Figure 1.
Transverse section of a whole juvenile oyster stained by H & E and MSI probes.
(A, B) H & E, (C, E) MSI31 probe; (F, G, H) MSI60 probe. B, E, and H are enlarged images of the areas enclosed by boxes in A, C, and F. (I, J) A piece of mantle for implantation stained by MSI31 (I) and MSI60 (J) probes.
Figure 2.
General images of the pearls examined in the present study.
(A) nacreous pearl; (B) prismatic pearl; (C) pearl without deposition; (D) complex pearl with a flaw and pigmentation; (E) nacreous pearl with unusual tablet formation. The areas shown by broken lines in D and E correspond to the pearl sac epithelium shown in Figs. 4A and 6A, respectively.
Figure 3.
Surface microstructure of pearls observed in the scanning electron microscope (SEM), and gene expression in the pearl sac detected by ISH.
(A, E) SEM images of a nacreous pearl and a prismatic pearl. (B, F, I) MSI31 and (C, G, J) MSI60 gene expression in the pearl sac epithelium. D, H, and K) are enlarged images of the regions outlined in C, G, and J, respectively. (I, J, K) MSI31 and MSI60 expression patterns in the pearl sac of the oyster which produced the pearl without deposition. The grade of the pearls was determined by eye and by scanning electron microscopy.
Table 1.
Summary of expression levels of MSI 31 and MSI 60 by ISH in the pearl sac epithelium.
Figure 4.
SEM images of a complex pearl with dark pigmentation and a flaw.
(A) Low power SEM image. The line (1, 30) and arrows indicate the plane of the sections of the pearl sac shown in Fig. 5. (B) The boundary region between the dark pigmentation site and the nacreous layer. The blue, red, and black boxes indicate the areas shown at higher magnification in panels B, C and D. The broken line outlines the area corresponding to pearl sac tissue examined by ISH and shown in Fig. 5. (C) Enlarged image of the protruded area shown by the red box in A. Inset shows the image at higher magnification. (D) Enlarged image of the nacreous layer shown as box D in Fig. 5A.
Figure 5.
Expression of MSI31 and MSI60 in pearl sac epithelium detected by ISH.
This oyster produced the pearl with pigmentation and a flaw (protrusion) shown in Fig. 4. Images in the upper and lower rows show the expression of MSI60 and MSI31, respectively. The numbers refer to sequence of serial sections. Sections 1 and 30 corresponded to the plane indicated by arrows on Fig. 4A (sections 27–30 are not shown). Scale bar is100 µm.
Figure 6.
SEM images of the nacreous pearl with unusual tablet growth.
(A) The surface of the pearl at low magnification. The numbers and arrows indicate the serial section sequence and the sectioning plane of the pearl sac tissue stained by ISH referred to in Fig. 8. (B) SEM micrograph of the surface near point B in panel A. The process of tablet growth was different from a typical nacreous pearl shown in Fig. 3A. (C) Enlarged image of the pearl surface around point C in panel A. (D) A partial cross-section showing the lamellar structure beside the scratch. (E) The surface structure of B shown at higher magnification. Note doughnut-shaped structures. (F) Ring like structure of the growing nacreous tablets between points B and C in panel A. Scale bars are 1 mm in A, 10 µm in B and C, and 100 µm in D–F.
Figure 7.
Expression of MSI31 and MSI60 in pearl sac epithelium detected by ISH in a nacreous pearl with unusual tablet growth.
This oyster produced the pearl shown in Fig. 6A. Upper and lower rows show the expression of MSI31 and MSI60, respectively. The numbers refer to the sequence of serial sections. Section 1 and 30 correspond to the plane indicated by arrows in Fig. 6A. Sections 29–30 are not shown.
Figure 8.
Schematic expression pattern of MSI31 and MSI60 based on the observations of the pearl sac of a complex pearl and a nacreous pearl with unusual tablet growth and the corresponding pearl surface observed in the SEM.
Nac and Pr refer to nacreous and prismatic layers, respectively. The intensity of the ISH signal is indicated: − undetectable; ± very weak; + positive; + + very strong. The numbers indicate the position of pearl sac sections.
Figure 9.
Schematic drawing and photograph of the region of the oyster including the pearl sac.
(A) The position of the pearl sac formed in the distal region of the oyster. (B) Schematic drawing of a vertical section of the pearl sac epithelium surrounding the pearl. (C) Dissection of the pearl sac tissue from the whole pearl sac for ISH. (D) After dissection of the pearl sac samples for ISH, we scratched the surface of pearl with a scalpel as a reference for SEM observation.