KEIRANS ET AL.: DIFFERENTIATION OF PRIONOTUS EGGS 



Arthus reactions following a single dose Subsequent 

 injections were accomplished intravenously using 

 Millipore (0.45 /mi) filtrates of PcPP and PePP. Titers 

 were monitored utilizing the standard complement 

 fixation assay because of the particulate consisten- 

 cy of the macerated antigen preparation (Kabat and 

 Mayer 1961). Maximum titers of 1,280 were obtain- 

 ed after about 10 wk with immunizations using PcPP 

 or PePP. 



Antiserum Specificity 



Antisera elicited in response to both soluble and 

 particulate antigens were multicompetent and ex- 

 hibited cross-reactions with heterologous antigens. 

 The presence of common antigens between the 

 northern searobin and striped searobin ovarian 

 material preparations required the specific adsorp- 

 tion of antisera with these shared antigens to render 

 a given antiserum monospecific (Eisen 1974). 

 Although antisera elicited in response to particulate 

 protein antigens exhibited precipitation reactions in 

 agar with both soluble antigens and extracts of par- 

 ticulate antigens from the two species under con- 

 sideration, they were not competent in reactions with 

 homologous fish eggs. Therefore, since the selected 

 method for analysis of planktonic eggs was immuno- 

 diffusion, only antisera elicited in response to solu- 

 ble antigens were used in all analyses of unknowns. 

 Specific adsorption of common antigens shared by 

 northern and striped searobins was accomplished by 

 adding PcSP to antisera elicited in response to PeSP 

 and vice versa. Adsorption lots of 1.5 mL anti-PeSP 

 antisera combined with 70 \xL PcSP (0.65 mg pro- 

 tein) were incubated at 4°C for 48 h prior to use. 

 This adsorption eliminated all reactivity of anti-PeSP 

 antisera with both PcSP and known ova of P. 

 carolinus, without significantly reducing activity 

 with ova of P. evolans. This specifically adsorbed anti- 

 PeSP, which reacted solely with known homologous 

 ova of P. evolans under controlled conditions, was 

 used as the basis for differentiation of northern and 

 striped searobin eggs. Species-specific anti-PeSP 

 antisera capable of 100% accuracy in differentiating 

 known ova of both searobins was the reagent selected 

 for use in all immunodiffusion analyses. 



Microimmunodiffusion Analysis 



Unknown planktonic fish eggs were analyzed with 

 monospecific anti-PeSP antiserum in a micromodi- 

 fication of the immunodiffusion technique (Ridgeway 

 et al. 1962). Microscope slides (2.5 x 8 cm) were 

 washed, rinsed first in distilled water and then 



methanol, and wiped dry. Two milliliters of 1% No- 

 ble Agar (Difco) in FA-Bacto buffer (Difco), pH 7.2, 

 were applied across each slide on a leveling table and 

 allowed to harden. Slides were then placed over a 

 template and wells cut using a Brewer needle with 

 beveled inner surface (Ridgeway et al. 1962). 



Agar plugs were removed from wells by aspiration. 

 Reagents were applied with either 1 mL syringes 

 (Burron) or sterile capillary pipettes, and 0.005 to 

 0.01 mL was required to fill each well. A typical 

 testing array appears in Figure 1, where corner wells 

 contain unadsorbed antiserum, the central well con- 

 tains adsorbed or monospecific antiserum, and re- 

 maining wells contain individual fish eggs which have 

 been broken using jeweler's forceps. FA-Bacto buf- 

 fer was applied to each well following egg disrup- 

 tion, and slides were allowed to incubate in moist 

 chambers for 18 h at 20°C. Slides were then washed 

 for 24 h in FA-Bacto buffer, and stained according 

 to the method of Crowle (1958). Results were always 

 recorded at a fixed time interval following slide 

 preparation to insure comparability from one deter- 

 mination to another. 



RESULTS AND DISCUSSION 



A total of 732 searobin ova were recovered from 

 plankton samples collected in the 1973-74 period. 

 The combined morphological characteristics of egg 

 diameter, number, color, and distribution of oil 

 globules, and embryo pigmentation when present, 

 allowed the separation of searobin eggs from those 

 of other species with reasonably high confidence 

 Preliminary classifications of Prionotus ova into 

 either evolans or carolinus species was based upon 

 differential oil globule distribution patterns reported 

 by Perlmutter (1939). Striped searobin, P. evolans, 

 eggs were placed into one grouping based upon a 

 polar or clustered oil globule distribution, and north- 

 ern searobin, P. carolinus, eggs placed into a second 

 group having oil globules generally dispersed across 

 the yolk sphere 



Each tentatively classified egg was then analyzed 

 in the microimmunodiffusion method illustrated in 

 Figure 1, to establish the immunochemical reactivity 

 of soluble egg antigens with adsorbed and unadsorb- 

 ed anti-PeSP antisera. When soluble P. evolans egg 

 antigens were sufficiently concentrated, a classical 

 line of identity was observed with fusion of precipitin 

 bands between adsorbed and unadsorbed anti-PeSP 

 wells. Identification of P. carolinus eggs was based 

 upon reactivity with unadsorbed anti-PeSP anti- 

 serum and no reactivity with adsorbed anti-PeSP. 

 Previously established reactivity of unadsorbed anti- 



65 



