THE APPLICATION OF PAPER CHROMATOGRAPHY IN 

 IDENTIFYING TUNA LARVAE 



By 



U. 



Walter M. Matsumoto 

 Fishery Research Biologist 

 Honolulu Biological Laboratory 

 S. Bureau of Commercial Fisheries 

 Honolulu, Hawaii 



The species identification of larval and 

 postlarval tunas has constituted an important 

 segment of the research program of the Bureau 

 of Commercial Fisheries, Honolulu Biological 

 Laboratory. The usual method of identifying 

 the larger juveniles and tracing certain diagnos- 

 tic characters through a decreasing size series 

 to the smallest available larva has been employ- 

 ed on Sonne species with general success (Matsu- 

 moto, 1958; 1959). However, the identification 

 of the younger larval stages becomes increas- 

 ingly difficult as smaller and smaller larvae 

 are examined. At the very earliest stages of 

 development, it maybe impossible to distinguish 

 one species from a closely related one. To 

 complicate nnatters further , there are a number 

 of species of tunas such as bigeye (Parathunnus 

 sibi), albacore ( Thunnus germo) ,bluefin ( Thunnus 

 thynnus, and Thunnus maccoyi), dogtooth (Gym- 

 nosarda nuda ), whose larval and postlarval 

 forms have not been identified positively. In an 

 attempt to find a faster and easier method of 

 identifying some of these larvae, techniques 

 ennployed in other fields of research were re- 

 viewed. One method which was considered wor- 

 thy of investigation was that of paper chromatog- 

 raphy. 



Paper chromatography is an important 

 technique used in the identification of chemical 

 compounds. Its merit, apart from simplicity, 

 is its extreme sensitivity, a small amount of a 

 substance being easily detected under suitable 

 conditions. Paper chromatography may be ei- 

 ther one- or two-dimensional (for details, see 

 Block, Le Strange and Zweig, 1952; Williams, 

 1954). In one-dimensional tests a sample ofthe 

 solution to be analyzed is placed near one end 

 of a strip of filter paper and that end of the pa- 

 per is dipped into an appropriate solvent. Cap- 

 illary action draws the solvent through the pa- 

 per and through the spot where the sample solu- 

 tion had been applied. Each substance in the 

 sample will move along with the solvent at a 

 unique rate, such that all the substances in the 

 sample will occupy a distinct position some- 

 where along the path of flow of the solvent. The 

 rate of movement of each substance, and there- 

 fore the final position attained by each substance, 



in a given time, depends upon its solubility in 

 the particular solvent used. Upon drying and 

 treating the paper with a suitable indicator, the 

 various substances appear as visible, colored 

 spots. Substances within the sample which can- 

 not be clearly separated or identified in one- 

 dimensional chromatography may sometimes be 

 separated by the two-dimensional method. In 

 this a large sheet of paper, about 20 x 20 inches, 

 is used. The paper is dried after the application 

 of the first solvent and the side nearest the sam- 

 ple is then dipped into a second solvent. Upon 

 completion of the development, the paper is 

 dried and treated with a suitable indicator. 



Initially the purpose of this study was 

 (1) to explore the usefulness of paper chromatog- 

 raphy in identifying the larvae and postlarvae 

 of several species of closely related tunas, es- 

 pecially yellowfin (Neothunnus macropterus), 

 bigeye and albacore, and then if possible, (2) 

 to apply the method under field (shipboard) 

 conditions. 



On the assumption that the free amino 

 acids in the muscle tissues of fishes are hered- 

 itary, the chromatograms obtained from sam- 

 ples of adults and larvae of the same species 

 should show similarities. The plan of work fol- 

 lowed, therefore, was first to determine the 

 differences or similarities among the various 

 species of adult tunas available for study; sec- 

 ond, to connpare the larval and adult samples; 

 and third, to determine species differences a- 

 nnong the tuna larvae. 



Ordinarily in chromatographic work the 

 concentration of specific substances in the sam- 

 ples tested would be determined by quantitative 

 analysis. This was not considered worthwhile 

 in this study, since, initially at least, I was in- 

 terested only in deternnining if there were sig- 

 nificant differences in the general chromatogra- 

 phic patterns of the amino acids found in tuna 

 flesh. Consequently, possible variations due to 

 locality of capture, between sexes, changes in 

 amino acid complex with length of time the fish 

 were kept in storage, and other variables were 

 not considered. 



