Hu KJ et al. (1999) Nematology "Nematicidal metabolites produced by Photorhabdus luminescens (Enterobacteriaceae), bacterial symbiont of entomopathogenic nematodes."

Hu KJ, Li JX, Webster JM

[Nematology, 1999]

The secondary metabolites, 3,5-dihydroxy-4-isopropylstilbene (ST) and indole, from the culture filtrate of Photorhabdus luminescens MD, were shown to have nematicidal properties. ST caused nearly 100% mortality of 54 and adults of Aphelenchoides rhytium, Bursaphelenchus spp. and Caenorhabditis elegans at 100 mu g/ml, but had no effect on J2 of Meloidogyne incognita or infective juveniles (IJ) of Heterorhabditis megidis at 200 mu g/ml. Indole was lethal to several nematode species at 300 mu g/ml, and caused a high percentage of Bursaphelenchus spp. (54 and adults), M, incognita (J2) and Heterorhabditis spp. (IJ) to be paralysed at 300, 100 and 400 mu g/ml, respectively. Both ST and indole inhibited egg hatch of M, incognita. ST repelled IJ of some Steinernema spp. but not IJ of Heterorhabditis spp., and indole repelled IJ of some species of both Steinernema and Heterorhabditis. ST, but not indole, was produced in nematode-infected larval Galleria mellonella. after 24 h infection.

Ciche TA et al. (2008) Appl Environ Microbiol "Cell Invasion and Matricide during Photorhabdus luminescens Transmission by Heterorhabditis bacteriophora Nematodes."

Nguyen KC, Ciche TA, Kaufmann-Daszczuk B, Hall DH, Kim KS

[Appl Environ Microbiol, 2008]

Many animals and plants are symbiotic 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 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 intra-uterine 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, 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 (PIVCs), helping 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 vs. invasion). Thus, Photorhabdus symbionts are maternally transmitted by an elaborate and infectious process involving multiple selective steps to achieve symbiont-specific transmission.

Liu, Ping et al. (2013) International Worm Meeting "Multiple innexins contribute to electrical coupling of C. elegans body-wall muscle."

Hall, David, Wang, Zhao-Wen, Chen, Bojun, Schuman, Benjamin, Liu, Ping, Altun, Zeynep, Gross, Maegan, Shan, Alan

[International Worm Meeting, 2013]

The C. elegans genome contains 25 innexin (INX) genes, which encode proteins for gap junctions (GJs) and hemichannels. A single type of cells often expresses two or more INXs. Little is known why multiple INXs are expressed in the same cells. To gain insight into this question, we assessed potential body-wall muscle expression for all the INXs by expressing promoter::GFP transcriptional fusions, and analyzed potential contributions of all but three INXs (INX-3, -12, -13) to electrical coupling by recording junctional current (Ij) between neighboring muscle cells. The expression of three INXs is observed in body-wall muscle cells, including INX-11, -18 and UNC-9. Comparisons of Ij between wild type and innexin mutants, however, suggest that six INXs contribute to muscle electrical coupling, including UNC-9, INX-1, -10, -11, -16, and -18. These INXs appear to contribute to the coupling cell-autonomously because the Ij deficiency in each specific INX mutant is rescued completely by expressing a corresponding wild-type INX specifically in muscle. Loss-of-function (lf) mutation of inx-1, -10, -11 or -16 inhibits Ij to a comparable degree (50~65%), and the Ij deficiency does not become more severe in various double mutants, raising the possibility that all the four INXs may contribute to the function of a single population of GJs. unc-9(lf) inhibited coupling by ~65%, which is not worsened by inx-18(lf) although Ij is decreased by ~40% in inx-18(lf) single mutant, suggesting that INX-18 and UNC-9 might be involved in the function of a single population of GJs. In double or triple mutants affecting both populations of GJs, Ij is essentially undetectable. Consistent with their roles in electrical coupling, five of the six INXs show punctate localization at muscle intercellular junctions when expressed as GFP fusion proteins. The remaining one (INX-11) shows diffuse expression in the cell membrane. These analyses present an unexpectedly complex picture about potential functions of INXs in C. elegans body-wall muscle.

Bennett MD et al. (2003) Ann Bot "Comparisons with Caenorhabditis (approximately 100 Mb) and Drosophila (approximately 175 Mb) using flow cytometry show genome size in Arabidopsis to be approximately 157 Mb and thus approximately 25% larger than the Arabidopsis genome initiative estimate of approximately 125 Mb."

Johnston JS, Bennett MD, Leitch IJ, Price HJ

[Ann Bot, 2003]

Recent genome sequencing papers have given genome sizes of 180 Mb for Drosophila melanogaster Iso-1 and 125 Mb for Arabidopsis thaliana Columbia. The former agrees with early cytochemical estimates, but numerous cytometric estimates of around 170 Mb imply that a genome size of 125 Mb for arabidopsis is an underestimate. In this study, nuclei of species pairs were compared directly using flow cytometry. Co-run Columbia and Iso-1 female gave a 2C peak for arabidopsis only approx. 15% below that for drosophila, and 16C endopolyploid Columbia nuclei had approx. 15% more DNA than 2C chicken nuclei (with gtoreq2280 Mb). Caenorhabditis elegans Bristol N2 (genome size approx. 100 Mb) co-run with Columbia or Iso-1 gave a 2C peak for drosophila approx. 75% above that for 2C C. elegans, and a 2C peak for arabidopsis approx. 57% above that for C. elegans. This confirms that 1C in drosophila is approx. 175 Mb and, combined with other evidence, leads us to conclude that the genome size of arabidopsis is not approx. 125 Mb, but probably approx. 157 Mb. It is likely that the discrepancy represents extra repeated sequences in unsequenced gaps in heterochromatic regions. Complete sequencing of the arabidopsis genome until no gaps remain at telomeres, nucleolar organizing regions or centromeres is still needed to provide the first precise angiosperm C-value as a benchmark calibration standard for plant genomes, and to ensure that no genes have been missed in arabidopsis, especially in centromeric regions, which are clearly larger than once imagined.

Cao, Mengyi et al. (2017) International Worm Meeting "Phenotypic switching between nematode mutualism and insect pathogenesis by the bacterium Xenorhabdus nematophila."

Hussa, Elizabeth A, Cao, Mengyi, Goodrich-Blair, Heidi, Stilwell, Matthew D, Weibel, Douglas B

[International Worm Meeting, 2017]

The entomopathogenic nematode Steinernema carpocapsae mutualistically associates with the symbiotic bacterium Xenorhabdus nematophila. During transmission stage, the infective juvenile (IJ) nematodes carry and release bacterial symbionts into insects. X. nematophila symbionts help kill the insect and convert its nutrients to support nematode reproduction. Upon nutrient depletion, progeny nematodes re-associate with the bacteria and develop into IJs that emerge from the cadaver and seek new insect hosts. X. nematophila phenotypic variation, termed VMO (virulence modulation), is characterized by a switch between two cell types: one type (V+M-) is virulent in insects but defective in nematode mutualism and the other (V-M+) is attenuated for virulence in insects, but engages in normal mutualism with nematodes. Varying levels of the global transcription factor Lrp (leucine responsive regulatory protein) control the VMO switch: High Lrp levels result in the V-M+ phenotype, while low Lrp levels cause the V+M- phenotype. To assess the population dynamics of VMO, we used an Lrp-dependent fluorescent reporter of gene expression. We discovered that nutrient limitation induces low-Lrp expression and heterogeneity within the bacterial population. Based on these data, we hypothesize that symbiotic bacteria in the nutrient-limiting nematode IJ express heterogeneous levels of Lrp, and that these cells are adapted to express virulence traits in the next life stage within the insect. To pinpoint the specific step of bacteria-nematode interaction in which the mutualism (high Lrp) to pathogenesis (low Lrp) switch occurs, we investigated the influence of bacterial Lrp on interactions at each nematode developmental stage. Our data revealed that high Lrp is optimal for early stages of bacteria-nematode association prior to the IJ stage. However, low-Lrp expressing cells, pre-adapted for virulence, arise during transmission: a low-Lrp expressing strain exhibits a higher number of bacteria CFU per IJ than does a high-Lrp strain. These data suggest the mutualism-to-pathogenesis switch occurs in the IJ nematodes. To track Lrp-dependent switching of symbiotic bacteria in the individual nematodes, we developed microfluidic devices and monitored colonization in multiple living IJ nematodes over 5 days. We will present our progress investigating the Lrp-dependent symbiosis switch in vitro and in vivo.

Michelle Rengarajan et al. (2002) West Coast Worm Meeting "Infection of Drosophila melanogaster with Heterorhabditis bacteriophora"

Takao Inoue, Michelle Rengarajan, Paul W Sternberg

[West Coast Worm Meeting, 2002]

Heterorhabditis bacteriophora is a parasitic nematode capable of infecting a wide variety of insects. The infectious juvenile (IJ, equivalent to the C. elegans dauer) harbors symbiotic bacterium Photorhabdus luminescens, which is used to kill the host. This is a potentially useful system for studying of bacteria/nematode symbiosis, bacteria/insect pathogenesis and nematode/insect parasitism.

Shih, Pei-Yin et al. (2013) International Worm Meeting "Transcription factors involved in dauer recovery."

Sternberg, Paul W., Shih, Pei-Yin

[International Worm Meeting, 2013]

In an adverse environment, the free-living nematode Caenorhabditis elegans becomes a dauer larva, a developmentally arrested stage. When the environment becomes favorable again, dauers recover and resume development. This post dauer is a dynamic period in that there are multiple structural, developmental and behavior modulations that occur. The analogous dauer states in some parasitic nematodes are called infective juveniles (IJ)s and dispersal juveniles (DJ)s. It has been shown that conserved neuron and signaling molecules control dauer/IJ recovery in C. elegans and parasitic nematodes Ancylostoma caninum and Heterorhabditis bacteriophora. However, how perception of the environmental change is converted to developmental changes is not well understood. In this study, we analyzed gene expression changes during dauer and dauer exit in C. elegans and found some transcription factors involved in the recovery process. We are now analyzing the transcription factors in more detail, including their roles in other parasitic nematodes. In addition, we are looking at the neural circuits that mediate the process of exiting dauer. Better understanding of the mechanism may help develop an evolutionary framework within the nematode phylum and potential novel treatment strategies by blocking IJ or DJ recovery.

Smith BS et al. (1990) Cryobiology "Cryopreservation of the entomogenous nematode parasite Steinernema feltiae (= Neoaplectana carpocapsae)."

Hodgson-Smith A, Popiel I, James ER, Smith BS, Minter DM

[Cryobiology, 1990]

Cryopreservation studies were conducted with the J1 juvenile and third stage infective juvenile (IJ) larval stages of the entomogenous parasitic nematode Steinernema feltiae (=Neoaplectana carpocapsae). The main parameters evaluated were (i) tolerance of the organisms to the cryoprotectants methanol, ethanediol, glycerol, and dimethyl sulfoxide; (ii) the incubation temperature and exposure period in cryoprotectant; and (iii) slow cooling (circa 1C min-1) vs rapid cooling (circa 5,100C min-1). The J1 stage was sensitive to all cryoprotectants at >20% (v/v). This sensitivity increased at 22 and 37C. Exsheathed IJ stage organisms tolerated exposure to 50% (v/v) ethanediol or Me2SO and 60% (v/v) methanol or glycerol. A proportion (3.4%) of J1s preincubated in 20% (v/v) methanol for 10 min at 0C survived slow cooling to -35C followed by plunge into liquid nitrogen. Preincubation in 60% (v/v) Me2SO for 45 sec at 0C followed by rapid cooling yielded a higher proportion (12.3%0 of surviving J1s. The infective IJ stage only survived rapid cooling. Preincubation for 20 min at 0C in either 60% (v/v) methanol or 45% glycerol, followed by rapid cooling at 5,100C min-1, yielded 30-34% viable IJs following thawing. These larvae were capable of further growth and development in vitro. The results suggest that the organisms are vitrified during the rapid cooling step. For routine maintenance of strains of S. feltiae and related

Elissa Hallem et al. (2007) International Worm Meeting "Nematodes, Bacteria, and Fruit Flies: a Tripartite Model System for Nematode Parasitism."

Michelle Rengarajan, Paul Sternberg, Todd Ciche, Elissa Hallem

[International Worm Meeting, 2007]

Nematode parasitism is a worldwide health concern: over a quarter of the world''s population is infected with nematode parasites, and over a hundred species of nematodes are parasites of humans. Despite the extensive morbidity and mortality caused by infection with nematode parasites, the biological mechanisms of host-parasite interactions are poorly understood. This is largely due to the lack of genetically tractable model systems for parasitism. We have demonstrated that the insect parasitic nematode Heterorhabditis bacteriophora, its bacterial symbiont Photorhabdus luminescens, and the fruit fly Drosophila melanogaster constitute a tripartite model for nematode parasitism and parasitic infection. We find that infective juveniles (IJs) of H. bacteriophora, which contain P. luminescens in their gut, can infect and kill D. melanogaster larvae. We show that infection activates a humoral immune response in D. melanogaster that results in the temporally dynamic expression of a subset of antimicrobial peptide (AMP) genes, and that this immune response is induced specifically by P. luminescens. We also investigated the cellular and molecular mechanisms underlying IJ recovery, the developmental process that occurs in parasitic nematodes upon host invasion. We find that the chemosensory neurons and signaling pathways that control dauer recovery in C. elegans also control IJ recovery in H. bacteriophora. In particular, IJ recovery is mediated by the ASJ neurons, a cGMP signaling pathway, and muscarinic acetylcholine receptors. Our results suggest conservation of these developmental processes across free-living and parasitic nematodes.

Noguez, Jaime H. et al. (2013) International Worm Meeting "A novel ascaroside controls the parasitic life cycle of the entomopathogenic nematode Heterorhabditis bacteriophora."

Nunnery, Joshawna K., Connor, Elizabeth S., Noguez, Jaime H., Ciche, Todd A., Ragains, Justin R., Zhou, Yue, Butcher, Rebecca A.

[International Worm Meeting, 2013]

Entomopathogenic nematodes survive in the soil as stress-resistant infective juveniles (IJs) that seek out and infect insect hosts. Upon sensing internal host cues, the IJs regurgitate bacterial pathogens from their gut that ultimately kill the host. Inside the host, the nematode develops into a reproductive adult and multiplies until unknown cues trigger the accumulation of IJs. Here, we show that the entomopathogenic nematode Heterorhabditis bacteriophora uses a pheromone to control IJ development. The pheromone, which likely increases in concentration at higher nematode densities, prevents IJ recovery to the J4 stage, allowing IJs to amass late in the infection process. Using activity-guided fractionation and NMR spectroscopy-based structure elucidation, we identify the chemical structure of the pheromone. The pheromone is structurally related to the dauer pheromone ascarosides that the free-living nematode Caenorhabditis elegans uses to control its development. However, none of the C. elegans ascarosides are effective in H. bacteriophora, suggesting that there is a high degree of species specificity. Our report is the first to show that ascarosides are important regulators of development in a parasitic nematode species.