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First published online May 1, 2006
Journal of Experimental Biology 209, 1859-1873 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02165
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Evolution of pharyngeal behaviors and neuronal functions in free-living soil nematodes

Jing-Tzyh Alan Chiang*, Mark Steciuk, Boris Shtonda and Leon Avery

Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA


Figure 1
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  		Fig. 1. Pharyngeal anatomy, behaviors, and neuronal stimulation in C. elegans. (A) The three muscle groups in the C. elegans pharynx. The corpus is large and anterior, the isthmus is narrow and in the middle, and the terminal bulb most posterior. There is a grinder in the terminal bulb for chewing bacteria. The lumen at rest is closed as shown. (B) Pumps are simultaneous contractions of entire muscle groups that open the pharyngeal lumen. Pumping occurs in the corpus, anterior isthmus, and terminal bulb in C. elegans (indicated by the open lumen). Peristalses are posteriorly moving contraction waves, and occur in the posterior isthmus in C. elegans (indicated by the arrows). (C) Stimulation of pumping and peristalsis by the two major excitatory pharyngeal neuron types in C. elegans, M4 and MC. M4 is required for posterior isthmus peristalsis. MC innervates the corpus and directly stimulates corpus pumping, which indirectly and simultaneously stimulates the anterior isthmus and terminal bulb to pump, since the anterior isthmus and terminal bulb are electrically coupled to the corpus via gap junctions.

 

Figure 2
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  		Fig. 2. Evolution of pharyngeal behaviors in free-living soil nematodes. Pharyngeal behaviors in four free-living soil nematode families and an outgroup species (Teratocephalus lirellus) are illustrated in A–E, where pumping is represented by open pharyngeal lumens and peristalsis is represented by arrows. The corpus pumped in all four families and pumped independently in all families except the Rhabditidae. The isthmus and terminal bulb (TB), however, exhibited significant differences regarding where pumping/peristalsis occurred, as well as how different parts were coupled in their contractions (see Results for more detailed descriptions). Thin grey lines are drawn between the corpus and isthmus/TB in each schematic to help visualize the largely conserved corpus behaviors and the varied isthmus/TB behaviors. (A) Rhabditidae family (including C. elegans): pumping occurred in the corpus, anterior isthmus, and TB, whereas peristalsis occurred in the posterior isthmus. Corpus, anterior isthmus, and TB pumping were coupled. (B) Diplogasteridae family: pumping occurred only in the corpus, whereas peristalsis occurred in the isthmus and TB. Isthmus and TB were coupled to conduct peristalsis. The Diplogasteridae TB lacks the grinder (Maggenti, 1981Go; Zhang and Baldwin, 1999Go). (C) Cephalobidae family: pumping occurred in the corpus and terminal bulb, whereas peristalsis occurred in the entire isthmus. Isthmus peristalsis and TB pumping were coupled. (D) Panagrolaimidae: pumping occurred in the corpus, posterior isthmus and TB, whereas peristalsis occurred in the anterior isthmus (AI). Corpus pumping, anterior isthmus peristalsis, and posterior isthmus/TB pumping all occurred independently. (E) T. lirellus: pumping occurred in the corpus and TB, whereas peristalsis occurred in the entire isthmus. Corpus pumping, isthmus peristalsis, and TB pumping all occurred independently. (F) A model of how pharyngeal behaviors evolved in the isthmus and terminal bulb of free-living soil nematodes, together with the currently accepted phylogenetic relationships between each family (Blaxter et al., 1998Go; Felix et al., 2000Go; Goldstein et al., 1998Go). Spatial pattern refers to where pumping/peristalsis occurred, whereas coupling pattern refers to how the different motions were coupled. By comparison to the outgroup species, T. lirellus, the spatial pattern in the Cephalobidae is ancestral, whereas the coupling pattern in the Panagrolaimidae is ancestral. Open circles and squares on the phylogenetic tree indicate that spatial and coupling patterns, respectively, evolved from an ancestral pattern to a derived pattern in the subsequent lineage. For example, the Rhabditidae lineage is marked with both a circle and a square to denote that both its spatial and coupling patterns are derived. AI, anterior isthmus; PI, posterior isthmus; TB, terminal bulb.

 

Figure 3
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  		Fig. 3. Identification of C. elegans pharyngeal neuron types in other nematodes. (A) The 20 neurons in the C. elegans pharynx. There are 14 neuronal types, 8 of which are single neurons (not shaded), and 6 of which are bilaterally symmetric left–right pairs (shaded). Paired neurons are more lateral, whereas single neurons are mostly near the midline, except that M1 is on the right side and I6 and M5 are on the left side. M1 and I6 are drawn as one nucleus because they occupy similar positions in the pharynx, even though they are two distinct neurons (M1 is right, I6 is left). (B–D) Nuclei of putative homologs of C. elegans pharyngeal neurons in Pristionchus pacificus PS312 (Diplogasteridae), Cephalobus sp. DWF1301 (Cephalobidae), and Panagrolaimus sp. PS1159 (Panagrolaimidae). Most C. elegans pharyngeal neurons were readily identifiable in these other species based on their sizes, shapes and relative positions on DIC microscopy, except for TB neurons in P. pacificus (Diplogasteridae), whose relationships to C. elegans TB neurons were not apparent. Additionally, assignments of homology in the ventral corpus were often tentative, as indicated by the asterisks, due to differences and inconsistencies in neuronal characteristics compared to C. elegans. (Even in C. elegans, positions of neurons in the ventral corpus are variable in adults, although they are reproducible in young larvae.)

 

Figure 4
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  		Fig. 4. M4 and M2 stimulated isthmus/terminal bulb behaviors in the Diplogasteridae, Cephalobidae, and Panagrolaimidae families. Laser ablations in select species from the Diplogasteridae, Cephalobidae, and Panagrolaimidae families. (A) Pristionchus pacificus PS312 (Diplogasteridae): M4 ablation caused a reduction in isthmus/TB peristalsis and also a decrease in corpus pumping. (B) Cephalobus sp. DWF1301 (Cephalobidae): M4 ablation caused a reduction in isthmus peristalsis/TB pumping. (C) Panagrolaimus sp. PS1159 (Panagrolaimidae): M4 ablation caused a reduction in posterior isthmus/TB pumping. M2 ablation caused a reduction in anterior isthmus peristalsis and also a reduction of corpus pumping. The effects on corpus pumping of M4 ablation in P. pacificus and M2 ablation in Panagrolaimus sp. PS1159 are probably secondary to the defects in peristalsis (see Results). Asterisks indicate statistically significant differences from controls by two-tailed t-test (**P<0.01, *P<0.05).

 

Figure 5
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  		Fig. 5. Schematic of neuronal stimulation in the isthmus and terminal bulb of free-living soil nematodes. The major sources of isthmus and terminal bulb stimulation, as revealed by laser ablations (see Fig. 4 and Results). (A) In the Rhabditidae (C. elegans), M4 stimulates posterior isthmus peristalsis, while anterior isthmus/TB pumping is stimulated via gap junction coupling to the corpus, as indicated by the broken arrows. (B) In the Diplogasteridae (P. pacificus), M4 stimulates isthmus/TB peristalsis. (C) In the Cephalobidae (Cephalobus sp. DWF1301), M4 stimulates isthmus peristalsis and TB pumping. (D) In the Panagrolaimidae (Panagrolaimus sp. PS1159), M2 stimulates anterior isthmus peristalsis while M4 stimulates posterior isthmus/TB pumping.

 

Figure 6
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  		Fig. 6. Loss of slo-1 in C. elegans activated M4-TB synapses. (A) Pceh-28::snb-1::gfp transgenic C. elegans animals showed punctate expression of SNB-1::GFP, a synaptic vesicle marker, along M4 axons in the isthmus and terminal bulb. White arrowhead points to the M4 cell body. Black arrow points to punctae in the TB. (B) A schematic of C. elegans EPGs (electropharyngeograms), a current recording from pharyngeal muscles during pumping, where positive and negative spikes correspond to muscle depolarizations and repolarizations, respectively. Neuronal stimulation by MC causes small positive spikes, which either remain as single positive spikes, i.e. MC EPSPs, if the MC stimulation did not induce muscle action potentials, or are followed by large positive and negative spikes if full muscle action potential was triggered. Inhibition by the M3 neurons sometimes occurs, which contributes to pharyngeal muscle repolarization during action potentials, and is seen as small trains of negative spikes on EPGs. Illustrated in the schematic are one full action potential on the left, and one single positive spike (resulting from an MC EPSP) on the right. The neurogenic spikes (i.e. due to MC and M3) are drawn in black, whereas myogenic spikes are drawn in gray. If MC and M3 are inactivated, then the resulting EPGs contain only myogenic spikes. (C) An example of a single positive spike due to an MC EPSP in wild-type C. elegans EPG, indicated by the black arrow. (D) eat-2; eat-4 EPGs lacked MC and M3 spikes, including single positive spikes (eat-2 and eat-4 remove MC and M3 functions, respectively). (E) eat-2; eat-4; slo-1 EPGs contained single positive spikes, indicated by the black arrows. (F) Quantification of single positive spikes (SPS) in wild-type and slo-1 mutant backgrounds, with and without MC/M3 function, either by laser ablations or the eat-2 and eat-4 mutations. (G) Laser ablation of M4 drastically reduced single positive spikes (SPS) in eat-2; eat-4; slo-1 animals, whereas ablation of another C. elegans neuron, M5, did not. Ablation of M4 and M5 entirely eliminated single positive spikes, suggesting slight activity by M5 as well. (H) The eat-5 mutation allows the TB to pump independently of the corpus (Starich et al., 1996Go), and slo-1 increased TB pumping in the eat-5 background. (I) Perinuclear expression of Pslo-1::SLO-1::GFP in the M4 neuron. The white arrows point to the M4 nucleus. The white arrowhead points to GFP signal surrounding the M4 nucleus. (J) eat-2 encodes a nicotinic channel subunit specific to the MC-corpus neuromuscular junction, whereas eat-18 is required for the surface expression of all pharyngeal nicotinic channels, including in the terminal bulb. eat-2; eat-4; slo-1 animals had single positive spikes, but eat-18; eat-4; slo-1 animals did not. (K) EPGs of eat-2; eat-4; slo-1 animals were recorded in standard conditions, followed by subsequent addition of d-tubocurarine, a nicotinic channel antagonist, to 100 µmol l–1, or control saline (see Materials and methods for details). 100 µmol l–1 d-tubocurarine decreased single positive spikes in eat-2; eat-4; slo-1 animals (compared to before treatment), whereas control treatment did not (compared to before treatment). Scale bars in C–E indicate 100 pA and 100 ms. Asterisks indicate statistically significant differences from controls by two-tailed t-test (**P<0.01); see Materials and methods for the statistical analysis performed in Fig. 6K. Dagger in G indicates statistical significance compared to M4 ablation by two-tailed U-test ({dagger}P<0.05).

 

Figure 7
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  		Fig. 7. Food density dependence of isthmus/TB pumping and peristalsis. When (A) C. elegans, (B) P. pacificus, (C) Cephalobus sp. DWF1301, or (D) Panagrolaimus sp. PS1159 animals were placed in dilute food conditions compared to normal conditions (see Materials and methods), isthmus/TB pumping/peristalsis rates were reduced whereas corpus pumping rates were unchanged or increased. Asterisks indicate statistically significant differences between dilute and normal conditions by two-tailed t-test (**P<0.01).

 

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© The Company of Biologists Ltd 2006