Influence of soil contaminated with cadmium on cell death in the digestive epithelium of soil centipede Lithobius forficatus (Myriapoda, Chilopoda)

Figures & data

Table I. Diagrammatic representation of the collection of adult specimens of L. forficatus. F – females, M - males

	Number of specimens examined
		Confocal microscope
Experimental group	TEM F/M	TUNEL assay	LysoTracker	Flow cytometry Annexin V-FITC	ADP/ATP ratio	AAS
C	5/5	5	5	6	5	5
Cd1	5/5	5	5	6	5	5
Cd2	5/5	5	5	6	5	5

Figure 1. Scheme of experimental setup described in 2.1.2. chapter

Figure 2. Cadmium concentration [mg ∙ l⁻¹] in the experimental groups: control, Cd1 and Cd2. Mean ± SE. Different letters (a, b) mean statistically significant differences at p ≤ 0.05. One-way ANOVA, Tukey’s test, n = 5–6

Figure 3. Diagrammatic representation of the midgut epithelium in L. forficatus. Digestive cells (dc), secretory cells (sc), regenerative cells (rc), basal lamina (bl), visceral muscles (vm), midgut lumen (l)

Figure 4. The midgut epithelium of L. forficatus – the control group (C group)

(a) The transverse section through the midgut epithelium (me). Midgut lumen (l), visceral muscles (vm). Light microscope. Scale bar = 12 µm. (b–d) The apical cytoplasm of the digestive cells. Midgut lumen (l), microvilli (mv), mitochondria (m), reserve material (rm), spherites (s), autophagosome (au), cisterns of rough endoplasmic reticulum (RER), vesicles with electron lucent content (white arrows). TEM. (b) Scale bar = 2 µm. (c) Scale bar = 3 µm. (d) Scale bar = 2.2 µm. (e) The perinuclear cytoplasm of the digestive cells: mitochondria (m), glycogen granules (g), nuclei (n), reserve material (rm), cisterns of RER (RER). TEM. Scale bar = 1.4 µm. (f) The basal and perinuclear cytoplasm of the digestive cells. Nuclei (n), mitochondria (m), basal lamina (bl), lipid droplets (ld), folds of the basal cell membrane (black arrows). TEM. Scale bar = 1.4 µm. (g) The perinuclear and apical cytoplasm of the digestive cells with electron dense spheres of reserve material (rm) and glycogen granules (g). Mitochondria (m), cisterns of RER (RER). TEM. Scale bar = 1.25 µm. (h) Regenerative cells (rc) located among basal regions of digestive cells (dc). Mitochondria (m), nuclei (n), cisterns of RER (RER). TEM. Scale bar = 1.6 µm.

Figure 5. The midgut epithelium of L. forficatus – animals treated with cadmium for 12 days (Cd1 group)

(a) The transverse section through the midgut epithelium (me). Midgut lumen (l), visceral muscles (vm). Light microscope. Scale bar = 20 µm. (b) The apical cytoplasm of the digestive cells. Microvilli (mv), midgut lumen (l), mitochondria (m), reserve material (rm), autophagosome (au), vacuoles (vc), apical cell membrane devoid of microvilli (black arrows). TEM. Scale bar = 2 µm. (c–d) The perinuclear region in digestive cells with some mitochondria (m), cisterns of RER (RER), vacuoles (vc) and autophagosomes (au). Nuclei (n). TEM. C. Scale bar = 1.1 µm. (d) Scale bar = 1.4 µm. (e–f) Basal cytoplasm of digestive cells. Basal lamina (bl), visceral muscles (vm), nuclei (n), mitochondria (m), lipid droplets (ld), cisterns of RER (RER), vacuoles (vc), folds of the basal membrane (black arrows). TEM. (e) Scale bar = 2.2 µm. (f) Scale bar = 2 µm. (g) Spherites (s) accumulated in the cytoplasm of digestive cells. TEM. Scale bar = 1.5 µm. (h) Regenerative cell (rc) located among basal regions of digestive cells (dc). Mitochondria (m), nucleus (n), basal lamina (bl), visceral muscles (vm). TEM. Scale bar = 1.5 µm. (i) Secretory cell (sc) located among basal regions of digestive cells (dc). Nucleus (n), electron dense granules (black arrows), basal lamina (bl), visceral muscles (vm). TEM. Scale bar = 1.1 µm.

Figure 6. The midgut epithelium of L. forficatus – animals treated with cadmium for 45 days (Cd2 group)

(a) The transverse section through the midgut epithelium (me). Midgut lumen (l), visceral muscles (vm). Light microscope. Scale bar = 7.5 µm. (b) The cytoplasm of numerous digestive cells is electron lucent with small amount of organelles. Mitochondria (m), nucleus (n), reserve material (rm). TEM. Scale bar = 1 µm. (c) The electron lucent basal cytoplasm of digestive cells with small amount of organelles. Mitochondria (m), reserve material (rm), basal lamina (bl), folds of the basal cell membrane (arrows). TEM. Scale bar = 1 µm. (d) The apical cytoplasm of the digestive cells with microvilli (mv) and large bulges lacking microvilli (black arrows). Glycogen granules (g), midgut lumen (l), vesicles (v), reserve material (rm). TEM. Scale bar = 2 µm. (e) The apical cytoplasm of the digestive cells with microvilli (mv). Midgut lumen (l), vesicles (v), fusion of the vesicles (black arrows), exocytosis (black arrowheads), reserve material (rm). TEM. Scale bar = 0.8 µm. (f) The cytoplasm of the digestive cells with the reserve material (rm) and the cytoplasm of the medium-electron density. Vesicles (v) and vacuoles (vc) in the digestive cells with the electron lucent cytoplasm. Microvilli (mv). TEM. Scale bar = 2 µm. (g) Electron lucent cytoplasm of the digestive cells with vacuoles (vc) and autophagic structures (au). Mitochondria (m). TEM. Scale bar = 1.2 µm. (h) Nuclei (n) in the digestive cells (dc). Spherites (s). TEM. Scale bar = 1.2 µm. (i) Regenerative cell (rc) among basal regions of the digestive cells (dc). Basal lamina (bl). TEM. Scale bar = 2.3 µm.

Figure 7. Autophagy in the midgut epithelium of L. forficatus.

(a) Digestive cells in animals from the control group. Autophagosomes (au), cisterns of endoplasmic reticulum (ER), mitochondria (m), nuclei (n). TEM. Scale bar = 1 µm. (b) Digestive cells in animals from Cd1 group. Autophagosomes (au), microvilli (mv), mitochondria (m), residual bodies (rb), vesicles with electron lucent content (v). TEM. Scale bar = 0.8 µm. (c) Digestive cells in animals from Cd2 group. Autophagosomes (au), microvilli (mv), mitochodnria (m), midgut lumen (l), reserve material (rm), vesicles with electron lucent content (v), vacuoles (vc). TEM. Scale bar = 1.7 µm. (d–f) 3D representation of the accumulation of lysosomes and autolysosomes (red signals) in the L. forficatus midgut. Nuclei (blue signals). LysoTracker Red and DAPI staining. Confocal microscope. (d) Control group. Scale bar = 6 µm. (e) Animals treated with cadmium for 12 days. Scale bar = 7 µm. (f) Animals treated with cadmium for 45 days. Scale bar = 7 µm.

Figure 8. Percentage of autophagic cells in intestine of L. forficatus from the control group (C) and exposed to cadmium (Cd1, Cd2). Different letters (a, b, c) indicate significant differences between experimental groups (x ± SE; Tukey’s test, p < 0.05); n = 6

Figure 9. Apoptosis and necrosis in the midgut epithelium of L. forficatus.

(a-c) 3D representation of the Tunel assay and DAPI staining. Nuclei of apoptotic cells (red signals), nuclei (blue signals). Confocal microscope. (a) Control group. Scale bar = 6 µm. (b) Animals treated with cadmium for 12 days. Scale bar = 6 µm. (c) Animals treated with cadmium for 45 days. Scale bar = 6 µm. (d–f) Diagrammatic representation of quantitative analysis of apoptotic and necrotic cells in the midgut epithelium of L. forficatus from the control group (C) and exposed to cadmium (Cd1, Cd2). Different letters (a, b) indicate significant differences between experimental groups within each parameter (x ± SE; Tukey’s test, p < 0.05); n = 5–6. Flow cytometry. (d) Percentage of early apoptotic cells (Annexin⁺, PI⁻). (e) Late apoptotic cells (Annexin⁺, PI⁺). (f) Necrotic cells (Annexin⁻, PI⁺).

Table II. Concentrations of ATP (x± SE) and ADP/ATP ratio (x± SE) in intestine of L. forficatus from the control group (C) and groups exposed to cadmium (Cd1, Cd2). The different letters (a, b) indicate significant differences between experimental groups within each parameter (Tukey’s test, p < 0.05); n = 4–5

Groups	ATP [nmol∙mg protein^−1]	ADP/ATP
C	0.321 ± 0.085a	0.375 ± 0.085a
Cd1	0.025 ± 0.011b	1.934 ± 0.326ab
Cd2	0.013 ± 0.005b	3.839 ± 0.925b

Figure 10. Hemocytes in the neighborhood of L. forficatus midgut. (a) Hemocytes (hc) in animals from the control group. Digestive cells (dc), basal lamina (bl), electron dense granules (black arrows), mitochondria (m), visceral muscles (vm), nucleus (n). TEM. Scale bar = 2 µm. (b) Hemocytes (hc) located among digestive cells. Animals treated with cadmium for 12 days. Digestive cells (dc), basal lamina (bl), nucleus (n), electron dense granules (black arrows). TEM. Scale bar = 1.5 µm. (c) Hemocytes (hc) located in the hemocoel in the neighborhood of visceral muscles (vm) and basal lamina (bl). Animals treated with cadmium for 45 days. Digestive cells (dc), electron dense granules (black arrows), mitochondria (m), cisterns of ER (ER). TEM. Scale bar = 0.6 µm