Application of enzyme-linked immunosorbent assay for studying of reproduction in the Manila clam Ruditapes philippinarum (Mollusca: Bivalvia) : I. Quantifying eggs

Abstract

A polyclonal antibody specific to Ruditapes philippinarum egg protein (ME-ab) was developed to quantify clam eggs using an enzyme-linked immunosorbent assay (ELISA). Western blots revealed that ME-ab reacted with egg proteins of molecular masses 475, 84, and 40 kDa under non-reducing conditions and 330, 96, 64, 50, and 31 kDa under reducing conditions. With ELISA, ME-ab detected between 0.23 and 15 μg ml −1 of clam egg protein; the number of eggs per clam was quantified by dividing the weight of the total egg protein by the average weight per egg. Reproductive output, expressed as the gonadosomatic index (GSI), was calculated as the ratio of the egg weight to the total tissue weight. Seasonal changes in reproductive output were measured in clams collected on a monthly basis from Gomso Bay, Korea. Clam egg protein was detected during all months except January. The monthly mean GSI varied from 0 (January) to 0.25 (August), and the highest GSI (0.389) was recorded from a clam collected in late July. ELISA indicated that clams in Gomso Bay spawned when the gonad accounted for 20% of the total tissue weight. The fecundity estimated from individual clams before spawning ranged from 0.94 to 11.79 million eggs, with a mean of 4.15 million. In conclusion, the ELISA used in this study was a sensitive and rapid method for quantifying eggs in individual clams and is considered the method of choice for quantifying the reproductive output.