Acute carbon dioxide avoidance in Caenorhabditis elegans

Elissa A. Hallem; Paul W. Sternberg

doi:10.1073/pnas.0707469105

New Research In

Edited by Martin Chalfie, Columbia University, New York, NY, and approved February 20, 2008 (received for review August 8, 2007)

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 TGFβ 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.

Carbon dioxide is a critical sensory cue for organisms as diverse as humans (1), rodents (2, 3), insects (4, 5), and bacteria (6). The ability to rapidly detect and respond to changing CO₂ concentrations is important for survival: high CO₂ concentrations cause hypoxia, anesthetization, and death. CO₂ perception may be particularly important for soil-dwelling organisms such as Caenorhabditis elegans, because bacteria-rich soil microenvironments are often subject to large fluctuations in CO₂ and O₂ levels (7). However, despite the fundamental importance of CO₂ for all living organisms, remarkably little is known about how CO₂ evokes behavioral responses in animals.

Many mammals, including humans, are capable of detecting CO₂. Humans detect CO₂ as a trigeminal stimulus, and different humans show different sensitivities to CO₂ (1). Mice detect and avoid near-atmospheric concentrations of CO₂ via a subset of olfactory sensory neurons that project to the necklace glomeruli in the olfactory bulb (2). Many insects also detect CO₂. For example, Drosophila is repelled by CO₂ via a single class of olfactory receptor neurons on the antennae (8), whereas mosquitoes are attracted to CO₂ via olfactory receptor neurons on the maxillary palps (9).

The phylum Nematoda contains both free-living and parasitic species, some of which have been shown to respond to CO₂. Many animal-parasitic nematodes, including the human-parasitic nematodes Ancylostoma duodenale and Necator americanus, are attracted to CO₂; for these nematodes, CO₂ exhaled by their hosts serves as a host-localization and host-invasion cue (10, 11). Similarly, many plant-parasitic nematodes use CO₂ emission by plant roots for host localization (12, 13). Some species of free-living marine nematodes are also attracted to CO₂, which is used as a sensory cue to locate the decaying animal and plant carcasses that serve as their food sources (14). However, nothing is known about the mechanisms underlying CO₂ perception in these nematodes, and whether free-living terrestrial nematodes respond to CO₂ was unclear (10, 15, 16).

The well studied model C. elegans is a free-living terrestrial nematode that navigates through its environment using a combination of chemosensory, thermosensory, and mechanosensory cues. The nervous system of the C. elegans hermaphrodite consists of 302 neurons, >10% of which are chemosensory (17, 18). The perception of volatile and water-soluble chemicals is mediated primarily by 11 pairs of chemosensory neurons that extend ciliated dendrites into the paired amphid sensilla of the head (17). C. elegans also senses O₂ via two ciliated and two unciliated sensory neurons that extend dendrites into the pseudocoelomic body fluid (17, 19).

We demonstrate that C. elegans display an acute avoidance response to CO₂. We then identify signaling pathways that affect CO₂ avoidance by conducting a screen of existing mutants with neurosensory defects. We find that CO₂ avoidance is mediated by cGMP signaling and requires the receptor guanylyl cyclase DAF-11 and the cGMP-gated channel TAX-2/TAX-4. CO₂ response is modulated by multiple neuronal regulatory molecules. Nutrient deprivation decreases CO₂ avoidance, and this response is mediated by the insulin and TGFβ pathways. Finally, acute CO₂ avoidance is mediated primarily by the paired BAG neurons of the head, and cGMP signaling is required in the BAG neurons to mediate CO₂ avoidance.

Results

An Acute Carbon Dioxide Response in C. elegans.

To determine whether C. elegans responds to CO₂, we developed a CO₂ avoidance assay based on the osmotic avoidance assay (20, 21). Specifically, the head of a forward-moving worm is exposed to an air stream containing CO₂, and a response is scored if the worm reverses direction within 4 seconds [Fig. 1A and supporting information (SI) Movies S1 and S2]. Reversals are characteristic of avoidance responses in C. elegans: exposure to 1-octanol, hyperosmolarity, and nose touch elicit rapid reversals (17, 22, 23).

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.

Wild-type N2 worms respond to CO₂; for example, 76% reverse in response to an air stream containing 10% CO₂, whereas only 23% reverse when the air stream does not contain CO₂ (Table S1). We calculated an avoidance index (a.i.) for CO₂ by subtracting the fraction of worms that reversed in response to an air stream that does not contain CO₂ from the fraction that reversed in response to an air stream containing CO₂; N2 worms show an a.i. of 0.53 in response to 10% CO₂ (Fig. 1B).

C. elegans responds to CO₂ in a dose-dependent manner (Fig. 1B). The response to 1% CO₂ was significantly different from the response to 0% CO₂, demonstrating that C. elegans can detect CO₂ concentrations as low as 1% above ambient levels (Fig. 1B). The average atmospheric concentration of CO₂ is ≈0.04%. A CO₂ concentration of 10% was used for subsequent experiments because of the robust response to this concentration.

Acute CO₂ Avoidance Varies Among Strains of C. elegans and Species of Free-Living Nematodes.

We next asked whether CO₂ aversion is conserved across six strains of C. elegans (24). Strains N2, CB3191, and TR389 show robust CO₂ avoidance, whereas strains AB1, CB4853, and CB4856 are essentially unresponsive to CO₂ in this assay (Fig. S1A). This suggests that CO₂ avoidance is a rapidly evolving behavior. Different strains of C. elegans have been isolated from diverse ecological niches (25, 26), raising the possibility that this behavior may be advantageous in only some niches.

We then examined CO₂ avoidance in five phylogenetically and ecologically diverse species of nematodes from the order Rhabditida (27). C. elegans and Pristionchus pacificus show robust CO₂ avoidance, whereas Caenorhabditis briggsae, Caenorhabditis species 3, and Panagrellus redivivus show little or no CO₂ avoidance (Fig. S1B). Thus, acute CO₂ avoidance is found in some but not all free-living nematode species. However, species that do not avoid CO₂ in our assay may exhibit CO₂ responses under conditions not tested here.

The Neuropeptide Y Receptor NPR-1 Modulates CO₂ Avoidance.

Different wild isolates of C. elegans show polymorphic feeding behavior: some are solitary feeders that disperse on bacterial lawns, and others are social feeders that aggregate into clumps at the edge of the bacterial lawn (28). The three CO₂-sensitive strains of C. elegans are solitary feeders, whereas the three CO₂-insensitive strains are social feeders (Fig. S1A) (28). Thus, solitary feeding correlates with CO₂ avoidance, suggesting that both behaviors may be subject to common regulatory mechanisms.

Feeding behavior is modulated by the neuropeptide Y receptor gene npr-1: loss-of-function mutations in npr-1 can convert a solitary strain into a social strain (28). We therefore investigated whether npr-1 also modulates acute CO₂ avoidance. A hypomorphic mutation in npr-1 in an N2 background results in reduced CO₂ avoidance, and a null mutation eliminates acute CO₂ avoidance (Fig. 2A). Thus, npr-1 modulates CO₂ avoidance.

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.

Differences in feeding behavior among strains appear to be due to differences in O₂ response, and npr-1 mutants display altered O₂ preference in the presence of food (29). The fact that O₂ and CO₂ responses are both modulated by NPR-1 raises the possibility that the same receptor proteins confer responses to both gases. O₂ receptors in C. elegans comprise a family of soluble guanylyl cyclases (sGCs) (29, 30). However, sGC mutants respond normally to CO₂ (Fig. S2A). Moreover, mutation of the transcription factor AHR-1, which regulates expression of the sGC genes, does not affect CO₂ avoidance (Fig. S2A). Thus, CO₂ and O₂ response are conferred by different receptors despite being subject to the same neuromodulatory control by npr-1.

Acute CO₂ Avoidance Is Mediated by cGMP Signaling.

To identify the signaling pathways that mediate CO₂ avoidance, we screened candidate mutants that had been previously isolated and that display a wide variety of defects in neuronal development and function. TAX-2 and TAX-4 are subunits of a cGMP-gated channel required for normal chemosensory and thermosensory responses (17). We found that mutations in tax-2 and tax-4 eliminate acute CO₂ avoidance (Fig. 2B). Also, mutation of the receptor guanylyl cyclase DAF-11 eliminates CO₂ response (Fig. 2B). Thus, a cGMP signaling pathway(s) that includes DAF-11, TAX-2, and TAX-4 is required for acute CO₂ avoidance.

Multiple Signaling Molecules Modulate Acute CO₂ Avoidance.

Our screen of candidate genes also identified a number of additional signaling molecules that modulate CO₂ avoidance: tax-6, cnb-1, rgs-3, and nhr-49. The calcineurin subunits TAX-6 and CNB-1 are required for CO₂ avoidance: tax-6 and cnb-1 mutants are unresponsive to CO₂ in our assay (Fig. 2C). These results suggest that CO₂ response requires calcium signaling.

rgs-3 mutants fail to avoid CO₂ (Fig. 2C). RGS-3 is a regulator of G protein signaling that is expressed in nine types of sensory neurons: ASH, ADL, AWB, AWC, ASI, ASJ, ASK, PHA, and PHB (31). The fact that rgs-3 mutants do not show acute CO₂ avoidance suggests that CO₂ response is modulated by signaling through G proteins acting in one or more of the neurons that express rgs-3. Animals with mutations in individual G protein subunits, as well as gpa-1 gpa-2 gpa-3 triple mutants, responded normally to CO₂ (Fig. S2B), suggesting that multiple G proteins act redundantly to regulate CO₂ response.

Mutation of the nuclear hormone receptor gene nhr-49 results in reduced CO₂ avoidance (Fig. 2C). NHR-49 regulates transcriptionally the response to starvation, including expression of fat and energy metabolism genes (32). Thus, acute CO₂ avoidance and the starvation response share a common regulatory mechanism involving nhr-49.

Mutants defective in the synthesis and reception of nonessential excitatory neurotransmitters, as well as mutants defective in neuropeptide synthesis and secretion, respond normally to CO₂ (Fig. S2 C and D). CO₂ avoidance may be mediated by both neuromodulators and neurotransmitters or by multiple neuromodulators or neurotransmitters acting in parallel.

Starvation Modulates CO₂ Sensitivity in C. elegans.

The regulation of CO₂ response by NHR-49 raised the possibility that starvation affects CO₂ avoidance. We therefore first asked whether CO₂ avoidance is affected by the presence of bacterial food. Many sensory behaviors in C. elegans are affected by food, including O₂ aerotaxis and 1-octanol avoidance (29, 33). However, worms show equally robust CO₂ avoidance in the presence and absence of food (Fig. 3A). We then asked whether CO₂ avoidance is modulated by starvation. Well fed worms tested off food showed an a.i. of 0.53; however, after 24 h of food deprivation the a.i. was reduced to 0.13 (Fig. 3B). When animals deprived of food for 24 h were placed back on food for 2 h, the a.i. was restored to the level of well fed animals (Fig. 3B). Thus, CO₂ response is reversibly modulated by nutritional status.

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.

The Insulin and TGFβ Pathways Modulate CO₂ Avoidance.

The insulin and TGFβ pathways are key regulators of starvation in C. elegans (34). Given that starvation reduces CO₂ avoidance, we asked whether insulin and TGFβ signaling might also affect CO₂ response. Mutation of the insulin receptor DAF-2 eliminates CO₂ avoidance, and mutation of the forkhead transcription factor DAF-16 suppresses the daf-2 phenotype (Fig. 4A). Thus, DAF-16 acts downstream of DAF-2 in the regulation of CO₂ response. Also, different alleles of daf-2 confer different CO₂ sensitivities (Fig. S3A). The strength of the different daf-2 alleles with respect to CO₂ avoidance correlates with the strength of the alleles with respect to hypoxia resistance (Fig. S3B) (35) but not lifespan and dauer formation (36, 37), suggesting that CO₂ avoidance and hypoxia resistance may be subject to similar mechanisms of regulation by daf-2.

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.

The TGFβ pathway also mediates acute CO₂ avoidance: mutations in the TGFβ ligand DAF-7, as well as the TGFβ receptors DAF-1 and DAF-4, show severely reduced CO₂ avoidance (Fig. 4B). The daf-7 phenotype is rescued by mutation of the SMAD gene daf-3, demonstrating that DAF-3 acts downstream of DAF-7 in the regulation of CO₂ avoidance (Fig. 4B). DAF-7 is thought to be expressed specifically in the ASI chemosensory neurons (38). However, ablation of the ASI neurons did not affect CO₂ avoidance (data not shown). This may be because daf-7 expression in ASI is required for CO₂ response only transiently during early development or because daf-7 is expressed at low levels in other cells required for CO₂ response.

To investigate the epistatic relationship between starvation and insulin and TGFβ signaling, we examined whether starved daf-16 and daf-3 mutants respond to CO₂. In contrast to wild type, daf-16 and daf-3 mutants that had been starved for 24 h responded normally to CO₂ (Fig. 4C). Thus daf-16 and daf-3 rescue the CO₂ response defect of starved worms. Starvation therefore modulates CO₂ response via the insulin and TGFβ pathways.

CO₂ Response Is Mediated by a Neural Circuit That Includes the BAG Neurons.

To gain insight into the neural circuitry underlying CO₂ perception, we examined the CO₂ sensitivity of mutants with sensory neuron defects. We first tested mutations that affect the development of ciliated sensory neurons. osm-3 and daf-19 mutants showed reduced CO₂ response, whereas a che-10 mutant was essentially unresponsive to CO₂ (Fig. 5A). osm-3 encodes a kinesin subunit required for normal formation of amphid cilia, and daf-19 encodes an RFX transcription factor required for the formation of all sensory cilia (39). These mutants implicate ciliated sensory neurons in CO₂ avoidance. A che-10 mutation causes degeneration of amphid and phasmid neurons; however, the IL1, OLQ, and BAG neurons are also affected (40). These results suggest that one or more of the amphid or phasmid neurons, as well as one or more of IL1, OLQ, and BAG, play a role in CO₂ perception. Mutations that affect development of specific subsets of sensory neurons do not affect acute CO₂ avoidance (Fig. S4).

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.

We also tested the tax-2 allele tax-2(p694), a cis-regulatory mutation that disrupts tax-2 expression in the AQR, AFD, ASE, and BAG neurons (19). tax-2(p694) mutants do not respond to CO₂ in our assay (Fig. 2B). Because mutations and transgenes that compromise the function of AQR, AFD, and ASE respond normally to CO₂ (Fig. 6 and Fig. S4), tax-2 is likely required in the BAG neurons (Fig. S5) for acute CO₂ avoidance.

To further test the role of the BAG neurons in CO₂ response, we ablated them using a laser microbeam and measured the ability of ablated worms to respond to CO₂. As a control, we ablated the AWC olfactory neurons, which mediate attraction (17). Ablation of the BAG neurons resulted in greatly reduced CO₂ avoidance (Fig. 5B). By contrast, mock-ablated and AWC-ablated animals responded normally to CO₂ (Fig. 5B). Therefore, the BAG neurons are important components of the neural circuit that mediates CO₂ response.

The fact that CO₂ response is severely reduced but not completely eliminated in BAG-ablated animals suggests that other sensory neurons play a role in acute CO₂ avoidance. In an attempt to identify these neurons, we ablated the ASH, ADL, and AWB neurons, which mediate olfactory repulsion (17). However, ablation of these neuron pairs individually did not affect acute CO₂ avoidance, and ablation of the ASH, ADL, AWB, and BAG neurons in the same animal resulted in a response that was not significantly different from the response of BAG-ablated animals (Fig. 5B). Thus ASH, ADL, and AWB play at most a minor role in acute CO₂ avoidance.

We then ablated the BAG neurons in osm-3 mutants. We found that the response of mock-ablated osm-3 mutants is reduced compared with mock-ablated wild-type animals, and the response of BAG-ablated osm-3 mutants is further reduced (Fig. 5C). These results suggest that, in addition to BAG, one or more of the ciliated sensory neurons affected by the osm-3 mutation play a role in CO₂ avoidance.

We note that acute CO₂ avoidance could be either a chemosensory or a nociceptive response. However, chemical nociception is mediated primarily by the ASH neurons (17), and ASH-ablated animals respond normally to CO₂ (Fig. 5B). Thus, acute CO₂ avoidance is likely to be a chemosensory response.

Finally, we generated dose–response curves for four mutants that showed defective CO₂ avoidance when tested with 10% CO₂: osm-3 and nhr-49, which showed reduced avoidance of 10% CO₂; and tax-4 and npr-1, which failed to avoid 10% CO₂. We found that the CO₂ response of all four mutants is defective across a broad range of concentrations (Fig. S6). These results suggest that acute CO₂ avoidance is mediated by the same signaling mechanisms across concentrations.

cGMP Signaling Is Required in the BAG Neurons for Acute CO₂ Avoidance.

tax-2 and tax-4 are coexpressed in 12 neurons: AWC, AFD, ASE, ASG, ASJ, ASI, AWB, ASK, BAG, AQR, PQR, and URX (41). To identify the neuron(s) in which TAX-2/TAX-4 is required for acute CO₂ avoidance, we performed a series of cell-specific rescue experiments with tax-4. We first tested whether CO₂ avoidance requires tax-4 expression in AQR, PQR, and URX because tax-4 is required in these neurons for normal O₂ response (19, 29, 42). We found that tax-4 mutants in which tax-4 is specifically rescued in AQR, PQR, and URX (42) do not respond to CO₂ (Fig. 6A). Moreover, animals containing a genetic ablation of AQR, PQR, and URX (42) respond normally to CO₂ (Fig. 6A). Thus, AQR, PQR, and URX do not mediate acute CO₂ avoidance.

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.

We then examined tax-4 mutants containing an odr-4::tax-4 transgene, in which tax-4 is expressed in AWA, AWB, AWC, ADF, ASG, ASH, ASI, ASJ, ASK, ADL, PHA, and PHB (19). These worms show essentially no CO₂ avoidance (Fig. 6B). However, expression of tax-4 in these neurons as well as AQR, PQR, URX, AFD, and BAG using the odr-4, gcy-8, gcy-32, and gcy-33 promoters (19) is sufficient to rescue the CO₂ response defect of tax-4 mutants (Fig. 6B). Given that ablation of the BAG neurons results in greatly reduced CO₂ avoidance, we then asked whether tax-4 expression in the BAG neurons is sufficient for CO₂ avoidance by expressing tax-4 under the control of only the gcy-33 promoter. We found that tax-4 expression in BAG rescues the CO₂ response defect of tax-4 mutants (Fig. 6B). Thus, a cGMP signaling pathway involving TAX-2/TAX-4 operates within the BAG neurons to mediate acute CO₂ avoidance.

Discussion

We have found that C. elegans exhibits acute avoidance of CO₂. This response requires a cGMP signaling pathway acting within the BAG neurons and is modulated by additional neuronal regulatory molecules as well as by insulin and TGFβ signaling (Fig. 6C).

Acute CO₂ avoidance is exhibited by some but not all wild isolates of C. elegans and some but not all species of free-living terrestrial nematodes. Thus, acute CO₂ avoidance is a rapidly evolving behavior that has either arisen or been lost multiple times during the course of nematode evolution, raising the possibility that it is advantageous only under some ecological conditions.

Starved worms show reduced CO₂ avoidance, and this effect is mediated by insulin and TGFβ signaling. The fact that CO₂ response is reduced by starvation contrasts with most olfactory responses in C. elegans, which are enhanced by starvation (17), presumably so as to maximize the worm's chance of finding food. The starvation-induced decrease in CO₂ avoidance may offer a similar ecological advantage: In nature, C. elegans presumably encounters CO₂ emitted by both bacterial food and predators. Under conditions of starvation it may be beneficial to down-regulate CO₂ avoidance so as to maximize the probability of encountering food, even if this incurs an increased risk of predation.

The sensitivity of CO₂ response to nutritional status is not universal among animals. For example, starved larvae of the bloodsucking insect Triatoma infestans respond as robustly to CO₂ as well fed larvae, even after 60 days of starvation (43). By contrast, many mosquitoes use CO₂ as their primary host-seeking cue, and host-seeking behavior is greatly reduced after a blood meal (44). Thus, CO₂ response may be subject to different regulatory mechanisms in organisms with different life cycles and behavioral repertoires.

Acute CO₂ avoidance is mediated primarily by the BAG neurons, and cGMP signaling mediated by TAX-2/TAX-4 is required in BAG for acute CO₂ avoidance. The BAG neurons are ciliated neurons of previously unknown function located in the head but not associated with the amphid sensillum (18). These neurons may sense CO₂ directly via one or more CO₂ receptors, or they may be indirect modulators of CO₂ response. It will be interesting to determine whether the BAG neurons also modulate O₂ response and also to identify additional signaling components that operate within the BAG neurons. Of the signaling molecules identified in this study as mediators of CO₂ avoidance, only TAX-2/TAX-4 are known to be expressed in the BAG neurons. In particular, expression of the guanylyl cyclase DAF-11 has not been observed in the BAG neurons (45), raising the possibility that the effect of daf-11 on CO₂ response is indirect and that a different guanylyl cyclase acts upstream of TAX-2/TAX-4 in the BAG neurons to mediate CO₂ avoidance.

A CO₂ receptor in C. elegans has not yet been identified. In Drosophila, CO₂ avoidance is mediated by two members of the gustatory receptor (Gr) family of serpentine receptors, Gr21a and Gr63a (46 ⇓–48), which are expressed in a single class of olfactory neurons on the fly antenna (5, 8, 46, 47). C. elegans does not contain orthologs of Gr21a and Gr63a, and thus Drosophila and C. elegans use different receptors for CO₂ detection.

The CO₂ avoidance we observed in some species of free-living terrestrial nematodes contrasts with the attraction to CO₂ exhibited by many parasitic and free-living marine nematodes (10 ⇓⇓⇓–14, 49). Our study provides a foundation for investigations into how the CO₂ response network may have evolved in nematodes with very different life cycles and ecological niches.

Materials and Methods

Standard techniques are listed in the SI Methods.

Population Assay for Acute CO₂ Avoidance.

For each assay, ≈10–30 C. elegans L4 hermaphrodites were placed onto assay plates overnight and tested as young adults. Assay plates consisted of NGM agar plates containing a thin lawn of OP50 bacteria grown for 1–2 days at room temperature. Gases were medical-grade certified mixtures (Air Liquide) of 0%, 0.2%, 1%, 2.5%, 5%, 10%, or 15% CO₂; 10% O₂; and the balance N₂. An O₂ concentration of 10% was chosen to closely approximate the preferred O₂ concentration of C. elegans (29). Ten percent CO₂ was used for all experiments unless otherwise indicated. For the avoidance assay, two 50-ml syringes were filled with gas, one with and one without CO₂. The mouth of the syringes were connected to tubes attached to Pasteur pipettes, and gases were pumped through the Pasteur pipettes by using a syringe pump (PHD 2000; Harvard Apparatus) at a rate of 1.5 ml/min. Individual worms were exposed to gases by placing the tip of the Pasteur pipette near the head of a forward-moving worm. A response was scored if the worm initiated backward movement within 4 seconds. The gas mixture to which each plate was exposed was alternated such that half of the plates were exposed to air and half were exposed to CO₂. Gases were delivered blindly, and worms were tested blindly. Each plate was considered one trial. Plates were assayed one to two times with at least 1 h between trials, except that worms tested off food were tested only once. An a.i. for each genotype was calculated by subtracting the fraction of worms that reversed in response to air from the fraction that reversed in response to CO₂. For assays involving other species, both males and females of dioecious species were tested. Values obtained for each genotype or treatment are listed in Table S1.

Single-Worm Assay for CO₂ Avoidance.

For each assay, individual L4 or young adult hermaphrodites were placed onto assay plates overnight. Worms were tested as described above, except that each worm was tested 15 times with >2 min between trials. No adaptation was observed during the course of these experiments. For each worm, an a.i. was calculated by subtracting the fraction of trials the worm reversed in response to air from the fraction of trials the worm reversed in response to CO₂. The a.i. for each genotype or treatment was calculated as the mean a.i. for each worm of the same genotype or treatment. Values obtained for each genotype or treatment are listed in Table S2. References for mutant strains are listed in Table S3.

Acknowledgments

We thank M. de Bono (MRC Laboratory of Molecular Biology, Cambridge, U.K.), C. Bargmann (HHMI and The Rockefeller University, New York, NY), J. Thomas (University of Washington, Seattle, WA), and the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN) for strains; D. Anderson (California Institute of Technology, Pasadena, CA) and members of our laboratory for insightful discussions; and J. Srinivasan (California Institute of Technology, Pasadena, CA) for discussion of other species. This work was supported by the Howard Hughes Medical Institute, of which P.W.S. is an investigator, and a Helen Hay Whitney postdoctoral fellowship (to E.A.H.).

Footnotes

↵*To whom correspondence should be addressed at: Division of Biology 156-29, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125. E-mail: ehallem{at}caltech.edu

Author contributions: E.A.H. and P.W.S. designed research; E.A.H. performed research; E.A.H. analyzed data; and E.A.H. and P.W.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0707469105/DCSupplemental.

Received August 8, 2007.

Freely available online through the PNAS open access option.

View Abstract

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Submit

[1] ↵
Bensafi M,
Frasnelli J,
Reden J,
Hummel T
(2007) The neural representation of odor is modulated by the presence of a trigeminal stimulus during odor encoding. Clin Neurophysiol 118:696–701.
.
OpenUrl CrossRef PubMed

[2] Bensafi M,

[3] Frasnelli J,

[4] Reden J,

[5] Hummel T

[6] ↵
Hu J,
et al.
(2007) Detection of near-atmospheric concentrations of CO₂ by an olfactory subsystem in the mouse. Science 317:953–957.
.
OpenUrl Abstract/FREE Full Text

[7] Hu J,

[8] et al.

[9] ↵
Youngentob SL,
Hornung DE,
Mozell MM
(1991) Determination of carbon dioxide detection thresholds in trained rats. Physiol Behav 49:21–26.
.
OpenUrl CrossRef PubMed

[10] Youngentob SL,

[11] Hornung DE,

[12] Mozell MM

[13] ↵
Bowen MF
(1991) The sensory physiology of host-seeking behavior in mosquitoes. Annu Rev Entomol 36:139–158.
.
OpenUrl CrossRef PubMed

[14] Bowen MF

[15] ↵
Suh GS,
et al.
(2004) A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila. Nature 431:854–859.
.
OpenUrl CrossRef PubMed

[16] Suh GS,

[17] et al.

[18] ↵
Hammer A,
Hodgson DR,
Cann MJ
(2006) Regulation of prokaryotic adenylyl cyclases by CO₂. Biochem J 396:215–218.
.
OpenUrl Abstract/FREE Full Text

[19] Hammer A,

[20] Hodgson DR,

[21] Cann MJ

[22] ↵
Sylvia DJ,
Fuhrmann JJ,
Hartel PG,
Zuberer DA
(1998) Principles and Applications of Soil Microbiology (Prentice Hall, Upper Saddle River, NJ).
.

[23] Sylvia DJ,

[24] Fuhrmann JJ,

[25] Hartel PG,

[26] Zuberer DA

[27] ↵
de Bruyne M,
Foster K,
Carlson JR
(2001) Odor coding in the Drosophila antenna. Neuron 30:537–552.
.
OpenUrl CrossRef PubMed

[28] de Bruyne M,

[29] Foster K,

[30] Carlson JR

[31] ↵
Lu T,
et al.
(2007) Odor coding in the maxillary palp of the malaria vector mosquito Anopheles gambiae. Curr Biol 17:1533–1544.
.
OpenUrl CrossRef PubMed

[32] Lu T,

[33] et al.

[34] ↵
Robinson AF
(1995) Optimal release rates for attracting Meloidogyne incognita, Rotylenchulus reniformis, and other nematodes to carbon dioxide in sand. J Nematol 27:42–50.
.
OpenUrl PubMed

[35] Robinson AF

[36] ↵
Haas W
(2003) Parasitic worms: Strategies of host finding, recognition and invasion. Zoology (Jena, Germany) 106:349–364.
.
OpenUrl

[37] Haas W

[38] ↵
Pline M,
Dusenbery DB
(1987) Responses of plant-parasitic nematode Meloidogyne incognita to carbon dioxide determined by video camera computer tracking. J Chem Ecol 13:873–888.
.
OpenUrl CrossRef PubMed

[39] Pline M,

[40] Dusenbery DB

[41] ↵
Klingler J
(1965) On the orientation of plant nematodes and some other soil animals. Nematologica 11:4–18.
.
OpenUrl

[42] Klingler J

[43] ↵
Riemann F,
Schrage M
(1988) Carbon dioxide as an attractant for the free-living marine nematode Adoncholaimus thalassophygas. Marine Biol 98:81–85.
.
OpenUrl CrossRef

[44] Riemann F,

[45] Schrage M

[46] ↵
Dusenbery DB
(1974) Analysis of chemotaxis in the nematode Caenorhabditis elegans by countercurrent separation. J Exp Zool 188:41–48.
.
OpenUrl CrossRef PubMed

[47] Dusenbery DB

[48] ↵
Dusenbery DB
(1985) Video camera-computer tracking of nematode Caenorhabditis elegans to record behavioral responses. J Chem Ecol 11:1239–1247.
.
OpenUrl CrossRef

[49] Dusenbery DB

[50] ↵
Bargmann CI
(10 25, 2006) Chemosensation in C. elegans. The C. elegans Research Community, WormBook, www.WormBook.org.
.

[51] Bargmann CI

[52] ↵
White JG,
Southgate E,
Thomson JN,
Brenner S
(1986) The structure of the nervous system of the nematode Caenorhabditis elegans. Philos Trans R Soc London B 314:1–340.
.
OpenUrl Abstract/FREE Full Text

[53] White JG,

[54] Southgate E,

[55] Thomson JN,

[56] Brenner S

[57] ↵
Coates JC,
de Bono M
(2002) Antagonistic pathways in neurons exposed to body fluid regulate social feeding in Caenorhabditis elegans. Nature 419:925–929.
.
OpenUrl CrossRef PubMed

[58] Coates JC,

[59] de Bono M

[60] ↵
Hart AC,
Kass J,
Shapiro JE,
Kaplan JM
(1999) Distinct signaling pathways mediate touch and osmosensory responses in a polymodal sensory neuron. J Neurosci 19:1952–1958.
.
OpenUrl Abstract/FREE Full Text

[61] Hart AC,

[62] Kass J,

[63] Shapiro JE,

[64] Kaplan JM

[65] ↵
Hart AC,
Sims S,
Kaplan JM
(1995) Synaptic code for sensory modalities revealed by C. elegans GLR-1 glutamate receptor. Nature 378:82–85.
.
OpenUrl CrossRef PubMed

[66] Hart AC,

[67] Sims S,

[68] Kaplan JM

[69] ↵
Kaplan JM,
Horvitz HR
(1993) A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans. Proc Natl Acad Sci USA 90:2227–2231.
.
OpenUrl Abstract/FREE Full Text

[70] Kaplan JM,

[71] Horvitz HR

[72] ↵
Culotti JG,
Russell RL
(1978) Osmotic avoidance defective mutants of the nematode Caenorhabditis elegans. Genetics 90:243–256.
.
OpenUrl Abstract/FREE Full Text

[73] Culotti JG,

[74] Russell RL

[75] ↵
Denver DR,
Morris K,
Thomas WK
(2003) Phylogenetics in Caenorhabditis elegans: An analysis of divergence and outcrossing. Mol Biol Evol 20:393–400.
.
OpenUrl CrossRef PubMed

[76] Denver DR,

[77] Morris K,

[78] Thomas WK

[79] ↵
Hodgkin J,
Doniach T
(1997) Natural variation and copulatory plug formation in Caenorhabditis elegans. Genetics 146:149–164.
.
OpenUrl Abstract/FREE Full Text

[80] Hodgkin J,

[81] Doniach T

[82] ↵
Barriere A,
Felix MA
(2005) High local genetic diversity and low outcrossing rate in Caenorhabditis elegans natural populations. Curr Biol 15:1176–1184.
.
OpenUrl CrossRef PubMed

[83] Barriere A,

[84] Felix MA

[85] ↵
Kiontke K,
Fitch DHA
(8 11, 2005) The phylogenetic relationships of Caenorhabditis and other rhabditids. The C. elegans Research Community, WormBook, www.WormBook.org.
.

[86] Kiontke K,

[87] Fitch DHA

[88] ↵
de Bono M,
Bargmann CI
(1998) Natural variation in a neuropeptide Y receptor homolog modifies social behavior and food response in C. elegans. Cell 94:679–689.
.
OpenUrl CrossRef PubMed

[89] de Bono M,

[90] Bargmann CI

[91] ↵
Gray JM,
et al.
(2004) Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue. Nature 430:317–322.
.
OpenUrl CrossRef PubMed

[92] Gray JM,

[93] et al.

[94] ↵
Cheung BH,
Cohen M,
Rogers C,
Albayram O,
de Bono M
(2005) Experience-dependent modulation of C. elegans behavior by ambient oxygen. Curr Biol 15:905–917.
.
OpenUrl CrossRef PubMed

[95] Cheung BH,

[96] Cohen M,

[97] Rogers C,

[98] Albayram O,

[99] de Bono M

[100] ↵
Ferkey DM,
et al.
(2007) C. elegans G protein regulator RGS-3 controls sensitivity to sensory stimuli. Neuron 53:39–52.
.
OpenUrl CrossRef PubMed

[101] Ferkey DM,

[102] et al.

[103] ↵
Van Gilst MR,
Hadjivassiliou H,
Jolly A,
Yamamoto KR
(2005) Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans. PLoS Biol 3:e53.
.
OpenUrl CrossRef PubMed

[104] Van Gilst MR,

[105] Hadjivassiliou H,

[106] Jolly A,

[107] Yamamoto KR

[108] ↵
Chao MY,
Komatsu H,
Fukuto HS,
Dionne HM,
Hart AC
(2004) Feeding status and serotonin rapidly and reversibly modulate a Caenorhabditis elegans chemosensory circuit. Proc Natl Acad Sci USA 101:15512–15517.
.
OpenUrl Abstract/FREE Full Text

[109] Chao MY,

[110] Komatsu H,

[111] Fukuto HS,

[112] Dionne HM,

[113] Hart AC

[114] ↵
Hu PJ
(8 8, 2007) Dauer. The C. elegans Research Community, WormBook, www.WormBook.org.
.

[115] Hu PJ

[116] ↵
Scott BA,
Avidan MS,
Crowder CM
(2002) Regulation of hypoxic death in C. elegans by the insulin/IGF receptor homolog DAF-2. Science 296:2388–2391.
.
OpenUrl Abstract/FREE Full Text

[117] Scott BA,

[118] Avidan MS,

[119] Crowder CM

[120] ↵
Gems D,
et al.
(1998) Two pleiotropic classes of daf-2 mutation affect larval arrest, adult behavior, reproduction and longevity in Caenorhabditis elegans. Genetics 150:129–155.
.
OpenUrl Abstract/FREE Full Text

[121] Gems D,

[122] et al.

[123] ↵
Kimura KD,
Tissenbaum HA,
Liu Y,
Ruvkun G
(1997) DAF-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. Science 277:942–946.
.
OpenUrl Abstract/FREE Full Text

[124] Kimura KD,

[125] Tissenbaum HA,

[126] Liu Y,

[127] Ruvkun G

[128] ↵
Schackwitz WS,
Inoue T,
Thomas JH
(1996) Chemosensory neurons function in parallel to mediate a pheromone response in C. elegans. Neuron 17:719–728.
.
OpenUrl CrossRef PubMed

[129] Schackwitz WS,

[130] Inoue T,

[131] Thomas JH

[132] ↵
Inglis PN,
Ou G,
Leroux MR,
Scholey JM
(11 27, 2006) The sensory cilia of Caenorhabditis elegans. The C. elegans Research Community, WormBook, www.WormBook.org.
.

[133] Inglis PN,

[134] Ou G,

[135] Leroux MR,

[136] Scholey JM

[137] ↵
Perkins LA,
Hedgecock EM,
Thomson JN,
Culotti JG
(1986) Mutant sensory cilia in the nematode Caenorhabditis elegans. Dev Biol 117:456–487.
.
OpenUrl CrossRef PubMed

[138] Perkins LA,

[139] Hedgecock EM,

[140] Thomson JN,

[141] Culotti JG

[142] ↵
Coburn CM,
Bargmann CI
(1996) A putative cyclic nucleotide-gated channel is required for sensory development and function in C. elegans. Neuron 17:695–706.
.
OpenUrl CrossRef PubMed

[143] Coburn CM,

[144] Bargmann CI

[145] ↵
Chang AJ,
Chronis N,
Karow DS,
Marletta MA,
Bargmann CI
(2006) A distributed chemosensory circuit for oxygen preference in C. elegans. PLoS Biol 4:e274.
.
OpenUrl CrossRef PubMed

[146] Chang AJ,

[147] Chronis N,

[148] Karow DS,

[149] Marletta MA,

[150] Bargmann CI

[151] ↵
Barrozo RB,
Lazzari CR
(2004) The response of the blood-sucking bug Triatoma infestans to carbon dioxide and other host odours. Chem Senses 29:319–329.
.
OpenUrl CrossRef PubMed

[152] Barrozo RB,

[153] Lazzari CR

[154] ↵
Klowden MJ
(1995) Blood, sex, and the mosquito. BioScience 45:326–331.
.
OpenUrl CrossRef

[155] Klowden MJ

[156] ↵
Birnby DA,
et al.
(2000) A transmembrane guanylyl cyclase (DAF-11) and hsp90 (DAF-21) regulate a common set of chemosensory behaviors in Caenorhabditis elegans. Genetics 155:85–104.
.
OpenUrl Abstract/FREE Full Text

[157] Birnby DA,

[158] et al.

[159] ↵
Jones WD,
Cayirlioglu P,
Kadow IG,
Vosshall LB
(2007) Two chemosensory receptors together mediate carbon dioxide detection in Drosophila. Nature 445:86–90.
.
OpenUrl CrossRef PubMed

[160] Jones WD,

[161] Cayirlioglu P,

[162] Kadow IG,

[163] Vosshall LB

[164] ↵
Kwon JY,
Dahanukar A,
Weiss LA,
Carlson JR
(2007) The molecular basis of CO₂ reception in Drosophila. Proc Natl Acad Sci USA 104:3574–3578.
.
OpenUrl Abstract/FREE Full Text

[165] Kwon JY,

[166] Dahanukar A,

[167] Weiss LA,

[168] Carlson JR

[169] ↵
Robertson HM,
Warr CG,
Carlson JR
(2003) Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster. Proc Natl Acad Sci USA 100:14537–14542.
.
OpenUrl Abstract/FREE Full Text

[170] Robertson HM,

[171] Warr CG,

[172] Carlson JR

[173] ↵
O'Halloran DM,
Burnell AM
(2003) An investigation of chemotaxis in the insect parasitic nematode Heterorhabditis bacteriophora. Parasitology 127:375–385.
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[174] O'Halloran DM,

[175] Burnell AM

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Acute carbon dioxide avoidance in Caenorhabditis elegans

Abstract

Results

An Acute Carbon Dioxide Response in C. elegans.

Acute CO₂ Avoidance Varies Among Strains of C. elegans and Species of Free-Living Nematodes.

The Neuropeptide Y Receptor NPR-1 Modulates CO₂ Avoidance.

Acute CO₂ Avoidance Is Mediated by cGMP Signaling.

Multiple Signaling Molecules Modulate Acute CO₂ Avoidance.

Starvation Modulates CO₂ Sensitivity in C. elegans.

The Insulin and TGFβ Pathways Modulate CO₂ Avoidance.

CO₂ Response Is Mediated by a Neural Circuit That Includes the BAG Neurons.

cGMP Signaling Is Required in the BAG Neurons for Acute CO₂ Avoidance.

Discussion

Materials and Methods

Population Assay for Acute CO₂ Avoidance.

Single-Worm Assay for CO₂ Avoidance.

Acknowledgments

Footnotes

References

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Acute carbon dioxide avoidance in Caenorhabditis elegans

Abstract

Results

An Acute Carbon Dioxide Response in C. elegans.

Acute CO2 Avoidance Varies Among Strains of C. elegans and Species of Free-Living Nematodes.

The Neuropeptide Y Receptor NPR-1 Modulates CO2 Avoidance.

Acute CO2 Avoidance Is Mediated by cGMP Signaling.

Multiple Signaling Molecules Modulate Acute CO2 Avoidance.

Starvation Modulates CO2 Sensitivity in C. elegans.

The Insulin and TGFβ Pathways Modulate CO2 Avoidance.

CO2 Response Is Mediated by a Neural Circuit That Includes the BAG Neurons.

cGMP Signaling Is Required in the BAG Neurons for Acute CO2 Avoidance.

Discussion

Materials and Methods

Population Assay for Acute CO2 Avoidance.

Single-Worm Assay for CO2 Avoidance.

Acknowledgments

Footnotes

References

Citation Manager Formats

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Articles

PNAS Portals

Information

Acute CO₂ Avoidance Varies Among Strains of C. elegans and Species of Free-Living Nematodes.

The Neuropeptide Y Receptor NPR-1 Modulates CO₂ Avoidance.

Acute CO₂ Avoidance Is Mediated by cGMP Signaling.

Multiple Signaling Molecules Modulate Acute CO₂ Avoidance.

Starvation Modulates CO₂ Sensitivity in C. elegans.

The Insulin and TGFβ Pathways Modulate CO₂ Avoidance.

CO₂ Response Is Mediated by a Neural Circuit That Includes the BAG Neurons.

cGMP Signaling Is Required in the BAG Neurons for Acute CO₂ Avoidance.

Population Assay for Acute CO₂ Avoidance.

Single-Worm Assay for CO₂ Avoidance.