Identification of sunfish species by muscle protein isoelectric focusing

Comp. Biochem. Physiol. Vol. 8411, No. 2, pp. 17%180, 1986 Printed in Great Britain

0305-0491/86 $3.00+0.00 Pergamon Journals Ltd

IDENTIFICATION OF SUNFISH SPECIES BY MUSCLE PROTEIN ISOELECTRIC FOCUSING D. H. WHITMORE Department of Biology, The University of Texas at Arlington, Arlington, TX 76019, USA (Tel: 817-273-2871)

(Received 16 October 1985)

Abstract--1. Muscle protein phenotypes of nine recognized Lepomis species were developed by isoelectric focusing. 2. Four classes were described based on putative parvalbumin patterns. 3. All nine species could be distinguished by their protein phenotypes. 4. Natural hybrids and their parentage were also identified, and the utility of this technique in studying natural sunfish hybridization is discussed.

INTRODUCTION Proteins, by virtue of their genetically controlled structural variations, have been studied extensively for some 30 years by electrophoretic techniques. Information about proteins and the genes controlling them has provided a huge data base for investigations of taxonomic and evolutionary relationships, development and population structure and dynamics. Newer methodologies such as isoelectric focusing, two-dimensional electrophoresis and D N A analysis are now beginning to find utility and will, undoubtedly, contribute significantly to the information provided by conventional electrophoresis. Isoelectric focusing (IEF) has been advocated as a powerful tool for fish species identification (Lundstrom, 1981). Protein profiles resulting from isoelectric focusing separations have provided Lundstrom with a library of species electrophoretograms that allows a highly successful rate in u n k n o w n identification. In the present study, the technique of isoelectric focusing is applied to muscle protein extracts of nine of the eleven species of Lepomis. The objectives of this study were two-fold: (1) to establish the utility of muscle protein phenotypes in the identification of these important freshwater species using a m i n i m u m a m o u n t of muscle tissue, and (2) to evaluate the potential use of isoelectric focusing in studies of Lepomis hybridization. MATERIALS AND METHODS Fish samples Samples of Lepomis macrochirus (bluegill), L. gulosus (warmouth), L. cyanellus (green sunfish), L. humilis (orangespotted sunfish), L. microlophus (redear sunfish), L. punctatus (spotted sunfish), L. auritus (redbreast sunfish) and L. megalotis 0ongear sunfish) were collected by seine or electrofishing in various lakes and streams in the vicinity of Dallas, Ft. Worth, Texas. Lepomis gibbosus was collected in Ontario, Canada. Isoelectric focusing procedure White muscle (400-500 mg) was dissected from the right lateral side or removed from the base of a clipped fin and 177

homogenized in an equal vol of distilled deionized water. Following centrifugation at 15,000g and 4°C for 20 min, the supernatant was retained for analysis by isoelectric focusing. Isoelectric focusing was performed on ultrathin (0.5 mm) agarose gels. A gel solution consisting of 0.8% agarose (Marine Colloids), and 10% sorbitol was heated in a boiling water bath until the agarose was completely melted. The temperature of this solution was reduced to 55°C in a water bath prior to the addition of 1% pH 34/ (Serva) and 1% pH 3.5-9.5 (LKB) ampholytes. This solution was injected between a glass plate sandwich. Both glass plates were lined with pieces of Gelbond (Marine Colloids), one with the hydrophobic surface up and the other with the hydrophilic surface up. A 0.5 mm plastic U-shaped spacer was placed between the pieces of Gelbond to complete the sandwich before it was clamped together. After the agarose solution had cooled to room temperature, the plates were carefully prised apart with a spatula and the gel with its Gelbond backing was placed in a humidity chamber (100%) at 5c'C for 1-24 hr. The IEF runs were performed on a flatbed isoelectic focusing apparatus (Hoeffer) which was thermoelectrically cooled to 7°C. Initial experimentation revealed that the pH 3-9.5 gradient employed in this study provided optimal separation of sunfish muscle proteins. Under these conditions, the anode solution of choice was 0.5 M acetic acid and the cathode solution was 1 M NaOH. Gels were run for 10min at 200V followed by 2hr at a constant 5W. Immediately following the completion of the run, electrode wicks were discarded and the gel was fixed for 20 min in a solution of 4% sulfosalicylic acid, 12.5% trichloroacetic acid, and 30% methanol. Fixation was followed by two 10 min washes in distilled water followed by drying. Protein bands were visualized by staining for 30 min in a solution containing 0.04% Coomassie Blue R-250, 40% methanol and 10% acetic acid. Gels were briefly destained in 10% methanol-15% acetic acid and dried. RESULTS Extracts of soluble muscle proteins subjected to isoelectric focusing result in phenotypic patterns of 40--50 proteins which can be utilized to identify sunfish species. Four classes (I-IV, Fig. IA) based on patterns of putative parvalbumin proteins are apparent. These highly acidic proteins have isoelectric points (pls) ranging from pH4.15--4.8 as extrapo-

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D.H. WHITMORE

Fig. 1. (A) Four Lepomis classes based on phenotypic patterns of putative parvalbumins. Isoelectric points of parvalbumins were estimated from co-focused standards. (B) Within each class, members can be further distinguished by specific proteins indicated with arrows.

lated from a co-focused mixture of standard proteins (Marine Colloids). It is probable that these proteins are parvalbumins since thay share two of the unique characteristics of these major fish muscle proteins;

low isoelectric points and low tool. wts (unpublished SDS-PAGE analysis). Confirmation of identification as parvalbumins awaits protein purification followed by analysis of calcium binding and u.v. absorption.

Sunfish muscle proteins Green, orangespotted and bluegill sunfishes all possess putative parvalbumins with pls of 4.5 and 4.4. This group is designated Class I. Redear and redbreast sunfishes constitute Class II, both species having proteins with pls of 4.8 and 4.4. Closely resembling the Class II parvalbumin phenotype is that of Class III which includes spotted, longear and pumpkinseed sunfishes. Class III fish have a parvalbumin with a pI of 4.4 like Class II fishes, however, the other major parvalbumin of Class III has a pI of 4.7 placing it just slightly anodal to the parvalbumin-4.8 of Class II. The final class, IV, has a single member, warmouth bass. While it has parvalbumin-4.5 like Class I fishes, it's second major parvalbumin has a pI of 4.15. Parvalbumin-4.15 is unique to the sunfishes examined and causes the warmouth to stand alone. Within each class, members may be distinguished from one another by careful examination of the protein profiles for shared and unique proteins. Arrows in Fig. 1B designate proteins that appear to be species-specific. During the course of this study, numerous specimens of several Lepomis species were analyzed to evaluate the extent of intraspecific variation of A muscle protein profiles. Preliminary results show some intraspecific variation, but not in the designated diagnostic proteins.

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In addition to species identification, protein phenotypes proved a useful tool for hybrid identification. Figure 2 illustrates the utility of protein phenotypes in the identification of hybrids between Class I and Classes III and IV. These hybrids are obvious by their composite parvalbumin patterns. Other diagnostic proteins are then used to identify the parental species. The three hybrid fish having bluegill genes are probable Fl generation because they appear to exhibit phenotypes that are combinations of parental patterns. Bluegills are known to hybridize in nature with at least seven of the ten congeneric species (Hubbs, 1955: Hubbs and Hubbs, 1932, 1933: Krumholtz, 1950; Childers and Bennett, 1961; Bailey and Lagler, 1938; Lagler and Steinmetz, 1957). The fourth hybrid protein phenotype (WM x OSPT) is more cryptic. It possesses some of the proteins of each parental type, but not all. There is an obvious asymmetry in the inheritance of one of the major parvalbumins from the orangespotted line. It is possible this specimen does not represent the F~ generation, but is the result of backcrossing or subsequent filial generations. Alternatively, this phenotype may be an example of nuclear-cytoplasmic incompatibility, resulting in allelic supression. It has been established that interspecific crosses can result in offspring that show allelic supression (Whitt et al.,

Fig. 2. Hybrid sunfish protein profiles and the parental species.

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1972), particularly if the species are not closely related. The resolution to this problem requires further experimentation, but it is clear that this is a hybrid fish with warmouth and orangespotted genes. Detection of intraclass hybridization is more subtle since the resulting hybrid protein phenotype will possess identical parvalbumins; however, there are sufficient numbers of diagnostic proteins to allow parental identification. DISCUSSION Species of Lepomis were originally described on the basis of their morphology. Adults of all 11 species can usually be accurately identified since they tend to remain morphologically distinct throughout their respective ranges. Errors in morphological identification, or at least uncertainty, occur in immature fish and where hybridization has occurred. In the present study, the utilization of isoelectric focusing of soluble muscle proteins easily permitted the accurate identification of each of the 9 Lepomis species examined (L. symmetricus and L. marginatus were not examined). Even small muscle samples taken from a fin clip can provide ample material for IEF analysis, thus avoiding the necessity of killing specimens as is frequently required in conventional electrophoresis. IEF offers several other advantages in problems of species identification from small tissue samples or dilute tissue extracts. Gels 0.5 mm or thinner can be run quickly at high voltages while being effectively cooled. Thin gels are more quickly stained and destained. Resolution of protein phenotypes can be excellent, with 40-50 scorable proteins from 5 or 10 #1 of extract. Since this is an endpoint technique, timing of the procedure is less critical than electrophoresis and comparisons between gel runs are facilitated by protein migration to specific pH points in a gradient that correspond to their isoelectric points. Gradient pH ranges may be altered using different ampholyte solutions such that zones containing particularly useful proteins can be selected for study. Finally, in addition to employing protein stains it is possible to study specific enzymes with procedures commonly employed with electrophoresis (Sevigny and Odense, 1985). The establishment of protein phenotypes of economically important fish surely can serve as fingerprints for identification. For these reasons, IEF may hold special attraction for law enforcement agencies which are often faced with questions of identification of unknown fish muscle. Not only were Lepomis species identifiable by their muscle proteins, but so were hybrid specimens. It is immature fish and hybrids that are frequently morphologically cryptic, often requiring biochemical techniques for accurate identification. It was once thought that natural hybridization was restricted to the Fj generation (Hubbs and Hubbs, 1933) but as results of artificial matings accumulated it became evident that many sunfish hybrids are not sterile and that F 2 and F 3 generations can be produced and successfully backcrossed to parent species (Childers, 1967). Studies of Lepomis population structure using electrophoresis (Avise and Smith, 1974a,b, 1977:

Felley and Avise, 1980) and DNA analysis (Avise et al., 1984; Avise and Saunders, 1984) have indicated that the question of the extent of natural hybridization among Lepomis species and its ramification on the evolution of this genus remains unanswered. These biochemical procedures, including isoelectric focusing have important roles to play in the resolution of this question. Acknowledgements--I wish to express my thanks to Drs Thomas Hellier and David Philipp and to Mr Allen Forshage for their assistance in collecting sunfish. REFERENCES

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