DOI: 10.5061/dryad.qv9s4mwk3
title: Data from: Parasitic fish embryos do a ‘front-flip’ on the yolk to resist expulsion from the host

description: Bitterlings are brood parasitic fish which complete their early
 development in the internal gill spaces of freshwater mussels. Bitterling
 embryos have wing-like yolk sac extensions that help prevent them from
 being expelled from the gills by the water flow. The ability to resist
 expulsion may be helped by the consistent ‘head-down’ position that all
 embryos adopt in the gills. The mechanism behind this positioning is
 unknown. We hypothesise here that it might lie in a process of unknown
 function, specific to bitterlings. That process is blastokinesis — the
 rotation of the embryo on the yolk ball before hatching. We used
 time-lapse imaging, histology, X-ray tomography, and expression profiling
 of the genes fgf8a, krt8, msx3 and ctslb by in situ hybridization in the
 pre-hatching and hatching stages of the rosy bitterling (Rhodeus
 ocellatus). We find that blastokinesis is a gastrulation process that has
 been ventralized by the shape of the yolk mass. Furthermore, we show that
 bitterlings, unlike other teleosts, hatch mechanically without hatching
 enzymes, and we provide evidence that this is mediated instead by the
 apical tubercles on the yolk sac extension. Finally, our data suggest that
 blastokinesis is functional, because it represents the mechanism behind
 the optimal, ‘head-down’ positioning of the embryo. Our study provides an
 example of how selection pressures can lead to a suite of dramatic and
 coordinated modifications of early development.
description: <em><strong>Rhodeus
 ocellatus</strong></em><strong>
 embryos</strong> <em>Rhodeus
 ocellatus</em> embryos of synchronized developmental age were
 collected at the Institute of Vertebrate Biology in Brno, Czech Republic,
 following <em>in vitro</em> fertilization (IVF). Embryos of
 various developmental stages (<em>SI Appendix</em>, Table S1)
 were fixed in 4% paraformaldehyde, dehydrated step-wise in methanol and
 stored in 100% methanol at -20°C. The developmental staging of the embryos
 was based on Yi <em>et al.</em> Two or three replicates were
 used for each stage. <em><strong>Danio
 rerio</strong></em><strong>
 embryos</strong> Embryos of the zebrafish
 <em>Danio rerio</em> (AB/TL line) were collected in the fish
 facility of the Institute of Biology Leiden. The eggs were fertilized by
 1:1 spawning (single crossing) at the beginning of the light period (14 h
 light, 10 h dark). The fertilized eggs were collected and incubated in egg
 water (containing 60 μg/ml “Instant Ocean” sea salts) at 28.5 °C. After
 collection, the embryos were immediately fixated in 4% paraformaldehyde.
 Fixed embryos were dehydrated step-wise in methanol and stored in 100%
 methanol at -20 °C. The developmental stages were determined according to
 Kimmel <em>et al</em>. (37). For each stage (<em>SI
 Appendix</em>, Table S1), ten replicates were used.
 <strong>Primer design</strong>
 Zebrafish (<em>Danio rerio</em>) is relatively
 closely phylogenetically related to the rosy bitterling (the same family,
 Cyprinidae). We BLAST searched the zebrafish genes of our interest
 (<em>fgf8a</em>, <em>msx3</em>,
 <em>krt8</em> and <em>ctslb</em>) since the entire
 zebrafish genome is known and aligned it to other species in the
 Cyprinidae<em> </em>(e.g., <em>Carassius auratus
 </em>and <em>Cyprinus carpio</em>). We searched for
 conserved regions. After the gene of interest was found, forward and
 reverse primers of approximately 20 base pairs were designed in the
 conserved regions that would provide fragments of at least 600 bp with a
 preferred melting temperature between 55°C and 60°C and a favourable CG
 percentage (around 30-40%). These primers were ordered from Sigma Aldrich
 and used to amplify the target gene from cDNA from reverse transcribed
 form <em>Rhodeus ocellatus</em> total RNA (see<em> SI
 Appendix</em>, Table S2 for primer sequences and accession number of
 target genes)<em>. </em>These PCR products were cloned in the
 TOPO-TA PCRII Vector (Invitrogen) and transformed into <em>E.
 coli</em> bacteria. <strong>Probe
 synthesis</strong> The probe templates were then
 obtained using PCR with M13-pUC primers and used for RNA polymerase suited
 to the sequence (T7/sp6, <em>SI Appendix</em>, Fig. S3).
 Afterwards these fragments were run through a 1% agarose gel to verify
 whether the fragment length was correct. The sequence of the template was
 determined by Sanger sequencing performed at Baseclear (Leiden) to verify
 that the probes contained the correct gene, and the product was compared
 using a BLAST with the family Cyprinidae. After this procedure, the probes
 were purified and made ready for use.
 <strong>Whole-mount
 </strong><em><strong>in
 situ</strong></em><strong>
 hybridization</strong> Whole-mount <em>in
 situ </em>hybridization (WISH) was performed as described for
 <em>Danio rerio</em>(38),<em> </em>with a few
 specific modification for <em>Rhodeus ocellatus</em>.
 Specifically, the Proteinase K (10 µg/ml) digestion time for <em>R.
 ocellatus</em> is longer than for <em>D. rerio</em>. For
 <em>R. ocellatus</em> embryo at blastula and gastrula stages,
 the digestion time was 1 min; for early somitogenesis stages, 5 min; for
 late somit <strong>Micro-computed tomography
 </strong> Micro-computed tomography analysis was
 carried out at Naturalis Biodiversity Center (Leiden, the Netherlands)
 using a Xradia 520 Versa 3-D X-ray microscope (Zeiss) based on described
 methods(8), and visualized with Avizo software (Avizo 9.5, Thermo
 Scientific™).
 <strong>Histology</strong> Sample
 were embedded in Technovit 7100 for routine histology followed by
 hameatoxylin and eosin, periodic acid Schiff, methenamine silver and oil
 red O staining. The section thickness was 0.5-0.7 µm. Samples were
 embedded in epon for toluidine blue staining and semi-thin sectioned with
 a glass knife. For scanning electron microscopy, we used critical-point
 drying, and scanned the samples with a JEOL SEM 7600. Ultra-thin sections
 were prepared for transmission electron microscopy and examined in a JEOL
 1400 transmission electron microscope.
 <strong>Time-lapse recording</strong>
 <em>R. ocellatus</em> embryos at the desired
 developmental stage were mounted in glass view chamber. Stereo microscope
 (Nikon SMZ1500) was used for recording and the focal level has been
 adjusted manually to keep the embryo in focus. Photographs were captured
 with a Nikon DS-Fi1-L2 camera. Time-lapse images were assembled into video
 recordings using Fiji (39). To quantitatively describe the movement of
 embryos during blastokinesis, we used the Manual Tracking plugin developed
 by F. Cordelières (http://rsbweb.nih.gov/ij/plugins/track/track.html) for
 Fiji. The ventral and dorsal margins of the blastoderm, and the head
 region and tail region of embryo were labeled and their trajectories
 during the blastokinesis movement were tracked. The distance and velocity
 information of each trajectory were then imported into Excel for
 statistical analysis. Velocimetric estimation of
 overall morphogenetic movements was performed by particle image
 velocimetry (PIV) analysis using the PIVlab software package (40). The
 size of the vectors is proportional with the velocity of tissue movements.
 The analysis spanned four periods: (1) Epiboly period until the yolk plug
 closure. (2) Front-flip period where the embryo's head region moved
 from the animal pole (12 o'clock position) to the vegetal pole (9
 o'clock position). (3) Elongate period with continued head migration
 towards the vegetal pole (7 o'clock position) and tail relocation
 from 3 o'clock to 12 o'clock. (4) Rostral protrude period
 concluding at the head's arrival at the 6 o'clock position
 before hatching. The deformation modes of each period were described by
 computing the temporal mean value of the velocity field. The resulting
 plots were imported to Adobe Illustrator to label the collective direction
 of vectors. Frame-by-frame PIV analysis was assembled into video
 (<em>SI Appendix</em>, Movie S3-S5).
description: Files can be accessed in Image J.
description: # Data For Parasitic fish embryos do a 'front-flip' on the yolk
 to resist expulsion from the host Raw data for time-lapse recording of
 embryo hatching process, the outing from chorion. Raw data for 3D
 reconstruction data from CT scans. ## Description of the data and file
 structure The data consists of two parts: raw data for time-lapse
 recording and 3D reconstruction data from CT scans. The time-lapse
 recording files are in JPG format, with filenames numbered sequentially
 according to time sequence. The 3D reconstruction data from CT scans
 includes four files named "host-mussel_dissected \_tomo-A", as
 well as a Header.txt file describing the scanning parameters. Among the
 four files named host-mussel_dissected \_tomo-A, one is an MPG video file,
 one is a compressed package of raw data, and two are preview images in JPG
 format. For time-lapse recording, *R. ocellatus* embryos at the desired
 developmental stage were mounted in glass view chamber. Stereo microscope
 (Nikon SMZ1500) was used for recording and the focal level has been
 adjusted manually to keep the embryo in focus. Photographs were captured
 with a Nikon DS-Fi1-L2 camera. Total recorded time was 1h of development.