'1- Comp. 8iod1em. Ph _niol. Vol. 72 B. pp. I t o~. 1981 0305-049 1 82 05000 1-05S03.00 0 Prin ted m Great Britain. ID 1982 Pergamon Press Ltd SERUM ELECTROPHORESIS AND SEA TURTLE CLASSIFICATION WAYNE fRAIR Department of Bi ology, The King's College, Briarcliff, NY 10510, U.S.A. (Received l3 August 1981) Abstract- !. Electrophoresis and immunoelectrophoresis reveal that Caretta, Eretmochelys and Lepido­ chelys share considerably simil ar blood serum proteins. 2. Proteins from Chelonia are more like those of Caretta and Lepidochelys than like Eretmochelys. 3. Dermochelys proteins are most distinct among living sea turtles and Dermochelys is classified either in the same family or superfamily with other sea turtles. INTRODUCTION as indicated in Frair (1979). In addition, the following are preserved with The American Museum of Na tural History In past decades discussion regarding taxonomy of sea (AMNH), University of Utah (UU) or my (WF) numbers­ turtles has centered mostly on the enigmatic leather­ Caretta c. caretra: AMNH 107110, 107111 , 107 112 ; WF back, Dermochelys, with its large size and lack of a 104 ; Chelonia m. mydas: AMNH 107114, 11 8638 ; WF 106; I Dermochelys coriacea : AMNH 118648 ; UU 11 794, 11795: hard shell. In more recent years the tendency has been to recognize a close affinity of the leatherback with Eretmochelys i. imbrica ra: AMNH 118642 ; Lepidochelys other marine turtles (see Frair, 1979). In the past, elec­ olivacea: AMNH 11 8644, 11 8645, 118646. trophoretic studies involving sea turtles have been limited by the number of specimens o r the number of species included (see Frair, 1969 ; M usq uera et a/., 1976; Smith et a/., 1977; Michael, 1978; Bashtar, RESULTS 1979). So this project was designed to add infor­ From the serum electrophoregrams pictured in Fig. mation gained from electrophoresis of a considerable 1 it is apparent that all sea turtles have some similar­ number of specimens from all five types of sea turtles. ly-migrating lines ; also they have relatively short pat­ terns as compared with human (and compared with many other turtles as well (Frair, 1964)). Although MATERIALS A D METHODS some intraspecific variability occurred, it more often Bl ood was obtained by aseptic cardiocentesis through was quantitative (heaviness of lines) rather than quali­ the mid-ventral plastral seam. Serum was separated and tative (position of lines). With the procedure utilized usually frozen until used for electrophoresis which was per­ consistent differences between sexes were not ob­ formed according to basic procedures fr om Helena Labor­ served. atories. Runs were in 25 em-wide cellulose acetate plate at constant 180 V for 30 min using Tris- ethylenediaminetetra­ The sea turtle genus with the fastest-moving anodal ceti c acid buffer at pH 8.8 and ionic strength of 0.05. Strips line (albumin) is Chelonia, this being followed closely were cleared in methanol, glacial acetic acid and polyethyl­ by Dermochelys. Other sea turtles tend to have simi­ ene glycol (70: 30:4). larly-positioned slower-moving leading albumin, a Most reactants used in immunoelectrophoresis (except in component which is important taxonomically. Preal­ anti-DE runs) were serum pools, the antiserums being from bumin lines were seen with a few specimens, for a mong those utilized in the work of Frair (1979). To ab­ instance, three Chelonia possessed leading anodal l sorb an anti serum I mixed a small volume of the absorbent triplets. erum wi th it , all owed time for reacti on, centrifuged and Not only do Caretta, Eretmochelys and L epido­ poured off the supernatant which was mixed with more absorbent and the process continued until no more precipi­ chelys have similarly-positioned leading albumins but tate was observed after centrifugation. also their total patterns, including position of the The number of specimens followed by straight-line cara­ wide cathodal line and central region, are more simi­ pace lengths in em were: Caretta c. caretta, 36 (7-100); lar to one another than to either Chelonia or Dermo­ Chelonia m. mydas, 35 (4.7-120); Dermochelys coriacea, 13 che/ys. The cathodal line of Chelonia is considerably (9-165) ; Eretmochelys i. imbricata, 26 (4.9-89); Lepidochelys more anodal than the cathodal line of the o ther hard­ kempi, 16 (2 1-62): Lepidochelys oli vacea, 22 (4.7-73). Using shelled specimens. serums from the a bove, 340 electrophoretic patterns were The L epidochelys kempi and L. olivacea patterns are employed in a comparative study which a lso included an similar, but among sea turtles L. kempi was the most additional 49 pa tterns from pools (each of 2- 8 specimens, averagi ng 4) composed of serum from the above and at variable electrophoretically; whereas L. olivacea is least an additional 6 specimens of Caretta and one of Che­ more like the other sea turtles, particularly Caretta lonia. More than 400 other plates were used in immuno­ and Eretmochelys. L. o/ivacea characteristica ll y has electrophoresis and protein identification. two close anodal lines (not obvious in Fig. I), but they Turtles were o btained from many locations but mostly are not as clearly separated as the two heavy anodal eastern United States, Mexico and Surinam and deposited lines of Chelonia. <. 11.1 '. 72 !11 " 2 WAYNE FRAIR + Caretta c. caretta Chelonia m. mydas Chelonia m. mydas Dennoche lys cori ace a Eretmochelys i. imbri cata Caretta c. caretta Lepidochelys kempi lepidochelys olivacea Human Lepidochelys ol ivacea Terrapene c. carolina ~ ­ Eretmochelys i. imbricata Fig. I. Photograph of ce llulose acetate plate electrophore­ grams of sea turtle serums."- DeYTJloche lys cori ace a Five L. oliuacea hatchlings were dug from sand 53 Chelydra s . serpentina days after the eggs were laid and they were bled 4 days later before being offered food. Their electro­ Terrapene c . carolina phoretic patterns lacked the wide cathodal band seen for mature males and females. Fig. 2. Immunoelectrophoresis employing an anti-Che/onia Some preliminary tests have been run to identify pool applied at upper edge of strip after electrophoresis proteins within the electrophoretic patterns studied of serum from list ed organisms. here. Included have been lactic dehydrogenase enzymes, glycoproteins including haptoglobins and lipoproteins. I have seen a strong and similarly­ reference organism, thus producing a series of final migrating glycoprotein line especially in Chelonia, values with unity for the reference organism and Dermochelys and Terrapene, whereas this line for Che­ higher values for organisms represented by weaker lydra is slightly anodal of them. Additional lines of patterns. For each species the final value for ranking these four and other turtles als o show the presence of was averaged with the final value from width-density­ carbohydrates. Three lipoprotein lines have been length calculations to produce the numbers given in identified especially for Caretta, Chelonia, Chelydra, Table 1. With values obtained after absorptions the Eretmochelys and Terrapene. larger drops (and thus greater elevations of Table 1 With immunoelectrophoretic strips each arc of a values) are indicative of greater similarity among pattern was given a numerical value after careful vis­ serum proteins of forms being considered. ual observation (with magnifying lens) of its width, Results from immunoelectrophoresis (Table 1) density and length. A sum was obtained for values of show Dermochelys to be the most diverse of sea turtles all arcs in each pattern. For instance, sums for pat­ and Chelonia not closest to Eretmochelys as popularly terns of a single run as showed in Fig. 2 ranged from believed. In many runs Caretta tested somewhat more 34 for Chelonia to 12 for Terrapene. The sums for all like Chelonia than did Lepidochelys which usua lly was the patterns of the reference antigens (Chelonia in Fig. close, however. Also Dermochelys absorption did not 2) were added and this grand total divided by the indicate closest similarity of Dermochelys to Lepido­ grand total for each of the turtles. This procedure chelys. Both Caretta and Lepidochelys olivacea gener­ resulted in a final value of unity for the reference ally appeared more similar to Chelonia than did Eret­ organism, slightly above unity for organisms tending mochelys. Although Terrapene reacted well with anti­ to have weaker patterns, and the highest value for the Chelonia, in many of the runs heavier arcs were seen organism with weakest patterns. for Chelydra (see Fig. 2). Both of the testudinids, Che­ Also each pattern in a run was given a rank value, lydra and Terrapene, reacted similarly. My evalu­ Number 1 for the heaviest (typically the reference ations using immunoelectrophoresis I consider only antigen's pattern) to 7 for the weakest pattern. All rough approximations because of the difficulties values for a species were added to obtain a sum for attending efforts to quantify these reactions (see Wil­ that species. Each sum was divided by the sum for the liams & Chase, 1971). Table I. Eva lu ation of immunoelectrophoretic strips Anti Absorbent No. Runs CA* CH DE ER LE cs TC CH 16 L76t 1.00 3.37 2.70 2.39 3.49 J72t CH cs 10 2.30 1.00 3.66 2.65 2.97 19. 39~ 6.87 CH DE 6 2.77 1.00 5.41 * 3.17 2.08 4.05 387 DE 3 2.28~ 1.24 1.00 2.26 3.03 * CA, Caretta c. caretta ; CH, Chelonia m. mydas; DE, Dermochelys coriacea; ER , Eret­ mochelys i. im!Jricata; LE , Lep idochelys olivacea; CS, Chelydra s. serpentina ; TC, Terrapene c. carolina. t Valu es obtained by averaging the visual evaluation means with means for rank in each run. t Only 12 runs. ~ Slight reaction in about half the runs. ~Patterns with CA , ER and LE were nearly identical.