APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2008, p. 2275–2287 Vol. 74, No. 80099-2240/08/$08.00�0 doi:10.1128/AEM.02646-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cell Invasion and Matricide during Photorhabdus luminescensTransmission by Heterorhabditis bacteriophora Nematodes�†

Todd A. Ciche,1* Kwi-suk Kim,1 Bettina Kaufmann-Daszczuk,1Ken C. Q. Nguyen,2 and David H. Hall2

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824,1 andThe Center for C. elegans Anatomy, Albert Einstein College of Medicine, Bronx, New York 104622

Received 21 November 2007/Accepted 8 February 2008

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

In most animals, healthy intestines are colonized by com-mensal and beneficial bacteria (8, 34). A fundamental questionis how beneficial interactions are established and maintainedwhile pathogenic interactions are resisted. To better under-stand symbiont discrimination in the animal intestine, trans-mission of the insect pathogen Photorhabdus luminescens bynematode hosts was investigated. Photorhabdus spp. are asso-ciated with entomopathogenic nematodes, such as Heterorhab-ditis bacteriophora (6). Knowledge about the relationship be-tween these two taxa and about the insecticidal toxinsproduced by the symbionts may prove to be useful for thecontrol of insect pests. An emerging human pathogen, Pho-torhabdus asymbiotica, was recently shown to be vectored byHeterorhabditis entomopathogenic nematodes (13). Coloniza-tion of developmentally arrested infective juvenile (IJ)-stageH. bacteriophora by P. luminescens is essential for both partnersto infect insects and reproduce in nature (7, 20). Germfree IJsinfect insects, but they do not cause insect mortality or repro-duce efficiently (20). H. bacteriophora IJs selectively vector P.luminescens or Photorhabdus temperata bacteria in their gutmucosa before regurgitating the symbionts into an insect host(7, 14). Since both partners of the symbiosis are required forinsect pathogenesis and insects are thought to be the preferredniche for both partners, there is likely strong selective pressureon symbiont-specific transmission.

The life cycle of H. bacteriophora is initiated by the environ-mentally resistant IJs usually inhabiting soil. After sensing an

insect host, the IJs enter the insect hemocoel through naturalopenings, such as the mouth or anus, or by using a buccal toothto slice through the cuticle and then regurgitate their intestinalsymbionts (7). Following symbiont release, insect mortalityoccurs rapidly (usually �48 h), and the IJs exit diapause andresume development, a process known as recovery. Symbioticbacteria that proliferate in the hemocoel preserve the insectcadaver by producing broad-spectrum antibiotics and/or sig-nals or nutrients essential for nematode growth and reproduc-tion (5, 33).

H. bacteriophora reproduces by either laying eggs or devel-oping eggs internally inside the maternal body cavity. Internaldevelopment of larvae ultimately causes matricide, a processtermed endotokia matricida, and the larvae develop into IJs(24). Nematodes that develop with symbiotic bacteria insideinfected insects or on agar media undergo two or three gen-erations before the IJs are generated en masse, and most of theIJs vector symbiotic bacteria to new insect hosts. It is reportedhere that symbiotic bacteria are maternally transmitted to IJsby the following elaborate sequence of events: (i) adherence tothe maternal posterior intestine, (ii) growth within the intesti-nal lumen, (iii) invasion of the rectal gland cells (RGCs), (iv)release into the maternal body cavity, (v) adherence to thepharyngeal intestinal valve cells (PIVCs), (vi) invasion of thePIVCs, and (vii) colonization of the IJ intestinal lumen.

MATERIALS AND METHODS

Media and culture conditions. The sources and descriptions of strains used inthis study are shown in Table 1. Photorhabdus spp. were grown in PP3salt–2%proteose peptone no. 3 (Difco, Detroit, MI) containing 0.5% NaCl (Sigma-Aldrich, St. Louis, MO), and agar (1.5%), gentamicin (0.75 �g/ml), streptomycin(40 �g/ml), and kanamycin (3.75 �g/ml) were added when they were required.Escherichia coli was grown in lysogeny broth (3) modified so that it contained 5g/liter NaCl, and agar (1.5%), gentamicin (5 �g/ml), ampicillin (50 �g/ml), anddiaminopimelic acid (300 �g/ml) were added when they were required. P. lumi-

* Corresponding author. Mailing address: Department of Microbi-ology and Molecular Genetics, Michigan State University, East Lan-sing, MI 48824. Phone: (517) 355-6463. Fax: (517) 355-8957. E-mail:ciche@msu.edu.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 15 February 2008.

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nescens subsp. laumondii was isolated from H. bacteriophora TT01 kindly pro-vided by Ann Burnell (NIU-Maynooth, Ireland) by placing IJs that were surfacesterilized for 5 min in 1% commercial bleach onto PP3salt.

Propagation of H. bacteriophora. Nematodes were propagated on 8 g of nu-trient broth with 10 �g/ml cholesterol (NA�chol) or on 8 g of nutrient broth with12 ml of corn oil (Mazola) per liter containing a lawn of Photorhabdus spp.pregrown for 18 to 24 h at 28°C in a culture dish (60 by 15 mm) or on one sideof a divided petri dish (100 by 15 mm). After growth for 10 to 14 days at 28°C,the IJs were washed off the lid of the culture dish with sterile saline (0.85%NaCl), or 12 ml of sterile saline was added to the empty side of the split petridish. When IJs form on lawns, they have dispersive behavior and become trappedon condensation on a culture dish lid or in the saline opposite the culture in asplit-well dish. Germfree IJs were first generated by growing the H. bacteriophoranematodes on P. temperata subsp. temperata isolated from Heterorhabditis megidisas described previously (21). However, better worm yields were obtained with P.temperata strain NC1 TRN16, a transmission-defective mutant that is unable tocolonize the IJs and normally is associated with H. bacteriophora nematodes. Aninbred strain of H. bacteriophora TT01, M31e, had been self-fertilized for 13generations prior to use in this study (9). H. bacteriophora was maintained as agermfree stock and added to P. luminescens subsp. laumondii TT01, P. temperatastrain NC1, or one of the strains labeled with Tn7-green fluorescence protein(GFP) as described previously (19). The bacteria were frozen in 4.5% dimethylsulfoxide in PP3salt and stored at �80°C, and the IJs were cryopreserved andstored in liquid nitrogen as described previously (31).

Analysis of worm development. Greater wax moth (Galleria mellonella) larvaewere obtained from Nature’s Way (Ross, OH). Larvae were infected by placing20 larvae in sterile a 100-mm Pyrex culture dish containing a 9.0-cm Whatmanno. 1 (Florham Park, NJ) filter, after which �1,000 monoxenic IJs were added in0.7 ml of sterile saline. Beginning 48 h postinfection, larvae were removed every24 h and disrupted in Ringer’s solution (100 mM NaCl, 1.8 mM KCl, 2 mMCaCl2, 1 mM MgCl2, 5 mM HEPES; pH 6.9) in a petri dish (100 by 15 mm), inwhich all worms, laid eggs, and external IJs were counted. For analysis ofnematode development on lawns of symbiotic bacteria, �25 IJs were placed onNA�chol preseeded with P. luminescens TT01 as described above. Beginning48 h after IJ addition, worms were washed off the lawns every 24 h with 1.8 mlof Ringer’s solution, centrifuged for 1 min at 2,000 rpm, and washed three timesin Ringer’s solution, after which all worms, laid eggs, and external IJs werecounted. When the number of worms exceeded the number which could becounted accurately (�300 worms), values were extrapolated from counts forthree aliquots containing 60 to 300 worms each. Worms were counted in tripli-cate in three independent experiments.

Assay for retention of intestinal symbionts by IJs after exit from diapause. Todetermine if IJs vertically transmit symbiotic bacteria directly to offspring, IJs

containing GFP-labeled bacteria were placed on lawns of unlabeled bacteria.Two to twelve hours after addition, nematodes were removed, and the presenceof symbiotic bacteria in the nematodes was determined by fluorescent and dif-ferential interference contrast or Nomarski microscopy using a Leica DM5000compound microscope (Leica Microsystems, Wetzlar, Germany) equipped withan X-cite 120 fluorescence illuminator (EXFO, Quebec, Canada), a Spot Pursuitcharge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI),and a GFP filter set (Leica). To determine if starvation stress triggered verticaltransmission, IJs in the process of regurgitating their intestinal symbionts wereremoved and starved for 12 h in Ringer’s solution before they were placed onlawns of unlabeled bacteria and imaged as described above.

Test for the presence of intestinal bacteria. H. bacteriophora IJs and Caeno-rhabditis elegans dauer larvae were each added to lawns of GFP-labeled P.temperata NC1, an H. bacteriophora symbiont that does not kill C. elegans onnematode growth media (data not shown), and observed 24 h after addition. Totest if nonsymbiotic bacteria are permitted in the H. bacteriophora intestine, IJswere incubated with TRN16, a P. temperata transmission-defective mutant, for24 h before transfer for 4 h to lawns of GFP-labeled Escherichia coli OP50 andthen directly observed. To determine if P. temperata persists in the H. bacterio-phora intestine in the presence of nonsymbiotic bacteria, H. bacteriophora IJswere added to lawns of DSRedexpress-labeled NC1 symbionts and incubated for24 h before transfer to GFP-labeled OP50 for 24 h and then observed.

Detection of symbiotic bacteria that have infected nematodes. To determinewhen nematodes are infected by symbiotic bacteria, nematodes were pulsed withGFP-labeled symbionts, which was followed by a 4-h chase with unlabeled sym-bionts. Most transient intestinal bacteria were defecated from the intestineduring the chase, so only GFP-labeled symbionts that had established persistentinfections were visible. Unless specified otherwise, these experiments were per-formed by adding ca. 25 germfree IJs to lawns of GFP-labeled symbionts andincubating the preparations for 12 to 144 h at 28°C. At least every 12 h during thisperiod, at least 15 worms were picked from the bacterial lawns into a drop ofRinger’s solution on a sterile PP3salt plate and then transferred to a lawn ofunlabeled bacteria for 4 h before imaging. At least three independent experi-ments were performed. To determine the growth of adherent bacteria on thematernal intestine, IJs were added to lawns of GFP-labeled bacteria, incubatedfor 12 h, removed, washed three times in Ringer’s solution, and placed on lawnsof unlabeled bacteria as described above. Nematodes were removed after 4, 12,or 24 h, and the growth of the initially adherent GFP-labeled bacteria wasanalyzed by counting fluorescent cells by epifluorescent microscopy. To deter-mine if GFP-labeled bacteria adhered to the male nematodes, first-generation(F1) adult males were removed, washed, chased with unlabeled bacteria, andanalyzed to determine the presence of fluorescent bacteria as described above.To determine if adherence occurred in a maternal intestine containing a biofilm

TABLE 1. Strains and plasmids used

Strain or plasmid Characteristics Reference(s) or source

NematodesHeterorhabditis bacteriophora TT01-M31e Inbred (self-fertilized 13 times) 9Caenorhabditis elegans N2 Wild type CGC

BacteriaPhotorhabdus luminescens subsp. laumondii TT01 Wild type (primary phase) Nematode hostP. luminescens subsp. laumondii TT01-GFP Labeled with Tn7-GFP This studyPhotorhabdus temperata NC1 Wild type (primary phase) ATCC 29304P. temperata NC1-GFP Labeled with Tn7-GFP This studyP. temperata NC1-DSRedExpress-a Labeled with Tn7-DSRedexpress This studyTRN16 (P. temperata NC1-GFP) Transmission-defective mutant This studyEscherichia coli OP50-GFP Labeled with Tn7-GFP This studyE. coli BW29427 dap auxotroph, tra pir K. A. Datsenko and B. L. WannerE. coli BW29427/pURR25 Tn7 PA1/04/03gfpmut3* D. Lies and D. NewmanE. coli BW29427/mini-Tn7 (Km, Sm) Tn7 PA1/04/03-DSRedexpress-a This study; 27E. coli BW29427/pUX-BF13 Tn7 transposase D. Lies and D. NewmanE. coli BW29427/pURE10 Mini-HimarGm D. Lies and D. Newman

PlasmidspURR25 Tn7 PA1/03/04gfpmut3* D. Lies and D. Newmanmini-Tn7 (Km, Sm) PA1/04/03-DSREDexpress-a Tn7 PA1/04/03-DSRedexpress-a 27pUX-BF13 Tn7 transposase 2pURE10 Mini-HimarGm D. Lies and D. Newman

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of unlabeled bacteria, germfree IJs were placed on unlabeled bacteria for 30 h,washed, and then placed on GFP-labeled bacteria and incubated for 4 h, afterwhich they were washed in Ringer’s solution, chased with unlabeled bacteria for4 h, and then imaged as described above. To determine when the window oftransmission was closed (i.e., the time at which new symbionts could no longerinfect the intestine), germfree IJs were propagated using either unlabeled sym-bionts or TRN16 that was unable to colonize the maternal intestine and 24, 36,48, and 60 h after IJ addition were placed on GFP-labeled bacteria for 4 h andthen chased with unlabeled bacteria as described above.

Transmission electron microscopy. For electron microscopy, live animals werefixed, rinsed, and stained using microwave irradiation to help with penetration ofthe animal’s thick cuticle. A Pelco Biowave oven (Ted Pella, Inc., Redding, CA)was tuned to very low power (70 W), and the animals were kept in a plasticculture plate containing liquid fixative that was placed on top of the PelcoColdspot device inside the oven chamber, which helped minimize sample heatingduring extended irradiation. A temperature probe was used, and the restrictiontemperature used was 39°C, although this temperature was rarely reached. Mul-tiple processing steps were done in the chamber, using irradiation to aid eachrinse step or staining. Samples were fixed first in aldehydes and then in osmiumtetroxide plus potassium ferrocyanide, stained en bloc in uranyl acetate, andfinally embedded in 3% agarose prior to dehydration and embedding in Em-bed812 plastic resin (18). The following conditions and temperature regimenwere used. (i) Samples in cacodylate buffer containing 3.5% glutaraldehyde and1.5% paraformaldehyde were microwaved twice (5 min on and 3 min off) andthen kept at room temperature for 60 min. The temperature ranged from 15 to39°C during irradiation. The 0.1 M cacodylate buffer (pH 7.2) included 2 mMCaCl2 and 50 mM NaCl. (ii) The samples were rinsed three times in 0.2 Mcacodylate buffer, microwaved for 1 min, and then kept at room temperature for10 min. (iii) The samples were placed in 1% OsO4-0.5% KFe(CN)6 in 0.1 Mcacodylate buffer, microwaved twice (5 min on and 3 in off), and then kept atroom temperature for 15 min. The temperature ranged from 15 to 35°C duringirradiation. (iv) The samples were rinsed three times in 0.2 M cacodylate buffer,microwaved for 1 min, and then kept at room temperature for 7 min. (v) Thesamples were rinsed three times in 0.2 M sodium acetate buffer (pH 5.2),microwaved for 1 min, and then kept at room temperature for 7 min. (vi) Thesamples were placed in 0.5% uranium acetate in 0.2 M sodium acetate buffer,microwaved twice (5 min on and 3 min off), and then kept at room temperaturefor 15 min. The temperature ranged from 15 to 33°C during irradiation. (vii) Thesamples were rinsed three times in 0.2 M sodium acetate buffer (pH 5.2),microwaved for 1 min, and then kept at room temperature for 7 min. (viii) Thesamples were rinsed three times in 0.2 M cacodylate buffer, microwaved for 1min, and then kept at room temperature for 7 min. (ix) Samples were embeddedin parallel in 3% type VII agarose and then kept at 4°C overnight. (x) Thesamples were cut into small agar cubes and transferred to snap cap vials in buffer.(xi) The samples were dehydrated at room temperature using the followingconditions: 70% ethanol for 10 min, 80% ethanol for 10 min, 90% ethanol for10min, three treatments with 100% ethanol (10 min each), and three treatmentswith 100% propylene oxide (PO) (10 min each). (xii) The samples were infil-trated at room temperature on a rotator using the following conditions: 2 partsPO to 1 part resin for 2 h, 1 part PO to 2 parts resin for 2 h, and four changesof 100% resin over 1 day. (xiii) Samples were arranged in a flat embedding moldand cured at 60°C for 65 h. Thin sections were obtained using a Power Tome XLultramicrotome (RMC, Boekeler Instruments, Tucson, AZ), stained with 2%uranyl acetate in 50% ethanol for 10 min and with lead citrate (Reynold’sformulation) for 15 min, and viewed with a JEOL100 CXII transmission electronmicroscope (Japan Electron Optics Laboratories Ltd., Tokyo, Japan) located atthe Michigan State University Center for Advanced Microscopy.

RESULTS

IJs develop inside maternal nematode body cavities and notfrom laid eggs. To determine if P. luminescens transmission tothe IJ vector directly involves maternal nematodes, it is impor-tant to determine if IJs develop inside maternal nematodes,outside nematodes from laid eggs, or both. Development ofthe IJs involves three developmental pathways and behaviors:reproduction by egg laying versus endotokia matricida (i.e.,intrauterine egg hatching); development of offspring into IJs(i.e., alternative developmentally arrested third-stage larvalnematodes that vector P. luminescens to insect hosts) versus

vegetative noninfective hermaphrodites, females, or males;and persistence of IJ diapause leading to dispersal behaviorversus exit from diapause (i.e., recovery) and resumption ofdevelopment (Fig. 1A). H. bacteriophora offspring arisingfrom endotokia matricida develop predominantly into IJs(24). Conversely, hermaphroditic nematodes presumed tobe pre-IJs develop from laid eggs (25), but whether suchprogeny can also develop into IJs has not been determinedpreviously.

To determine if IJs develop from laid eggs and/or via en-dotokia matricida, nematode development was monitored bothinside infected insect larvae (G. mellonella) and on agar-basedmedia seeded with symbiotic bacteria by determining the num-bers of laid eggs, IJs, and total worms (laid eggs plus IJs plusother stages) present 2 to 11 days after IJs were added (Fig. 1Band 1C). Most IJs that were added to the symbiont lawns orthat infected insects recovered and resumed development.Two days after IJ addition, 13 recovered IJs (standard error ofthe mean [SEM], 7.9 IJs) and 13 recovered IJs (SEM, 6.7 IJs)were present (recovered IJs were distinguished from nonre-covered IJs on the basis of feeding behavior and morphology)inside infected insects and on symbiont lawns, respectively(Fig. 1B and 1C). Two days later (4 days after IJ addition), theanimals had molted twice and developed into adults that werelaying eggs, and 1,650 laid eggs (SEM, 1,315 eggs) and 1,852laid eggs (SEM, 1,979 eggs) were present in insects and onsymbiont lawns, respectively (Fig. 1B to 1D). First- and second-stage larvae (L1 and L2, respectively) derived from hatchedeggs were also observed (Fig. 1D and data not shown). Theoffspring derived from laid eggs developed into vegetative(non-IJ) stages, based on morphology, the fact that no IJs werepresent on the symbiont lawns prior to day 8, and the fact thatIJs accounted for a minority of the total worms (17 and 30% ondays 6 and 7, respectively) inside insects (Fig. 1B and 1C). Inaddition, no IJs appeared on the symbiont lawns before day 8following removal of parental (P0) nematodes on day 6 (datanot shown). After the initial egg-laying period 3 to 5 days afterIJ addition, reproduction occurred exclusively by intrauterineegg hatching (endotokia matricida) because few or no eggswere observed either on symbiont lawns or inside insects (Fig.1B and 1C). Offspring arising from endotokia matricida devel-oped into IJs (which were distinguished from vegetative stagesby morphology and behavior). IJs arising from endotokia ma-tricida were first detected 6 days after addition of IJs to insects,but most IJs recovered and resumed development, as shown bythe increase in the total worm numbers from day 5 (7,403worms; SEM, 8,745 worms) to day 6 (15,984 worms; SEM,21,511 worms), whereas only a minority were IJs (3,567 worms;SEM, 5,173 worms) that remained in diapause (Fig. 1B). F1 IJsalso developed via endotokia matricida on symbiont lawns(data not shown), but all of them recovered and resumeddevelopment after they left the maternal body cavity (and thuswere not scored as IJs), as shown by the complete absence ofIJs before day 8 on symbiont lawns (Fig. 1C). These datasuggest that during the first generation of reproduction anddevelopment both in insects and on symbiont lawns maternalnematodes first laid eggs that developed into vegetative (non-IJ) stages and then reproduced via endotokia matricida, whereoffspring developed into IJs but most IJs recovered and re-sumed development.

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In contrast to P0 nematodes, F1 nematodes reproduced only viaendotokia matricida; essentially no laid eggs were observed afterday 5 and during days 7 to 11, when IJs developed en masse (Fig.1 B, 1C, and 1E). On average, 405,000 IJs (SEM, 77,800 IJs) and

28,500 IJs (SEM, 5,600 IJs) were observed on day 11 in insectsand on day 10 on symbiont lawns, respectively (Fig. 1B and 1C).Egg laying occurred too early and the numbers of eggs wereinsufficient to account for the large numbers of IJs that devel-

FIG. 1. IJs develop inside maternal body cavities and not from laid eggs. (A) H. bacteriophora makes three key developmental or behavioral choicesrelated to IJ formation: (i) egg-laying behavior versus intrauterine egg hatching (endotokia matricida); (ii) development of offspring to IJs versusvegetative larval growth and adulthood; and (iii) recovery versus dispersal of IJ offspring. The black lines indicate observed developmental pathways orbehaviors, and the burgundy lines indicate alternative pathways or behaviors (see panels B to E). Green ovals indicate development by endotokiamatricida. (B) Nematode development inside G. mellonella larvae. The mean numbers of total nematodes (Œ), IJs (f), 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 andthen 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, reproductionoccurred via endotokia matricida, ultimately producing more than 500,000 IJs in a single insect larva. (C) Nematode development and behavior on lawnsof 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 byP0 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 40,000IJs 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 onday 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.

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oped. These data suggest that IJs develop exclusively in maternalnematodes reproducing via endotokia matricida.

Intestinal symbionts are completely released when IJs exitdiapause. Because F1 IJs developed inside the body cavities ofP0 nematodes that were originally IJs harboring bacteria, somesymbionts might have been retained by the IJ-derived P0 nem-atodes and directly transmitted to IJ progeny. To determine ifIJs retained symbionts after recovery, IJs harboring GFP-la-beled symbionts were added to lawns of unlabeled bacteria,and assayed to determine whether they retained GFP-labeledsymbionts. Prior to recovery, each IJ contained approximately130 symbiont bacterial cells that were predominantly in theanterior intestine (see Fig. S1A in the supplemental material).During recovery on lawns of symbiotic bacteria, IJs regurgi-tated the intestinal symbionts, as previously reported for IJsimmersed in insect hemolymph (7). At 4 h after IJ addition tosymbiont lawns, IJs regurgitating symbiotic bacteria were ob-served (data not shown), and �75% of the IJs contained re-sidual GFP-labeled symbiont cells in the posterior intestine(Fig. S1B), possibly due to the progressive release of the sym-bionts from the anterior intestine to the posterior intestine. At8 h, no GFP-labeled symbionts were found in any IJs that hadresumed development (see Fig. S1C in the supplemental ma-terial).

To determine if stress could induce direct symbiont trans-mission, IJs which were in the process of recovery and whichstill contained residual intestinal symbionts (at 4 h) were re-moved, starved, and assayed to determine whether they re-tained GFP-labeled intestinal symbionts. No fluorescent bac-teria were retained by the starved IJs (data not shown).

Feeding H. bacteriophora contains viable P. luminescens inthe intestinal lumen. H. bacteriophora is a bacteriovore like C.elegans and utilizes bacteria as its primary food source. C.elegans feeds on bacteria by efficiently grinding them using achitin grinder located in the basal bulb of the pharynx and byproducing lytic proteins and enzymes. Recovered H. bacterio-phora IJs develop by feeding on symbiotic bacteria and mac-romolecular components provided by the bacteria. Becauseintestinal symbionts are completely released during IJ recov-ery, we sought to determine if the conditions in the intestine offourth-stage H. bacteriophora larvae are conducive for symbi-ont survival or growth compared with the conditions in C.elegans. To test this, H. bacteriophora and C. elegans werepropagated on GFP-labeled P. temperata NC1, since this straindoes not kill C. elegans when it is grown on nematode growthmedia and is symbiotically associated with H. bacteriophora(data not shown). Intact P. temperata cells were present only inthe intestines of H. bacteriophora (see Fig. S2A and S2B in thesupplemental material). To determine if nonsymbiotic bacteriaare also permitted in the H. bacteriophora intestine, H. bacte-riophora IJs were recovered on TRN16, a transmission-defec-tive mutant of P. temperata that is unable to colonize maternalnematodes (see below), and transferred to GFP-labeled E. coliOP50. Labeled OP50 cells were found to survive or grow in theH. bacteriophora intestine to an extent similar to or greaterthan that of symbiont bacteria (see Fig. S4C in the supplemen-tal material). When H. bacteriophora nematodes were allowedto recover for 24 h on lawns of DSRedexpress-labeled P. tem-perata symbionts and then transferred for 24 h to lawns ofGFP-labeled OP50, DSRedexpress-labeled symbionts were

found to persist in the posterior intestine (see below), whileGFP-labeled OP50 cells were found throughout the intestine(see Fig. S4D in the supplemental material). Compared to theC. elegans intestine, the H. bacteriophora intestine appears tobe more permissive for both symbionts and nonsymbionts, andsuch cells are available to infect the intestine and other tissues.

Symbiont infection of the posterior maternal intestine. Todetermine if and when symbiont transmission is initiated inmaternal nematodes, a series of pulse-chase experiments wereperformed. Germfree (i.e., axenic) IJs were transiently ex-posed to GFP-labeled symbionts and then chased with unla-beled symbionts (for 4 h) before an assay to determine thepresence of GFP-labeled symbionts by fluorescence micros-copy was performed. IJs exposed to GFP-labeled symbionts forless than 6 h were not colonized by GFP-labeled bacteria (datanot shown). However, symbionts were detected in �25% of theworms following 8 h of exposure of IJs to GFP-labeled symbi-onts (data not shown). At 12 h, all IJs that exited diapausecontained 1 to 3 GFP-labeled symbiont cells (mean, 2.2 cells;SEM, 0.7 cells) adhering only to the two most posterior intes-tinal epithelial cells (left and right intestinal epithelial cells[INT9L and INT9R, respectivel]) (Fig. 2A). More GFP-la-beled symbiont cells (mean, 12.2 cells; SEM, 2.3 cells) werefound adhering to the posterior intestinal cells following 24 hof exposure (Fig. 2B), and still more were found following 36 hof exposure (Fig. 2C).

To determine if and when second-generation progeny ac-quired symbionts, maternal nematodes were placed onto lawnsof GFP-labeled symbionts and allowed to lay eggs for 5 h, afterwhich the adults were removed. At 12 h after eggs were laid,most L1 and L2 nematodes contained symbionts in the poste-rior intestine (data not shown). Like recovered IJs, the L1 andL2 nematodes initially contained one to three symbiont cells inthe posterior intestine. Symbiont cells were also found adher-ing to the adult male posterior intestine in an anatomicallocation corresponding to that of the INT9 cells, as seen inhermaphrodites (Fig. 2D), but these cells usually did not per-sist into late adulthood (data not shown).

To determine if adherent symbiont cells prevent additionalsymbiont cells from adhering, nematodes containing a biofilmof unlabeled symbionts were exposed to labeled symbionts (for4 h), chased with unlabeled symbionts (for 4 h), and observedto determine the presence of adherent GFP-labeled symbionts.Adherence of GFP-labeled bacteria was still possible in ma-ternal nematodes containing a mature community of attachedsymbionts 36 to 42 h after IJ addition (Fig. 2E). However, at 42to 48 h after IJ addition, no GFP-labeled bacteria colonizedthe maternal intestine (Fig. 2F). Thus, symbiont adherence tothe maternal intestine can occur 8 to 42 h after IJ addition tosymbiont lawns.

Growth of adherent bacteria on the maternal intestinal ep-ithelium. Since adherence of new bacteria occurred duringthe entire time that bacteria were found to be attached to theposterior maternal intestine, it is not clear to what extent thesymbionts grow while they are attached to the maternal intes-tine. To determine the growth of adherent bacteria, IJs werepulse-chased as described above (12-h pulse and 4-h chase),which resulted in one to four adherent symbiont cells (see Fig.S3A in the supplemental material). The equivalent worms 20 hlater contained 16 to 25 adherent GFP-labeled symbiont cells,

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suggesting that there were three or four doublings of adherentbacteria during this time (see Fig. S3B in the supplementalmaterial). These data suggest that the mass of symbiont bac-teria present as a biofilm on the maternal posterior intestinewas a result of both growth and new adherence.

Invasion of maternal RGCs. Because the symbiont biofilmpersists for up to 36 h before it disappears, the fate of theadherent bacteria was determined during the abrupt transition.At 42 h after addition of IJs to GFP-labeled symbionts, a massof �50 cells was found to be attached to the posterior intestinallumen (Fig. 2C). At 42 to 48 h after addition of IJs, GFP-labeled cells appeared to migrate, cluster around the rectum,and invade the cytoplasm of RGCs (Fig. 3A). At 48 h afteraddition of IJs, GFP-labeled cells formerly attached to theposterior intestine began breaching the gland epithelium andinvading the RGCs (Fig. 3B). Actively invading symbiont cellswere recognized by the appearance of a symbiont-containing

vacuole in contact with the luminal (apical) face of the INT9cells (Fig. 3B). Active invasion is also shown in Fig. 3C and 3D,in which in one focal plane symbionts are visible both adheringto the intestinal epithelium and invading the RGCs (Fig. 3C)and in another focal plane above the intestinal lumen a sym-biont-containing vacuole is visible (Fig. 3D). The likely routefor invasion is through the apical surface of the RGCs, whichare exposed to the intestinal lumen near the rectum (Fig. 3A to3C). Most or all of the adherent symbionts appeared to invadethe RGCs, because when a few GFP-labeled symbionts wereloaded onto an almost mature unlabeled biofilm (36 h after IJaddition), the labeled cells invaded the RGCs (data notshown). By invading the RGCs, the symbionts partiallybreached the glandular epithelium, but never the intestinalepithelium (see below).

Intracellular symbiont behavior. To determine the growthand behavior of the intracellular symbionts, the RGCs were

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FIG. 2. Adherence of P. luminescens to maternal and adult male nematode intestines. Transient GFP-labeled cells were chased from theintestine so that only labeled symbiont cells that had established persistent infections were visible. (A) Single GFP-labeled P. luminescens celladhering to posterior intestinal (INT9) cells of a fourth-stage H. bacteriophora larva following 8 h of exposure to labeled symbionts. (B) Severaladherent 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 adheringto the posterior maternal intestine containing unlabeled adherent symbionts after 36 h of exposure of IJ to unlabeled symbionts, followed by 4 hof exposure to labeled symbionts. (F) GFP-labeled symbionts no longer adhered to the maternal intestine following 48 h of exposure of IJs tounlabeled symbionts, followed by 4 h of exposure to GFP-labeled symbionts. i, intestinal lumen; r, rectum.

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observed after symbiont invasion. Recently invaded maternalRGCs (60 h after IJ addition) contained one to three symbi-ont-containing vacuoles per cell (Fig. 4A). Twenty-six hourslater (96 h after IJ addition), the symbiont-containing vacuoleshad multiplied and there were 12 to 30 vacuoles per cell (Fig.4B). The cloverleaf appearance of three RGCs, each filled withsymbiont-containing vacuoles and tethered to the rectum, isevident in Fig. 4B. The intracellular symbionts also appearedto multiply during this time (compare Fig. 4A and 4B).

Since symbiont-containing vacuoles multiply inside theRGCs, the possibility that the intracellular symbionts induce

vacuole multiplication is plausible. To test this possibility, ma-ternal nematodes were propagated using P. temperata TRN16,which was unable to infect and invade the maternal intestine.Only a few (�3) enlarged vacuoles were observed in the RGCswithout intracellular symbionts 96 h after addition of IJs toTRN16 (Fig. 4D). Thus, intracellular symbionts influence thesize and number of RGC vacuoles.

Symbiont invasion and growth inside RGCs preceded andoccurred during egg-laying behavior of recovered IJs (P0 nem-atodes) (80 to 96 h, equivalent to day 4 in Fig. 1C). However,after 96 h after IJ addition, egg laying ceased and new IJs

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FIG. 3. Invasion of maternal RGCs in maternal nematodes 48 h after IJs were added to GFP-labeled lawns and then chased with unlabeledsymbionts for 4 h. The intestinal lumen (i), rectum (r), and RGC vacuole (v) are indicated. (A) Adherent GFP-labeled symbionts, most of whichare still adhering to the intestinal lumen, beginning to invade an RGC (white arrows). Transient unlabeled symbionts (black arrow) were visiblein the intestinal lumen, but most of them did not invade the RGCs. (B) Site of invasion (arrow), where a symbiont-containing vacuole appearedto form at the basal surface of the RGC in close proximity to GFP-labeled symbionts attached to the intestinal lumen. The INT9 posterior intestinalcells were not invaded. (C) GFP-labeled symbionts adhering to the intestinal epithelium and invading RGCs (arrow). (D) Same worm as the wormin panel C at a focal plane ca. 5 to 10 �m above that in panel C where a RGC vacuole was apparent.

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developed via intrauterine hatching (endotokia matricida).During this time (96 to 108 h), pre-IJs (L2s developing into IJs)began consuming maternal protoplasm and developing insidethe maternal body cavity (Fig. 4C). At 100 h after IJ addition,while the maternal intestine remained intact, the RGCs lysed,and the symbiont-containing vacuoles were liberated into thematernal body cavity but not into the intestinal lumen (Fig.4C). The symbiont-containing vacuoles lysed shortly after re-lease (data not shown), which made their bacterial contentsavailable to the pre-IJs present there. The intracellular symbi-onts were the primary or sole inoculum for the IJs becausematernal nematodes propagated on unlabeled bacteria untilshortly after RGC invasion (�48 h after IJ addition) and trans-ferred to GFP-labeled symbiont lawns did not transmit anyGFP-labeled symbionts to the IJs (data not shown). These datasuggest that both biofilm formation on the maternal intestineand subsequent RGC invasion are required for symbionts tobreach the glandular epithelium and infect the pre-IJs devel-oping in the maternal pseudocoelom. Otherwise, the intestinalepithelium is well protected against transit of live bacteria tothe pseudocoelom.

Ultrastructure of symbiont-containing vacuoles. To betterunderstand the behavior of intracellular symbionts in RGCs, theultrastructure of symbiont-containing vacuoles was analyzed usingtransmission electron microscopy. Symbiont-containing vacuoleswith a granular appearance were observed in RGCs (Fig. 5A).The nearby INT9 cells were easily recognized and never showedany sign of invasion (not shown). The symbiont-containing vacu-oles occurred in chains and may have been dividing or expanding,as shown by granular connections between the globular intracel-lular compartments (Fig. 5A and 5B). Several bacteria were vis-ible inside each RGC vacuole, and a vacuolar membrane wasalways visible (Fig. 5B and 5C). Numerous membranous blebswere apparent inside the symbiont-containing vacuoles (Fig. 5C)and were often directly attached to the intracellular symbionts(Fig. 5D). The inner and outer membranes of the intracellularbacteria, as well as unusual compartments with an unknown func-tion, were clearly visible (Fig. 5D).

Symbiont infection of IJs developing inside the maternalbody cavity. To monitor the symbiont infection of IJs inmaternal nematodes undergoing endotokia matricida, GFP-labeled bacteria were observed in the intestines of pre-IJs

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FIG. 4. Behavior of intracellular symbionts. (A) Recently invaded RGCs each contained one to three vacuoles following 54 h of exposure ofIJs to labeled symbionts. (B) Multiplication of symbiont-containing vacuoles was evident 38 h later, and each RGC contained 12 to 30symbiont-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 cavityof worms undergoing endotokia matricida, while the intestine and basal surface of the RGCs appeared to be intact. (D) Morphological differencesof vacuoles in maternal nematodes grown on a mutant GFP-labeled symbiont unable to invade the RGCs. Only a few large vacuoles were presentin nematodes following 112 h of exposure of IJs to this mutant symbiont. n, nucleus; i, intestine; r, rectum; v, vacuole.

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developing inside the body cavity. Symbiont infection of thepre-IJs occurred after symbiont release from the RGCs andwas apparent before the maternal intestine was disrupted bythe pre-IJs developing inside the body cavity (Fig. 6A). Largenumbers of free GFP-labeled bacteria were usually observed inthe maternal pseudocoelom (evident in the body cavity in Fig.6A), suggesting that symbiont replication occurs in thepseudocoelom. Infection of the pre-IJs was initiated inside thematernal body cavity ca. 120 h after IJ addition, and a singlesymbiont cell usually adhered to the PIVCs located betweenthe pharynx and the intestine (Fig. 6B). After adherence, thesymbiont cell appeared to invade the PIVCs and multiply (Fig.6C). Definitive ultrastructural evidence, such as the presenceof a vacuolar membrane, was not observed for the intracellularsymbionts in PIVCs, and it was also not clear whether theinitial adherent cells replicated before invasion or while theywere inside the PIVCs. However, 16 h later, multiple cells wereagain found in the intestinal lumen of IJs (Fig. 6D); 24 to 72 hlater, bacteria were growing in the anterior intestine (Fig. 6E);and about 7 days later, bacteria were observed throughout theintestine. This full colonization of the IJ intestine completedthe transmission cycle.

Ultrastructure of IJ intestinal symbionts. To infect a newinsect host, IJs must vector viable symbionts to the insecthemocoel. To better understand how the symbionts are main-tained in a semidormant state, sometimes for several monthsbetween insect hosts, we compared the ultrastructure of sym-biotic IJs and the ultrastructure of germfree IJs (see Fig. S4 inthe supplemental material). The IJs were enclosed by a spe-cialized outer (“dauer”) cuticle protecting the nematode fromthe environment (see Fig. S4A in the supplemental material);the animal’s mouthparts were closed (1, 11). The symbionts

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FIG. 5. Ultrastructure of symbiont-containing vacuoles. (A) Transmission electron micrograph of a cross-section of a maternal nematodefollowing 96 h of exposure to symbiont lawns. Symbiont-containing vacuoles are indicated by thick arrows. A few bacterial cells (b) inside thevacuoles are also indicated (thin arrows). (B) Two connected vacuoles possibly in the process of division. The vacuolar membrane (vm) and a fewbacteria (b) are indicated. (C) Granular contents of symbiont-containing vacuoles. Bleb-like structures are indicated. (D) Bleb in contact with abacterial 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) Fluores-cent micrograph of GFP-labeled symbiont cells (arrows) in the bodycavities of two nematodes undergoing endotokia matricida and insidepre-IJs (arrows) (the image was not pseudocolored or overlaid likeother fluorescent micrographs). (B) One or two GFP-labeled symbi-onts (arrow) adhering to the PIVCs 120 h after the maternal nematodewas placed on labeled bacteria. No chase was necessary since transientsymbionts 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) SeveralGFP-labeled symbiont cells (arrow) in the intestinal lumen of the IJswere visible 16 h later. (E) Growth of intestinal symbionts (arrows) inearly stages of IJ colonization 24 to 72 h later.

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were present the intestinal lumen in a presumed semidormantstate, replicated little, and apparently were not digested by thehost. The intestinal lumen contained microvilli that sometimeswere in close contact with bacteria (see Fig. S4B and S4E in thesupplemental material). No differences in the density of mi-crovilli were observed when germfree and symbiotic nema-todes were compared, although an electron-dense matrix wasmore uniform and diffuse in the intestinal lumen of germfreeIJs than in the intestinal lumen of symbiotic IJs (compare Fig.S4C to Fig. S4B and S4D in the supplemental material). Adirect physical connection between the symbiotic bacteria andthe IJ intestinal epithelium was usually not apparent (see Fig.S4E in the supplemental material). In some IJs, the bacteriaappeared to be surrounded by a thin electron-lucent zonedistinct from a thick electron-dense matrix in the intestinallumen (see Fig. S4F in the supplemental material), suggestingthat these bacteria were enclosed in a protective acellular ma-trix or biofilm.

DISCUSSION

A simple model can explain how mutualistic associations(i.e., associations beneficial to both partners) are establishedand maintained to the exclusion of nonsymbiotic or pathogenicassociations. Visualization of labeled bacteria inside the trans-parent nematode body revealed an infectious developmentalprogram that ensures symbiont-specific transmission to the IJnematode. Notably, transmission involved (i) IJ developmentonly inside the maternal nematode body cavities, (ii) symbiontinfection of both maternal and IJ offspring intestines, and (iii)temporal and spatial specific host and symbiont cell behaviors.

The majority of the information concerning host-bacteriuminteractions has been obtained by studying pathogens thatcause acute disease, even though many organisms are associ-ated with commensal or mutualistic bacteria, some of whichare close relatives of pathogens (29). Recent findings for themodel mutualism between the Hawaiian bob-tailed squid (Eu-prymna scolopes) and the bacterium Vibrio fischeri have re-vealed a selective gauntlet employed by the squid to ensuresymbiont-specific colonization of the light organ (32). Thisassociation has revealed the role of virulence-like factors andinnate immunity in establishing a symbiotic relationship (16,26). Similarly, virulence-like genes and innate immunity areinvolved in the colonization of legumes by symbiotic rhizobia(12). The nematode C. elegans has been used extensively tostudy the host-bacterium interactions of a variety of pathogens

(17, 36). Here we investigated mutualistic host-bacterium in-teractions with another rhabditid nematode, where transmis-sion of P. luminescens is essential for the insect-pathogeniclifestyle of the two organisms (21). Due to the obligate natureof the association for both the nematode and the symbiont, itis not surprising that the elaborate selective process describedin this paper is employed for symbiont transmission.

However, the process of transmission of Photorhabdus to H.bacteriophora IJ nematodes was notably sophisticated. Trans-mission of a related insect pathogen, Xenorhabdus nemato-phila, by Steinernema carpocapsae IJs, although far from sim-ple, seems to involve selective binding, growth, and survival ofsymbionts only in the IJ intestine (15). In the P. luminescens-H.bacteriophora symbiosis, the symbionts are maternally acquiredand maintained both extracellularly and intracellularly duringmost of the nematode life span. Furthermore, symbiont acqui-sition occurs shortly after IJ recovery and after hatching fromlaid eggs in vegetative stages destined to produce IJs in sub-sequent generations via endotokia matricida.

Since symbiont transmission is essential to both the nema-tode and the bacterium in nature (where each organism de-pends on the other to infect insects), it is interesting that IJscompletely release their intestinal symbionts during IJ recov-ery. One possible advantage of symbiont release is to avoidMueller’s ratchet, a phenomenon where deleterious mutationstend to accumulate in some vertically (e.g., strictly transovar-ian) transmitted symbionts (30). In addition, release of theintestinal symbionts into insect hemolymph might select forinsect virulence or at least survival in hemolymph that containsefficient humoral and cellular innate immune effectors (28).This selection might also eliminate cheaters which do not con-tribute to insect virulence. Because a single symbiont cell (andsometimes pairs of cells) was observed adhering to the pre-IJPIVCs, symbiont transmission is highly clonal. Clonal symbionttransmission might be an effective mechanism to eliminatecheaters (cells that are transmitted but rely on other cells forinsect pathogenicity), because in single IJ infections symbiontclones are directly selected for insect virulence.

Symbiont transmission is initiated soon after expulsion fromIJs or after hatching from eggs and proceeds through a seriesof infectious steps (Fig. 7). Symbiont infection of the maternaland IJ intestines is a developmental process, where symbiontcells adhere to, invade, and exit specific nematode cells atspecific times in nematode development (Fig. 7). For example,symbiont adherence to nematode cells occurs on only two

FIG. 7. Model of the transmission cycle. Symbionts that have colonized the maternal intestine (top panel) or pre-IJs or IJs (bottom panel) areshown 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) ison 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 areindicated by green ovals, and the green arrows indicate regurgitation or ingestion of symbiont cells. The time (t) is the time (in hours) after IJaddition to lawns of symbiont bacteria (zero time). Dormant symbionts are not adherent to the IJ intestinal lumen. At �8 h intestinal symbiontsare 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 to110 h symbionts grow intracellularly in the RGCs and stimulate vacuole formation. At 106 to 112 h symbionts are released from RGCs after lysisand 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) developingwithin the maternal pseudocoelom. At 110 to 120 h symbionts exit the pre-IJ intestinal lumen, possibly invade PIVCs, and multiply. At 120 to 288 hsymbionts exit the PIVCs and colonize the IJ intestinal lumen. Note that vegetative progeny acquire symbionts 5 to 12 h after they hatch from laideggs also by symbiont adherence to the INT9 cells but otherwise exhibit similar symbiont transmission.

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posterior INT9 intestinal cells in a �40-h window in the ma-ternal intestine and only to the PIVCs in the pre-IJ anteriorintestine (Fig. 7). Similarly, invasion of nematode cells occursonly in specific cells at specific stages of nematode develop-ment. Like metazoan development, symbiont transmissionlikely involves surface components to order cells in space andsignals that affect cell behavior. For the symbiont, expression of1 of 10 predicted fimbrial genetic loci is required for the sym-biont to adhere to the maternal intestine (B. Kaufmann-Daszc-zuk and T. A. Ciche, unpublished data).

The signals mediating the changes in symbiont behavior arecurrently not known. The global second messenger, cyclicdiguanylate, is a key signaling molecule for social behavior andvirulence in many pathogenic bacteria (10, 37). It is surprisingthat P. luminescens, a highly virulent insect pathogen as well asa mutualistic symbiont, appears to lack proteins containingGGDEF, EAL, and PilZ domains involved in the synthesis,degradation, and sensing of cyclic diguanylate, respectively(22). Other proteins and/or signals might regulate P. lumine-scens behavior while this bacterium is infecting nematode andinsect hosts. It is clear from these studies that the P. lumine-scens behaviors exhibited during infection of the nematode arealso behaviors important to other pathogens. Because P. lumi-nescens is an insect pathogen and a mutualistic symbiont, themechanisms underlying these infectious behaviors can be di-rectly compared in the two hosts.

H. bacteriophora has adapted what in C. elegans is a stressresponse behavior and developmental choice, namely, egg lay-ing and dauer formation, for symbiont transmission. Becauseendotokia matricida occurs in every maternal hermaphroditeand female nematode and results in the development of sym-biont-containing IJs, it is clear that endotokia matricida is anadaptation for symbiont transmission. Furthermore, giantnematodes also undergo endotokia matricida. Among the larg-est nematodes that we observed was a nematode retrievedfrom an infected G. mellonella larva, which was 6,000 �m longand contained 472 IJs. In C. elegans, egg-laying behavior ismodulated by environmental conditions, such as food availabil-ity (35), and may result in the preferential development ofoffspring to the environmentally resistant dauer stage (4).However, the total absence of egg laying is rare in C. elegansand has been the subject of mutant screens to identify genesinvolved in behavior and tissue development (23). In contrast,H. bacteriophora nematodes always reproduced by endotokiamatricida even after an initial period of laying by maternal P0

nemotodes, despite high food (symbiont) availability and lowworm densities. Therefore, egg laying might be an electivebehavior in H. bacteriophora or may involve different sensitiv-ities to environmental cues, such as dauer pheromone.

It is striking that the majority of offspring that develop viaendotokia matricida develop into IJs (analogous to the dauerstage) in H. bacteriophora. In C. elegans, a transforming growthfactor � pathway (Daf-1, -3, -4, -7, -8, and -14) regulates bothegg laying and dauer formation, and mutants defective in thisegg-laying pathway develop into dauer larvae (35). Egg layingand IJ formation might be regulated in H. bacteriophora by ahomologous pathway or by other stress response pathways(e.g., insulin- and mitogen-activated protein kinase pathways).The host biology related to symbiont transmission will likelybecome amenable to genetic analysis soon, as RNA interfer-

ence in H. bacteriophora has recently been developed (9), andan H. bacteriophora genome project at the National HumanGenome Research Institute is currently in progress (R. Wilson,personal communication).

ACKNOWLEDGMENTS

We acknowledge Alicia Pastor at the Center for Advanced Micros-copy at Michigan State University for her exceptional patience andexpertise in assisting with transmission electron microscopy. We ac-knowledge John Breznak, David Brian Butvill, Elissa Hallem, and PaulSternberg for advice and insightful comments on the manuscript.

This work was supported in part by the Center for Microbial Patho-genesis at Michigan State University and by grant NIH RR 12596 toD.H.H.

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