The author is also indebted to the follow- 

 ing persons and organizations: O.E. Sette of 

 the Pacific Oceanic Fishery Investigations, Dr. 

 M.B. Schaefer, Inter- American Tropical Tuna 

 Commission, andE.K. Holmberg, Fish Com- 

 mission of Oregon, for directing the collection 

 of many samples of the frozen whole tuna blood. 

 Vernon E. Brock, Division of Fish and Game, 

 Board of Agriculture and Forestry, Honolulu, 

 Hawaii, Dr . Robert Hiatt, Department of 

 Zoology, University of Hawaii, Dr . Howard 

 Buroughs, and the staff of the Hawaii Marine 

 Laboratory assisted the author in many ways. 

 The author is also indebted to Dr. Lionel A. 

 Walford, U .S . Fish and Wildlife Service, and 

 Dr. Carl L. Hubbs, Scripps Institution of 

 Oceanography for much advice and encourage- 

 ment during these studies. The collection and 

 identification of tuna during the author's stay at 

 the Hawaii Marine Laboratory was made poss- 

 ible through the assistance of the following men: 

 Georges Gilbert, skipper of the Makua, re- 

 search vessel of the Hawaiian Division of Fish 

 and Game, Lester Zukeran, skipper of the 

 Salpa, research vessel of the Hawaii Marine 

 Laboratory, and Harry Yagi, owner and skip- 

 per of the aku boat Venus , Honolulu . Dr . Leon 

 E. Mirmose of the Blood Bank of Hawaii con- 

 tributed samples of human blood. Hyland 

 Laboratories, Los Angeles, have donated human 

 blood-typing serums. Dr. George Ridgway, 

 U.S. Fish and Wildlife Service, contributed 

 suggestions on the manuscript. 



MATERIALS AND METHODS 



TTie tuna bloods used in these studies 

 were obtained either as frozen whole bloods 

 transported to Santa Barbara by air, or as 

 fresh bloods collected in the vicinity of Oahu. 

 Bloods were taken from living fish by various 

 methods such as heart puncture, tail bleeding, 

 or cutting the conus arteriosus at its narrowest 

 constriction beneath the gills. The author's 

 experience was that the most practical method 

 was the latter, cutting the vessel with a knife 

 and collecting the blood in a wide mouth screw - 

 cap glass bottle (plastic is recommended for 

 future work). Twenty five to 100 milliliters of 

 blood could readily be obtained in this way from 

 fish averaging two to three feet in length. 



In addition to the frozen and fresh 

 bloods studied, a collection of nine skipjack 

 bloods was taken from fish being unloaded at 

 the dock after a day's fishing. These fish had 

 been dead and iced for approximately 6 to 10 

 hours . The bloods taken were kept at refriger - 

 ation temperatures for approximately 36 hours 

 or longer while the author went collecting fresh 

 bloods on the Venus . This series of bloods 

 showed varying degrees of hemolysis, but eight 

 of the nine samples upon washing yielded sus- 

 pensions of erythrocytes that appeared in ex- 

 cellent condition. 



For reason of time, further study of 

 this material from dead fish was dropped in 

 favor of working on fresh cells, but the obser- 

 vations just reported suggest that it may be 

 feasible to sample catches of tuna several hours 

 after they have been taken. Erythrocytes col- 

 lected from living fish and kept in whole blood 

 at ordinary refrigeration temperatures lasted 

 as long as a week . 



This stability, coupled with the rela- 

 tively large amounts of blood obtained from 

 single fish, proved of great basic value in the 

 studies to be reported. Tuna bloods did not 

 seem to clot to the extent that many other fish 

 bloods do, relatively small clots being formed 

 in a given sample . Two -percent washed cell 

 suspensions were prepared fresh each day, 

 (even though such preparations often kept well 

 for 3 or 4 days in the refrigerator) . They were 

 made by washing the erythrocytes contained in 

 a half milliliter of whole blood 3 or more times 

 in at least 15 ml. of 1.5 percent sodium-chlor- 

 ide solution. This salt solution proved so 

 satisfactory that no comparative studies were 

 made on other types of solutions. 



Agglutination tests were made by putting 

 2 drops of 2 -percent cell suspension together 

 with 2 drops of suitably diluted antiserum in 

 test tubes (10 mm. X 70 mm.), allowing them 

 to stand at room temperature for 15 minutes, 

 centrifuging at 1, 000 r.p.m. for 30 seconds, 

 and observing the degree of agglutination upon 

 resuspending. The conventional method of 

 recording by estimating degree of agglutination 

 in terms of pluses was employed, with 4+ 



