(October-November 1983). Because albacore have 

 a high titer of red blood cells (Alexander et al. 1980), 

 it was expedient to separate the lymphocytes from 

 the erythrocytes. The lymphocytes were isolated 

 from the blood on a density gradient of ficoll-sodium 

 diatrizoate solution using a modification of the tech- 

 nique developed by Boyum (1968), which is specific 

 for the concentration of lymphocytes. We found that 

 it was necessary to isolate the lymphocytes and place 

 them in culture within a couple of hours after blood 

 samples were collected. The ficoll gradient pro- 

 cedure was not successful using undiluted hepari- 

 nized blood that was retained for more than a few 

 hours. 



Two albacore, three skipjack tuna, and four 

 yellowfin tuna were sampled. All fish were juveniles 

 which have virtually no sexual dimorphic character- 

 istics, and no sex determinations were made. The 

 estimated fork lengths of the fish ranged from 65 

 to 85 cm for albacore, 80 to 120 cm for yellowfin, 

 and 45 to 55 cm for skipjack. 



From each fish, an 8-10 mL sample of blood was 

 withdrawn via sterile intracardial puncture into a 

 syringe coated with 1,000 units/mL of heparin. Two 

 mL aliquots of blood were pipetted into each of the 

 four 15 mL centrifuge tubes, and 4 mL of cell culture 

 medium 2 was added. The mixture was centrifuged 

 at 20 g for 5 min, and the white cells and plasma 

 were transferred to another centrifuge tube. This 

 procedure for the separation of the plasma and white 

 cell mixture was repeated three times following the 

 suggestions given by Blaxhall (1981). 



Five mL of the white cell-plasma mixture were 

 layered over 3 mL of ficoll-sodium diatrizoate solu- 

 tion and centrifuged at 572 g for 30 min. The over- 

 laying plasma was removed carefully with Pasteur 

 pipets, and the lymphocytes below were transferred 

 to a culture tube containing 5 mL of marine teleost 

 cell culture medium (Michael and Beasley 1973). This 

 procedure resulted in an erythrocyte free culture of 

 lymphocytes having a higher mitotic index. The 

 cultures were incubated at 25 °C for 3-5 d, at which 

 time they were terminated and the cells harvested. 

 The techniques for chromosomal analysis were pat- 

 terned after those of Nowell (1960) for mammals 

 because tuna are also endothermic (Graham and 

 Dickson 1981). This work is an extension of the pro- 

 cedures developed by Kelly and Laurs (1983 3 ). 



Prior to harvesting the cells, 0.5 \ng colcemid was 

 added to 5 mL of culture medium and incubated for 

 2 h at 25° C. The culture was then centrifuged for 

 5 min at 180 g and the supernatant was replaced 

 with 5 mL 0.075 M KC1 for 10 min. The culture tubes 

 were centrifuged again for 5 min at 180 g, and the 

 supernatant was replaced with 3 mL of freshly 

 prepared cold fixative which consists of 3 parts 

 methyl alcohol to 1 part glacial acetic acid and mixed 

 for 1 h. The tubes were again centrifuged at 180 g 

 for 5 min, decanted, and fixed. The cell pellet plus 

 0.5 mL of fixative was retained for slide preparation. 



Precleaned slides dipped in methanol and then in 

 deionized water were used for slide preparations. 

 Two drops of cell suspension were placed on the slide 

 and 4 drops of fixative were immediately added. The 

 slide was dried on a slide warmer at 37 °C and stored 

 at room temperature for 24-72 h prior to C-banding. 

 The C-banding procedures were patterned after the 

 work of Pardue and Gall (1970) and Arrighi and Hsu 

 (1971). 



In preparation for C-banding, the slides were 

 placed in 0.2 N HC1 for 15 min at 37°C, rinsed in 

 deionized water, treated with saturated Ba(OH) 2 at 

 room temperature for 7 min, and rinsed in deionized 

 water. They were then immediately dipped again in 

 0.2 N HC1 for 10 s and rinsed in deionized water. 

 After the final rinsing the slides were incubated in 

 2x sodium chloride-sodium citrate solution at 60 °C 

 for 90 min and then stained for 90 min in Giemsa 

 diluted with 1:10 Sorenson's buffer pH 6.8. Suitable 

 metaphase figures were photographed at 1,008 x 

 magnification using a Zeiss 4 microscope equipped 

 with a phase planapochromat 63/1.4 oil immersion 

 lens. 



Results 



Chromosome Numbers 



Kelly and Laurs (fn. 3) found that the diploid 

 number of chromosomes for albacore was 48. We 

 have confirmed this observation and have found that 

 the diploid numbers for yellowfin and skipjack tuna 

 are also 48. The modal frequencies of about 90 cells 

 containing 48 chromosomes were 82.2% for alba- 

 core, 92.6% for yellowfin, and 80.5% for skipjack. 

 Kelly and Laurs also observed that 85% of albacore 

 cells had 48 chromosomes. Two polyploid cells with 



2 RPMI-1640 Sigma Cat. No. R6504. 



3 Kelly, Raymond M., and R. Michael Laurs. 1983. Summary 

 of methods developed for investigations of albacore chromosomes 

 and of findings made on number of chromosomes. Unpubl. field 

 and laboratory notes and results (April 1983). [Raymond M. Kelly, 

 School of Medicine, University of California, La Jolla, CA; R. 



Michael Laurs, National Marine Fisheries Service, NOAA, La Jolla, 

 CA.] 



4 Reference to trade names does not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



470 



