Cytological defects during embryogenesis in heat-induced tetraploid Kuruma shrimp Penaeus japonicus Andrew Foote a, b, c, * , Melony Sellars a, b , Greg Coman a, b , David Merritt c a CSIRO Food Futures National Research Flagship, 5 Julius Avenue, North Ryde, NSW 2113, Australia b CSIRO Marine and Atmospheric Research, 233 Middle Street, Cleveland, QLD 4163, Australia c The University of Queensland, School of Biological Sciences, Brisbane QLD 4072, Australia article info Article history: Received 15 September 2009 Accepted 17 December 2009 Keywords: Tetraploid Shrimp Intracellular body Anucleate Tetraploar spindles abstract Tetraploid shrimp embryos have been induced; however, in all cases no postlarvae were produced. This study determined when tetraploid Penaeus japonicus became non-viable and identified unique abnor- malities to aid in elucidating the causes of lethality. Embryonic development was analyzed using flow cytometry to determine ploidy and laser scanning confocal microscopy for cytological examination of embryogenesis. Abnormalities exclusive to tetraploids were identified from the 1-cell stage: an off-centre pronucleus, polypolar spindles, delayed time to first mitosis and polypolar cleavage. Following first mitosis in the tetraploids, 50% of the cells did not contain DNA. This unique abnormality was not resolved later in development and is therefore believed to be a lethal trait. Causes of this phenomenon likely stemmed from abnormal mitotic spindle regeneration following the mitotic heat shock. Consequently, the findings of this study indicate that current methods of tetraploidy induction using heat shock appear unsuitable for viable tetraploid shrimp production. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Successful domestication and genetic improvement of several penaeid species worldwide has increased the reliability and effi- ciency of shrimp farming, resulting in high value stocks (Hetzel et al., 2000; Coman et al., 2006). Various methods have been investigated to genetically protect elite lines from unlicensed breeding such as: ionizing irradiation (Sellars et al., 2005, 2007; Sellars and Preston, 2005), polyploidy (Dumas and Ramos, 1999; Li et al., 2003c; Norris et al., 2005; Sellars et al., 2006b) and gene regulation (Sellars et al., 2006b). However, no method trialed to date can guarantee total reproductive sterility in 100% of progeny. Polyploid organisms have been induced experimentally through techniques involving chemical, temperature and pressure shocks timed to disrupt embryo development at specific developmental stages (El Gamal et al., 1999; Eudeline et al., 2000; Li et al., 2003c; Norris et al., 2005). Triploidy (3N) has been the most successful reproductive sterilization method reported in shrimp; commonly reducing or completely impairing the reproductive capacity of the animal (El Gamal et al., 1999; Allen et al., 2005). Triploid shrimp have been induced via inhibition of polar body I (PBI) or polar body II (PBII) extrusion during meiosis (Dumas and Ramos, 1999; Li et al., 2003c; Sellars et al., 2006b). Heat shocks have been successful in inducing triploids in the penaeid shrimps Penaeus japonicus (Norris et al., 2005; Sellars et al., 2006b), Penaeus chinensis (Li et al., 2003c), Penaeus vannamei (Dumas and Ramos, 1999) and Penaeus monodon (CSIRO, unpublished). Chemical (6-dimethylaminopurine) shocks have also been successful in inducing triploid P. japonicus (Norris et al., 2005; Sellars et al., 2006b) and P. monodon (CSIRO, unpublished). In shrimp species where triploids have been successfully produced, triploid individuals are either all female or have a gender ratio skewed towards the female. Additionally, triploid individuals have been found to be reproductively sterile (Li et al., 2003b; Xiang et al., 2006; Coman et al., 2008; Sellars and Preston, 2008). Given the faster growth rate of female penaeids (Sellars, 2007), triploidy has the potential to increase farm yields, while also providing the genetic protection benefits of reproductive sterility. However, the success of triploid induction varies due to the lack of synchrony in treatment application; this is a consequence of the protracted duration of egg release and fertilization (Li et al., 2003c; Norris et al., 2005). Variability in the range of optimal induction para- meters between species and spawnings, has also contributed to inconsistent levels of triploidy (Li et al., 2003c; Norris et al., 2005; Sellars et al., 2006a). * Corresponding author. CSIRO Food Futures National Research Flagship, 5 Julius Avenue, North Ryde, NSW 2113, Australia. E-mail address: andrew.foote@csiro.au (A. Foote). Contents lists available at ScienceDirect Arthropod Structure & Development journal homepage: www.elsevier.com/locate/asd 1467-8039/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2009.12.001 Arthropod Structure & Development 39 (2010) 268–275