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Fishery Bulletin/U S Dept of Commerce National ELECTROPHORETIC INVESTIGATION OF THE FAMILY SCORPAENIDAE ALLYN G. JOHNSON, FRED M. UTTER, AND HAROr..o O. HODGINS' ABSTRACT Thirty-one species of three genera of the family Scorpaenidae were separated into 17 groups based on starch gel electrophoretic comparison of muscle proteins and six enzy­ matic systems. This study concluded that relatively greater similarity existed between the Pacific Sebastes and the Atlantic Sebastes than between either and the other genera. Ten of the 27 species of Pacific Sebastes tested had unique biochemical profiles which may be useful for identification of specimens. 'I'he family Scorpaenidae contains several ge­ in two out of eight species of Sebastodes. Altuk­ nera in the Pacific and Atlantic Oceans. On the hov and Nefyodov (1968') demonstrated serum Pacific coast of North America there are four protein differences between Sebastes marinus genera-Sebastes,' Sebastolobus, Scorpaena, and S. mentella using agar gel electrophoresis. and Scorpaenodes. The genus of Pacific This paper reports the findings of our inves­ Sebastes contains over 50 species (Tsuyuki tigation of proteins and six enzyme systems et al., 1968). In this genus new species and ex­ found in the skeletal muscle or liver of members tensions of known distribution ranges have been of the family Scorpaenidae. Our study involved described in recent years (Westrheim, 1965; 27 species of Pacific Sebastes, 2 of the Atlantic Westrheim and Tsuyuki, 1967; Nishimoto, 1970; Sebastes, and 1 each of Sebastolobus and Helico­ 'I'suyuki and Westrheim, 1970). At present lenus. We present information on the relative there are difficulties in showing taxonomic re­ biochemical similarity between genera and a ~ations and, in some instances, in making positive key which separates 10 of the 27 Pacific Sebastes ldentification of specimens using morphometric species studied. This was not a genetic study and meristic methods, although taxonomic re­ per se but a research which demonstrated repeat­ lations can be obtained by biochemical methods. able biochemical differences between species. ~tarch gel electrophoresis-developed by Smith­ The observed constancy of biochemical charac­ les (1955)-coupled with histochemical proce­ ters examined within a species in samples taken dUres (Hunter and Markert, 1957) is one of the at different ages, depths, and geographic loca­ best biochemical techniques for taxonomic tions is evidence that the reported differences stUdies. between species are, indeed, genetic. Alternate Scorpaenid muscle proteins and hemoglobin explanations for such repeatable expression of Were investigated by starch gel electrophoresis proteins under the above conditions seem much by Tsuyuki et al. (1968). They suggested that less likely. the electrophoretic evidence did not support the separation of the two genera Sebastodes and MATERIAL AND METHODS Sebastes. Chu (1968), using disk electrophore­ sis of muscle proteins, found different patterns Sampling data including location, species, and number of individuals collected are given in e ~ National Marine Fisheries Service, Northwest Fish- Table 1. ./les Center, Seattle Laboratory, a725 Montlake Boule- Most samples were frozen quickly after cap­ --a;d East, Seattle, WA 98102. ture, but in some instances were kept on ice for 1 In this paper we follow the designation of Bailey b( 970) and Chen (1971). considering Sebastodes as Se­ short periods; all samples were kept frozen at I Q8te8. Members of the genus Sebastes that were col­ ~.cfited along the Pacific Coast of North America are sig- -20°C after receipt at the laboratory until I ed in this paper as Pacific Sebastes. tested. Extracts were prepared by mixing equal ~ FlSHscnpt accepted November 1971. ERY BULLETIN, VOL. 70, NO.2, 1972. 403 FISHERY BULLETIN: VOL. 70, NO.2 TABLE l.-Location and number of specimens of Scor­ phosphoglucomutase were best resolved by using paenidae collected, 1968-70. the buffer system described by Ridgway, Sher­ Location' Total burne, and Lewis (1970). Gels consisted of 35 g Species number B G 0 of fish starch plus 250 ml of buffer. A voltage of 300 Pacific S~basttJ was applied for 10 min; sample inserts were S. allutianu, 10 6 16 removed and 400 v applied until indicator dye S. alutu, 217 843 1,060 S. auriculatul 76 76 markers reached a point 6 to 9 cm anodal to the ~~~ 3 3 origin. The gels were cooled during electro­ S. brtvi,pini, 5 40 45 S. cQlnarmaticus 3 phoresis by placing ice packs on glass plates on S. caurinu, 283 283 top of the gels. After electrophoresis, bands re­ S. chIorostictuJ 1 S. cramlri 2 16 18 flecting enzyme activity were detected by the fol­ S. diploproa 14 14 lowing methods: S. dongatu, 297 96 393 S. Intomda' 2 2 4 . Tetrazolium oxidase (TO) (after Brewer, 1967, S. lIavidu, 8 8 S. Atlvomaculatu, 5 19 24 and Johnson, Utter, and Hodgins, 1970b): S. Ilvi' J 5 mg phenazine methosulfate (PMS) ~m~~ e e S. mdanops 28 28 3 mg p-nitro blue tetrazolium (NET) S. pauci,pini, 2 15 18 40 ml tris-citrate buffer (0.03 M tris, 0.005 M S. pinniglr 24 24 S. proriglr 9 100 109 citric acid, pH7.0) S. mdi 1 110 111 S. rublrrimus 5 27 5 37 L-alpha-glycerophosphate dehydrogenase S. rubivinctus 5 34 39 (aGPDH) (after Nyman, 1967, and Johnson, S. JQxico/a 5 6 Utter, and Hodgins, 1970a): S. wil'oni I S. varilgatu, I 5 mg PMS S. ","ntrus 37 38 3 mg NET Atlantic Slba,t" S. marin.uJ 9 9 5 mg NAD+ S. viviparous 10 10 Stbast%buJ 100 mg L-alpha-glycerophosphate alascanu! 100 100 40 ml tris-citrate buffer H eliCO!lnUJ dactyloptlru! 10 10 Lactate dehydrogenase (LDH): 1 A = Pacific Coast of Washington and Orogon, 1968-70, B = Pugot 10 mg PMS ~9~od, ~~\;.,~~6~~2~t ~f Bri~~~o~n~hf;~I~~d,SAuu~~stB'f;;70~aEa~, ~~i7~ 5 mg NBT Beech, Calif., October 1970, F = Cape Ommaney, Alaska, April 1970. 5 mg NAD+ 20 ml of 0.5 M sodium lactate solution volumes of tissue and phosphate-buffered phys~­ 40 ml tris-citrate buffer ological saline solution (pH 7.4) into uniform Peptidase A (after Lewis and Harris, 1967, and pastes with glass rods. The extracts were tested Lewis and Truslove, 1969): by electrophoresis without further treatment by 10 mg DL valyl-Ieucine (1) drawing them into %,-inch X 3/16-inch filter 1 mg horseradish peroxidase paper inserts (Schleicher and Schuell grade S 5 mg O-dianisidine in 10 ml acetone and,S No. 470)', placed on the surface of the tis­ 0.5 ml M MgC12 sue-saline mixture, and (2) placing the inserts 1 mg Bothrops atrox venom into starch gels. 40 ml tris-citrate buffer Electrophoresis in starch gel followed the Phosphoglucomutase (PGM) (after Spencer, methods of Kristjansson (1963). All but two Hopkinson, and Harris, 1964): of the biochemical systems were resolved well 100 mg ,glucose-I-phosphate (dipotassium using a buffer system described by Markert and salt) Faulhaber (1965). Lactate dehydrogenase and 5 mg NADP 5 mg PMS , Reference to trade names in this publication does not imply endorsement of commercial products by the Na­ 3 mg NBT tional Marine Fisheries Service. 20 units glucose-6-phosphate dehydrogenase 404 JOHNSON, UTfER and HODGINS: ELECTROPHORETIC INVESTIGATION TABLE 2.-Classification of species of Scorpaenidae into various groups by means of biochemical characteristics. Biochemical characteristics Spedes Peptldas. A ,Biochemical Muscle group 1 TO GPDH LDH pattern ,I (Fast zone) II (Slow zone) PacIfic Stbastu S. dongatus 2 F E C I" S. tntomtlas 2 S E C c c II" S. aurora 3 F E C b d III" S. chiarostictul ~ F E B a IV" S. aleutianus ~ F E C c V S. zauntru! 4 F E C c V S. (aurinu! 4 F F, S C d c VI S. dip/oproa 4 F E C b b VII" S. lulvomaculatus 4 F E B c c VIII" S. "'alig" 4 F F C d c VI S. rubtrrimus 4 F E C c c V S. rubrivinctu! 4 F E C c c V S. saxicola 4 F F C c c IX" S. auriculatus 4 F E, F C d c VI S. buvispinis 4 F E C c c V S. Ifavidus 4 S E C c c X S. mtlanops 4 S E C c c X S. pinnigtr 4 S E C c c X S. proTig!r 4 S E C c c X S. wilsoni 4 S S. tlaritgatuJ 4 S E C a XI" S. eaenatmaticu! 4 S E C X S. alutus 4 S F, S C XII" S. crameri 4 VS E C c XIII S. Paucispinis 4 VS E C c XIII S. rudi 4 VS E C c XIII S. Itvis _a F E C a XIV" Stbast%bus alascanuJ A VS 0 A b 0, • XV' Atlantic Stbastts S. marinu1 B S E, F B c XVI S. viviparous B S E B XVI 1lt/i'o/tnus dacty[oPttrus G S,VS C B • b XVII" -; Modified rifter Tsuyuki et aI., 196B. Species with unique biological characteristics. 3 Pattern of the single specimen tested has not been described. 0.5 ml 1 M MgCh and destained with a 1: 4: 5 solution of acetic 40 ml tris-citrate buffer acid, methanol, and water. Isocitrate dehydrogenase (ICDH): (a) NADP dependent (after Opher, Leonard, ENZYME AND PROTEIN PHENOTYPES and Miller, 1969): 30 mg DL sodium isocitrate TETRAZOLIUM OXIDASE (TO) 5 mg NADP 0.5 ml 1 M MgCb Interspecific variation of TO was previously 5 mg PMS reported in the genus Sebastes (Pacific) by 5 mg NBT Johnson et al. (1970b), where three anodal mo­ 40 ml tris-citrate buffer bilities were observed in 15 species studied: (b) NAD + dependent: Fast (F), Slow (S), and Very Slow (VS).
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