Figure 1.
Molting and calcium bodies in Hyloniscus riparius.
(A) An individual shortly after anterior ecdysis. Exuviae of both body-halves are visible in the proximity. (B) Dark-field image of the posterior (pcb) and anterior (acb) calcium bodies in situ. (C) An illustration of the position of the digestive system and the calcium bodies in the body. Each calcium body consists of a medial tube and a lateral extension (ext). (D) 3D reconstruction of calcium bodies from serial paraffin sections, dorsolateral view. h – hepatopancreas.
Figure 2.
Structure and contents of the posterior calcium bodies in intermolt stage.
(A) Semithin section through a posterior calcium body. A single-layered epithelium (e) encloses the layered, bacteria-containing matrix (ma). (B) Transmission electron micrograph showing bacteria (ba) in the proximity of a lamina (la) within a posterior calcium body. (C) Scanning electron micrograph (aldehyde/OsO4 fixation) of the bacteria within the posterior calcium body. An extracellular lamina (la) is visible among the bacteria (D) Scanning electron micrograph of the matrix within the posterior calcium bodies (methanol fixation). Layers of crystals (cr) and numerous bacterial casts (bc) are visible. (E) Scanning electron micrograph (methanol fixation) of crystals (cr) within the posterior calcium bodies. g – gut, h – hepatopancreas.
Figure 3.
Ultrastructure of the posterior calcium body epithelium in intermolt stage.
(A) Section through the epithelium (apical side up). An extracellular envelope (en) lines the folded apical plasma membrane (pm). Mitochondria (m) and electron dense vesicles (v) are present in the cytoplasm. (B) The apical surface of the posterior calcium body epithelium. The apical plasma membrane forms folds (mf). The envelope (en) consists of a proximal electron dense and a distal electron lucent layer. A lamina (la) is visible inside the lumen. (C) Rosettes of electron dense particles (most likely glycogen) in the cytoplasm of the epithelial cells. (D) An autophagic vacuole (av) in the cytoplasm of an epithelial cell. (E) Junction between two epithelial cells. The subapical adherens junction (aj) and the more basally positioned septate junction (sj) are visible. g – glycogen, n – nucleus.
Figure 4.
Anterior calcium bodies in intermolt stage.
(A) Semithin section through an anterior calcium body (acb). An epithelium (e) forms the wall of the organ. An agglomeration of laminae (la) is visible in the lumen. Mineral content was lost during tissue preparation. (B) Mineral concretion from the medial tube of an anterior calcium body. (C) Scanning electron micrograph of a fractured mineral concretion from an anterior calcium body (methanol fixation) with a central cavity (ca). (D) Scanning electron micrograph of a fractured anterior calcium body (methanol fixation) showing a continuous layer of mineralized matrix (ma) formed around the central cavity (ca) with folded laminae. (E) Higher magnification scanning electron micrograph showing small spherules (sp) on the surface of laminae (la) within the central cavity. (F) Transmission electron micrograph of the laminae (la) within an anterior calcium body. (G) Cross-section through the anterior calcium body epithelium. The cytoplasm of epithelial cells contains numerous mitochondria (m), electron dense vesicles (v) and autophagic vacuoles (av). The apical plasma membrane is not folded. (H) The apical surface of an epithelial cell (e). The apical plasma membrane is lined by an envelope (en) consisting of a proximal electron dense and a distal electron lucent layer. Numerous laminae (la) are visible in the lumen. h – hepatopancreas, n- nucleus.
Figure 5.
Calcium bodies in premolt stage.
(A) Synthesis of the new envelope (arrows) in an anterior calcium body. The new envelope is discontinuous and is deposited over short protrusions of the apical plasma membrane. The old envelope (oe) is detached. (B) Synthesis of the new envelope (arrows) in the posterior calcium bodies. (C) Synthesis of the tergal epicuticle (arrow) in early premolt stage. A forming cuticular scale (sc) is visible. (D) The apical surface of the anterior calcium body epithelium in late premolt stage. The folded apical plasma membrane (pm) is lined by the new envelope (ne). (E) Differential interference contrast image of a posterior calcium body in late premolt stage (eosin stain). A glassy layer (gl) of mineralized matrix is present between the bacteria-containing central matrix (cm) and the epithelium (e). (F) Scanning electron micrograph of the glassy layer (gl) in a fractured posterior calcium body (methanol fixation). Small spherules are visible on the laminae (la) in the outer parts of the central matrix. (G) Differential interference contrast image of a premolt stage anterior calcium body (eosin stain). The old envelope (oe) is embedded in the mineralized matrix (ma). The new envelope (ne) lines the epithelium (e). ba – bacterium, es – ecdysial space, h – hepatopancreas, m – mitochondrion.
Figure 6.
Calcium bodies in intramolt stage.
(A) The posterior calcium body epithelium is unaltered with respect to the intermolt stage. (B) The anterior calcium body epithelium forms deep apical membrane folds (mf). Electron dense granules (arrows) are present in intercellular spaces between neighboring cells. Mineral components were lost during tissue preparation. (C) Formation of electron dense granules in intercellular spaces of the anterior calcium body epithelium on short protrusions (arrows) of the plasma membrane. (D) The mineralized matrix (ma) in an anterior calcium body. The outer surface of the mineral concretion (upper left side of image) appears etched (arrowhead). The embedded old envelope (oe) separates two layers of the mineralized matrix (ma). (E) Higher magnification of the etched surface of the mineralized matrix within an anterior calcium body showing granular material arranged in radial threads. The surface of the concretion is to the upper left side of the image. bl – basal lamina, j – cell junction, l – calcium body lumen, m- mitochondrion, ne – new envelope, oe – old envelope.
Figure 7.
Calcium bodies one day after anterior ecdysis.
(A) The posterior calcium body epithelium (apical side up) with apical membrane folds (mf) and electron dense granules (arrow) in intercellular spaces between neighboring cells. (B) The anterior calcium body epithelium (apical side up). Folds (mf) of the apical plasma membrane reach deeply into the epithelium. Electron dense granules of varying sizes (arrows) are present in intercellular spaces. (C) Oblique section through the apical part of the anterior calcium body epithelium showing numerous folds of the apical plasma membrane. (D) Oblique section through the apical part of the posterior calcium body epithelium with numerous membrane folds and mitochondria (m). (E) High magnification image of the electron dense granules in intercellular spaces of the anterior calcium body epithelium. (F) Paraffin section of the posterior calcium body (eosin stain) showing the glassy layer (gl), which is still present after anterior ecdysis. (G) Paraffin section of the anterior calcium body (eosin stain) showing the presence of mineralized matrix (ma) within it. cm – central matrix, e – epithelium, l – lumen, n – nucleus.
Figure 8.
Calcium bodies two days after anterior ecdysis.
(A) The posterior calcium body epithelium. Epithelial ultrastructure is similar to that of the intermolt stage epithelium. (B) The anterior calcium body epithelium. The apical surface of epithelial cells is no longer folded and adjacent cells are in close contact. (C) Paraffin section (eosin stain) of a posterior calcium body (pcb). No glassy layer is present. (D) Paraffin section (eosin stain) of an anterior calcium body (acb). The lumen is devoid of mineral and only organic laminae (la) are visible within it. ba – bacteria, e – epithelium, en – envelope, h – hepatopancreas, j – cell junction, m – mitochondrion, mu – muscle, n – nucleus.
Figure 9.
Raman spectra of mineralized matrices within calcium bodies.
(A) Raman spectrum of the bacteria-containing central matrix in a posterior calcium body. (B) Raman spectrum of the glassy layer developed during the premolt stage in a posterior calcium body. (C) Raman spectrum of the concretion within an anterior calcium body. Positions of the most prominent peaks are given in cm−1. Asterisks denote spikes.