Schultz et al.: Immunologic methods for species identification of early-life-stage lutjanid fishes 



737 



FPLC in lane 3, and the 0.32 M NaCl eluate of the 

 highly purified 66 kDa protein from a Mono Q anion 

 exchange column after FPLC in lane 4. Although the 

 biotinylated goat anti-GSE antiserum reacted with 

 many different proteins in the saline extract, it did 

 not contain antibodies that were reactive with the 

 trace 66 kDa protein in the Western blot (B, 1-4). In 

 contrast, where the goat anti-66 kDa IgG was used, 

 the protein was weakly positive with the saline ex- 

 tract (CD, but was strongly positive in the other 

 lutjanid preparations (C, 2-4). 



The purification procedure for the 66 kDa protein 

 was repeated 4 times, with an average yield of 119 

 ug/g of the whole L. griseus saline extract. The pro- 

 tein was determined to be a single-chain polypep- 

 tide under reducing conditions in SDS-PAGE. The 

 trace glycoprotein was not albumin (Hartmann et al, 

 1994), and under nonreducing, nondenaturing con- 



ditions, the lutjanid glycoprotein migration differed 

 from human or bovine albumin (mol. weight 66 kDa, 

 data not shown). 



After purification of the 66 kDa protein and pro- 

 duction of a specific goat antiserum, saline extracts 

 of different life stages of L. griseus were tested for 

 the presence of the protein. Western blot analysis 

 (Fig. 2) showed that the protein was present in all 

 stages: oocyte (lane 2), juvenile (lane 3), and adult 

 (lane 4). 



Saline extracts of 14 species of adult lujanids and 

 the hybrid (L. synagris x O. chrysurus) were prepared 

 by the same method (see Materials and Methods sec- 

 tion), and each extract was adjusted to 235 ug total 

 protein. After Western blot analyses of the 15 differ- 

 ent specimens, each blot was evaluated for the 66 

 kDa protein by visual inspection as shown in Figure 

 3, and the species were classified, as shown in Table 

 2. The species judged strongly positive were L. 

 griseus, L. jocu, and L. apodus because for these three 

 species the staining intensity of band patterns was 

 approximately equal. Weak reactors were L. buccan- 

 ella, O. chrysurus, P. aquilonavis, L. synagris, L. 

 analis,A. dentatus, andL. campechanus; L. vivanus, 

 L. mahogoni, L. cyanopterus, E. oculatus, and the 

 hybrid exhibited no reaction. 



Because the staining patterns of the 66 kDa pro- 

 teins of L. griseus, L. jocu, and L. apodus were of 

 similar intensity and because seven other species 

 reacted weakly with the anti-66 kDa IgG for West- 

 ern blots (Table 2; Fig. 3), an important question was 

 raised: Was the protein from the different species 

 identical but present in diminished concentrations, 

 or were dissimilar determinants being detected? To 

 investigate these questions, we selected L. griseus, 

 L.jocu, and L. apodus because their 66 kDa proteins 

 were strongly reactive with the specific antiserum. 

 In addition, other studies have shown close phyloge- 

 netic relationships among these three species 

 through morphological and biochemical data (Rivas, 

 1966; Vergara, 1980; Chow and Walsh, 1992) and by 

 analysis of mitochondrial DNA sequences (Chow et 

 al., 1993; Sarver et al., in press). Figure 4 shows the 

 results of adsorbing the goat anti-66 kDa IgG with 

 gluteraldehyde-insolubilized L. apodus saline extract 

 for 24 h at 4°C. Comparing the Western blot in A 

 (preadsorption) with B (postadsorption), we found 

 that the antiserum reacted equally well with the L. 

 griseus marker protein (lane 1, A and B) and the L. 

 griseus saline extract (lane 4, A and B). The pre- 

 adsorption antiserum also reacted with equal potency 

 with L.jocu (lane 2A) and L. apodus (lane 3A). After 

 adsorption of the antiserum with the insolubilized 

 L. apodus saline extract, bands in the Western blots 

 for L. jocu (lane 2B) and L. apodus (lane 3B) were 



