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Protein variation of some pacific euphausiids in relation to environmental stability : An intraspecific… Bromley, Gregory J. 1972

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Cl PROTEIN VARIATION OF SOME PACIFIC EUPHAUSIIDS IN RELATION TO ENVIRONMENTAL STABILITY: AN INTRASPECIFIC AND INTERSPECIFIC STUDY by Gregory J. Bromley A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of ZOOLOGY and INSTITUTE OF OCEANOGRAPHY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1972 In present ing th is thes is in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y sha l l make i t f r e e l y a v a i l a b l e for reference and study. I fu r ther agree that permission for extensive copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion . The U n i v e r s i t y of B r i t i s h Col Vancouver 8, Canada Department of ABSTRACT i The degree of biochemical v a r i a b i l i t y i s , i n many cases, thought to be related to environmental s t a b i l i t y . Intraspecific protein varia-tion" was examined i n Euphausia pacifica using starch gel electrophoresis. Specimens were collected from eight oceanic and three n e r i t i c stations over a wide geographic area. The interspecific aspect of this study ex-amined the general protein patterns of five species of North Pacific euphausiids. Temperature, salinity and oxygen were monitored, at the time of biological collections, i n order to distinguish between water types. Two major protein patterns characterize E_. pacifica from the d i f -ferent regions. The pattern possessing the greatest number of bands occurs i n highest frequency i n areas of greatest environmental i n s t a b i l i t y (as characterized by temperature, s a l i n i t y , and oxygen); the highest fre-quency occurrence of the pattern with the lowest number of bands i s i n environmentally stable regions. The expression of each pattern appears related to the degree of fluctuation of the physical parameters monitored, but these parameters are not suggested as the direct cause of which pattern i s expressed. The physical parameters serve only as a tool for character-izing the s t a b i l i t y of a region. Analysis of the electrophoretic protein patterns from five species of euphausiids, Euphausia pacifica, E_. gibboides, Thysanoessa spinifera, T. inspinata and Nematoscelis d i f f i c i l i s , revealed that each species i i possesses a unique protein pattern. The interspecific and intergeneric similarities of protein patterns parallel the existing morphological taxonomy. i i i TABLE OF CONTENTS Page I. GENERAL INTRODUCTION 1 II . GENERAL MATERIALS AND METHODS 7 Field Procedures 7 Laboratory Procedures 11 Tissue Extraction Preparation 11 Electrophoresis ....... 12 General Proteins and General Esterases 13 Lactate Dehydrogenase, Malate Dehydrogenase, Alpha-Glyceral Phosphate Dehydrogenase 14 Data Analysis 16 II I . RESULTS 18 Neritic Stations 18 Oceanic Stations 20 Electrophoretic Results 25 Interspecific Study 25 Intraspecific Study 29 Specific Enzymes (MDH, LDH, -X-GPD) 31 IV. DISCUSSION 33 Intraspecific 37 Interspecific 41 i v TABLE OF CONTENTS (Cont'd) Page V. SUMMARY AND CONCLUSIONS 44 VI. REFERENCES 47 V LIST OF TABLES Table Page 1 Depths of Hydrographic Observations and Clarke-Bumpus Net Tows 10 v i LIST OF FIGURES Figure Subject Page 1 l.O.U.B.C. Cruise 71/34—Hydrographic and Biological Data Stations 8 2 Hydrographic and Biological Data Stations in Neritic Waters 9 3 Electron Transport System Important in the Staining of Dehyrogenases 15 4 Oxygen Profiles at Stations Representative of Each Sampled Water Type (Environment) 19 5 Temperature-Salinity Curves of Neritic Stations at the Time of Biological Collec-tions 21 6 Temperature-Salinity Curves of Oceanic Stations at the Time of Biological Collec-tions 22 7 Electrophoretic Patterns of General Pro-teins of Five Species of North Pacific Euphausiids 27 8 Electrophoretic Patterns of General Pro-teins from Euphausia pacifica Inhabiting Different Water Types (Environments) 28 9 Frequencies of General Protein Patterns at Stations Sampled 30 10 Patterns of General Proteins, General Esterases, Alpha-Glycerol Phosphate Dehydrogenase, (CK-GPDH), Lactate dehyro-genase (LDH), and Malate dehydrogenase (MDH), from Euphausia pacifica 32 v i i ACKNOWLEDGEMENT S The author wishes to extend thanks to a l l the members of my advisory committee. Sincere thanks must go to Dr. A.G. Lewis for supporting this project and for his encouragements and criticisms. I would also l i k e to thank Dr. H. Tsuyuki for his aid i n modifying the electrophoretic technique as well as for his wealth of knowledgeable help i n this f i e l d . I am also indebted to Dr. C. Wehrhahn for his encouragement throughout the project. Several others deserve recognition in helping with this project. Especially Mr. J. Fulton for the identification of several euphausiid species, and Dr. P. Dehnel and Mr. G. Gardner for reading and c r i t i c i -zing this manuscript. Finally, I wish to extend a cheerful thanks to the officers and crew of the C.S.S. Vector and the C.S.S. Parizeau for their patience and assistance that made the cruises a very enjoyable experience. Class Order Family Genus Species Crustacea Euphausiacea Euphausiidae Euphausia  Euphausia pacifica I. GENERAL INTRODUCTION Natural variation, both morphological and functional, has long intrigued biologists. Contemporary theories believe that variation between and within species i s a direct result of biochemical compensa-tion for environmental changes. Technological advances have enabled evaluation of biochemical variants at taxonomic levels below the species level. Consequently, differences within populations, and the biochemical nature of physiological races and ecotypes, have recently been examined. However, there are no documented biochemical studies of zooplankton populations, and only a few concerning biochemical species differentia-tion. Past investigations by Nemoto (1966) revealed allomorphic varia-tions within a single species of euphausiid (Crustacea, Euphausiacea) inhabiting one water mass. More recently i t has been suggested that physiological variations may occur within a local species, Euphausia  pacifica ( G i l f i l l a n , 1970). The present study was initiated to determine i f biochemical differences, of both an intraspecific and interspecific nature, could be resolved i n local and north Pacific euphausiids. Although the biochemical aspects of zooplankton populations have been neglected, the general aspects of zooplankton ecology are better documented. Oceanographers recognize the. existence of "water masses" (Helland-Hansen, 1916) and i t i s common practice to correlate the appear-ance of an organism with the presence of a particular water mass (Russell, 1935, 1939). This association has been further developed so that the - 2 -presence or absence of planktonic organisms can be used to determine the boundaries of a body of water (Marumo, 1957; Bary, 1959; Fager and McGowan, 1963), and to follow currents (Glover, 1952; Marumo, 1957). Bary (1961 and 1964) correlated the distribution of numerous members of the plankton with particular "water bodies", several of which may be contained within a single water mass. He concluded that an unknown pro-perty, unique to the water body, limited the distribution of the organ-ism; thus the plankton could be used as an indicator of the presence of the "water body." Although studies on the indicator species—water body concept are documented, they have not considered that organisms may exist as populations, races, or ecotypes. Expressions of intraspecific biochemi-cal or morphological variations are usually initiated by some environ-mental change or stress, and may f i r s t be detected as individual biochemi-cal deviations from the biochemical constitution of a species. During the past twenty years techniques have been developed that permit the analysis of organisms at the molecular level. Chromatography, peptide fingerprinting, amino acid analysis, serology and electrophoresis are the most commonly used tools in molecular studies. The basis for such studies, and for electrophoresis in particular, l i e s in the specificity of the amino acid sequence (Crick, 1966), and i n the configuration and net charge of the proteins (Sibley, 1962; Shaw, 1965; Tsuyuki et a l . , 1965; Kaplan, 1968). Alterations in electrophoretic mobility can occur as the - 3 -result of single amino acid substitutions (Shaw, 1965). McCleur (from Shaw, 1965), however, has calculated that only 27.56 percent of amino acid substitutions modify the net charge of the molecule. This anomaly can arise from a variety of causes. For example, different mutations of the same gene may cancel any alterations in mobility, thus two variants may be recorded as the original single electrophoretic variant (Gooch and Schopf, 1970). Originally the basis for electrophoresis concurred with the one-gene, one-enzyme hypothesis and thus a single molecular form of an enzyme was expected (Shaw, 1965; Markert, 1968). Lately, however, investigations indicate that enzymatic activity may be retained even though the structure of the protein has been altered (Markert, 1968). Multiple forms of the same eizyme, known as isozymes, or of other homologous proteins, are now frequently observed (Manwell, et a l . , 1967; Bailey, et a l . , 1970; Schwabl, et a l . , 1970; Selander,et a l . , 1970 a,b; Sprague, 1970; Barlow,et a l . , 1971; Gooch,et a l . , 1971; Nyman, 1971) and in many cases are the rule and not the exception (Selander, et a l . , 1971). Geneticists have lately been referring to genetic variants as different isozymes, thus causing a degree of ambiguity in the usage of the term. Prakash et a l . , (1969) proposed that genetic variants be known as allozymes and that isozymes refer to different polymers produced from a monomeric protein. Protein polymorphism can be useful as genetic markers i n many biological areas of investigations such as identifications at the - 4 -subspecies, species and higher levels of organization, taxonomic rela-tionships, and in hybrid analysis. Frequency distributions of a l l e l i c proteins have found extensive application in population analyses (re-viewed by De Ligny, 1969). Studies on marine benthic organisms have uncovered significant geographic correlations between allozyme d i s t r i -butions and physical parameters, suggesting that the polymorphisms ob-served are direct responses to climatic conditions (Wium-Andersen, 1970; Gooch and Schopf, 1971; Koehn and Mitton, 1972). The same pattern i s observed i n mammals (Ashton, 1958) even though Kaplan (1960) contends that invertebrates show more diversity than vertebrates. Gooch and Schopf (1970) suggest that population structure and amount of genetic v a r i a b i l i t y between marine and terrestial organisms are fundamentally homologous. Koehn and Mitton (1972) suggest that identical environmen-tal conditions i n i t i a t e similar modifications i n the electrophoretic patterns of two species of mussels. Freeman, et a l . (1971) report that genotype-environment interactions exist and that monitoring the perfor-mance of various genotypes in differing environments usually reveals a linear relationship. Fitness values may then be extrapolated from the concurrence of a genotype and surrounding environments. The mode of heterogeneity provides the molecular basis for specia-tion. Once an environmental stress i s imposed, a population must move in one of two directions, towards extinction or acclimation (Levin, 1970). Undoubtedly extinction i s the more common occurrence as climatic changes - 5 -are usually considerable (Lipps, 1970). Nevertheless, some organisms are able to acclimate, either through behavioral or physiological com-pensations; and i t i s this phenomenon that permits organisms to survive in new environments, or to exploit habitats from which they were for-merly absent. Physiological compensation occurs only in somatic tissues (Prosser, 1967) and can only operate within genetically preset tolerance limits of the organism. If the environmental stress i s not outside of these tolerance l i m i t s , there may be "qualitative" changes in the bio-chemistry of the organism. For example, "qualitative" changes of trout result i n lower activation threshold of many enzymes and this may be initiated by changes in temperature (Hochachka and Somero, 1971). Such modifications are only active as long as the immediate environmental influence i s present, and are reversible should the environment revert to the original state. Prosser (1967) defines genetic variation, as opposed to acclima-tion, as the result of a mutational alteration in the primary structure of the protein. Random genetic alterations, by mutation or gene recom-bination, are continuously occurring and when one occurs in the direction of increasing tolerance limits i t i s incorporated into the gene pool. Not only is the population better able to exploit a new niche, but i t may also extend or shift i t s acclimation limits. In time the populations may become geographically or ecologically isolated. Ultimately the populations - 6 -become reproductively incompatible and new species arise. It i s trends of this nature that the present study hopes to elucidate i n some members of the Euphausiidae. I I . GENERAL MATERIALS AND METHODS A. Field Procedures E_. pacifica i s a vertical migrant that inhabits an extensive geographic range. To increase detection of intraspecific biochemical variation, i t was f e l t advisable to sample over as wide a geographic area as possible and to sample many different environments within the study area. The majority of the specimens used i n this project were collected between October 25 and November 14, 1971, from stations occupied on two transects between the Queen Charlotte Islands, British Columbia, and Gaudalupe Island, Mexico (Fig. 1). One transect was 200 miles offshore while the other was 20 miles offshore. Other samples, those from rela-tively neritic stations (Fig. 2) , were collected during the period of November, 1971 to January, 1972. Specimens and hydrographic data were collected at each station. As E_. pacifica i s a vertical migrant, i t was f i r s t necessary to determine the depth of the organisms at the time of sampling. Horizontal tows using Clarke-Bumpus opening/closing samplers (Clarke and Bumpus, 1950) were made at specified depths (Table I ) . Frequency echo sounders were used to locate a scattering layer as Pieper (1971) demonstrated that euphausiids were an important constituent of this layer. Specimens were collected with a one meter diameter ring net (1.0 mm average mesh diame-ter) hauled ve r t i c a l l y , obliquely, or horizontally, as dictated by the - 8 -Figure 1. 1.0.U.B.C. Cruise 71/34 Hydrographic and biological data stations in oceanic waters. Stations Type of Water 2, 10, 15, 24. 32, 54, 67, 61 44, 48 Subarctic Transitional Subtropical Figure 1 - 9 -Figure 2. Hydrographic and biological data stations i n Neritic waters. IA-9 Indian Arm Saa-3 Saanich Inlet GS1 Strait of Georgia Figure 2 - 10 -Table I Depths of Hydrographic Observations and C-B Net Tows (Depth in meters) Neritic Stations Oceanic Stations 0 0 10 10 * 20 25 30 50 50 75 75 100 100 150 125 200 150 300 *** 175 400 200 500 250 300 350 375 390 * depths that only Hydrographic data were collected. ** this depth sampled only in Indian Arm-9 *** this depth not sampled at Georgia Strait-1 Note: Saanich Inlet and Indian Arm were sampled only to a depth of 200 m. - 11 -vertical distribution and concentration of the euphausiids. After capture, the specimens were placed in glass or plastic screw top vials using stainless steel forceps. The samples were then labelled, and quick-frozen either i n an isotherm containing dry ice or in a freezer (Danby/INGLIS-Model 100) at -26 C. The frozen samples were stored at -26°C u n t i l analy-sis (not longer than 6 months). No apparent loss of relevant biochemical activity occurred during storage. Hydrographic parameters, specifically temperature, s a l i n i t y , and oxygen, were monitored at several depths at each station (Table I ) . Water, at depth, was collected with National Institute of Oceanography (N.I.O.) sampling bottles, and simultaneous temperature measurements were obtained with attached, reversing thermometers. Furthermore, at every station and at every 30" of latitude during the Mexico cruise, a bathythermograph (BT) was lowered to the depth of deepest sampling or to 275 m, whichever was shallower. Dissolved oxygen content of the water was determined by Winkler t i t r a t i o n (Carrit and Carpenter, 1966) while on board the ship. Salinities were determined by the conductivity method using an inductively coupled salinometer. Data i s tabulated in I.0.U.B.C. data reports. B. Laboratory Procedures (a) Tissue Extract Preparation Manwell et a l . (1967) have pointed out that the method of protein extraction i s of cardinal importance to taxonomic protein studies. The - 12 -extraction buffer concentration and pH are especially c r i t i c a l . Super-natant proteins were extracted in a 0.1 M Tris (Hydroxymethyl Aminome-thane) buffer adjusted to pH 7.0 with 1 N HC1. A freshly thawed specimen was checked under a dissecting microscope to confirm species identifica-tion, and was then placed i n a polypropylene test tube (10.9 x 77 mm) con-taining a volume of the extraction buffer approximately equal to the volume of the animal. This volume averaged 0.03 ml with maximum variation occurring between oceanic and neritic individuals. The specimen was macerated for 25-30 seconds with an elect r i c a l l y rotated teflon pestle. The macerate was subsequently centrifuged at 12,000^ for 15 minutes and the supernatant collected i n a non-heparinized micro-hematocrit capillary tube (1.1-1.2 mm I.D.) that had previously been f i r e drawn to a fine t i p . Tissue extract preparations were maintained at low temperatures in ice or in a cold room (4 C). Each sample was labelled and quick frozen (-26UC) unt i l analysis. Storage time from extraction u n t i l analysis varied from 1 to 3 days. (b) Electrophoresis Electrophoretic analysis was conducted using a horizontal starch gel method described by Tsuyuki et a l . (1965). The buffers used depended on whether general proteins or specific enzymes were to be stained. In either case, clear bands resulted only when the buffers were of a pH be-tween 8.0 and 8.6. The isotonic nature of euphausiids with sea water (pH 8.3) necessitates the use of pH's of at least 8.0. At lower pH's - 13 -many bands do not appear, and resolution i s poor. Only the resolution obtained from the general proteins was sufficiently reproducible for analysis but the techniques and the results for other enzymes are included for the sake of completeness. (i) General Proteins and General Esterases The best results for proteins and esterases were obtained with a 12% hydrolyzed starch gel (Connaught) i n an 0.023 M Sodium borate buffer (pH 8.5). The buffer consisted of 0.023 M boris solution adjusted to pH 8.5 with 1 N sodium hydroxide. The electrode buffer of 0.3 M Sodium borate (pH 8.0) was made in a similar fashion. Electrophoresis was con-ducted for 2.5 hours at 4°C with a voltage gradient of 8 v/cm. For general proteins, the starch gel strips were stained for 5 minutes with amido black. This non-specific protein stain was comprised of 0.3% buffulo black NBR i n a solution of methanol: d i s t i l l e d water: acetic acid (5:5:1;v:v:v). The gels were then washed in several suc-cessive solutions of methanol: d i s t i l l e d water: acetic acid (5:5:5;v:v:v). This procedure removed much of the background stain and revealed the denatured proteins as dark blue bands. General esterases were resolved with the following stain: 3 mis of substrate, 10 mis of 0.5 M Tris-HCl (pH 7.0), 100 mgs fast blue RR and 87 mis of d i s t i l l e d water. - 14 -Substrate 0.5 M Tris-HCl (pH 7.0) o< -Napthai acetate lgm Tris 64.6 gms Acetone 50 mis HC1 42 mis H20 ..50 mis H20 to 1000 mis The gels were stained for one hour or u n t i l dark bands appeared. ( i i ) Lactate Dehydrogenase, Malate Dehydrogenase, Alpha-Glycerol Phosphate Dehydrogenase The gel medium used in resolving dehydrogenase consisted of 12% hydrolyzed starch i n an 0.003 M Tris-citrate (pH 8.0) buffer. This solu-tion was made of 0.003 M c i t r i c acid adjusted to pH 8.0 with 1 N t r i s . The electrode buffer was 0.042 M Tris citrate (pH 8.0). The activity of dehydrogenase stains depends on the transfer of electrons (Fig. 3). The specific enzyme stained thus depends on the sub-strate used. The stains used for each dehydrogenase are as follows: LDH MDH ©<-GPD Tris (0.1 M) 10 mis (pH 8.0) 10 mis (pH 8.5) 10 mis (pH 8,5) Substrate 20 mis lactate 10 mis malate 225 mgs -GP NAD1 (DPN) 30 mgs 30 mgs 30 mgs 2 PMS 1 mg 1 mg 1 mg NBT3 15 mgs 15 mgs 15 mgs EDTA4 40 mgs 1. Nicotinamide adenine diphosphate 2. Phenazine methosulphate 3. Nitro-blue tetrazolium 4. Disodium Ethylenediamine tetraacetate - 15 -substrate enzyme substrate red. ox. DPN*, (TPN ) DPNH' (TPNH) PMS red. ±etrazolium salt ox. PMS px. tetrazolium salt red. dark blue precipitate Fig. 3 Electron transport system important i n the staining of dehydrogenases. - 16 -The substrates were made as follows: 1 M Na-DL-Lactate (pH 7.0) 88% DL-Lactic acid . ... 10.6 mis 1 M Na2C03-H20 49 mis Di s t i l l e d water to 1000 mis 1 M Na-L-Malate (pH 7.0) L-Malic acid 13.4 gms 2 M Na2C03-H20 49 mis Di s t i l l e d water to 1000 mis The gels were stained u n t i l dark bands appeared, usually completed i n 2-4 hours. A l l gels were subsequently photographed, recorded, labelled and stored i n sealed poly-bags (whirl-pak). Chemicals were obtained from A l l i e d Chemical Co. and Sigma Chemical Co. C. Data Analysis Examination of the electrophoretic results by common s t a t i s t i c a l methods proved unsatisfactory. Therefore, combinations of relative fre-quencies of protein band patterns and the water masses in which they occurred were plotted. The order of the water masses on Fig. 9 was deter-mined by the estimated hydrographic v a r i a b i l i t y of the water masses. The water mass with the least fluctuations (i.e. the most stable) i s on the le f t portion of the graph, while those estimated to have successively larger fluctuations are plotted to the right. The determination of the - 17 -of the order of stations within each water mass was from the T-S curves (Fig. 5 and Fig. 6). The percent occurrence of the patterns consisting of the most bands and of the least number of bands were plotted for each station. Although the general trends hoped for do appear with this tech-nique, i t i s f u l l y realized that the fine detail i s lost. - 18 -I I I . RESULTS Eight oceanic and three neritic stations were sampled. Station position was determined by the environments (a p r i o r i ) , the range of the ship, and the available time. NERITIC STATIONS Three stations, Indian Arm (IA-9) , Georgia Strait (GS-1), and Saanich Inlet (Saa-3), represent the nerit i c complement of the study (Fig. 2). GS-1 and IA-9 show typical annual estuarine fluctuations in temperature and salinity. These cyclic fluctuations are related to changes in runoff and insulation. However, Saa-3, although typically a fjord i n shape, does not undergo a normal annual cycle. Herlinveaux (1962) observes that complete flushing of this inlet may occur only once in several years and, subsequent to flushing, an oxygen deficient layer forms below 100 m (Fig. 4). This layer i s a direct result of the lack of runoff and the high s i l l (rising to a depth of 73 m) that retards mixing from the Strait of Georgia. In essence, a very stable environment results. Figure 5 depicts the temperature-salinity (T-S) curves of the neritic stations at the time of specimen collection. G i l f i l l a n (1970) documented the T-S envelopes for annual fluctuations in these stations and observed that the monthly fluctuations of the neritic stations in many cases exceeded the annual variations of oceanic stations. These - 19 -Figure 4. Oxygen profiles at stations representative of each water type (environment) sampled. Station Type of Water GS-1 Neritic 2 Subarctic 48 Subtropical 32 Transitional between Subarctic and Subtropical Saa Saanich Inlet with oxygen-poor deep water Figure 4 fluctuations predominated in surface layers, but even the deep water of of the neritic stations never approximated the oceanic waters. Further-more, because of these large annual fluctuations, oceanographers have had some d i f f i c u l t y in applying the water body concept of Bary (1961, 1963) to these areas. On the other hand, the more stable oceanic waters are more adaptable to such a concept (Johnson and Brinton, 1963). Figure 6 demonstrates the relatively narrow range of environments of the oceanic habitats, as compared to the nerit i c stations. Closer examination of neritic environment reveals many trai t s common to estuaries in the Briti s h Columbia region. Surface s a l i n i t i e s are low, and the range within the water column can be large. The oxygen content of the water column i s i n a state of constant flux (Fig. 4). But even more striking are the annual fluctuations in hydrographic parameters (temperature and salinity) that can occur within the fjord. There i s no typical T-S curve (Fig. 5) comparable to that seen at oceanic stations (Fig. 6). OCEANIC STATIONS Oceanographic data was collected from ten oceanic, stations during the perxod of October 25 to November 14, 1971. The T—S curves identify the three water masses, the subarctic, the transitional, and the subtropi-cal, that were sampled. The subarctic water mass occupies much of the north Pacific ocean (Uda 1963; Dodimead et a l . 1963) and supplies two - 21 -Figure 5. Temperature-salinity curves of neritic stations at the time of biological collections. - 22 -Figure 6. Temperature-salinity curves of oceanic stations at the time of biological collections. Figure 6 - 23 -major currents as i t approaches the west coast of Vancouver Island. One current turns north and forms the Alaskan gyre while the southern flowing component, the California current, follows the west coast of the United States. Uda (1963) has defined subarctic water as a l l water with the termination of the halocline at a sal i n i t y of 33.8%* (±0.1%0. Thus stations 2, 10, 15, and 24 are in subarctic water (Fig. 6). A permanent halocline at 100 to 150 m reaffirms that these stations are subarctic (Dodimead et a l . 1963). The subtropical water mass i s situated to the south of the sub-arctic mass and i s characterized by the lack of a permanent halocline (Dodimead et a l . 1963). Two stations, 44 and 48, are in the subtropical region, although station 44 might be confused with transitional waters from the T-S properties of the deep water. It i s suggested that station 44 i s i n the subtropical region, but that a tongue of colder, transition-a l water may be intruding beneath the subtropical surface layer. This station i s situated in an area of continuous boundary shifting between water masses (Brinton, 1962 a,b). A region of mixing occurs between subtropical and subarctic wa-ters and i s generally termed the transitional zone (Dodimead et a l . 1963; Uda, 1963). Stations 32, 54, 61 and 67 are representative of this regime. The temperature and sal i n i t y curves are. generally more erratic in form and may differ greatly between two adjacent stations. The oxygen pro-f i l e of this zone i s intermediate to the subarctic and subtropical - 24 -curves and might be considered as an index of the amount of mixing (Fig. 4). Stations 32 and 54 were the two most southerly stations where E_. pacifica was found. Brinton (1962) has captured specimens as far south as 32°N, but that was at times when the Davison current was weak (June to September). Cross and Small (1967) report that the northward flowing Davison current i s well established by October. This current i s located very near to the coast and tends to destroy any southward flow-ing component of the California current in i t s path(Sverdrup et a l . 1942). Thus, this impedes any southern dispersion of euphausiids in this region during the winter months. Mauchline and Fisher (1969) report the southern limit of E_. pacif ica to correspond to the 9.5°C isotherm at 200 m. Such is the case at station 48, but at station 44, where no specimens were obtained the temperature was 9°C. It i s further interesting to note that the surface waters of two stations, 61 and 67, are as cold as the surface waters of station 2, ten degrees of latitude to the north. Warmer surface temperatures were re-corded between these stations. This anomaly i s best explained by the phenomenon of upwelling. Dodimead et a l . (1963) reports that upwelling i s a common occurrence along the western coast of California and Oregon, and i s a direct result of the prevailing northwesterly winds. During upwelling, which usually occurs within 27 km of the Oregon coast, surface temperature may drop below 9°C and surface sa l i n i t i e s may be in excess of 33%. (Hebard, 1966). - 25 -For a more detailed oceanographic background of the area studied, Waldichuk (1957), Gilmartin (1962), Herlinveaux (1962), Aron (1962), Dodimead et a l . (1963) and Uda (1963) may be consulted. ELECTROPHORETIC RESULTS The electrophoretic aspect of this study investigated both intra-specific and interspecific differences within the family Euphausiidae. The interspecific study was conducted with two aims. The primary objec-tive was to determine whether the method at hand could differentiate be-tween species and thus could detect sibling speciation should i t be pre-sent i n the intraspecific study. The secondary objective of the inter-specific study was to give a possible insight into any evolutionary trends, within the family Euphausiidae, that might be suggested by simi-l a r i t i e s and differences within the electrophoretic patterns. The intra-specific part of the study was designed to examine possible populations of E_. pacifica, and to attempt to associate them with a change in the nature of the environment. INTERSPECIFIC STUDY Five species from three different genera were chosen because of their abundance, and because they were of a size sufficient for adequate protein extraction. The species used include Euphausia pacifica, E_. gibboides, Thysanoessa spinifera, T_. inspinata, and Nematoscelis d i f f i c i l i s . - 26 -At least f i f t y specimens of each species were secured at offshore sta-tions 15 and 24 (Fig. 1). The general protein pattern of each species was found to be unique (Fig. 7). Species specific patterns have also been observed in invertebrate sera (Woods et a l . 1958; Cowden and Coleman, 1962), i n several enzymes of copepods (Manwell et a l . 1967), and in several enzymes of marine fishes (Tsuyuki et a l . 1968; Johnson et a l . 1972). Two bands (B^ and D) are common to a l l species while each of the remaining bands i s absent from at least one of the species (Fig. 7). Furthermore, the B^ and D bands are the darkest staining, possibly i n d i -cating the relative quantity and importance of these proteins. Specimens from the genus Euphaiisia have protein patterns consist-ing of at least eight bands. E_. pacif ica i s variable in that i t may possess a complement of six to nine bands. These variations are dis-cussed later in the manuscript. Only one species i s represented from the genus Nematoscelis. Nematoscelis d i f f i c i l i s consistently possesses seven protein bands, one of which i s the A^ band. The only other species that displays this band i s E.. pacif ica, and then only in isolated specimens (Fig. 8 c,d). Two members of the genus Thysanoessa have been analyzed. T. inspinata and T_. spinifera, have comparatively few bands and both lack the C band (Fig. 7). T. inspinata consistently has a six banded protein pat-tern while T. spinifera possesses only four bands. The B^ band i s unique to T_. inspinata. - 27 -Figure 7. Electrophoretic patterns of general proteins of five species of north pacific euphausiids. Euphausia pacifica Euphausia gibboides Nematoscelis difficilis Thysanoessa inspinata Thysanoessa spinifera A, A, B,B& C D E,E2EjE4 F i II I ! i! 1 1 II! 1! 11 1 1 1 ! 11! i S i 1 1! Figure 7 - 28 -F i g u r e 8. E l e c t r o p h o r e t i c p a t t e r n s o f g e n e r a l p r o t e i n s o f E u p h a u s i a p a c i f i c a i n h a b i t i n g d i f f e r e n t v a t e r t y p e s ( e n v i r o n m e n t s ) . a-d., l o c a t e d a t a l l s t a t i o n s e x c e p t Saa-3 and ocean s t a t i o n 2 e - f o n l y l o c a t e d a t Saa-3 g-h o n l y l o c a t e d a t ocean s t a t i o n 2 rr (Q -+> CD C L O cr co > *n rrrro nrcra rasas isass rsaaa rasra cwag rasen _r + INTRASPECIFIC STUDY _E. p a c i f i c a , due to i t s wide d i s t r i b u t i o n , lends i t s e l f w e l l to i n t r a s p e c i f i c studies. Specimens from eleven s t a t i o n s were analyzed for protein v a r i a t i o n s . Four d i f f e r e n t e l e c t r o p h o r e t i c patterns ( F i g . 8, a-d) commonlyappeared at nine sta t i o n s while the remaining two stations each revealed an a d d i t i o n a l two patterns unique to the s t a t i o n . The unique spec-imens form s t a t i o n 2 d i f f e r from the normal populations i n that the C band i s absent (Fig. 8, g,h). Woodhouse (1971), while examining the d i s t r i b u t i o n of two species of calanoid copepods, over e s s e n t i a l l y the same geographic area, found that the species composition i n the region of s t a t i o n 2 was d i f f e r e n t from that i n other areas. He concluded that this was a r e s u l t of a d i f f e r e n t type of water. S i m i l a r l y , the unique specimens from Saanich I n l e t lack a band that i s common to a l l other populations. The B 2 band was not observed suggesting a difference i n the biochemical composition of these specimens from this area. T r a d i t i o n a l l y used s t a t i s t i c a l methods y e i l d e d l i t t l e information. To allow some evaluation, the rank order of frequencies of each pattern at each s t a t i o n was examined. The frequencies of the patterns with the l e a s t bands ( F i g . 8, a) and with the most bands (Fig. 8, d) are p l o t t e d f or each s t a t i o n i f Figure 9. The remaining two patterns, those with intermediate numbers of bands (Fig 8, b and c) and which fluctuate randomly, do not involve a s i g n i f i c a n t number of specimens and are not p l o t t e d . 30 -Figure 9. Frequencies of general protein patterns at stations sampled. Simple band pattern i s "a" i n Fig. 8. Complex band pattern i s "d" in Fig. 8. Stations are li s t e d in order of de-creasing environmental s t a b i l i t y (i.e. increasing environmental fluctuations). Refer to Data Analysis section of Materials and Methods for a detailed explanation. Figure 9 - 31 -The pattern frequencies ( F i g . 9) show a d i s t i n c t trend. The pattern with the smallest number of bands occurs with the lowest frequency i n the n e r i t i c environment and with highest frequency i n s u b a r c t i c waters. The reverse of t h i s trend i s observed i n the pattern possessing the high-est number of bands. Station 10 and Saa-3 show frequencies opposite to the observed trend. For the sake of d i s c u s s i o n , the patterns with the lowest and highest number of bands are l a b e l l e d simple and complex r e s p e c t i v e l y (Fig. 9). There i s , however, no inference that any evolutionary rank or molecular advancement i s implied by the number of bands. SPECIFIC ENZYME SYSTEMS Results from s p e c i f i c enzymes and general esterases are shown i n Figure 10. The pattern shown for each enzyme system i s the pattern most t y p i c a l of the e l e c t r o p h o r e t i c analysis although v a r i a t i o n s were present, e s p e c i a l l y i n the esterases. Esterases, i n mammals, are as a c l a s s more highly polymorphic than other enzymes (Selander at a l . 1971). Results with i n d i v i d u a l enzyme systems were not c o n s i s t e n t l y reproducible and, as such, were not considered r e l i a b l e . - 32 -Figure 10. Patterns of general proteins, general esterases, alpha-glyceral phosphate dehydrogenase (<X-GPDH), lactate dehydrogenase (LDH), and Malate dehydro-genase (MDH) from Euphausia pacifica. Protein Esterase <*-GPDH IDH MDH A,A 2 B,Bi C. D E3 F II I! I I I I S I I I I I I I 1 I I I ! 1 I I i I + Figure 10 - 33 -IV. DISCUSSION Euphausiids are one of the more common members of the oceanic zooplankton. Euphausia pacifica i s ubiquitous in the Pacific subarctic and transitional water masses (as defined by Dodimead et a l . 1963), and i s an especially prominent constituent of the plankton in the eastern north Pacific (Hebard, 1966). The southern extent of i t s range coincides with the appearance of the subtropical water mass (Brinton, 1962). Fur-thermore, large numbers of E_. pacif ica are found in the coastal estuaries of Brit i s h Columbia. These areas are neritic and have temperatures and sa l i n i t i e s modified by runoff and insolation. Characterization of the marine environments i s one of the major stumbling blocks in oceanographic studies. Terrestial biologists can easily classify differing habitats as changes in vegetation and topography are natural boundaries. The apparently homogeneous oceanic environment does not have the abrupt barriers that characterize te r r e s t i a l biomes but, nevertheless, barriers are present. Small changes in the habitat are significant i n oceans because the organisms are physiologically attuned to the existing environment (David, 1963). Temperature and sal i n i t y are two universal oceanic parameters that are easily measured and, as such, are ut i l i z e d to classify marine environments. These parameters are not neces-sarily the range limiting factors but serve only as a useful tool i n differentiating habitats. - 34 -The extensive geographic area and wide range of environments over which E_. pacif ica i s found suggests that some variation may exist between specimens from different regimes. Regan (1968) suggested the E_. pacifica has a large tolerance range for changes i n temperature and salinity. With the support of respiration data, G i l f i l l a n (1970) put forward the idea that several physiological races of E_. pacifica exist within morphologi-cally identical populations. Brinton (1962) observed two morphological variations of Thysanoessa longipes, spined and unspined, within the sub-arctic water mass. The above sequence of concepts, from tolerance of en-vironments, to physiological races, to morphologically distinct populations, might imply that the evolutionary process can be observed i n the oceans. It i s at the molecular level that detection of minute variations is most l i k e l y , because proteins are a basic constituent of body materials. Variations at this level could account for physiological races and might infer the general pattern of speciation. The object of the present study was to compare, biochemically, intraspecific and interspecific variations in Pacific euphausiids. Contemporary theories of phenotypic and genotypic variation i n a fluctuating environment tend to follow a theme that i s comprised of two major stages. The f i r s t stage i s demonstrated by studies with Drosophila, which indicate the highest degree of polymorphism i s observed in popula-tions i n the central habitat (Lakavaara and Savoa, 1971). Under marginal conditions a balancing selection maintains the alleles in a given frequency - 35 -and the net result i s a reduction i n polymorphism. Prakash et a l . (1969) further contend that populations isolated from the central range would show even lower polymorphism. Thus, only the organisms that can survive within narrow tolerance limits can survive in the marginal and isolated populations. The second stage of the theme believes that an increase in insta-b i l i t y (i.e. increased fluctuations) w i l l lead to biochemical compensation in the form of either increased protein v a r i a b i l i t y or of a more complex functional protein make-up. Natural selection, in a fluctuating environ-ment, would preserve a number of phenotypes; for a single genotype, no matter how versatile, cannot be expected to function effi c i e n t l y at a l l levels of the fluctuations. Hence, polymorphic populations could accom-modate a fluctuating habitat to a greater degree than monomorphic popula-tions. It has been suggested that i f two populations are exploiting the same environments, i t w i l l be the population with greater reserve i n gene-t i c variation that w i l l survive an environmental change ( Cain and Sheppard, 1954). In addition, Levin (1970) proposed that peripheral iso-lates may have a source of phenotypic variation not immediately exploitable by most central populations whose genetic and physical environments are less harsh. Genetic homeostatis i s a result of environmental uniformity in both time and space, and i n such homogeneous environment vi r t u a l l y any mutation would be disadvantageous. - 36 -If there i s a system where the input from the immediate environ-ment (water mass) or from external forces (weather, etc.) creates a history of continual fluctuations within the habitat, then i t might be expected that a variable phenotype and in some cases genotype, might be propagated. The primary stipulation for genotypic variation in this environment i s long-term i n s t a b i l i t y of the habitat. Recent studies have indicated that the variants i n salmonid lactate dehydrogenase are suited to function at particular temperatures (Hochachka and Somero, 1968), and that, i n response to eivironmental adaptation these variants may be incorporated into the genetic complement of a population. Thus, where fluctuations are charac-t e r i s t i c of the habitat, i t might be expected that the populations are more biochemically variable than in a stable environment. Moreover, i t seems reasonable that i f an organism has a greater variety of isozymes, then the enzyme may well be able to function optimally over a wider environ-mental range or flux. Furthermore, populations inhabiting areas of insta-b i l i t y would be expected to possess a larger number of allozymes in their genome. Individuals and populations with these attributes would be selec-ted for i n areas prone to consistent fluctuations. Salmonids, which evolved by gene duplication of their entire genome (Bailey et a l . 1969), possess large numbers of isoenzymes (e.g. hemoglobins) and presumably other "isoproteins" conferring upon them a wide range of biochemical spe-c i f i c i t i e s consistent with their variable habitat and behavior (Tsuyuki. pers. comm.). - 37 -Closer examination of this hypothesis, in conjunction with the electro-phoretic and oceanographic data from this study, suggests that some planktonic organisms may adhere to the above postulate, particularly those i n areas of fluctuating parameters. INTRASPECIFIC STUDY The nerit i c stations (GS-1 and IA-9) undergo characteristic annual fluctuations of temperature and sal i n i t y . In essence, one of the prime trai t s of n e r i t i c waters i s the i n s t a b i l i t y of the system. The electro-phoretic data associated with the neri t i c stations shows that the pattern comprised of the greater number of bands appears most frequently (Fig. 9). GS-1 and IA-9 are typical examples. Runoff i s seasonal as i s the net insolation, bjth in peak occurrence during the spring to summer months. The annual fluctuations in these regions are a product of the local clima-tology and topography. The one station (Saa-3) that does not show the expected occurrence of banding patterns is not subjected to the same extent of fluctuation as are the other neritic stations. Annual runoff i s negligible and a large influx of water from the Strait of Georgia may not occur for several suc-cessive years (Herlinveaux, 1962). It is presumed that such conditions would lead to a modified neri t i c habitat, somewhat more stable and as such with organisms that have adapted to narrower tolerance limits. The relative s t a b i l i t y of the inlet is further supported by the formation of - 38 -an oxygen deficient layer below 100 m (Fig. 4) which implies very l i t t l e mixing. Euphausiids in Saanich Inlet may be influenced by the lack of environmental flux of the system and thus may be adjusted biochemically to the patterns observed i n oceanic waters. In the oceanic environments the same degree of fluctuation found in ne r i t i c habitats i s not observed. For the most part, the physical parameters of an oceanic water column are quite stable, especially below the upper 100 m. Only in areas where intermixing of water masses occurs, i s the s t a b i l i t y consistently disrupted. A water mass, with characteris-t i c parameters, mixing with a differing water mass w i l l produce a transi-tion zone. Johnson and Brinton (1963) believe such a transitional zone can be defined by a sharp temperature gradient, and that these regions may cover expanses of several hundreds of miles. Regimes of this variety and size, which are usually found i n the eastern half of the oceans, are large enough to contain endemic species (Beiri, 1959). It i s thus reasonable to postulate the occurrence of populations unique to the transitional region. The electrophoretic data for the oceanic stations most influenced by mixing, 32 and 54, supports the above theory. At both stations, the pattern with the most bands predominates; to the south no animals were caught because the stations were i n subtropical waters. As successively more northern stations were sampled, a cline i n the predominance of band-ing patterns was observed, eventually resulting in the pattern with the fewest bands predominating in the subarctic water mass. - 39 -Although the rank occurrence between each band pattern from the transitional regime was similar to that in neri t i c areas, the relative frequency of each pattern differed. The spread between the occurrence of the types of patterns was much greater in the neritic areas (Fig. 9). Presumably this was a product of the amount of s t a b i l i t y i n the habitat. Fluctuations i n parameters are much more extensive in n e r i t i c regions than in the transitional regions, and this i s reflected i n the percentage occurrence of a band pattern. Stability i n the oceans i s most pronounced in the central regions of a water mass. Fluctuations are negligible below 100 m and those in the surface layers are relatively small. According to the predicted hypothe-sis (p. 36), increased s t a b i l i t y would result in an increased percentage of organisms with the lower number band pattern. The electrophoretic data for the stations i n subarctic water (2, 10, 15, and 24) generally support the hypothesis. Also, as the stations progress southward, away from the center of the subarctic mass, the percentage of patterns with the least bands decreases. It might be suggested that, as members of the popu-lation are carried into a less stable environment, the members with the low number band patterns are differentially selected against. The result-ing population would thus show a preponderance of the patterns with high band number. It i s intriguing that at station 10, near the Columbia River plume, there i s a large deviation from the predicted occurrence of band patterns. - 40 -One possible explanation i s the influence of the Columbia River plume. Although water from the plume has been recorded 463 km offshore (Cross and Small, 1967), any increase in fluctuations that the Columbia River water may exert on station 10 (380 km offshore) i s expected to be slight, although closer inshore i t i s known to be significant (Hebard, 1966). It is also possible that the Columbia River water i s transporting a near shore population of euphausiids into a more stable oceanic environment. Such a phenomenon could lead to a spurious interpretation of the electro-phoretic data. In summary, the results suggest that the numbers of bands observed in the protein complement of a population of E_. pacifica varies directly with the amount of environmental fluctuation. An alternative view that phenotypic expression i n teleost fish increases with rising temperature (Tsuyuki, pers, comm.) does not appear applicable in the present study as neritic areas, although colder than transitional regimes, possess greater v a r i a b i l i t y . Furthermore, the temperatures of most oceanic stations (Fig. 6) are approximately equal although the va r i a b i l i t y in phenotypic expres-sion at these stations i s not. In the most stable environment samples, the central subarctic water mass, the pattern with the least number of bands predominates. As the stations sampled moved into transitional water, a shift occurred from lower to higher number of bands. The result was a predominance of the pattern with the highest number of bands i n the transi-tional area. Two possible hypotheses can explain such a phenomenon. - 41 -F i r s t , patterns with the low number of bands may have been selectively eliminated as the California current carried a population towards the transitional zone, or the transitional zone may be large enough to es-tablish a biochemically endemic population. It i s this latter postulate that i s favored, as i t can also explain the observed predominance of high number band patterns in neritic areas. INTERSPECIFIC STUDY The most widely accepted view on speciation suggests that varia-b i l i t y and isolation are the two most important factors i n species forma-tion (Kohn, 1960). Variation i n i t i a l l y may be exhibited either pheno-typically or genotypically, depending on whether the modification has been incorporated into the genome or i f the variation i s an acclimatization to a short term environmental parameter. The genotypic variation i s the more important since i t is only possible to propagate a new characteristic i f i t i s i n the gene pool. Such a phenomenon i s suggested by Johnson and Brinton (1963, p. 403) who believe that "thermal fluctuations occurring simultaneously within a series of intercommunicating oceans could have led to the rise of groups of euphausiid species." Such a theory has come under considerable criticism as mixing between oceans i s a common occur-rence (McGowan, 1963), and i t i s unlikely that separate daughter popula-tions would be maintained. Several other theories depend on barriers for isolation, but not to the same extent. Fluctuations between favorable - 42 -and unfavorable habitats lead to refugial pockets of organisms and to isolation of a genome (Brown, 1957). Hybridization between species occurs in fresh-water plankton (Brooks, 1957) and has been suggested as one mode of speciation in the oceanic environment. Buzzati-Traverso (1958) has put forward a hypothesis that does not require spatial i s o l a -tion but instead u t i l i z e s a genetic mechanism and differential survival as the basis for speciation. A mechanism of this variety has been shown to cause variation in Limacina helicina inhabiting the North Pacific (McGowan, 1963). Taxonomists classically have util i z e d morphological features of organisms as a basis for categorizing groups. Differentiation at phylo-genetic levels above species i s , for the most part, quite w i l l defined, but i t i s below the species level that many classification problems occur. Frequently populations, races, and sibling species are hopelessly indis-tinguishable using the classical morphological approach. It i s only within the last two decades that problems of this nature are being solved and this i s die largely to biochemical techniques u t i l i z e d in taxonomic studies. Manwell and Baker (1963) differentiated two sibling species of sea cucum-ber and later (1967) solved the taxonomic problem of distinguishing two morphologically similar copepods. Recent investigations i n speciation and population genetics of other marine animals—such as ectoprocts (Gooch and Schopf, 1971; Schopf and Gooch, 1971), pelecypods (Koehn and Mitten, 1972), - 43 -periwinkles (Wium-Andersen, 1970), and crabs (Inoue et a l . 1969), provide an insight into the relatively unexplored domain of marine invertebrates. The basis of biochemical taxonomy l i e s i n the generic relation-ships between species, and the degree of similarity or differentiation i n the protein patterns between species serves as an index of speciation (Sibley, 1962; Tsuyuki, et a l . , 1965, 1968). As Lim and Lee (1970) state: "this i s not unexpected, since protein synthesis in l i v i n g organisms are controlled by fundamental genetic systems, and their expressions lead to. structural differentiation, the products of which are a measure of genetic differences that could be employed to provide data for systematic studies." Moreover, closely related species tend to be similar in behavioral, develop-mental, functional, physiological and genetic background, a l l of which are reflected in the protein constitution. If we accept Lim's (1970) viewpoint, then i t becomes evident that each of the five species examined i s distinct, and that there i s a cline in the number of bands between species. Examination of the patterns (Fig. 7) reveals that the electrophoretic results parallel the existing morpho-logical classification. Both members of the genus Euphausia are biochemi-cally closely associated, as are the patterns cf Thysanoessa species, although these two genera show the greatest divergence. The variation between species of a genus is greater than that found within a species and the existing individual variation in E_. paci^'i£a i s never so extreme as to obscure the species specificity of the pattecns. - 44 -SUMMARY AND CONCLUSIONS Contemporary theories on i n t r a s p e c i f i c and i n t e r s p e c i f i c v a r i a -t i o n contend that environmental stresses are the underlying causes of biochemical, p h y s i o l o g i c a l and morphological v a r i a t i o n s . The number of modifications, and thus the v a r i a b i l i t y w i t h i n a genome, would be expec-ted to be greater i n a population that i s subjected to a wide range of environmental st r e s s e s . The objectives of t h i s study were to examine biochemically, populations of Euphausia p a c i f i c a from several d i f f e r i n g habitats and to examine biochemical taxonomic r e l a t i o n s h i p s between f i v e species of P a c i f i c euphausiids. Specimens were c o l l e c t e d from eight oceanic and three n e r i t i c s t a t i o n s between the Queen Charlotte Islands, B r i t i s h Columbia, and Gaudalupe Island, Mexico. To allow c l a s s i f i c a t i o n of the environments a number of p h y s i c a l parameters, s p e c i f i c a l l y temperature, s a l i n i t y and oxygen were monitored at each s t a t i o n . The temperature-salinity (T-S) p r o f i l e s i n d i c a t e that three oceanic water masses, the s u b a r c t i c , the t r a n s i t i o n a l , and the s u b t r o p i c a l , were sampled. There i s no T-S curve c h a r a c t e r i s t i c to the n e r i t i c s t a t i o n s due to large f l u c t u a t i o n s i n the p h y s i c a l parameters. Fluctuations i n the n e r i t i c environment are caused by seasonal changes while, most of the f l u x observed i n the oceanic re-gions i s a r e s u l t of mixing between d i f f e r i n g water types. The general proteins of one hundred specimens of E_. p a c i f i c a from each s t a t i o n were analyzed by h o r i s o n t a l starch gel e l e c t r o p h o r e s i s . - 45 -The general protein patterns of f i f t y specimens of each of Euphausia  pacifica, E_. gibboides, Thysanoessa spinif era, T_. inspinata and Nematos- celis d i f f i c i l i s were analyzed by the same technique. The electrophoretic results of the general proteins from E_. paci-f i c a indicate that two protein patterns predominate in the commonly occur-ring patterns, although minor patterns do exist at some stations. These two major patterns possess the least number of bands and the greatest number of bands (seven and nine respectively). A six banded pattern may be observed at ocean station 2 and i n Saanich Inlet, but these are exceptions to the normally observed patterns. A general trend i s evident between the frequencies of the two major patterns and the degree of environmental i n s t a b i l i t y (environmental fluctuation). The greatest environmental s t a b i l i t y i s in the central region of the subarctic water mass and i t i s here that the highest fre-quency of the major pattern with the least number of bands i s observed. The pattern with the greatest number of bands occurs in highest frequency in the very unstalbe neritic regions. A cline in the frequency of each pattern can be observed as the samples progress from one extreme (e.g. very unstable-neritic) to the other (very stable-subarctic). Thus, the relationships observed between electrophoretic and oceanographic data are in accord with contemporary theories on protein v a r i a b i l i t y . Anomalies occur i n the data that contradict these theories and explanations for - 46 -these anomalies are suggested. It must be reiterated that any variants in the populations are not directly related to any specific environmental parameter, but are thought to be associated with the degree of environ-mental s t a b i l i t y . Each of the five euphausiid species possess a unique electrophor-etic pattern for general proteins. The degree of biochemical similarities between species and genera parallel the existing taxonomic classification. Intraspecific variations are never so great as to obscure interspecific or intergeneric relationships. - 47 -VI. REFERENCES ARON, W. 1962. The Distribution of Animals in the Eastern North Pacific i n Relation to Physical and Chemical Conditions. J. Fish. Res. Bd. Can. 19 (2) :271-314. 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