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A reproducible method of culturing leukocytes from rainbow trout (Salmo gairdneri) has been found. Using an atmosphere of 100 percent oxygen, we stimulated peripheral blood leukocytes to divide at 19 degrees C. Cultured cells were used for a karyological study; they had a modal chromosome number of 60 and a constant arm number of 104, a value previously reported for other tissues in these fish.

Evolutionary and immunological relationships among the different genes specifying synthesis of rainbow trout (Salmo gairdneri) LDH subunits were demonstrated. A procedure was devised for the purification and concentration of specific rainbow trout isozymes which were then used for antisera production. The B4 • and B42 autotetramer isozymes and the Group "a" isozyme system were shown by double agar diffusion to produce distinctly different antibodies. Antisera against specific isozymes showed degrees of cross-reaction depending on the relatedness of the isozymes. The LDH-C gene locus is more closely related to the LDH-B2 gene than to the LDH-B• gene. Electrophoresis was demonstrated to be an efficient additional method to spectrophotometric assays to show inactivation of LDH by antibodies. LDH isozymes underwent rapid conformational changewhen purified and stored. • Based on part of a thesis submitted by J. R. Heckman in partial fulfillment of the requirements for the Ph.D. degree, The Pennsylvania State University, (1975) used antisera against purified chinook saltnon (Oncorhynchus tshawytscha) to study the immunochemical relationship of isozymes in brook and brown trout (Salmo trutta). The specific objectives of the present study in this regard were: (1) to devise a purification procedure to isolate rainbow trout LDH tetramers; (2) to prepare antisera against certain specific tetramers; (3) to deterthine the relatedness of rainbow trout LDH isozymes by use of these antisera. Markert and Faulhaber (1965) were the first to deal extensively with LDH patterns of fish. They found greater diversity in these organistns than had previously been reported for thatrituals or birds. The exceptional complexity of sahnonid LDH has led to much confusion in the classification and notnenclature of the various isozytne systetns. The present study utilizes the Wright et al. (1975) unifying system of LDH notnenclature in sahnonids (Table 1) which considers subunits, groups, and allelic variants as previously designated by Klose et al. (1968), Massaro and Markert (1968), and Utter and Hodgins (1972). Also, the LDH e gene described in the literature is now referred to as LDH-C (Shaklee et al. 1973). This notnenclature takes into account the genetic convention of designating loci with numbered subscripts. However, conventional 295

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The bluegill, Lepomis macrochirus Rafinesque, and the pumpkinseed, Lepomis gibbosus (Linnaeus), were crossed by an artificial fertilization technique. This resulted in two types of offspring; that is, Lepomis macrochirus X macrochirus and Lepomis macrochirus X gibbosus. A comparative study was made: (1) to determine the incubation time, (2) to determine the per cent hatched, (3) to compare standard length of the early larva, and (4) to compare heart development. The results were: (1) the hybrid cross, under the specific circumstances of the experiment, had a 3.1 hour faster mean incubation time than the pure cross, (2) the hybrid cross gave the higher per cent hatched, 63.4% (625) as compared to 51.7% (834) for the pure cross, (3) no significant standard length difference was found, and (4) heart differentiation began with a tubular heart and ended with the development of a series of four chambers. No morphological difference in heart development was observed. There were, however, differences in heart positioning and maturation.