The morphology of the AI and AR P. monodon sperm are shown in Figure 1A. Transmission electron microscopy revealed the presence of electron-dense materials in the anterior spike, and moderately electron-dense materials in the acrosome and subacrosomal region of the AI sperm (Fig. 1A, a). Within 5 min of EW addition, the anterior spike completely disappeared along with the rupture of the acrosome (Fig. 1A, b). The subacrosomal region of the AR sperm became somewhat larger and more electron dense compared with that of the AI sperm (Fig. 1A, b versus Fig. 1A, a). There was no evidence for the acrosomal filament formation (as seen in S. ingentis) [22], even at the longer incubation time period (>10 min) (data not shown). At the light microscopic level, the disappearance of the anterior spike and the presence of dark subacrosomal materials in the AR sperm were evident. These features were thus used to differentiate between AI and reacted sperm in all experiments described below.