Cleavage and gastrulation in the shrimp Penaeus (Litopenaeus ...

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456 of 4-cell stage blastomeres; (2) an invariant cleavage pattern, including mirror-image patterns and two interlocking semicircles of cells through the early cleavages; (3) the cleavage arrest at the 32-cell-stage and subsequent ingression of two cells Xd and Xv at the vegetal pole, which identify the dorsal–ventral axis, and may give rise to endoderm and germ line (Taube, 1909, 1915) or mesendoderm and germ line (Zilch, 1978, 1979; Hertzler and Clark, 1992; Hertzler, 2002); and (4) the radial division of cleavage-retarded crown cells (Kranzzellen) in a ring around Xd and Xv, which later forms the naupliar mesoderm. Differences between euphausiaceans and dendranchiates include the cell lineage of the crown cells and position of the dorsal–ventral axis (Alwes and Scholtz, 2004). The classification of penaeoidean shrimp is controversial at present, centered on the question of whether six subgenera of Penaeus senso lato are natural groups that should be raised to the generic level (Pérez Farfante and Kensley, 1997; Baldwin et al., 1998; Maggioni et al., 2001; Lavery et al., 2004). A recent study of 26/28 of the recognized species of genus Penaeus s.l., based on the mitochondrial 16S and COI genes, found support for the monophyly of some subgenera, e.g. Litopenaeus and Farfantepenaeus, but not for others, e.g. Penaeus s.s. and Marsupenaeus (Lavery et al., 2004). The first comprehensive phylogeny of Penaeus provides an opportunity to ask questions about the evolution of development in the Dendrobranchiata. Embryological characters can be mapped onto the phylogeny to produce hypotheses about homology versus convergent evolution and the direction of evolutionary change (Raff, 1996). While the details of dendrobranchiate development are best known for the ridgeback prawn Sicyonia ingentis, incomplete data are available for other species (Table 1), including the sergestoidean shrimp Lucifer (Brooks, 1882), the kuruma shrimp Penaeus (Marsupenaeus) japonicus (Hudinaga, 1942; Kajishima, 1951), the caramote shrimp Penaeus (Melicertus) kerathurus (ZP. trisulcatus) (Heldt, 1938; Zilch, 1978, 1979), the Green tiger shrimp Penaeus semisulcatus (Kungvankij et al., 1980), and the Indian white shrimp Penaeus (Fenneropenaeus) indicus (Morelli and Aquacop, 2003). Two embryological characters can be compared from the literature: the stage at which mesendoderm cell arrest occurs, which is 64-cells for P. japonicus and 16-cells for P. kerathurus, and the initial number of crown cells, which is 8 for both P. japonicus and P. kerathurus (Kajishima, 1951; Zilch, 1979). In addition, there are claims of embryos with ‘64-cells’ and ‘128-cells’ for P. kerathurus and P. semisulcatus (Heldt, 1938; Kungvankij et al., 1980), which do not account for the arrest of the mesendoderm cells. It was therefore of interest to examine the pattern of cleavage and gastrulation in detail in another representative of the Dendrobranchiata, the Pacific white shrimp Penaeus (Litopenaeus) vannamei. This species is the most commonly farmed shrimp species in P.L. Hertzler / Arthropod Structure & Development 34 (2005) 455–469 Table 1 Partial classification of relevant malacostracan crustaceans, with references referred to in the text Class Malacostraca Superorder Eucarida Order Euphausiacea Euphausia sp. (Taube, 1909, 1915) Meganyctiphanes norvegica (Alwes and Schultz, 2004) Order Decapoda Suborder Dendrobranchiata Superfamily Penaeoidea Family Penaeidae Penaeus (Fenneropenaeus) indicus (Morelli, 2003) P. (Litopenaeus) vannamei (present study) P. (Marsupenaeus) japonicus (Kajishima, 1951) P. (Melicertus) kerathurus (Zilch, 1978, 1979) Family Sicyoniidae Sicyonia ingentis (Hertzler and Clark, 1992; Hertzler, 2002) Family Solenoceridae Solenocera koelbeli (Lavery et al., 2004) Superfamily Segestoidea Family Luciferidae Lucifer sp. (Brooks, 1882) Suborder Pleocyemata After Martin and Davis (2001) and Pérez Farfante and Kensley (1997). Note that recent studies have questioned the close relationship of the Euphausiacea to the Decapoda (Jarman et al., 2000; Richter and Schultz, 2001). the western hemisphere and thus represents an important agricultural model. A comparison with S. ingentis showed an identical pattern of cleavage, time of mesendoderm cell arrest, and number of initial crown cells formed. In addition, development of the mesendodermal derivatives in P. vannamei was identical to that described for S. ingentis. The primordial endoderm cell derived from the ventral mesendoblast X v in both P. vannamei and S. ingentis; in contrast, the primordial endoderm cell is reported to derive from the dorsal mesendoblast Xd in P. kerathurus. 2. Material and methods 2.1. Broodstock and spawning Penaeus vannamei broodstock for this study were maintained at the Hawaii Oahu Suisan, Inc. hatchery in Kahuku, Hawaii in March 2002, as described in Wyban and Sweeney (1991). A 12-ft diameter recirculating maturation tank, containing about 35 females and 45 males, was placed on a reverse photoperiod so that ‘sunset’ occurred at 6 a.m. to induce mating. Mated females were sourced 2 h later and placed in a bucket with 5-L of seawater with gentle aeration at 27 8C, and monitored for spawning activity. Animals were allowed to spawn for 1 min then were removed from the bucket, which was then swirled periodically to keep the oocytes in suspension while the jelly was extruded. The
culture was maintained at 27 8C during the sampling period, through 8 h post-spawning (ps). Samples were taken every 15 min, passed through a 500 mm nylon mesh to remove the jelly, briefly pelleted with a hand centrifuge, and fixed and stored in 90% methanol-50 mM EGTA, pH 8.0. 2.2. Fluorochrome staining and microscopy For nuclear labeling, eggs or embryos were stained with 2.5–5 mM Sytox Green (Molecular Probes Inc.) as described in Hertzler (2002). Observations were made with an Olympus Fluoview 300 laser scanning confocal microscope at Central Michigan University, using argon laser excitation with a 20X Plan Apo 0.7 NA objective lens with digital zoom of 2.5–3. Other scanning conditions and image manipulations were performed as in Hertzler (2002). 2.3. Cell nomenclature The cell nomenclature in Fig. 1 is based on that proposed for the dendrobranchiate shrimp Sicyonia ingentis (Hertzler and Clark, 1992; Hertzler, 2002) and the euphasiacean Meganyctiphanes norvegica (Alwes and Scholtz, 2004). The four capital letters A, B, C, and D are used for the 4-cell blastomeres, with the D cell containing the vegetal pole and giving rise to the mesendoderm cells. 3. Results 3.1. Fertilization and zygote Oocytes are spawned arrested at meiosis I and sperm immediately bind and enter. Meiosis is resumed with the formation of two polar bodies, one outside and one inside a hatching envelope (not shown). No attempt was made to follow the location of the polar bodies during cleavage, to determine the relation of the polar axis to the first cleavage plane. Development proceeds within the hatching envelope, from which the nauplius larvae hatch within 12 h. While newly-spawned oocytes are oblongate, the fertilized egg becomes perfectly spherical, with a diameter of 230 mm and a uniform distribution of yolk granules (Fig. 2(A)). Sperm nuclei were present in oocytes by 12.5 min ps, and pronuclear migration occurred from 15–25 min ps (data not shown), in a similar fashion as in S. ingentis (Hertzler and Clark, 1993). 3.2. First division and 2-cell stage, second division and 4-cell stage The first cleavage occurred at about 40 min ps at 27 8C. The cleavage pattern of P. vannamei was similar to that described previously for S. ingentis (Hertzler and Clark, 1992). A revised nomenclature (Fig. 1) was used to label the early blastomeres, based on Hertzler and Clark (1992) and a P.L. Hertzler / Arthropod Structure & Development 34 (2005) 455–469 457 recent study on the krill M. norvegica (Alwes and Scholtz, 2004). First cleavage was complete, resulting in a 2-cell stage of approximately equally sized blastomeres, AB and CD (Fig. 2(C) and (D)). If the relation of these cells to the polar axis is the same as that determined for S. ingentis, the CD blastomere contains the vegetal pole (Hertzler and Clark, 1992). The second division planes formed synchronously at a 458 angle to each other, resulting in a 4-cell stage with blastomeres oriented in a close packing arrangement (Fig. 2(E)–(H). All four blastomeres appeared to be the same size in intact embryos, so that no asymmetries could be detected at the 4-cell stage. The cell identities and sister cell relationships in Fig. 2 were inferred by tracing spindle orientations back from the 32-cell stage, and from live embryo lineage tracing in S. ingentis (Hertzler and Clark, 1992; Hertzler et al., 1994). Blastomeres A and C contacted each other at a cross-furrow running in the mid-sagittal plane on the dorsal side (Fig. 2(F)), while blastomeres B and D touched in a transverse furrow on the ventral side (Fig. 2(H)). A and C thus became left and right territories while B and D were anterior and posterior, respectively. 3.3. Third division and 8-cell stage The third division, from 4- to 8-cells, was now orthogonal to the preceding one, with A and C spindles and B and D spindles orienting end to end. As in S. ingentis and M. norvegica, this resulted in two interlocking semicircles of cells, AC and BD. A divided into the dorsal cell A I and ventral A II, while C divided into the dorsal cell CI and ventral CII (Fig. 2(J) and (L)). AI and CI thus met at the AC cross-furrow, while AII and CII lay at the ends of the AC band. From left ventral to right ventral, the cells in the AC band consisted of AII, AI, CI and CII. B divided into the dorsal BII and ventral BI, while D divided into the dorsal DII and ventral DI (Fig. 2(J) and (L)). BI and DI met at the BD cross-furrow, while B II and D II lay at the ends of the BD band. From the dorsal, anterior to the dorsal, posterior, the cells in the BD band consisted of BII, BI, DI, and DII. 3.4. Fourth division and 16-cell stage The fourth division was synchronous, with spindles in the AC and BD bands in a row of four side-by-side spindles, yielding a 16-cell stage (Fig. 2(J) and (L). The AC band divided in the anterior–posterior direction, with A II forming AIIa and AIIp, AI forming AIa and AIp, CI forming CIa and CIp and AII forming AIIa and AIIp(Fig. 2(N)). The BD band divided left and right, with BII forming BIIr and BIIl, BI forming BIr and BIl, DI forming DIr and DIl, and DII forming DIIr and DIIl (Fig. 2(N) and (P)). 3.5. Fifth division and 32-cell stage In the synchronous fifth division, the spindles of the AC
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