Fig. 1. The identification of MAP as a dual component of M. galloprovincialis semen. (A—C) Sequential phases of spawning (bars, 1.6 cm). (A) Whitish-colored semen streams are emitted by the male. (B) 5-10 min later, one portion of the sperm (II) is dispersed into seawater (asterisk), whereas the other portion (I) forms compact thread-shaped structures (black arrow), which are precipitated at the bottom of the tank (III, white arrow) 30-40 min after sperm emission (C). (D,E) The sperm suspension (II) and thread-shaped semen (I and III) were sampled, separated into cell-free seminal fluid (sf) and sperm (sp) fractions and studied by SDS-PAGE (D) followed by western blot (E) with anti-MAP antibodies. The protein loading for each lane is indicated. Lane 1, extract from the total male gonad (15 µg). Lanes 2 and 6, sperm fraction of the semen (15 µg each). Lanes 3-5, cell-free seminal fluid (2.0 µg, 0.3 µg and 2.0 µg, respectively). In both the soluble phase (Lanes 3-5) and in the cell fraction (Lanes 2, 6) of each semen sample, the 39 kDa band was detected on the gel (D) and it was positively stained by anti-MAP antibodies on the blot (E). The positions of marker proteins (kDa) are shown.