Ammonium-acetate is sensed by gustatory and olfactory neurons in Caenorhabditis elegans

Abstract

Background: Caenorhabditis elegans chemosensation has been successfully studied using behavioral assays that treat detection of volatile and water soluble chemicals as separate senses, analogous to smell and taste. However, considerable ambiguity has been associated with the attractive properties of the compound ammonium-acetate (NH(4)Ac). NH(4)Ac has been used in behavioral assays both as a chemosensory neutral compound and as an attractant.

Methodology/main findings: Here we show that over a range of concentrations NH(4)Ac can be detected both as a water soluble attractant and as an odorant, and that ammonia and acetic acid individually act as olfactory attractants. We use genetic analysis to show that NaCl and NH(4)Ac sensation are mediated by separate pathways and that ammonium sensation depends on the cyclic nucleotide gated ion channel TAX-2/TAX-4, but acetate sensation does not. Furthermore we show that sodium-acetate (NaAc) and ammonium-chloride (NH(4)Cl) are not detected as Na(+) and Cl(-) specific stimuli, respectively.

Conclusions/significance: These findings clarify the behavioral response of C. elegans to NH(4)Ac. The results should have an impact on the design and interpretation of chemosensory experiments studying detection and adaptation to soluble compounds in the nematode Caenorhabditis elegans.

Figure 1. Odorant chemotaxis to NH₄Ac.

(A) Diagram of the assay. Droplets of attractant and negative control solutions were placed on opposite sides of plate at the locations indicated by the eccentric black dots. To manipulate effective NH₄Ac concentrations, rather than changing NH₄Ac concentration we changed the droplet volumes as indicated in panels B and C. NH₄Ac concentration was 7.5 M and adjusted to pH = 6.0 with acetic acid. Sodium-azide was added to immobilize worms reaching either location. Worms were placed at the center of plate and allowed to move for one hour after which worms in zones A, B, and C were counted and the chemotaxis index (C.I.) was computed as shown. (B) Chemotaxis index vs. droplet volume for NH₄Ac placed directly on the assay plate immediately before the assay. (C) Chemotaxis index vs. droplet volume for NH₄Ac suspended from the Petri plate lid immediately before the assay. (D) Chemotaxis index for equal volumes (10 µL) of different attractants suspended from the lid. In all panels, H₂O refers to a negative control in which only water was spotted on the plate. The concentration of NH₄Ac in the droplets was 7.5 M (pH = 6.0). Each bar represents the mean of at least 8 independent assays. Statistics: * p<0.05 and ** p<0.01 in a one way ANOVA with Dunnet's post test comparing all means to the negative control (H₂O at both spots).

Figure 2. Genetic analysis of chemotaxis to NH₄Ac presented in water soluble or odorant form.

(A) Water soluble chemotaxis assays. Chemotaxis index is plotted vs. strain for assays in which radial gradients of NH₄Ac were established by diffusion in the agar. (B) Odorant NH₄Ac assays. Chemotaxis index is plotted vs. strain for assays in which a droplet of NH₄Ac (10 µL, 7.5 M) was suspended from the lid of the plate. In A and B, each bar represents the mean of at least 8 independent assays; n.d. means no data. Wild type (neg. con) is a negative control assay with no attractant on plate. Statistics: * p<0.05 and ** p<0.01 in a one way ANOVA and Dunnet's post test comparing all means to the wild-type (N2) mean; # p<0.05 in a one-way ANOVA with Dunnet's post test comparing che-2(e1033), che-3(e1124), and osm-3(p802) to the negative control.

Figure 3. Genetic analysis of ammonium and acetate signal transduction pathways.

(A–C) Water soluble chemotaxis assays for NaCl, NH₄Cl, and NaAc. In each case, radial gradients of attractant were established by diffusion in the agar. Each bar represents the mean of at least 4 independent assays. Statistics: * p<0.05 and ** p<0.01 in a one-way ANOVA and Dunnet's post test comparing all mutants to the che-1(p679), which serves as a control in that it has normal chemotaxis to NH₄Ac but no chemotaxis to NaCl; § p<0.01 in a one sample t-test comparing the observed mean to a mean of zero.

Figure 4. Genetic analysis of the relative ionic contributionsto water soluble chemotaxis assays.

Attractants and uniform background compositions are indicated below each group of bars; the no-background conditions are indicated by “-” and n.d. means no data. Each bar represents the mean of at least 8 independent assays. Statistics: * p<0.05 and ** p<0.01 in a one way ANOVA and Dunnet's post test comparing all means to the wild-type (N2) mean.