all of the species. Larvae of two species of 

 tunas, skipjack and yellowfin, measuring from 

 5. 5 mm. to 10. 5 mm. were used for these tests. 

 Four skipjack samples consisting of 1 , 2, 3 and 

 5 larvae, and three yellowfin samples consist- 

 ing of 1 , 2 and 3 larvae were tested. In addi- 

 tion, samples of muscle tissue fluid from adults 

 of skipjack and yellowfin were used as controls. 

 Each larval sample was mashed on the filter 

 paper with the end of a glass rod, and the tis- 

 sue was removed to prevent streaking around 

 the point of application. 



In addition to the three solvents mentioned, 

 n-butanol:acetic acid:water was also tried and 

 excellent separation was obtained with them. 

 Of the four solvents used, the two n-butanol 

 solutions provided the best results, m-cresol 

 was fair, and phenol was not satisfactory. 



Another series of chromatograms was 

 prepared in or.der to determine the proper size 

 of the larval sample required for clear separa- 

 tions. In this series n-butanol:methylethylketone : 

 17 N ammonia:water , which had proved success- 

 ful for the adult samples, was used as the sol- 

 vent. There was good separation in the 2- and 

 3-larvae samples, but only faint traces were 

 obtained from samples with 1 and 5 larvae. 

 Thus, it was decided that a sample size of about 

 3 larvae was adequate. 



Results 



Interesting results were obtained in the 

 chromatograms developed in the two n-butanol 

 solvents (fig. 1). In solvent A (n-butanol:acetic 

 acid:water) it was possible to distinguish the 

 adults of the two frigate mackerels from the 

 other tunas by the smaller number (5) and posi- 

 tion of the spots. However, the two species did 

 not differ much from each other. It is not pos- 

 sible to state how much of this difference was 

 due to the long period (about 1 year) the frigate 

 nnackerels had been kept frozen. Of the other 

 species, albacore showed 7 spots and was easily 

 separated from bigeye, yellowfin and skipjack, 

 which had nearly identical patterns of 8 spots 

 each. All of these sannples, except skipjack 

 were obtained from the fish market. 



Somewhat different results were obtained 

 when the same samples -were run in solvent B 

 (ji- but anol:nnethylethylke tone: ammonia: water). 

 The two frigate mackerels had more spots (8) 

 than in solvent A. The positions of the spots 

 differed, indicating the presence of additional 

 amino acids not shown by solvent A. Of the 

 other species, bigeye and albacore had fewer 

 spots (6 and 7, respectively) and their positions 

 differed, but there was no change in the number 

 of spots in yellowfin and skipjack. 



Figure 1 . --One-dimensional chromatograms of 

 adult tuna samples developed in two different 

 solvents. 



Considering the number and location 

 of amino acid spots revealed with the two sol- 

 vents, it is possible to differentiate the adult 

 albacore and bigeye from the others. The two 

 frigate mackerels can be distinguished from 

 the other species by the smaller number of 

 spots in solvent A and from each other by the 

 position of the spots in solvent B. The yellow- 

 fin and skipjack can be distinguished from other 

 species, but not from each other. 



Chromatograms of the larvae of yellow- 

 fin and skipjack were generally comparable to 

 that of the adults, but exhibited wide variations 

 in the number of spots obtained. Five out of 9 

 sample applications (1- and 3-larvae samples) 

 produced good separation, showing 7 or 8 spots, 

 2 sample applications (1-larva samples) pro- 

 duced no spots and 2 sample applications ( 1 - 

 and 5-larvae samples) produced 3 or 4 spots. 

 In all instances where a sufficient number of 

 spots (8) were obtained, the positions of the 

 spots generally coincided with those of the re- 

 spective adult traces (fig. 2). However, inas- 

 much as the adults of these two species yielded 

 similar results, it was not surprising to find no 

 significant difference in the larval chromato- 

 grams of the two species. 



