ISSN: 1687-4285 Egyptian Journal of Aquatic Research, 2009, 35(3), 385-403 Improving triploidy induction in the hard clam Mercenaria mercenaria notata: cytological evaluation of fertilized eggs *Eman El-Wazzan a , and John Scarpa b a Division of Aquaculture, National Institute of Oceanography and Fisheries, Qaiyet-Bay, El-Anfoushi, Alexandria, 21556, Egypt. b Aquaculture and Stock Enhancement Program, Harbor Branch Oceanographic Institute at Florida Atlantic University, 5600 U.S. 1 North, Fort Pierce, FL, 34946, USA. * corresponding author email: emanelwazzan@yahoo.com Received 30 th September 2009, Accepted 15 th November 2009 Abstract Triploid organisms contain three sets of chromosomes instead of the usual two, thereby rendering them essentially sterile, which may improve growth and stress resistance. Triploidy may be induced by inhibiting extrusion of the first polar body (PBI) or second polar body (PBII) during meiosis using chemicals, such as cytochalasin B (CB). In bivalves, which release large quantities of pre-meiotic eggs, producing a high proportion of triploids relies upon synchrony of meiotic events after fertilization. Therefore, timing of meiotic stages of fertilized eggs of the hard clam Mercenaria mercenaria notata was examined to improve triploid induction yield. Spawned eggs from three females were sampled before insemination and intermittently afterwards up to 90 min post-insemination (PI). Meiotic stages of eggs (avg. 90 eggs/sample) were visualized with the DNA-specific flurochrome Hoechst 33258 and epifluorescence microscopy. The maximum proportion of eggs with PBI and PBII appeared on average at 16 min and 28 min PI, respectively. First cell cleavage (i.e., first mitosis) was greatest on average at 60 min PI. Using these data, triploid induction in hard clams was evaluated in ten trials using cytochalasin B (CB) at 0.67 or 1mg/L egg suspension. CB treatment started at 6.5 to11 min PI with a 10-12 minutes exposure for PBI inhibition and at 15.5 to 21 min with a 13-15 minute exposure for PBII inhibition. The proportions of eggs synchronized at stages just before PBI or PBII extrusion in CB-treated eggs (i.e., potential triploid proportion) were compared to triploid proportions in D-stage larvae as measured by flow-cytometry. Potential triploid proportions from eggs ranged from 0-100%, whereas triploid proportions from larvae ranged from 0- 93%; however, triploid values from both methods were not always similar for each spawn. Parameters that resulted in lower triploid proportions included low CB concentration (0.67 mg/L), pre-fertilization that also severely reduced larval survival (1-2% compared to control), and early or late addition or removal of CB. Depending on environmental conditions, especially temperature that hastens development, induction of triploidy in hard clams using CB should begin 10 minutes PI for PBI inhibition and at 17 min PI for PBII inhibition. CB exposure should not go beyond 25 minutes PI for PBI inhibition or 40 min PI for PBII inhibition. Keywords: Polyploidy, Triploid, Clam, Mercenaria, Bivalve, Cytology 1. Introduction Hard clam culturists in southwest and west Florida, USA report below average survival during the prolonged hot summers (Leslie Sturmer, University of Florida, pers. comm.). It has been suggested that bivalve mortality increases after spring spawning, which decreases tissue glycogen levels (Perdue et al. 1981), and is followed by hot (30 o C) water temperatures, which are associated with lower phytoplankton concentration and oxygen concentrations, as well as large salinity fluctuations from rainfall (Goldman and Ryther 1976, Ohgai et al. 1982, Surge and Lohmann 2002, Weiss et al. 2007). Bivalves that are deprived of energy needed for their maintenance after investment in gametogenesis and spawning are more susceptible to stressors that amplify the already present reproductive cost than those with lower reproductive effort or metabolic needs (Perdue et al. 1981, Eversole 1987, Shpigel et al. 1992, Myrand and Gaudreault 1995). Triploid bivalves may have an energetic advantage over diploids at elevated temperature as triploids are sterile or have severely retarded gametogenesis (e.g., Tabarini 1984, Allen et al. 1986, Lee 1988, Shpigel et al. 1992, Eversole et al. 1996, Utting et al. 1996, Brake et al. 2004, Maldonado- Amparo et al. 2004). Therefore, triploids may be more resistant to summer mortality as they would have higher body mass and more energy reserves (Perdue et al. 1981, Allen et al. 1989, Shpigel et al. 1992, Hand et al. 2004). Successful induction of triploidy in hard clams would provide viable triploids as a first step towards examining their performance in Florida waters, as has been accomplished with other molluscan species (e.g., Baron et al. 1989, Utting and Child 1994, Toro and Sastre 1995). Inducing triploidy in bivalves relies upon EJAR