Interspecific nematode signals regulate dispersal behavior

Abstract

Background: Dispersal is an important nematode behavior. Upon crowding or food depletion, the free living bacteriovorus nematode Caenorhabditis elegans produces stress resistant dispersal larvae, called dauer, which are analogous to second stage juveniles (J2) of plant parasitic Meloidogyne spp. and infective juveniles (IJ)s of entomopathogenic nematodes (EPN), e.g., Steinernema feltiae. Regulation of dispersal behavior has not been thoroughly investigated for C. elegans or any other nematode species. Based on the fact that ascarosides regulate entry in dauer stage as well as multiple behaviors in C. elegans adults including mating, avoidance and aggregation, we hypothesized that ascarosides might also be involved in regulation of dispersal behavior in C. elegans and for other nematodes such as IJ of phylogenetically related EPNs.

Methodology/principal findings: Liquid chromatography-mass spectrometry analysis of C. elegans dauer conditioned media, which shows strong dispersing activity, revealed four known ascarosides (ascr#2, ascr#3, ascr#8, icas#9). A synthetic blend of these ascarosides at physiologically relevant concentrations dispersed C. elegans dauer in the presence of food and also caused dispersion of IJs of S. feltiae and J2s of plant parasitic Meloidogyne spp. Assay guided fractionation revealed structural analogs as major active components of the S. feltiae (ascr#9) and C. elegans (ascr#2) dispersal blends. Further analysis revealed ascr#9 in all Steinernema spp. and Heterorhabditis spp. infected insect host cadavers.

Conclusions/significance: Ascaroside blends represent evolutionarily conserved, fundamentally important communication systems for nematodes from diverse habitats, and thus may provide sustainable means for control of parasitic nematodes.

Figure 1. Dispersal assay.

(A) Naturally dispersing infective juveniles (IJ) of S. feltiae from an insect cadaver. (B) Approximately 300 IJs were placed on an agar plate in water. (C) IJs were treated with either water (control) or insect cadaver extract. Images are representative of six experiments for each treatment. (D) Dispersal assay on the same plate. The illustration represents two experiments. Behavior is temperature and season dependent. Assays were conducted at RT (22±0.5°C) or under temperature controlled conditions.

Figure 2. Structures of ascarosides.

Structures of published ascarosides.

Figure 3. An ascaroside blend regulates C. elegans dispersal behavior, and the dispersal blend is recognized by other nematodes.

(A) Identification of the dispersal blend. Images (∼250 nematodes) are representative of 9, 10, and 11 experiments of control (0.25% E. coli (HB101)), synthetic blend with 0.25% E. coli (HB101) and dauer supernatant, respectively. (B) Quantification using Image J (http://rsbweb.nih.gov/ij/download.html). Control vs synthetic blend student's t-test unpaired (p<0.02). (C) S. feltiae IJs (∼250) response to the dispersal blend; four experiments for each treatment. (D) Response of root-knot J2s (Meloidogyne spp., mixture of M. incognita, M. javanica, and M. floridensis) to the C. elegans dispersal blend. The data represent 19 and 20 experiments from control water and C. elegans dispersal blend, respectively. At 2 h, using a student's t-test, unpaired, p<0.007.

Figure 4. Activity guided fractionation of the S. feltiae dispersal pheromone.

(A) Reverse phase (C18) chromatography of insect cadaver extract. The image represents two independent experiments. The estimated physiologically relevant concentration was used in the assays; Frc, fraction. (B) Testing physiologically relevant concentration of ascarosides found in Frc A (ascr#9, 40.3 pmol/µl and ascr#11, 1.3 pmol/µl). Image represents three experiments. (C) Ascr#2 and ascr#9 are structural analogs. Natural and synthetic ascr#9 were tested in combination with fraction B and C. Image represents four experiments. (D) Structure of ascr#11. It (1.3 pmol/µl) is also sufficient to cause dispersal in combination with fractions B and C. Image represents three experiments.