Acute carbon dioxide avoidance in Caenorhabditis elegans

Abstract

Carbon dioxide is produced as a by-product of cellular respiration by all aerobic organisms and thus serves for many animals as an important indicator of food, mates, and predators. However, whether free-living terrestrial nematodes such as Caenorhabditis elegans respond to CO2 was unclear. We have demonstrated that adult C. elegans display an acute avoidance response upon exposure to CO2 that is characterized by the cessation of forward movement and the rapid initiation of backward movement. This response is mediated by a cGMP signaling pathway that includes the cGMP-gated heteromeric channel TAX-2/TAX-4. CO2 avoidance is modulated by multiple signaling molecules, including the neuropeptide Y receptor NPR-1 and the calcineurin subunits TAX-6 and CNB-1. Nutritional status also modulates CO2 responsiveness via the insulin and TGFbeta signaling pathways. CO2 response is mediated by a neural circuit that includes the BAG neurons, a pair of sensory neurons of previously unknown function. TAX-2/TAX-4 function in the BAG neurons to mediate acute CO2 avoidance. Our results demonstrate that C. elegans senses and responds to CO2 using multiple signaling pathways and a neural network that includes the BAG neurons and that this response is modulated by the physiological state of the worm.

Fig. 1.

Acute CO₂ avoidance in C. elegans. (A) When the head of a forward-moving N2 worm is exposed to an air mixture, the worm continues forward locomotion (Upper). When the worm is exposed to 10% CO₂, the worm halts forward locomotion and rapidly reverses (Lower). (B) The response of N2 worms to CO₂ is dose-dependent. ***, P < 0.001. n = 22–44 trials. For all graphs, error bars represent SEM.

Fig. 2.

Signaling pathways that affect acute CO₂ avoidance. (A) NPR-1 modulates CO₂ response. npr-1(ky13) hypomorphs show greatly reduced CO₂ avoidance, and npr-1(ad609) null mutants show essentially no CO₂ avoidance. ***, P < 0.001. n = 12–44 trials. For all graphs, error bars represent SEM. (B) cGMP signaling is required for CO₂ response. Mutants of tax-2, tax-4, and daf-11 do not show acute CO₂ avoidance. ***, P < 0.001. n = 11–44 trials. (C) The cnb-1, rgs-3, and tax-6 mutants do not show acute CO₂ avoidance, whereas the nhr-49 mutant shows reduced CO₂ avoidance. **, P < 0.01; ***, P < 0.001. n = 11–44 trials. Data for N2 are from Fig. 1.

Fig. 3.

The response to CO₂ is modulated by starvation. (A) Avoidance of CO₂ is not modulated by the presence of food. No significant differences were observed between worms tested on OP50, the Escherichia coli strain typically used as a food source for C. elegans; HB101, a different strain of E. coli; or off food. n = 12–44 trials. For all graphs, error bars represent SEM. The data for N2 tested on OP50 are from Fig. 1. (B) Starvation results in a reversible decrease in CO₂ avoidance. *, P < 0.05; **, P < 0.01. n = 7–13 trials. As a control for the 24-h time point off food, N2 worms were left on assay plates for an additional 24 h and then tested on food; no difference was observed between these worms and worms left on assay plates overnight and then tested.

Fig. 4.

Insulin and TGFβ signaling modulate CO₂ response. (A) daf-2(e1370) mutants do not respond to CO₂. This defect is rescued by mutation of daf-16. ***, P < 0.001. n = 12–44 trials. For all graphs, error bars represent SEM. (B) The TGFβ pathway mutants daf-7, daf-1, and daf-4 show little or no CO₂ avoidance. The daf-7 phenotype is rescued by mutation of daf-3. ***, P < 0.001. n = 12–44 trials. Data for N2 are from Fig. 1. (C) Epistatic interactions between insulin and TGFβ signaling and starvation. A 24-h starvation decreases acute CO₂ avoidance. The daf-16(mgDf47) and daf-3(e1376) mutations restore CO₂ response to starved animals, indicating that starvation acts via the insulin and TGFβ pathways. **, P < 0.01. n = 7–15 trials. Error bars represent SEM. Data for N2 are from Fig. 3.

Fig. 5.

CO₂ response is mediated primarily by the BAG neurons. (A) Among mutants with defects in ciliary structure, osm-3 and daf-19 mutants show reduced CO₂ avoidance, whereas che-10 mutants show essentially no CO₂ avoidance. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n = 11–44 trials. For all graphs, error bars represent SEM. Data for N2 are from Fig. 1. (B) BAG-ablated animals show greatly reduced CO₂ avoidance, whereas mock-, AWC-, ASH-, ADL-, and AWB-ablated animals respond normally to CO₂. No significant difference was observed between BAG-ablated animals and animals in which ASH, ADL, AWB, and BAG neurons were ablated. ***, P < 0.001. n = 13–36 animals for each condition. (C) osm-3 mock-ablated animals show reduced CO₂ avoidance compared with N2 mock-ablated animals, and osm-3 BAG-ablated animals show reduced CO₂ avoidance compared with osm-3 mock-ablated animals. Data for N2 mock-ablated are from B. No significant difference was observed between N2 BAG-ablated and osm-3 BAG-ablated animals. *, P < 0.05. n = 22–33 animals for each condition.

Fig. 6.

cGMP signaling is required in the BAG neurons for acute CO₂ avoidance. (A) The URX, AQR, and PQR neurons are not required for acute CO₂ avoidance. Animals containing a gcy-32::tax-4 transgene in the tax-4(ks28) mutant background, in which tax-4 is expressed specifically in URX, AQR, and PQR (42), do not respond to CO₂. Animals containing a gcy-36::egl-1 transgene, which kills URX, AQR, and PQR (42), respond normally to CO₂. ***, P < 0.001. n = 11–44 trials. For all graphs, error bars represent SEM. (B) tax-4 is required in the BAG neurons for acute CO₂ avoidance. Animals containing an odr-4::tax-4 transgene in the tax-4(p678) mutant background (19), in which tax-4 is expressed in 12 sensory neurons, do not respond to CO₂. Animals containing odr-4+gcy-8+gcy-32+gcy-33::tax-4 transgenes in the tax-4(p678) mutant background (19), in which tax-4 is expressed in 17 neurons including BAG, respond normally to CO₂. Animals containing a gcy-33::tax-4 transgene in the tax-4(p678) mutant background, in which tax-4 is expressed specifically in the BAG neurons, also respond normally to CO₂. ***, P < 0.001. n = 12–44 trials. Data for N2 are from Fig. 1, and data for tax-4 mutants are from Fig. 2. (C) A model for acute CO₂ avoidance in C. elegans. CO₂ avoidance is mediated by a cGMP signaling pathway involving TAX-2/TAX-4 acting within the BAG neurons. This response is modulated by NPR-1. CO₂ response is decreased by starvation, which acts via the insulin and TGFβ pathways.