Cell Invasion and Matricide during Photorhabdus luminescens Transmission by Heterorhabditis bacteriophora Nematodes

Abstract

Many animals and plants have symbiotic relationships with beneficial bacteria. Experimentally tractable models are necessary to understand the processes involved in the selective transmission of symbiotic bacteria. One such model is the transmission of the insect-pathogenic bacterial symbionts Photorhabdus spp. by Heterorhabditis bacteriophora infective juvenile (IJ)-stage nematodes. By observing egg-laying behavior and IJ development, it was determined that IJs develop exclusively via intrauterine hatching and matricide (i.e., endotokia matricida). By transiently exposing nematodes to fluorescently labeled symbionts, it was determined that symbionts infect the maternal intestine as a biofilm and then invade and breach the rectal gland epithelium, becoming available to the IJ offspring developing in the pseudocoelom. Cell- and stage-specific infection occurs again in the pre-IJ pharyngeal intestinal valve cells, which helps symbionts to persist as IJs develop and move to a new host. Synchronous with nematode development are changes in symbiont and host behavior (e.g., adherence versus invasion). Thus, Photorhabdus symbionts are maternally transmitted by an elaborate infectious process involving multiple selective steps in order to achieve symbiont-specific transmission.

FIG. 1.

IJs develop inside maternal body cavities and not from laid eggs. (A) H. bacteriophora makes three key developmental or behavioral choices related to IJ formation: (i) egg-laying behavior versus intrauterine egg hatching (endotokia matricida); (ii) development of offspring to IJs versus vegetative larval growth and adulthood; and (iii) recovery versus dispersal of IJ offspring. The black lines indicate observed developmental pathways or behaviors, and the burgundy lines indicate alternative pathways or behaviors (see panels B to E). Green ovals indicate development by endotokia matricida. (B) Nematode development inside G. mellonella larvae. The mean numbers of total nematodes (▴), IJs (▪), and laid eggs (⧫) are indicated. The time (in days) is the time after IJ addition to insect larvae. The P₀ nematodes (nematodes derived from recovered IJs) laid eggs on days 3 to 5 and then underwent endotokia matricida on days 5 to 7 and produced the F₁ IJs on 6 day. Essentially no eggs were laid by the F₁adults. Instead, reproduction occurred via endotokia matricida, ultimately producing more than 500,000 IJs in a single insect larva. (C) Nematode development and behavior on lawns of symbiotic bacteria is similar to nematode development and behavior in insect larvae. The symbols are the same as those in panel B. Eggs were laid only by P₀ nematodes at 3 to 5 days. Reproduction by the P₀ nematodes and by the F₁adults occurred only via endotokia matricida on days 5 to 8, when more than 40,000 IJs were produced. (D) Egg-laying behavior of P₀ nematodes on day 4 on lawns of P. luminescens. (E) Reproduction of F₁ offspring via endotokia matricida on day 8 on lawns of P. luminescens. Maternal body cavities were packed with IJs, and one cavity is outlined in red; no laid eggs were observed.

FIG. 2.

Adherence of P. luminescens to maternal and adult male nematode intestines. Transient GFP-labeled cells were chased from the intestine so that only labeled symbiont cells that had established persistent infections were visible. (A) Single GFP-labeled P. luminescens cell adhering to posterior intestinal (INT9) cells of a fourth-stage H. bacteriophora larva following 8 h of exposure to labeled symbionts. (B) Several adherent GFP-labeled P. luminescens cells on the posterior intestine of an adult H. bacteriophora following 20 h of exposure to labeled symbionts. (C) More adherent GFP-labeled symbionts on the posterior adult intestine following 38 h of exposure to labeled symbionts. (D) GFP-labeled P. luminescens adhering to an adult male H. bacteriophora intestine exposed for 36 h to labeled symbionts. (E) GFP-labeled symbionts still adhering to the posterior maternal intestine containing unlabeled adherent symbionts after 36 h of exposure of IJ to unlabeled symbionts, followed by 4 h of exposure to labeled symbionts. (F) GFP-labeled symbionts no longer adhered to the maternal intestine following 48 h of exposure of IJs to unlabeled symbionts, followed by 4 h of exposure to GFP-labeled symbionts. i, intestinal lumen; r, rectum.

FIG. 3.

Invasion of maternal RGCs in maternal nematodes 48 h after IJs were added to GFP-labeled lawns and then chased with unlabeled symbionts for 4 h. The intestinal lumen (i), rectum (r), and RGC vacuole (v) are indicated. (A) Adherent GFP-labeled symbionts, most of which are still adhering to the intestinal lumen, beginning to invade an RGC (white arrows). Transient unlabeled symbionts (black arrow) were visible in the intestinal lumen, but most of them did not invade the RGCs. (B) Site of invasion (arrow), where a symbiont-containing vacuole appeared to form at the basal surface of the RGC in close proximity to GFP-labeled symbionts attached to the intestinal lumen. The INT9 posterior intestinal cells were not invaded. (C) GFP-labeled symbionts adhering to the intestinal epithelium and invading RGCs (arrow). (D) Same worm as the worm in panel C at a focal plane ca. 5 to 10 μm above that in panel C where a RGC vacuole was apparent.

FIG. 4.

Behavior of intracellular symbionts. (A) Recently invaded RGCs each contained one to three vacuoles following 54 h of exposure of IJs to labeled symbionts. (B) Multiplication of symbiont-containing vacuoles was evident 38 h later, and each RGC contained 12 to 30 symbiont-containing vacuoles at that time. The three RGCs indicated are ventral left (RGC VL), ventral right (RGC VR), and dorsal (RGC D) cells. One RGC nucleus is also indicated. (C) Apical lysis of RGCs and liberation of symbiont-containing vacuoles into the maternal body cavity of worms undergoing endotokia matricida, while the intestine and basal surface of the RGCs appeared to be intact. (D) Morphological differences of vacuoles in maternal nematodes grown on a mutant GFP-labeled symbiont unable to invade the RGCs. Only a few large vacuoles were present in nematodes following 112 h of exposure of IJs to this mutant symbiont. n, nucleus; i, intestine; r, rectum; v, vacuole.

FIG. 5.

Ultrastructure of symbiont-containing vacuoles. (A) Transmission electron micrograph of a cross-section of a maternal nematode following 96 h of exposure to symbiont lawns. Symbiont-containing vacuoles are indicated by thick arrows. A few bacterial cells (b) inside the vacuoles are also indicated (thin arrows). (B) Two connected vacuoles possibly in the process of division. The vacuolar membrane (vm) and a few bacteria (b) are indicated. (C) Granular contents of symbiont-containing vacuoles. Bleb-like structures are indicated. (D) Bleb in contact with a bacterial cell (white arrow). Outer (om) and inner (im) gram-negative membranes and unknown subcellular structures (black arrows) are indicated.

FIG. 6.

Colonization of pre-IJ second-stage juveniles. (A) Fluorescent micrograph of GFP-labeled symbiont cells (arrows) in the body cavities of two nematodes undergoing endotokia matricida and inside pre-IJs (arrows) (the image was not pseudocolored or overlaid like other fluorescent micrographs). (B) One or two GFP-labeled symbionts (arrow) adhering to the PIVCs 120 h after the maternal nematode was placed on labeled bacteria. No chase was necessary since transient symbionts were not visible at this time. The pharynx (p) and intestine (i) are also indicated. (C) Several GFP-labeled symbiont cells (arrows) possibly in the PIVCs were visible <8 h after adherence. (D) Several GFP-labeled symbiont cells (arrow) in the intestinal lumen of the IJs were visible 16 h later. (E) Growth of intestinal symbionts (arrows) in early stages of IJ colonization 24 to 72 h later.

FIG. 7.

Model of the transmission cycle. Symbionts that have colonized the maternal intestine (top panel) or pre-IJs or IJs (bottom panel) are shown in the context of select nematode cells in the same orientation on the upper left, where the anterior (A) is on the left, the posterior (P) is on the right, dorsal (D) is up, ventral (V) is down, left (L) is out, and right (R) is into the page. Nematode cells are abbreviated as follows: INT, intestinal cells; RGC-D, dorsal RGCs; RGC-VL, ventral-left RGCs; and PIVCs, pharyngeal intestinal valve cells. Colonized symbionts are indicated by green ovals, and the green arrows indicate regurgitation or ingestion of symbiont cells. The time (t) is the time (in hours) after IJ addition to lawns of symbiont bacteria (zero time). Dormant symbionts are not adherent to the IJ intestinal lumen. At <8 h intestinal symbionts are completely released during IJ recovery and regurgitation. At 8 to 42 h symbionts adhere to and grow within the maternal posterior intestine, corresponding to INT9L and INT9R cells. At 42 to 48 h adherent symbionts invade the RGCs and no longer adhere to the INT9 cells. At 48 to 110 h symbionts grow intracellularly in the RGCs and stimulate vacuole formation. At 106 to 112 h symbionts are released from RGCs after lysis and gain access to the pre-IJs developing in the maternal pseudocoelom. At 100 to 112 h symbionts adhere to the PIVCs of pre-IJs (L2) developing within the maternal pseudocoelom. At 110 to 120 h symbionts exit the pre-IJ intestinal lumen, possibly invade PIVCs, and multiply. At 120 to 288 h symbionts exit the PIVCs and colonize the IJ intestinal lumen. Note that vegetative progeny acquire symbionts 5 to 12 h after they hatch from laid eggs also by symbiont adherence to the INT9 cells but otherwise exhibit similar symbiont transmission.