42 .2 .2 60.8 56.1 Fasting 21 days, held at 200C. Same, but dirunal hypoxia. 35. 36. 62.7 63.9 Fed, held at 18°C. Same, but exposed to 3 ppm nickelous nickel for 5 days. 28. 34. 71.8 65.5 Fasting, held at 180C. Same, but exposed to 3 ppm nickelous nickel and diurnal hypoxia for 72 hours. 44. Same, but stressed and al- lowed to recover for 48 hours. 46. 53.9 124 stressful conditions, albeit slightly increased. Whereas hypoxia increased the levels of fraction I, hypoxia had little effect when the fish had fasted 21 days. Stressful conditions always increased the coef— ficients of variation for the levels of fraction 1. The average amount of protein in hemolyzate fraction II was 60.8 grams / 100 grams of extracted protein in fed fish held at 23 C (Table 27). Long term fasting and lower acclimation temperatures were associated with higher amounts of fraction II. However, the experimental stresses in the previous experiments usually induced a significant reduction in the levels of this fraction, as well as altering its coef— ficients of variation. Although fraction I was consistently increased by stressful conditions, the use of hemolyzates incurs the same problems that are generally found in studies of blood. Therefore, the use of hemolyzate fractions in stress studies is not recommended. Liver succinic dehydrogenase activity Liver succinic dehydrogenase activity was very simi- lar between the two control lots that were tested. The normal activity appears to be very close to 70 units per grams of water soluble liver protein. Both of the experi— mental stresses that were tested increased SDH activity 125 approximately 30% to 40%. After the two day recovery period, the SDH activity was even higher than before, indicating that recovery was not yet completed. Plasma enzyme activity The activities of lactic dehydrogenase (LDH), glutamic-oxalacetic transaminase (GOT), and glutamic-pyruvic transaminase (GPT) were investigated only in one experiment, hence only a general description of their response to stress is warranted. The level of LDH in the plasma of rock bass was high for higher vertebrates, but not high in comparison to other species of fish. Rainbow trout had slightly higher amounts of plasma LDH activity and this was not influenced by capture (by hook and line method) (unpublished data, Bouck). The response of rock bass to stress in experiment four was an increase in the plasma activity of LDH, indicating tissue destruction (Hess, 1963). However, rainbow trout that were poisoned by DDT decreased their plasma LDH activity; one would have expected them to increase their plasma LDH. Plasma transaminase activity in rock bass was also influenced by stressful conditions. GOT was decreased and GPT activity was increased immediately after the stress period, but after two days of recovery, the transaminase levels appeared to be returning to normal levels. 126 Evidence for Homeostasis in the Rock Bass The data obtained in these experiments would be of little value if the various parameters under study varied without regard to a steady state level. If homeostatic mechanisms control these parameters, then coordinated physio— logical processes should return these parameters to their pre-stress levels (Hugh, 1964). To ascertain if this was the case for rock bass, comparisons of the levels of the various parameters were made between control, stressed, and specimens that were stressed and then allowed to recover for two days. The results are depicted in Figures 7-12 (listed previously in Tables 18-23) and are presented in this form only for graphic emphasis. These comparisons strongly indicate that homeostatic mechanisms do control the levels of these parameters and will return them to their pre—stress steady state. However, a definative analysis was not possible because additional specimens weren't available to allow additional observations at other times during the period of stress and recovery. During recovery, the levels of the various parameters probably oscillate both above and below the normal values, and finally reach their normal steady state. Recovery seems to occur at different rates for the various parameters and normal blood composition is regained slower than that in muscle. Perhaps this is because blood 127 Figure 7. Progressive changes in the hematocrits, hemoglobin'concentrations, and plasma protein concentrations before, during stress and during recovery from stress in rock bass (Ambloplites rupestris). ML PACKED CELLS / 100 ML OF WHOLE BLOOD GRAMS / 100 ML GRAMS / 100 ML 40 35 3O 25 128 l Hematocrits l I | l Hemoglobin l | l l I Plasma x\\\\\\\\\\\\\\1/////////fi Protein STRESS % RECOVERY __ _ -’ I r l I I r O 1 2 3 -1 -2 129 Figure 8. Progressive changes in the distribution of protein in the plasma fractions during, be- fore, and during recovery from stress in rock bass (Ambloplites rupestris). GRAMS / 100 GRAMS OF PLASMA PROTEIN 10:] 5 35- 30.. 15 10 3O 25 15‘— 10—4 10 130 Fraction I Fraction II Fraction III Fraction IV Fraction V Fraction VI --' - "a 131 Figure 9. Progressive changes in the circulating concentrations of protein in the plasma fractions before, during stress, and during recovery from stress in rock bass (Ambloplites rupestris). MILLIGRAMS OF PROTEIN / 100 ml OF PLASMA 210.. 200-— 190.. 900 700—, 500 4 350 340 750— 700* 650‘ 6001r 400m 350‘ 300— 250— 200~ 150— 132 l Fraction I Fraction II I Fraction III Fraction IV Fraction V Fraction VI RECOVERY _ ,_ _, 133 Figure 10. Progressive changes in the distribution of protein in the plasma fractions before, during stress, and during recovery from stress in the rock bass (Ambloplites rupestris). GRAMS / 100 GRAMS OF EXTRACTED PROTEIN 30 25 20 50 45 4O 15 10 15 10 134 —- I Fraction I _' I Fraction II Fraction III ./[\ I Fraction IV ‘ STRESS .L RECOVERY __ _’ l l I I l 2 3 2 I I O - 1 - DAYS Figure 11. Progressive changes in the activities of enzymes before, during stress, and during recovery from stress in the rock bass (AmblOplites rupestris). ACTIVITY UNITS / GRAM OF SOLUBLE PROTEIN / HOUR ACTIVITY UNITS / ML OF PLASMA / 30 MINUTES 100 90 80 70 I'—' U1 0 O 1000 25 20 15 10 20 15 10 136 Liver Succinic I Dehydrogenase I Plasma Lactic l Dehydrogenase Plasma Glutamic— Oxalacetic Transaminase Plasma Glutamic— Pyruvic Transaminase STRESS RECOVERY__ _ I *1 —1 —2 I “I 137 receives contributions from all the tissues of the body and the slow recovery of blood may indicate that significant amounts of cellular proteins were still entering the blood. On the other hand, the slow recovery of normal blood compo- sition may reflect impaired functions in the tissues which regulated blood composition. Both stress and recovery syndromes are indicated by the data and the knowledge that they exist may alert other investigators to the changes associated with them. For ex- ample, each testing Of the blood or myogen composition should be followed by further analyses which would indicate whether or not the values are returning to their normal steady state. Additional testing is necessary because stress is not always ended with the cessation of adverse environmental conditions. Rather, stress or its latent effects continue until recovery is complete. This may occur rapidly or it may require a long period of time. These syndromes may account for much of the variability in the serum protein values noted by Booke (1964). The effects of stress on homeostasis are also evi- dent in the coefficients of variation. Most of the para— meters became more variable during the stress period and then became less variable after the recovery period. Hence stresses not only affected the levels of the steady state, but also stresses adversely affected the degree of control over those steady state levels. 138 Causes of Change in Blood and Tissue Protein Several factors can be suggested as possible causes of the responses that were Observed, and these include anti- body production, cellular leakage of protein, steroid hormones, and other factors. Probably none of these factors are solely responsible for the change; rather, these factors probably act concurrently, perhaps with interaction between them. But whatever the causes of the response, the result— ing changes may directly influence both the value of fish flesh as food and limit the ability of sport fish to survive in mildly polluted waters. Antibody production One might suggest that the changes in protein compo- sition are the result of antibody production. However, several lines of evidence suggest that this is not the case. Stressful conditions such as hypoxia activate the hypothalamic-hypophyseal axis in higher vertebrates, causing the release of adrenal corticotrOphic hormone (Van Liere and Stickney, 1963). According to Turner (1960), this would lead to a suppression of antibody formation, possibly in fish as it does in higher vertebrates. Secondly, the experi— ments did not directly involve foreign proteins, thus there would seem to be little stimulus for antibody production. Finally, the production of a significant antibody titer 139 doesn't occur rapidly in fish (PCst, 1963). Hence it seems unlikely that antibody production was involved here. Cellular leakage of proteins A second and more likely cause of these changes is the leakage of proteins from affected cells. Conditions which interfere with cellular metabolism reduce the energy available for cellular maintenance (Hess, 1963). As a re- sult, diffusion of intracellular proteins can occur across the cell membranes. These proteins then pass via the lymphatics, into the general circulation and appear in one or more of the several plasma protein fractions. To ascertain which plasma protein fractions might re— ceive the proteins lost from cells, the mobilities of the myogens and other tissue proteins were compared to that of the plasma protein fractions. The results were depicted previously in Figure 4 which shows that even the fastest mi— grating tissue fractions have very low mobilities (at pH 8.6 in vernol buffer). If cellular proteins should leak into the blood, they would contribute mainly to the lower mo- bility fractions and thus tend to produce the classical "stress pattern" of Dunn and Pearce (1961). It is possible that skeletal muscle is a prime source of the proteins which appear during stress in the plasma of rock bass. This possibility is suggested by the concurrent alteration of the myogen fractions during 140 stressful conditions. .Also, the myogens that are reduced by stress have mobilities Similar to the lower mobility plasma proteins which increased. The implication is that myogen proteins leaked from the muscle into the blood, thereby con- tributing to the changes in the plasma protein. If such leakage did occur from muscle, the plasma activity of lactic dehydrogenase would be increased and the molecular form of LDH would be identical to that in muscle. Data pertinent to these considerations showed that the plasma activity of lactic dehydrogenase was elevated by stressful conditions, and that the form of LDH in the plasma matched that in skeletal muscle. This seems to confirm cellular leakage during stress in the rock bass. Another observation concerning LDH is worth noting here because it contributes to the biological significance of these findings. LDH activities of the various non-bony tissues in rock bass were determined and used to estimate the total body LDH activity. The results are listed in Table 28 and indicate that as much as 3% of the total body LDH activity had leaked into the blood following a 72 hour ex— posure to multiple stresses. If such a loss of enzyme activity had occurred to vital organs such as the heart, kidney, or brain, these organs would probably be severely debilitated. However, the bulk of this enzyme probably comes from the skeletal muscle which contained approximately 80% of the total body LDH activity. 141 Table 28. Comparison of the lactic dehydrogenase activity in tissues of the rock bass (Ambloplites rupestris). Activity / gram Estimated‘% wet weighta of total body activity Muscle 7,900 81.88 Kidney 109,800 4.20 Liver 29,400 3.16 Stomach 9,400 2.45 Brain 80,200 1.32 Midgut 11,700 1.25 Heart 117,000 1.24 Hindgut 25,200 1.10 Pyloric ceace 9,000 0.96 Gills 20,700 0.89 Spleen and erythrocytes 34,700 0.26 Estimated leakage into the blood = 3% aPooled samples from three fish. Activity is ex- pressed as the micrograms Of pyruvate transformed in 30 minutes. 142 The fate of enzymes that leak into the blood is un— known in fish, but studies of mammalians provide some sug— gestions. Zierler (1959) points out that when enzymes (of cellular metabolism) have leaked into the blood, they repre- sent debris and rarely re-enter the cells to any significant extent. Fleisher and Wakim (1963) found that splenectomy did not alter the disappearance rate of glutamic—pyruvic transaminase from the blood, nor did injections of india ink or cortisone. Hess (1963) believes that proteins which enter the blood are broken down within the blood by proteo- lytic enzymes. Therefore, cellular leakage of proteins (enzymes) probably represents a biologically significant loss, if not to the biochemical pathways, then because proteolysis destroys the peptide bonds which required con- siderable energy for their biosynthesis. Another possible cause of the changes in the plasma proteins is also related to decreased cellular maintenance. The blood plasma normally contains still other enzymes (called plasma specific enzymes) which are produced within cells, liberated into the blood, and have their normal function in the blood plasma (Hess, 1963). When cellular integrity is reduced by stressful conditions, the production and liberation of these enzymes is reduced (Hess, 1963; Lawrence, 1964) and their plasma levels are decreased. The reduced levels of plasma specific enzymes could account in part for changes in the plasma protein composition. 143 Effects of hormones Another possible explanation for the changes in protein composition is that they are mediated by steroid hormones. Studies by Knox and Auerbach (1955), Feigelson (1961), Goldstein, Stella, and Knox (1962), and Caffery, Wichard, and Irvin (1964) have shown that steroid hormones increase the activity of many enzymes in the liver. Perhaps liberation of steroid hormones account for the increased levels of succinic dehydrogenase activity in the livers Of stressed fish. Cordier and Barnoud (1957) have also shown that steroid hormones can alter the total concentration of protein in fish serum, as well as change its composition. Rasquin and Rosenbloom (1954) noted tissue hyperplasia during en— docrine imbalance in teleosts. Idler etyal. (1963) suggest that the extensive degenerative changes in spawning pacific salmon were due to impaired clearance of steroid hormones which promoted the mobilization of tissue proteins for gluconeogenesis. Clearly, the steroid hormones can exert a profound influence on the protein composition of blood and tissues. Other causes Several other factors may be associated with the stress reSponses of rock bass as well as the levels of these 144 parameters in control Specimens. Responses to reproductive deveIOpment, acclimation temperatures, seasonal growth periods (Gross, Fromm, and Roelofs, 1965), and nutritional deficiencies (vitamin E, Baechtel, Allen, and Dobson, 1957) may have important influences on protein composition. Several other factors have been summarized by Booke (1964). Prognosis for ElectrOphoretic Studies Many improvements in electrophoretic technic, its equipment, and the understanding of the resulting data have occurred since this work began in 1962. Separations that previously required 20 pl of plasma now require less than Z‘pl and these are now completed in 20 minutes rather than 16 hours. New procedures and buffer systems also allow the investigation of protein composition among invertebrate species. For example, crayfish blood is difficult to pre— pare for electrOphoretic studies and even more difficult to separate into subsequent fractions. However, the muscle myogens in the tails Of crayfish do not present these diffi— culties (Figure 12). Clam blood has such a low protein concentration that it is worthless for electrOphoretic studies, but the myogens in the adductor muscles can be ex— tracted and analyzed (Figure 12). Still other improvements in electrOphoretic analysis include the use of molecular sieves. Proteins which cannot 145 Figure 12. Electropherogram and muscle myogen fractions of crayfish Orconectes immunis. I 23 24 CM. mm srmco ANALYTROI. noon n on: mm or Imscmnou - 0.1 seen. I II 18 19 20 21 I 17 eweve... o;.4.,.,, I I I I 1 O 147 be separated on the basis of a mobility difference can often be separated because of differences in molecular size. Judging from the above improvements in technic, and from the responses of rock bass to stressful conditions, electrophoretic analyses Of proteins can be useful in the study of stream ecology. SUMMARY The composition of plasma, myogen, and hemolyzate protein in fed rock bass were significantly altered by diurnal hypoxia (3 ppm oxygen for eight hours per day for nine days). Similar results were Obtained when fasting fish were subjected to the same conditions, except that the compo— sition of hemolyzates was not changed by hypoxia in fasting fish. Hematocrit and hemoglobin values were increased by low oxygen in both the fed and the fasting fish. During the periods of low oxygen hypoxic fish had reduced swimming activity, increased ventilation rates, and lost-their normal body color. -Among fed fish, hypoxia was also associated with reduced gain in body weight, and vomiting of previously ingested food. Hypoxic fish usually did not feed until ap- proximately three hours after the daily low oxygen conditions had ended. Fed rock bass that were exposed five days to 3 ppm of divalent nickel, had significantly higher hematocrits and and hemoglobin concentrations, but significantly lower concentrations of protein in their plasma. Nickel toxicity also significantly altered the composition Of plasma, myogen, and hemolyzate proteins, and increased the succinic dehydro— genase activity in liver homogenates. 148 149 Fasting rock bass were also tested after being ex- posed to the combined effects of divalent nickel (3 ppm), low oxygen (diurnal, 5 ppm), high levels of carbon dioxide (30 ppm) and low pH (6.8). Other specimens were exposed to these conditions but they were allowed to recover for two days before they were tested. Immediately after the experi- mental conditions ended the physiological parameters noted above were altered significantly, and the responses were similar to those in the previous experiments. Additional tests for enzymes in the blood plasma indicated that as much as 3% of the total lactic dehydrogenase activity in tissues had leaked into the blood during the stress period. After two days of recovery, most of the physiological parameters were either at or were returning to their pre—stress level. The physiological stress responses of the rock bass proceed as a syndrome and include a stress phase and a re— covery phase (if allowed). Whether or not physiological con— dition was deteriorating or ameliorating strongly influenced the level of each parameter. 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