558 



Fishery Bulletin 102(3) 



(type II). The remaining 91 unidentified eggs were di- 

 vided into three groups (types I, III, and IV) based on 

 diameters. Of these 91, 51 type-II eggs reacted with 

 MT-1. This finding is compatible with the possibility 

 that the eggs were P. major, because the size was simi- 

 lar to that of P. major and each contained a single oil 

 globule of a similar size (Tables 1 and 4). Another 43 

 eggs were collected from another area of Wakasa Bay 

 (Table 5). None of the eggs fixed just after collection 



Table 3 



Reactivity of monoclonal antibodies to fish eggs. + repre- 

 sents positive reaction; - represents negative reaction. 



Reactivity 



Egg no. 



Species 



MT-1 MT-2 MT-3 



were morphologically identifiable. But, after incuba- 

 tion at 20°C for 24 hours until the late stage, all six 

 eggs identified as P. major were reactive with the an- 

 tibody MT-1, whereas the others were not. These find- 

 ings strongly suggest that the method developed in this 

 study is useful for identifying P. major eggs in seawater. 

 Although only late stage eggs were used in this experi- 

 ment, early stage eggs are also detectable because the 

 antibody recognized both stages of P. major eggs from 

 several sea farming centers (Table 2). 



Compared to genetic analysis of fish eggs, this method 

 has the advantage of being able to assay many eggs 

 simultaneously without the need to separate individual 

 eggs in tubes and without extracting DNA from the in- 

 dividual egg in each tube. Further, this method works 

 with formalin-fixed eggs, whereas extraction of DNA 

 from formalin-fixed material is problematic. Plankton 

 samples from field studies are typically fixed in forma- 

 lin-seawater solution. 



There was no problem obtaining a large amount of 

 the monoclonal antibody required when identifying P. 

 major eggs. The antibody can be easily obtained by 

 large-scale cultures of hybridoma cells. About 50 mL 

 of antibody solution was obtained after two weeks of 

 cultivation. There was no technical problem assaying 43 

 or 102 eggs from natural waters. However, one assay of 

 a field sample cost about 20 U.S. dollars. To keep costs 

 down an assay kit cheaper than the VECTASTAIN R 

 Elite ABC kit is needed when a large number of field 

 samples are analyzed. 



Acknowledgments 



We would like to thank the following sea farming centers 

 and universities for providing the fish eggs used in this 

 study: Fukui Prefectural Sea Farming Center; Kyoto 

 Prefectural Sea Farming Center; Faculty of Agriculture, 

 Kyushu University; Osaka Prefectural Fisheries Station; 

 Sea Farming Center of the Japan Sea-Farming Associa- 

 tion; Fisheries Laboratory of Kinki University. We also 

 thank Jeffrey M. Leis, Australian Museum, Sydney, 

 Australia, for his kind advice during the writing of this 

 manuscript. 



Table 4 



Reactivity of monoclonal antibody MT-1 to the pelagic eggs fixed with formaldehyde just after collection from Wakasa Bay. 

 O.G. diameter = oil globule diameter 



Fish egg type 



Egg diameter imm) 



O.G. diameter imm) 



Reactivity (%) 

 ( positive egg no./ total egg no.) 



I 

 II 



III 

 IV 



0.72-0.79 

 0.75-0.82 

 0.81-1.02 

 1.07 



0.16-0.19 

 no oil globule 

 0.19-0.28 

 0.21 



0(0/2) 



0(0/11) 



58(51/88) 



0(0/1) 



