variable number of 10 to 25 unequal-sized oil globules 

 scattered over the yolk surface which showed some 

 tendency toward aggregation with progressing 

 development. The diameter range was extended 

 from 0.94 mm to 1.20 mm by Bigelow and Schroeder 

 (1953) and Wheatland (1956), respectively. The up- 

 per diameter limit extension was verified by Her- 

 man (1963). Prionotus evolans ova have never been 

 positively identified. Perlmutter (1939) made a ten- 

 tative identification, later accepted by Marshall 

 (1946), from ripe ova stripped from gravid females 

 collected in Long Island Sound and described as 

 having similar appearance and diameter as northern 

 searobin eggs, but with oil globules clustered at one 

 pole rather than dispersed across the yolk sphere 

 surface This singular observed morphological dif- 

 ference of oil globule distribution pattern has beer 

 used as the primary distinguishing characteristic 

 between ova of Prionotus carolinus and Prionotus 

 evolans. 



MATERIALS AND METHODS 



Conventional Identifications 



Field-collected, buffered Formalin 3 -preserved 

 plankton samples were physically sorted for all 

 ichthyoplankton using forceps under a dissecting 

 microscope, and the criterion of oil globule distribu- 

 tion differences established by Perlmutter (1939) was 

 used to tentatively separate P. carolinus from P. 

 evolans eggs. The annual cycle and species composi- 

 tion aspects of the field-collected samples using con- 

 ventional means for egg and larval identifications 

 have been submitted elsewhere for publication. 



3 Reference to trade names does not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



FISHERY BULLETIN: VOL. 84, NO. 1 



Immunochemical Identifications 



Antigens and Immunizations 



Antigen preparations from both species of 

 searobin eggs were generated using the techniques 

 developed by Orlowski et al. (1972) with ovarian 

 tissue from ripe adults and immature individuals. 

 The four antigen preparations presented in detail in 

 Table 1 were each used to elicit immune responses 

 in at least two New Zealand white rabbits to improve 

 the probability of obtaining useful antisera. Preim- 

 mune serum samples were obtained from each 

 animal to establish that no reactivity with antigen 

 existed prior to immunization. 



The soluble protein antigens of Prionotus evolans 

 (PeSP) and Prionotus carolinus (PcSP) were injected 

 intravenously in 4.7 and 4.8 mg protein doses (stan- 

 dard biuret analysis), respectively, to begin the im- 

 munization program. Maintenance injections of 2 mg 

 protein followed on a weekly basis. Blood samples 

 were obtained by cardiac puncture 3 wk following 

 the first injection and the presence of precipitating 

 antibody was demonstrated by the standard precip- 

 itin ring test (Abramoff and LaVia 1970). Additional 

 monthly cardiac puncture samples were monitored 

 by quantitative double diffusion (Feinberg 1957) until 

 after about 12 wk; a titer of 32 was reached in all 

 animals receiving soluble antigens when sera were 

 tested with 40 ^g homologous antigen. 



Particulate protein antigens from macerated 

 ovarian tissue of northern (PcPP) and striped (PePP) 

 searobins were prepared in a 1:1 emulsion with 

 Freund's complete adjuvant (Cappell Laboratories). 

 PcPP (8 mg) and PePP (10 mg) protein preparations 

 were injected subcutaneously along several bilateral 

 dorsal sites on New Zealand white rabbits. Rabbits 

 injected with Freund's complete adjuvant developed 



Table 1. — Antigen characterization and nomenclature. 



64 



