A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans

Abstract

Homeostasis of internal carbon dioxide (CO2) and oxygen (O2) levels is fundamental to all animals. Here we examine the CO2 response of the nematode Caenorhabditis elegans. This species inhabits rotting material, which typically has a broad CO2 concentration range. We show that well fed C. elegans avoid CO2 levels above 0.5%. Animals can respond to both absolute CO2 concentrations and changes in CO2 levels within seconds. Responses to CO2 do not reflect avoidance of acid pH but appear to define a new sensory response. Sensation of CO2 is promoted by the cGMP-gated ion channel subunits TAX-2 and TAX-4, but other pathways are also important. Robust CO2 avoidance in well fed animals requires inhibition of the DAF-16 forkhead transcription factor by the insulin-like receptor DAF-2. Starvation, which activates DAF-16, strongly suppresses CO2 avoidance. Exposure to hypoxia (<1% O2) also suppresses CO2 avoidance via activation of the hypoxia-inducible transcription factor HIF-1. The npr-1 215V allele of the naturally polymorphic neuropeptide receptor npr-1, besides inhibiting avoidance of high ambient O2 in feeding C. elegans, also promotes avoidance of high CO2. C. elegans integrates competing O2 and CO2 sensory inputs so that one response dominates. Food and allelic variation at NPR-1 regulate which response prevails. Our results suggest that multiple sensory inputs are coordinated by C. elegans to generate different coherent foraging strategies.

Fig. 1.

C. elegans avoids elevated levels of CO₂. (A and B) Distribution of N2 animals in microfluidic devices after 10 min without a CO₂ gradient (A) or with a 5% to 0% CO₂ gradient (B). Assays are in the absence of food. Gases pumped into the chamber are indicated at the top. (C and D) Distribution of N2 animals in CO₂ gradients in the absence (C and Table S1) or presence (D) of E. coli food (see also Fig. S1). Bin numbers refer to different portions of the microfluidic chamber. High CO₂ is to the left, as indicated by the wedge. Distribution of animals in all CO₂ gradients shown was significantly different from 0–0% CO₂ (P < 0.0001). Distribution of animals in all CO₂ gradients shown on food was significantly different from that off food (P < 0.0001). In this and all subsequent figures measurements were taken 10 min after the assay began.

Fig. 2.

Behavioral mechanisms involved in avoidance of CO₂. (A–D) Fraction of animals reversing (A and C) or executing a turn (B and D) after a switch in CO₂ concentration. A and B show responses on food, and C and D show responses off food. Events are binned into 15-s time intervals. Gas switches (indicated by an arrow) occur at time 0. Blue bars represent animals subjected to an increase in CO₂, from 0% to 3%; red bars represent animals subjected to a decrease in CO₂ from 3% to 0%. “pre” indicates responses in a 15-s interval immediately before the gas switch. Asterisks indicate significances compared with responses before the gas switch (pre). In this and all subsequent figures, *** or +++ indicates P < 0.001, ** or ++ indicates P < 0.01, and * or + indicates P < 0.05. (E) Feeding N2 animals respond to high CO₂ by increasing their movement. Animals were subjected to a rise in CO₂ (indicated by the first arrow) from 0% to 5% followed by a fall in CO₂ (indicated by the second arrow) from 5% to 0%. “pre” refers to speed before the first gas switch. The gas stimulus regime is indicated below the graph. Speed was measured for each animal every second and then binned into 50-s intervals. Asterisks indicate the significance compared with speed before the up step (“pre”). + indicates significance compared with the 50-s interval before the down step. (F) The average speed of feeding N2 animals exposed to 5% CO₂ remains elevated as long as CO₂ levels are high. Animals were exposed to 0% CO₂ for 4 min, switched to 5% CO₂ for 30 min, and then returned to 0% CO₂ for 4 min. Bars represent the average speed of animals during 50-s intervals just before increasing CO₂ levels, just before decreasing CO₂ levels, and 3 min after return of CO₂ levels to 0%. Fifty-second intervals are indicated by shaded boxes in the gas stimulus regime displayed below the graph. Asterisks indicate significance compared with speed at 0% CO₂. (G) In the absence of food, N2 animals respond to a rise in CO₂ by reducing their speed. Speeds were averaged over the 50-s intervals indicated by shaded boxes in the gas stimulus regime displayed below the graph. (H) CO₂ is potentially a complex stimulus. Aqueous CO₂ as well H⁺ and HCO₃⁻ could be sensory cues for the nematode. Because nematodes are gas-permeable, CO₂ detection could involve both external and internal sensors. Double-headed arrows indicate equilibration of CO₂ among gas, liquid, worm, and agar phases. (I) Avoidance of 5% CO₂ persists with little or no change in magnitude across a broad range of external pH. All pairwise comparisons of chemotaxis indices at different pH values are not significantly different.

Fig. 3.

Multiple signal transduction pathways contribute to CO₂ avoidance. (A) Disrupting the cGMP-gated ion channel encoding genes tax-2 and tax-4 reduces avoidance of 5% CO₂ both on and off food, whereas loss of osm-9, which encodes a TRPV-related channel, does not. In this and all other panels of this figure, a 5% to 0% CO₂ gradient was used to test behavior. * and + indicate significance compared with N2. Alleles used were tax-4(p678), tax-2(p691), and osm-9(ky10). “N2 (air)” represents a negative control with no CO₂ gradient. (B) N2 animals deprived of food gradually reduce CO₂ avoidance. Asterisks indicate significance compared with unstarved N2. (C) Disrupting DAF-2 insulin-like receptor signaling results in strong defects in CO₂ avoidance. Reduced DAF-2 signaling inhibits CO₂ responses by activating the DAF-16 Forkhead transcription factor. Alleles used were daf-2(e1370) and daf-16(mgDf47). Because daf-2(e1370) is a temperature-sensitive allele, animals were grown at 15°C and assayed at 22°C. *, significance compared with N2; ns, not significant compared with N2; +, significance compared with daf-2. (D) Mutations that disrupt pdk-1 3-phosphoinositide-dependent protein kinase 1 or akt-1 protein kinase B also disrupt avoidance of CO₂. PDK-1 and AKT-1 link activation of DAF-2 to inhibition of DAF-16. Alleles used were pdk-1(sa709) and akt-1(mg306). *, significance compared with N2. (E) egl-9 mutants grown in 21% O₂ exhibit attraction to high CO₂. This switch in CO₂ response requires HIF-1. ns, not significant compared with N2; *, significance compared with N2; +, significance compared with egl-9 (sa307). (F) Exposing feeding N2 animals to hypoxia (1% O₂) for 1 h inhibits CO₂ avoidance in a HIF-1-dependent manner. ns, not significant compared with N2; *, significance compared with N2; +, significance compared with N2 conditioned in 1% O₂ for 1 h on food. (G) Genetic pathways contributing to CO₂ avoidance and its modulation.

Fig. 4.

The naturally polymorphic NPR-1 receptor promotes CO₂ avoidance. (A) Mutations in npr-1 reduce avoidance of 5% CO₂ both in the presence and in the absence of E. coli; this phenotype is rescued by an npr-1 215V transgene. N2 animals carrying the npr-1 215F natural allele also exhibit reduced avoidance of CO₂ in the presence of food but maintain avoidance in its absence. ns, not significantly different compared with N2; nd, not determined. (B) The CO₂ avoidance defect in npr-1 mutants is not a consequence of their aggregation behavior. npr-1 animals grown in isolation (GII) retain a strong defect in avoidance of 5% CO₂ compared with similarly reared N2 animals. The weighted chemotaxis index was calculated by recording the position of each animal in a CO₂ gradient at 1-s intervals for 5 min and weighting this according to location in the CO₂ gradient (see Methods). “N2 air” represents a negative control with no CO₂ gradient. *, significance for comparisons between N2 and npr-1; +, significance between N2 GII and npr-1 GII.

Fig. 5.

C. elegans integrates antagonistic gradients of O₂ and CO₂ according to food availability and genotype at the npr-1 locus. Data show distribution of N2 and npr-1(ad609) animals in simple and mixed gradients of O₂ and CO₂ when food is present (A–C) or absent (D–F). The gas gradients are indicated below each set of panels: 5% to 0% CO₂ in A and D; 11% to 21% O₂ in B and E; and a combined gradient of 5% to 0% CO₂ and 11–21% O₂ in C and F. N2 animals strongly avoid CO₂ both on and off food, even if this requires migration to high-O₂ environments. In contrast, the behavior of npr-1 mutants and animals bearing the npr-1 215F allele (see Fig. S3) depends on context. These animals accumulate at low O₂/high CO₂ if food is present (C): an adverse CO₂ gradient does not appear to affect their avoidance of high O₂. Conversely, if food is absent, they tend to migrate to high O₂/low CO₂.