Timing of meiotic maturation and first mitosis in the Pacific white shrimp Litopenaeus vannamei

Intense efforts are currently underway to develop polyploid strains of cultured shrimp species, both for enhanced culture performance and protection of intellectual property (Fast and Wyban 2001). Triploidy induction has been reported for several species of Superfamily Penaeoidea, including the Chinese shrimp Penaeus chinensis = Fenneropenaeus chinensis (Bao et al. 1994), the Pacific white shrimp Litopenaeus vannamei (Dumas and Ramos 1999), and the Indian white shrimp Penaeus indicus = Fenneropenaeus indicus (Morelli and Aquacop 2003), all members of Family Penaeidae, and Sicyonia ingentis, a member of Family Sicyoniidae (Hertzler 2002; for new genera see PCrez Farfante and Kensley 1997). Gonadal development has been shown to be impaired in triploid shrimp, and a higher ratio of females to males develops (Li et al. 2003b). Current efforts are focused on producing tetraploid strains carrying desirable traits such as disease resistance or enhanced growth, which can then be mated to diploids to produce 100% triploid (presumably sterile) offspring, thereby protecting the financial investment of those who developed the strains. Tetraploidy induction has been reported for E chinensis (Xiang et al. 1991; Li et al. 2003a). Polyploidy induction typically uses either temperature shock (heat or cold) or drugs such as cytochalasins or 6-dimethylaminopufine (6-DMAP) to suppress cell division while allowing extra chromosome sets to be retained. The optimal procedures are determined empirically and depend on a critical synchronization with the events of meiotic maturation, DNA synthesis, and first mitosis that accompany fertilization. However, the details of these processes in penaeoidean shrimp have only been described for the ridgeback prawn S. ingentis (Lindsay et al. 1992; Hertzler and Clark 1993) and recently for E indicus (Morelli and Aquacop 2003). In S. ingentis (Superfamily Penaeoidea, Family Sicyoniidae), egg jelly release and meiotic maturation are triggered by exposure to seawater at spawning, and occur in both fertilized and non-fertilized oocytes (Pillai and Clark 1987; Lindsay et al. 1992). The hatching envelope develops after first polar body formation but before second polar body formation, so that the first polar body lies outside of the hatching envelope and the second polar body lies inside of it (Lynn et al. 1991; Hertzler 2002). Sperm are first detected in eggs after about 35 min post-spawning, following an elaborate acrosomal reaction which includes the formation of an acrosomal filament. Pronuclear migration requires microtubules, and the first cleavage occurs at about 90-min postspawning at 22 C (Hertzler and Clark 1993). In contrast, in many species of Family Penaeidae, the time to first cleavage is much faster since most live in warm water. For example, in E indicus first cleavage occurs by 35 min at 29.5 C (Morelli and Aquacop 2003), while in L. vannamei first cleavage occurs within 50 min of spawning at 27 C (Wyban and Sweeney 1991). Furthermore, sperm of the Penaeidae do not form an acrosomal filament (Alfaro et al. 1993; Pongtippatee-Taweepreda et al. 2004). This report describes the events of fertilization and first mitosis in the Pacific white shrimp L. vannamei, using fluorescent probes for DNA and tubulin and confocal microscopy. While S. ingentis and L. vannamei are in different families and have different thylecum types, meiotic maturation, pronuclear migration, and first mitosis proceed in a similar way but with different timing. Knowledge of the timing of these events may be useful for efforts aimed at