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, 115 (8), E1819-E1828

Bacteriocyte Cell Death in the Pea Aphid/ Buchnera Symbiotic System

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Bacteriocyte Cell Death in the Pea Aphid/ Buchnera Symbiotic System

Pierre Simonet et al. Proc Natl Acad Sci U S A.

Abstract

Symbiotic associations play a pivotal role in multicellular life by facilitating acquisition of new traits and expanding the ecological capabilities of organisms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells (bacteriocytes) or organs (bacteriomes) have evolved for harboring beneficial microbial partners. The processes regulating the cellular life cycle of these endosymbiont-bearing cells, such as the cell-death mechanisms controlling their fate and elimination in response to host physiology, are fundamental questions in the biology of symbiosis. Here we report the discovery of a cell-death process involved in the degeneration of bacteriocytes in the hemipteran insect Acyrthosiphon pisum This process is activated progressively throughout aphid adulthood and exhibits morphological features distinct from known cell-death pathways. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of nonautophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, activation of reactive oxygen species, and Buchnera endosymbiont degradation by the lysosomal system. We showed that this multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization spread throughout the entire cytoplasm and surrounding Buchnera aphidicola endosymbionts. Our findings provide insights into the cellular and molecular processes that coordinate eukaryotic host and endosymbiont homeostasis and death in a symbiotic system and shed light on previously unknown aspects of bacteriocyte biological functioning.

Keywords: Acyrthosiphon pisum; Buchnera aphidicola; bacteriocytes; cell death; symbiosis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Degeneration of bacteriocyte tissue and cells during aphid aging. (AC) Representative images of H&E-stained whole-aphid sections from N3 (A), reproductively active adults (B), and senescent adults (C) demonstrating a progressive dissociation of bacteriocyte clusters. (A′C′) Enlarged images of H&E-stained bacteriocyte clusters showing the appearance of disaggregated bacteriocytes and the occurrence of morphological abnormalities with aphid aging. Rectangles correspond to the locations of the following magnified images. (D, F, and H) Magnified views of H&E-stained bacteriocytes. Note the presence of large unstained areas in adult degenerating bacteriocytes (black arrows), previously referred to as “low symbiont-density zones” (25). (E, G, and I) FISH of B. aphidicola symbionts (green) and nuclear DNA staining (blue) in bacteriocyte cells demonstrating the absence of symbionts within the low symbiont-density zones (white arrows). Note the progressive changes in bacteriocyte nuclear shape in aphid adults compared with nymphs (yellow arrows). Symbionts or DNA were stained with the Alexa 488-Buch probe specifically targeting B. aphidicola 16S rRNA or DAPI, respectively. Bact, bacteriocyte; Ct, cuticle; Emb, embryo; Gt, gut; Hd, head. (Scale bars: 200 µm in AC, 100 µm in A′C′, and 20 µm in DI.)
Fig. 2.
Fig. 2.
TEM of A. pisum degenerative bacteriocytes. (A) Low-magnification image of a degenerative bacteriocyte cell exhibiting extensive cytoplasmic vacuolation and an abnormal nuclear shape. (B) Enlarged image of the border between the peripheral cytoplasmic zone filled with B. aphidicola symbionts and the central heavily vacuolated zone extending around the nucleus. (C) Magnified view of the bacteriocyte nucleus showing loss of its round shape but no major ultrastructural modifications. (Inset) Enlarged view of chromatin structures and nucleolus. (Magnification of Inset: 6.4-fold.) Note the absence of the characteristic features of apoptosis such as chromatin condensation, nuclear fragmentation, and apoptotic body formation. (D and E) Magnified images of large cytoplasmic electron-lucent vacuoles (D) and B. aphidicola symbionts (E). Additional high-magnification images of nuclear structures, vacuoles, and symbionts are available in Fig. S1. Ec, euchromatin; Hc, heterochromatin; No, nucleolus; Nu, nucleus; S, symbiont; Sz, symbiont-dense zone; V, vacuole; Vz, vacuole-dense zone. (Scale bars: 20 µm in A, 10 µm in B, 5 µm in C, and 2 µm in D and E.)
Fig. 3.
Fig. 3.
Activation of autophagy during aphid bacteriocyte degeneration. (A) Representative TEM images of autophagic figures accumulating in the intervacuolar space of degenerative bacteriocytes. Black and white arrows denote autophagosomes containing membranous whorls and remnants of cellular organelles, respectively. (Scale bars: 1 µm.) (B) Schematic overview of the ATG pathway involved in autophagosome formation in A. pisum. Pea aphid ATG homologs are listed with their A. pisum (ACYPI) accession number referring to the A. pisum genomic database AphidBase (70), and paralogs are shown in different colors (black or gray). (C) Induction of Atg gene expression in bacteriocyte cells in the course of aphid aging revealed by qRT-PCR. Atg gene-expression levels in bacteriocytes at different life stages are expressed relative to the third-instar nymph levels. The rpl7 gene was used for data normalization. Results are reported as means ± SD (error bars) from three independent experiments. Data were analyzed by one-way ANOVA followed by a post hoc multiple-comparisons test (Tukey’s HSD test). Life stages labeled with different letters are significantly different (P < 0.05). Paralogs of Atg1 and Atg3 genes are displayed in distinct colors (black or gray), according to those used in the ATG pathway (B). N3 and N4, third and fourth nymphal stages, respectively; A9, A15, A23, adult time points at days 9, 15, and 23, respectively.
Fig. 4.
Fig. 4.
Organization of the bacteriocyte microtubule network during the degeneration process. (AD) Confocal images of the microtubule network (yellow) and B. aphidicola symbiont (green) distribution in whole-mount immunohistochemical (IHC)-stained bacteriocytes isolated from N3 (A), young adults (B), reproductively active adults (C), or senescent aphids (D). Arrows show a dense microtubule network in the degenerative vacuolated zone. (A′D′) Enlarged views of the perinuclear area. (EK) Magnified images of the microtubule network in the symbiont-dense zone (E, F, H, and J) and vacuole-dense zone (G, I, and K). Aphid symbionts and microtubules were labeled with anti-Buchnera GroEL and anti–β-tubulin antibodies, respectively. Z-stack imaging and 3D rotation reconstructions of degenerative bacteriocytes (C and D) can be observed in Movies S1–S4. Nu, nucleus. (Scale bars: 20 µm in AD, 10 µm in A′D′, and 5 µm in EK.)
Fig. 5.
Fig. 5.
ER-derived origin of hypervacuolation in degenerative bacteriocytes. (AE′) Confocal images (AE) and magnified views of ER organization (A′E′) in whole-mount IHC-stained gut (controls; A and A′) and bacteriocytes from N3 (B and B′), young adults (C and C′), reproductively active adults (D and D′), and senescent aphids (E and E′). ER (magenta), microtubule network (yellow), and B. aphidicola symbionts (green) were labeled with anti-KDEL, anti–β-tubulin, or anti-Buchnera GroEL antibodies, respectively. White arrows show the ER+ labeling in the degenerative bacteriocyte vacuolated zone. Note the discrepancy of ER organization between gut and bacteriocyte cells, with structured in cisternae around nuclei in the gut or forming a reticular network enveloping symbionts in bacteriocytes. Z-stack imaging and 3D rotation reconstructions of degenerative bacteriocytes (D and E) can be observed in Movies S5–S8. (FH) Representative TEM images of intervacuolar spaces in degenerative bacteriocytes revealing the ER origin of vacuoles. The black arrow shows a nascent vacuole originating from ER. Black and white arrowheads denote ribosomes that label the cytosolic face of the vacuole membrane and swollen mitochondria, respectively. Nu, nucleus; Vz, vacuole-dense zone. (Scale bars: 20 µm in AE, 5 µm in A′E′, 1 µm in F, and 0.5 µm in G and H.)
Fig. 6.
Fig. 6.
Activation of the lysosomal system during aphid bacteriocyte degeneration. (AE′) Confocal images (AE) and magnified views (A′E′) of lysosomes in whole-mount IHC-stained gut (controls; A and A′) and bacteriocytes from N3 (B and B′), young adults (C and C′), reproductively active adults (D and D′), and senescent aphids (E and E′). B. aphidicola symbionts (green) and lysosomes (red) were labeled with anti-Buchnera GroEL and anti-RAB7 antibodies, respectively. (E, Inset) Enlarged image of the lysosomal membrane enclosing a B. aphidicola symbiont. B. aphidicola symbionts (asterisk) engulfed in lysosomes in degenerative bacteriocytes. (Magnification of Inset: 5.3-fold.) Note the presence in degenerative bacteriocytes (D and E) of lysosome-positive signals only in symbiont-dense zones and not in vacuole-dense zones. Nu, nucleus; Vz, vacuole-dense zone. (Scale bars: 20 µm in AE, and 5 µm in A′E′′.) (F) Induction of lysosomal gene expression in bacteriocytes throughout aphid aging as revealed by qRT-PCR. Expression profiles of representative genes for major lysosomal activities: lysosomal acid hydrolases (CtsL), glycosidases (Lsz), and nucleases (DNaseII); activators (PSAP); or major (LIMP) and minor (LAPTM) membrane proteins (for a comprehensive overview of lysosomal gene-expression analysis, see Fig. S6). Gene-expression levels in bacteriocytes at different life stages are expressed relative to the third-instar nymph level. The rpl7 gene was used for data normalization. Results are reported as means ± SD (error bars) from three independent experiments. Data were analyzed by one-way ANOVA followed by a post hoc multiple-comparisons test (Tukey’s HSD test). Life stages labeled with different letters are significantly different (P < 0.05). Gene names: CtsL, cathepsin-L; DNaseII, DNase II; LAPTM, lysosomal-associated protein transmembrane; LIMP, lysosomal integral membrane protein; Lsz, lysozyme i-1; PSAP, prosaposin. A9, A15, A23, adult time points at days 9, 15, and 23, respectively; N3 and N4, third and fourth nymphal stages, respectively. (G) Representative TEM image of lysosomes containing B. aphidicola symbionts (asterisks) in degenerative bacteriocytes. Unaffected symbionts are marked with an “S.” Additional high-magnification images are available in Fig. S1. (Scale bar: 2 µm.)
Fig. 7.
Fig. 7.
Major phases of aphid bacteriocyte cell death. (A) Phase I: induction of ER-derived hypervacuolation. (B) Phase II: induction of bacteriocyte stress responses (autophagy activation and mitochondria swelling in the intervacuolar spaces). (C) Phase III: lysosome-mediated degradation of Buchnera. Blue, bacteriocyte nucleus; brown, autophagosome; dark green, B. aphidicola; light green, bacteriocyte cytoplasm; magenta, ER-derived vacuole membranes; peach, mitochondria; red, lysosomes.

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