For the insect pathogen Photorhabdus luminescens, which end of a nematode is out?

Abstract

The nematode Heterorhabditis bacteriophora is the vector for transmitting the entomopathogenic bacterium Photorhabdus luminescens between insect larvae. The dauer juvenile (DJ) stage nematode selectively retains P. luminescens in its intestine until it releases the bacteria into the hemocoel of an insect host. We report the results of studying the transmission of the bacteria by its nematode vector. Cells of P. luminescens labeled with green fluorescent protein preferentially colonized a region of the DJ intestine immediately behind the basal bulb, extending for various distances toward the anus. Incubation of DJ nematodes in vitro in insect hemolymph induced regurgitation of the bacteria. Following a 30-min lag, the bacteria migrated in a gradual and staggered movement toward and ultimately exited the mouth. This regurgitation reaction was induced by a low-molecular-weight, heat- and protease-stable, anionic component present in arthropod hemolymph and in supernatants from insect cell cultures. Nematodes anesthetized with levamisole or treated with the antihelmenthic agent ivermectin did not release their bacteria into hemolymph. The ability to visualize P. luminescens in the DJ nematode intestine provides the first clues to the mechanism of release of the bacteria during infection of insect larvae. This and the partial characterization of a component of hemolymph triggering release of the bacteria render this fascinating example of both a mutualistic symbiosis and disease transmission amenable to future genetic and molecular study.

FIG. 1.

Location of GFP-labeled P. luminescens cells in the intestines of H. bacteriophora DJ nematodes. (A) Differential interference contrast micrograph showing cells (arrow) located anterior to the nematode basal bulb. (B) Epifluorescent micrograph of same nematode as in panel A, showing GFP-labeled P. luminescens cells present throughout the DJ nematode lower intestine. (C) Epifluorescent micrograph taken with higher magnification, showing bacteria in the DJ intestine. (D and E) Epifluorescent micrographs of several nematodes freshly harvested (D) and after 30 days of incubation in saline (E). a, anterior orientation of nematode; b, posterior orientation of nematode.

FIG. 2.

Association of GFP-labeled P. luminescens with non-DJ nematodes. (A, C, and D) Digestion of GFP-labeled bacteria by adult nematodes indicated by a bright and diffuse fluorescence in the intestine (i). Intact bacteria (arrows) are found to be associated with the vulva (v) opening (A and C) and rectum (r) (C and D). Eggs (e) are indicated in panel C. (B) Four nematodes undergoing endotokia matricida containing variable amounts of bacteria in their body cavities (arrows). Two nematodes are brightly fluorescent due to large numbers of bacteria in their body cavities (>1,000 bacteria/nematode). Two other nematodes are weakly fluorescent due to a small population of bacteria (<200 bacteria/nematode). Bacteria are also present in the remnants of the pharynx (p) and rectum (r). A DJ nematode outside the adult nematodes is also indicated.

FIG. 3.

Release of GFP-labeled P. luminescens by DJ nematodes incubated in M. sexta hemolymph. In all panels the anteriors of the nematodes are orientated to the left of the field of view. (A) Phase-contrast micrograph showing bacterial cells that were released from a DJ nematode. (B) Epifluorescent micrograph of the same nematode clearly shows bacteria in the intestine and pharynx. (C) A DJ nematode incubated for 20 min, prior to releasing GFP-labeled P. luminescens. (D, E, and F) Nematodes incubated for 30, 40, and 60 min, respectively. Note progressive movement of bacteria toward the nematode mouth. (G and H) A nematode observed under higher magnification, showing individual bacterial cells leaving a cluster of bacteria in the posterior region of the intestine after 90 min of incubation (G) and showing bacteria poised to exit the mouth after 90 min of incubation (H).

FIG. 4.

Numbers of GFP-labeled P. luminescens organisms released by DJ nematodes incubated in M. sexta hemolymph or Grace's insect cell culture medium. CFU counts were made, and the numbers of bacteria released were normalized to those DJ nematodes that epifluorescence microscopy showed to be actively releasing bacteria at each experimental point. Error bars indicate standard deviations.