Saccoglossus kowalevskii (Agassiz, 1873)

Wanninger, Sabrina Kaul-Strehlow Makoto Urata Takuya Minokawa Thomas Stach Andreas, 2015, Neurogenesis in directly and indirectly developing enteropneusts: of nets and cords, Organisms Diversity & Evolution 15 (2), pp. 405-422 : 407-408

Saccoglossus kowalevskii (Agassiz, 1873)

Adult specimens of S. kowalevskii were collected from intertidal flats near Woods Hole (Massachusetts, USA) in September 2007 and 2011. Animals were transported into the laboratory, separated according to sex, and kept individually in finger bowls. Animals were kept at 18 °C on a seawater table. Freshly spawned eggs were mixed with active sperm isolated from a ripe male and diluted in seawater ( Lowe et al. 2004). Fertilization envelopes were ruptured using fine forceps, and embryonic and juvenile stages were collected using Pasteur pipettes. Embryos were relaxed in a mix of 7 % MgCl 2 and seawater (1:1) for 5–10 min prior to fixation for transmission electron microscopy ( TEM) and scanning electron microscopy ( SEM) .

Terminology of names of developmental stages

For indirectly developing enteropneusts such as B. misakiensis , we follow the terminology sensu Nielsen and Hay-Schmidt (2007). Accordingly, the Spengel stage is characterized by a regression of size of the larva and the fusion of the preoral ciliary feeding band, the neotroch. The Spengel stage is followed by the Agassiz stage. Agassiz larvae have a cone-shaped anterior body region with a smooth surface, the prospective proboscis region. The overall size is further decreased, and the anterior eye spots begin to disappear. The Agassiz larvae are competent for settlement.

For the direct developer S. kowalevskii , we followed the terminology of Lowe et al. (2003, 2006). Accordingly, the Kink stage is characterized by a dorsal bending of the future trunk region.

Electron microscopy

TEM and SEM embryos and juveniles of S. kowalevskii were fixed with ice-cold 2.5 % glutaraldehyde (GA) in 0.2 M sodium cacodylate buffer (pH 7.2), adjusted to an osmolarity of approximately 800 mosm with the addition of NaCl (for B. misakiensis , 2.5 % GA in 0.05 M phosphate buffer + 0.3 M sodium chloride ( PBS)). Primary fixation was stopped after 45 min (30 min, B. misakiensis ) with three buffer rinses of 10, 15, and 20 min, respectively (five rinses with PBS for 10 min each for B. misakiensis ). Primary fixation was followed by 30 min of postfixation with 2 % OsO 4 in sodium cacodylate buffer ( PBS). Postfixation was stopped with three buffer rinses (15, 30, 30 min) followed by two rinses with ddH2O (15, 30 min). After dehydration through a graded series of ethanol, specimens were embedded in Epon resin for TEM and light microscopy. For SEM, specimens were critical point dried in a CPD 030 (Balzers Union, Liechtenstein) or Leica CPD 300 (Leica, Germany). Dried specimens were sputter coated with gold in a SCD 040 (Balzers Union, Liechtenstein) sputter coater and viewed with a Fei Quantum 200 SEM at 15 kV ( FEI Co., The Netherlands) or a Philips XL 30 ESEM (Philips, The Netherlands). Complete longitudinal and transverse semithin serial sections (0.5 μm) for light microscopy and ultrathin serial sections (~ 55 nm) for TEM from three stages of S. kowalevskii (56, 132, and 432 h pf) were produced on a Leica Ultracut S microtome and from five stages of B. misakiensis (B Spengel stage^: 13 days pf, B Agassiz stage^: 14 days pf, 12-h postsettlement (ps), 1 ay ps and 3 days ps) sectioned on a Leica UC 7 microtome. Semithin sections were stained with 1 % toluidin blue for 3 min at 63 °C. Ultrathin sections were stained with 2 % uranylacetate and 2.5 % lead citrate, either in an automatic stainer (NanoWlm Technologie GmbH, Göttingen, Germany) or manually. Light microscopic images were recorded with a digital camera (Olympus BX-UCB) mounted on an Olympus BX51 compound microscope or Nikon Eclipse E800. TEM pictures were documented with a Philips EM 208 electron microscope at 70 kV equipped with a Nikon digital camera. Images were optimized by using Adobe Photoshop and Illustrator CS 3 (Adobe, San José, CA, USA).

Immunolabeling and confocal laser scanning microscopy

For immunocytochemistry embryos and juveniles of S. kowalevskii (dorsal kink = 76 hpf, 1-gill-slit hatchling = 156 hpf, 3-gill-slit juvenile = 750 hpf) and larvae and juveniles of B. misakiensis (B Spengel stage^ = 13 days pf, B Agassiz stage^ = 14 days pf, early settled stage = 12-h postsettlement (ps), 1-gill-slit juvenile = 1 day ps, and 2-gill-slit juvenile = 3 days ps) were fixed with 4 % paraformaldehyde ( PFA) in phosphate buffer ( PB). Specimens were washed three times and permeabilized in PB containing 2 % Triton X-100 ( PBT). Subsequent blocking was carried out in PBT containing 6 % normal goat serum (Jackson Immuno Research) for 2 h at room temperature ( RT). Primary antibodies were diluted in blocking solution in PBT and applied at a final concentration of 1:500 (anti-serotonin, Sigma, Cat. # S5545; anti-acetylated α- tubulin Sigma, Cat. # T 6793) overnight at RT. Then, animals were washed six times for 20 min each with PBT on a rocker table. Secondary antibodies were diluted in blocking solution in PBT and applied at a final concentration of 1:600 (goat anti-rabbit Alexa Fluor 633, Invitrogen, Cat. # A21070; goat anti-mouse Alexa Fluor 568, Invitrogen, Cat. # A11004) together with a nuclei marker (1:600, DAPI, Invitrogen, Cat. # D1306) and a marker for filamentous actin (1:150, Alexa fluor 488 phalloidin, Invitrogen, Cat. # A12379) for 4 h at RT. Afterward, the samples were rinsed three times for 20 min each in PBT and twice in PBS. Subsequently, samples were stepped gradually into 70 % glycerol and eventually mounted in Fluoromount G (SouthernBiotech) on glass slides. The strong signal resulting from the epidermal cilia and mesodermal muscles made it impossible to unambiguously identify neural structures such as neurites in the tubulin and phalloidin stainings; yet, the detected cilia and muscles were used as positional markers. Analysis and digital image acquisition were performed on a Leica TCS SP5 II confocal laser scanning microscope (Leica Microsystems, Germany). Optical sections were taken at a step size between 0.3 and 0.6 μm in Z-resolution. The resulting image stacks were digitally merged into maximum Z-projections and further processed with the open source image software Fiji (Max Planck Society for the Advancement of Science e. V., Germany), Adobe Photoshop, and Illustrator CS 3 (San José, CA, USA). At least ten specimens per stage were stained and scanned and rendered identical results for each staining per stage.

Negative controls were performed by excluding either the primary or the secondary antibody. No signal was detected in any specimens of these experiments. In order to test for unspecific binding of the anti-serotonin (5- HT) antibody, additional negative controls with preadsorbed antibodies were performed on developmental stages of B. misakiensis and S. kowalevskii . For this, the rabbit anti-serotonin (5- HT) antibody (polyclonal; ImmunoStar) was incubated in PBT blocking solution overnight at 4 °C together with serotonin (5-HT-)-BSA conjugate (ImmunoStar) reconstituted in PBT block with a final dilution of the antibody of 1:500 and a final concentration of the serotonin-BSA conjugate of 20 μg/ml. This solution was subsequently used as primary antibody solution according to the protocol described above, and none of the individuals showed any signal.