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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
Evolution of pharyngeal behaviors and neuronal functions in free-living soil nematodes
Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA
* Author for correspondence (e-mail: alan{at}eatworms.swmed.edu)
Accepted 8 February 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Key words: evolution, nematode, Caenorhabditis elegans, behavior, neuronal function
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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). Using these well-studied C. elegans behavioral
paradigms as starting points, comparative studies of evolutionary differences
in other nematode species may provide useful models for studying the
mechanisms by which behaviors evolve. Comparative studies between C.
elegans and other nematodes have been previously conducted. However, they
often focused on evolutionary similarities, with the aim of demonstrating the
applicability of C. elegans knowledge towards understanding other
nematodes, and vice versa. For example, studies in the parasite
Ascaris focused on anatomical and functional similarities to C.
elegans in the ventral cord locomotory neurons
(Johnson and Stretton, 1980;
Stretton et al., 1978;
Walrond et al., 1985), and
studies in the parasite Strongyloides focused on similarities to
C. elegans in the control of chemotaxis by amphid sensory neurons
(Ashton et al., 1999;
Forbes et al., 2004). We were
interested instead in investigating nematode behaviors and their neuronal
regulation explicitly for their evolutionary differences.
Nematodes feed using a neuromuscular organ in the head termed the pharynx.
Pharyngeal behaviors in C. elegans consist of well-defined,
stereotyped patterns of muscle contractions, which are regulated by a small,
self-contained pharyngeal nervous system. The neuronal and molecular bases of
pharyngeal feeding behaviors in C. elegans have been well
characterized (Avery and Thomas,
1997), but in other nematode species, they are not as well
understood. In particular, C. elegans is a free-living soil nematode
(nematodes can be classified ecologically as free-living or parasitic, and
soil-dwelling or aquatic). Characterization of pharyngeal behaviors in other
free-living soil nematodes has been rather limited, and generally did not
examine the regulatory functions of pharyngeal neurons
(Doncaster, 1962;
Mapes, 1965;
von Lieven, 2003).
Thus, as a model for studying behavioral evolution in C. elegans
and related nematodes, we examined how pharyngeal behaviors and their neuronal
regulation evolved in free-living soil nematodes. Four main families
constitute the large majority of free-living soil nematode species: the
Rhabditidae (which includes C. elegans), the Diplogasteridae, the
Cephalobidae, and the Panagrolaimidae. We began with direct observations of
pharyngeal behaviors from these four families, to obtain a systematic model of
how their pharyngeal behaviors evolved. Then, to understand what changes in
neuronal function corresponded with the behavioral differences, we used laser
ablations (Bargmann and Avery,
1995) to define the functions of homologous pharyngeal neurons.
And finally, we analyzed a C. elegans mutant, whose phenotypes
suggested that changes in synaptic strength may have contributed to the
functional evolution of a specific pharyngeal neuron. Overall, our study
provides a basic, broad characterization of evolutionary differences in
pharyngeal behaviors and neuronal functions in free-living soil nematodes, and
demonstrates the potential utility of using C. elegans and related
nematodes as a system for studying behavioral evolution.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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In addition to the above species, we also used Teratocephalus
lirellus PDL0011 from the family Teratocephalidae, as an outgroup
species. An outgroup species is a species outside of the group of interest
(i.e. free-living soil nematodes, in this study) used to determine the
evolutionary polarity of specific characters within the group of interest.
That is, assuming the principle of parsimony, a character in the group of
interest is either ancestral or derived, based on whether the character is
also present or absent in the outgroup species, respectively
(Ridley, 1996). Usually, close
relatives of the group of interest are selected as outgroup species. We chose
T. lirellus because of its close relation to the free-living soil
nematodes (Blaxter et al.,
1998), and also because T. lirellus had previously been
used as an outgroup to the free-living soil nematodes
(Zhang and Baldwin, 2001).
Of the above strains, T. lirellus PDL0011 was maintained at
19°C on soil agar plates consisting of 1% agar, 1% soil and 5 µg
ml–1 cholesterol (De Ley
and Mundo-Ocampo, 2004), and seeded with E. coli HB101
(Boyer and Roulland-Dussoix,
1969). All other strains were maintained at 19°C on NGMSR
plates (Davis et al., 1995)
seeded with E. coli HB101, except DWF1301, PS1146, PS1197, DF5017 and
DF5019, which were maintained at 24°C.
For C. elegans experiments, we used the wild-type strain N2, as
well as mutant strains that contained the following alleles:
slo-1(js379) (Wang et al.,
2001), eat-2(ad465)
(McKay et al., 2004),
eat-4(ky5) (Lee et al.,
1999) and eat-5(ad1402), which is a putatively null
deletion (835–2274 bp of F13G3.8) that removes exons 2 through 4. All
C. elegans strains were maintained at 19°C on NGMSR plates seeded
with E. coli HB101.
Visual observations of pharyngeal behaviors in free-living soil nematode species
Using video microscopy, we observed free-living soil nematode species for
differences in pharyngeal behaviors. Bacterial suspensions were made by
scraping E. coli from 1–2 seeded HB101 plates into 100 µl of
M9 medium followed by thorough mixing. Animals were then transferred to 2.5
µl of the bacterial suspension, placed either on an unseeded NGMSR plate or
a 
1 mm thick NGMSR agar pad, and coverslips placed on top. We typically
waited 30–60 min before making observations because feeding motions
were often erratic and inconsistent immediately after application of the
coverslip, presumably because the animals were disturbed by the protocol (this
problem was more pronounced in some species). Pharyngeal motions were then
observed using a Zeiss Axiophot microscope with a 40x, 63x or
100x objective. Videos were taken with a Hitachi kP-160 CCD camera and
digitized using Adobe Premiere v6.5. Compression of videos into MPEG files was
done via SmartVideoConverter v1.5.15 (DoEase Software). Adults and
larvae of each species generally displayed similar pharyngeal behaviors, and
movies provided as supplementary material were typically taken from larvae,
since the smaller larvae permitted easier focusing and tracking for video
recordings.
Laser ablations in free-living soil nematodes
We chose P. pacificus PS312, Cephalobus sp. DWF1301 and
Panagrolaimus sp. PS1159 as representatives of the Diplogasteridae,
Cephalobidae and Panagrolaimidae families, respectively, for laser ablations
because they have large broods, are easily anesthetized, and are easy to
handle. Laser ablations were performed on newly hatched larvae less than 4 h
old as previously described for C. elegans
(Bargmann and Avery, 1995),
except for the anesthetic conditions. For PS312, the larvae were anesthetized
on 10 mmol l–1 NaN3 pads, as in C.
elegans. For PS1159, the larvae were anesthetized on 20 mmol
l–1 NaN3 pads. For DWF1301, larvae were placed in
1 µl of 100 mmol l–1 NaN3 on top of 10
mmol l–1 NaN3 pads for 30 s until the animals
relaxed by curling up, followed by application of the coverslip. We assayed
pumping and peristalsis rates of operated animals after they reached
adulthood, determined by the presence of laid eggs on the plate. The animals
were observed by microscopy as described above, and their pumping and
peristalsis rates were determined by counting the number of pumps/peristalsis
in 1 min. For MC ablations (see Results), the Panagrolaimidae species P.
redivivus DA1711 was also used, but MC could not be distinguished from I2
in DA1711 so both nuclei types had to be killed. P. redivivus DA1711
was anesthetized on 10 mmol l–1 NaN3 pads, and
adulthood was determined by the presence of oocytes in females or spicules in
males.
Two types of controls were performed to verify that the changes in pharyngeal function were specific to the neuron ablated. We performed (1) mock ablations, where the larvae were anesthetized but no neurons were ablated, to control for nonspecific effects of anesthetization, and (2) nearby ablations, where nearby neurons were ablated (e.g. M3 or NSM for M4), to control for nonspecific effects of laser damage. In general, the effects of mock and nearby ablations were indistinguishable, and the control results, presented in the figures were derived from mock ablations.
In the outgroup species, T. lirellus, culture difficulties prevented laser ablations from being performed. Culture of T. lirellus was generally challenging due to long generation times and sensitivity to growth conditions, and single animal cultures, which are important for laser ablation experiments, were invariably lost (the animals either died or could not be found on the plates).
Food density dependence assays
Normal bacterial suspensions were prepared as above (i.e. 1–2 HB101
plates/100 µl M9), whereas dilute bacterial suspensions were prepared by
diluting the normal suspensions 50-fold for Cephalobus sp. DWF1301
and Pristionchus pacificus PS312, or 20-fold for Panagrolaimus
sp. PS1159 (PS1159 fed inconsistently or not at all when placed in
50-fold or more dilute suspensions). We then placed coverslips over each
animal in both normal and dilute bacterial suspensions, and counted their
pumping and peristalsis rates.
PS1159 and DWF1301 animals were often hyperactive and hard to track under
the microscope in the dilute condition. Roaming behaviors in C.
elegans can be reduced if they are grown on poor quality food (B. Shtonda
and L. Avery, manuscript submitted for publication), such as the E.
coli strain DA837 (Davis et al.,
1995). We decreased the hyperactive motions of PS1159 and DWF1301
animals in the dilute condition by placing them on DA837 plates for one night
prior to the assay. Both the normal and dilute pumping/peristalsis rates
reported in the food density dependence experiments for PS1159 and DWF1301
animals are from DA837 preconditioned animals, to ensure consistency in the
comparisons. Pumping/peristalsis rates at normal food density in
preconditioned PS1159 and DWF1301 animals are similar to those of
unconditioned animals, indicating that DA837 preconditioning does not
significantly affect pumping/peristalsis rates, at least at normal food
density. For PS312, DA837 preconditioning was not necessary.
Transgenic C. elegans
We made Pceh-28p::SNB-1::GFP constructs by overlap extension PCR
(Hobert, 2002). We amplified
Pceh-28, 2.4 kb of sequence upstream of the ceh-28 start
ATG, from genomic DNA, and SNB-1::GFP from pSB120.65 (courtesy of M.
Nonet). Then we fused the two together by additional PCR. The resulting
products were coinjected with either the pRAK3 [rol-6(d)] plasmid
(Davis et al., 1995) or
Podc-1::GFP (PCR product), which expresses in intestinal cells (J. T.
A. Chiang, unpublished observations), as transformation markers. We made
Podc-1::GFP by amplifying Podc-1, 3.0kb sequence upstream of
the start ATG of odc-1 (Macrae et
al., 1995), from genomic DNA, and GFP from pPD95.75 (A.
Fire, Fire Lab Vector Kit–June 1995,
ftp://www.ciwemb.edu/pub/FireLabInfo/FireLabVectors/1995_Vector_Kit/Vec95_Docs/Vec95Doc.rtf.),
then fusing the two by additional PCR. Injections for transformation were
performed following standard procedures
(Mello and Fire, 1995).
Pictures were taken on a Zeiss Axiophot microscope using a MaxCam CM7-2E CCD
camera (Finger Lakes Instrumentation, Lima, NY, USA).
Two independent Pslo-1::SLO-1::GFP lines were gifts from the lab of Michael Nonet. They were generated by the coinjection of Pslo-1::SLO-1::GFP and pJM23 (lin-15+) into unc-64(e246); slo-1(js100); lin-15(n765ts) mutants. Pslo-1::SLO-1::GFP is a fusion of a 5.2 kb Pslo-1 promoter with SLO-1 and GFP cDNAs. Pictures were taken as described above.
Electropharyngeograms
Electropharyngeograms (EPGs) on exposed pharynxes bathed in Dent's saline
and 1 µmol l–1 serotonin (5HT) were performed as
previously described (Davis et al.,
1995), with one exception. Although novel single positive spikes
in slo-1 mutant EPGs (see Results) were detectable using standard
Dent's saline containing 3.0 mmol l–1 Ca2+, those
single positive spikes were larger in amplitude and easier to distinguish when
we increased [Ca2+] to 5.0 mmol l–1. Therefore,
all results presented were derived from EPGs using modified Dent's containing
5.0 mmol l–1 [Ca2+], although the results were
qualitatively similar when using standard Dent's saline. EPG currents were
digitally sampled and recorded as previously described
(Davis et al., 1995).
Digitized EPG recordings were then viewed and analyzed using Igor Pro 4.05
from Wave Metrics (Lake Oswego, OR, USA). Quantification of single positive
spikes on EPGs was assisted by Igor Pro procedures (available upon request),
which scanned for positive spikes that are not followed by full pharyngeal
action potentials.
Laser ablations in slo-1 mutants
MC and M3 ablations in wild-type and slo-1 mutants were performed
using standard procedures (Bargmann and
Avery, 1995). Mock, M4, and M5 laser ablations in eat-2;
eat-4; slo-1 animals were also performed using standard procedures,
except that all animals were subsequently grown on HB101 seeded plates
containing 5 mmol l–1 arecoline. We added arecoline because
M4 ablation normally causes larval arrest due to the loss of isthmus
peristalsis (Avery and Horvitz,
1987), but growth of M4 ablated worms can be improved with 5 mmol
l–1 arecoline in the culture plates (in M4– animals,
10% grow to adulthood on HB101 plates, whereas 70% reach adulthood on HB101+5
mmol l–1 arecoline plates; L. Avery, unpublished
observations). The mechanism by which arecoline improves growth of M4–
animals is unknown; it may be via general depolarization of the
pharyngeal membrane, as that is the mechanism by which eat-6 improves
growth of M4– animals (Davis et al.,
1995). Importantly, EPGs of mock ablated eat-2; eat-4;
slo-1 animals grown on 5 mmol l–1 arecoline were similar
to EPGs of animals grown under standard conditions, indicating that growth on
5 mmol l–1 arecoline had no major effects on EPGs. In
particular, single positive spike rates between eat-2; eat-4; slo-1
animals grown on standard conditions were indistinguishable from eat-2;
eat-4; slo-1 animals grown on 5 mmol l–1 arecoline.
d-tubocurarine studies
For the 100 µmol l–1 d-tubocurarine studies, EPGs were
recorded for 2 min in 100 µl of 5.0 mmol l–1
[Ca2+] Dent's+5HT as described above, followed by the addition of
another 100 µl of either the identical saline (control) or the same saline
with 200 µmol l–1 d-tubocurarine added (experiment), to
achieve a final concentration of 100 µmol l–1
d-tubocurarine. Single positive spike rates before and after drug addition
were analyzed as described. Statistical significance between d-tubocurarine
and control treatments was tested on differences in single positive spike
rates (i.e. after treatment minus before treatment) in individual animals.
Pumping rate assays of slo-1 mutants
We examined eat-5 and eat-5; slo-1 pumping rates in L1
larvae selected within 6 h of hatching because the eat-5 phenotype of
electrically isolated terminal bulb (TB) is virtually 100% penetrant in
larvae, but significantly less so in adults
(Starich et al., 1996). We
measured eat-5 and eat-5; slo-1 TB pumping rates by counting
for 1 min at 19°C under a Zeiss Axiophot microscope using a 20x
objective.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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; Avery and
Thomas, 1997). Pharyngeal behaviors consist of stereotyped
patterns of two motions, termed `pumping' and `peristalsis'. Pumps are
simultaneous contractions of entire muscle groups that open the pharyngeal
lumen, and peristalses are posteriorly sweeping contraction waves
(Avery and Horvitz, 1987;
Avery and Horvitz, 1989;
Avery and Thomas, 1997).
Anteriorly in the C. elegans pharynx, pumping occurs in the corpus
and anterior isthmus to suck in bacteria, and posteriorly, pumping occurs in
the terminal bulb to break up ingested bacteria with the grinder
(Fig. 1B). Peristalsis occurs
only in the posterior isthmus, and transports food between the two regions of
pumping (Fig. 1B). Corpus,
anterior isthmus and terminal bulb pumping are coupled in C. elegans
due to gap junctions (see below), such that all three regions contract
together with each pump (Fig.
1B). In contrast, posterior isthmus peristalsis occurs separately
from pumping.
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).
Interestingly, only a small subset of these neurons is known to have readily
observable functions. Specifically, only two are known to be major excitatory
neurons in C. elegans (schematized in
Fig. 1C): M4, which is required
for posterior isthmus peristalsis (Avery
and Horvitz, 1987), and MC, which stimulates pumping
(Avery and Horvitz, 1989;
Raizen et al., 1995). The
corpus muscles are stimulated to pump via direct MC innervations
(McKay et al., 2004;
Raizen et al., 1995), whereas
anterior isthmus and TB pumping are indirectly stimulated via gap
junctions between the corpus and isthmus, and isthmus and terminal bulb. Each
time MC stimulates the corpus, the excitations spread rapidly to the anterior
isthmus and terminal bulb and cause the anterior isthmus and terminal bulb
muscles to pump simultaneously with the corpus
(Starich et al., 1996; M.
Steciuk, unpublished observations).
The isthmus and terminal bulb evolved differences in their spatial and coupling patterns of pumping and peristalsis
Using video microscopy, we examined pharyngeal behaviors in a broad sample
of free-living soil nematode species from each of the four major families (see
Materials and methods for the complete list). Additionally, to help determine
the ancestral pattern of pharyngeal behaviors, we observed a close relative,
Teratocephalus lirellus, as the outgroup species
(Blaxter et al., 1998).
Anatomically, the pharynxes in all of these species were similarly divided
into the corpus, isthmus and terminal bulb (albeit with minor anatomical
variations; see Chitwood and Chitwood,
1974; Maggenti,
1981; von Lieven,
2003; von Lieven and Sudhaus,
2000; Zhang and Baldwin,
1999). Yet behaviorally, each family, as well as T.
lirellus, exhibited significant differences. Species within the same
families had largely similar behaviors.
Overall, pharyngeal behaviors differed both with respect to (1) where
pumping and peristalsis occurred, and (2) how these motions were coupled,
which we term their spatial and coupling patterns, respectively. In the
Rhabditidae family, as in C. elegans, pumping occurred anteriorly in
the corpus and anterior isthmus and posteriorly in the terminal bulb (TB),
with peristalsis in the posterior isthmus
(Fig. 2A). Further, as in
C. elegans, corpus, anterior isthmus and TB pumping were all coupled
in the Rhabditidae family (Fig.
2A). In the Diplogasteridae family, however, pumping occurred only
anteriorly in the corpus with no posterior region of pumping, and peristalsis
occurred throughout the isthmus and TB
(Fig. 2B). The Diplogasteridae
TB also lacks the grinder (Maggenti,
1981; von Lieven and Sudhaus,
2000). Furthermore, in the Diplogasteridae, while the isthmus and
TB were coupled for peristalsis, corpus pumping occurred independently
(Fig. 2B). In the Cephalobidae
family, pumping occurred anteriorly in the corpus and posteriorly in the TB,
whereas peristalsis occurred in the entire isthmus
(Fig. 2C). While corpus pumping
occurred independently, isthmus peristalsis and TB pumping were coupled in the
Cephalobidae, such that each isthmus peristalsis always occurred with a TB
pump and vice versa (Fig.
2C). In the Panagrolaimidae family, pumping occurred anteriorly in
the corpus and posteriorly in the posterior isthmus and TB, whereas
peristalsis occurred only in the anterior isthmus
(Fig. 2D). Furthermore, in the
Panagrolaimidae, corpus pumping, anterior isthmus peristalsis, and posterior
isthmus/TB pumping all occurred independently of each other
(Fig. 2D). In one
Panagrolaimidae species, Panagrellus redivivus, some posterior
isthmus/TB pumps were coupled to corpus pumps, although posterior isthmus/TB
pumping also occurred independently. Finally, in the outgroup species T.
lirellus, pumping occurred anteriorly in the corpus and posteriorly in
the TB, and peristalsis occurred in the entire isthmus
(Fig. 2E), which is similar to
the Cephalobidae family. However, in T. lirellus, corpus pumping,
isthmus peristalsis, and TB pumping all occurred independently
(Fig. 2E), which is more
similar to the Panagrolaimidae family. Representative videos of these
different patterns in each family are included as supplementary material, and
can also be viewed at
http://eatworms.swmed.edu/~alan.
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Differences in stimulation of isthmus and TB behaviors by the M4 and M2 neurons
Having characterized how pharyngeal behaviors evolved in free-living soil
nematodes, we were then interested in how their neuronal regulation may have
correspondingly evolved. In C. elegans, M4 is required for posterior
isthmus peristalsis, whereas MC and gap junction coupling are important for
stimulating corpus pumping and anterior isthmus/TB pumping, respectively
(schematic in Fig. 1C). Since
corpus pumping occurred independently of isthmus and TB motions in the
Diplogasteridae, Cephalobidae and Panagrolaimidae, gap junction coupling to
the corpus is unlikely to be important for isthmus and TB excitation in those
families. On the other hand, nematode nervous systems are generally well
conserved anatomically (Martin et al.,
2002), and we were similarly able to identify homologs to most
C. elegans pharyngeal neurons in the other species (see
Fig. 3; identification of
ventral corpus neurons was generally less certain, and TB neurons of the
Diplogasteridae could not be identified at all). Thus, how might have M4 and
MC functions evolved?
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), we
examined M4 functions in select species from the Diplogasteridae,
Cephalobidae, and Panagrolaimidae families. In Pristionchus pacificus
of the Diplogasteridae family, M4 ablation caused a reduction in isthmus/TB
peristalsis (Fig. 4A). In
Cephalobus sp. DWF1301 of the Cephalobidae family, M4 ablation caused
a reduction in isthmus peristalsis/TB pumping
(Fig. 4B). In Panagrolaimus
sp. PS1159 of the Panagrolaimidae family, M4 ablation interestingly had
no effect on anterior isthmus peristalsis, but caused a specific reduction in
posterior isthmus/TB pumping (Fig.
4C). Thus, outside of C. elegans and the Rhabditidae
family, we found M4 to be an important excitatory neuron as well in the other
three free-living soil nematode families. In fact, with the exception of
anterior isthmus peristalsis in the Panagrolaimidae, and anterior isthmus/TB
pumping in the Rhabditidae, all isthmus/TB behaviors in the four families
appeared to respond to M4 stimulation. Yet, M4 varied in its detailed function
between each family, both with regards to (1) the exact region of isthmus and
TB affected by M4 stimulation, and (2) whether M4 stimulated pumping and/or
peristalsis.
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Based on these laser ablation studies, we saw that the isthmus and TB in each family evolved significant differences in their patterns of neuronal stimulation (schematics in Fig. 5). In the Rhabditidae, M4 stimulates posterior isthmus peristalsis, whereas gap junction coupling to the corpus is important for anterior isthmus/TB pumping; in the Diplogasteridae, M4 stimulates isthmus/TB peristalsis; in the Cephalobidae, M4 stimulates isthmus peristalsis/TB pumping; and in the Panagrolaimidae, M4 stimulates posterior isthmus/TB pumping whereas M2 is important for anterior isthmus peristalsis. Overall, M4 appears to be the central excitatory neuron for isthmus/TB behaviors, whereas M2 and gap junction coupling have specific roles in particular families.
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In addition to the changes in isthmus and TB stimulation by the M4 and M2 neurons, we were also interested in evolutionary differences in the function of the MC neurons. Systematic examination of MC function was much more difficult, however, because the homologs of MC and other ventral corpus neurons were harder to identify in non-C. elegans nematodes (see above and Fig. 3). Putative MC ablations in P. pacificus PS312, a Diplogasteridae species, and P. redivivus DA1711, a Panagrolaimidae species (data not shown) suggested that MC may be generally conserved to stimulate corpus pumping, but this hypothesis awaits further future testing.
Killing M4 in P. pacificus and M2 in Panagrolaimus sp.
PS1159 led to moderate decreases in corpus pumping
(Fig. 4A,C). While these
results might suggest that M4 and M2 contribute to corpus pumping in the
Diplogasteridae and Panagrolaimidae families, an alternative explanation is
that the effects are actually secondary to the peristalsis defects. In C.
elegans, accumulation of bacteria in the corpus due to defective
peristalsis after M4 ablation can cause reduced corpus pumping
(Raizen et al., 1995), and we
think a similar effect occurs in M4/M2 ablated P. pacificus and
Panagrolaimus sp. PS1159 animals.
Loss of slo-1, a negative regulator of synaptic transmission, activated M4-TB synapses in C. elegans
We wished to determine underlying mechanisms that may be responsible for
these differences in pharyngeal behaviors and neuronal functions. As shown
above, the exact isthmus/TB regions stimulated by M4 varied in each family. In
particular, while M4 stimulation affected terminal bulb behaviors in the
Diplogasteridae, Cephalobidae, and Panagrolaimidae families, M4 had no effect
on the terminal bulb in the Rhabditidae family. Why might this be? We
addressed this question by examining the relationship between M4 and the TB in
more detail in the Rhabditidae, using C. elegans.
One possible reason could be that M4 does not innervate TB muscles. We thus
characterized M4's synaptic pattern in C. elegans using
Pceh-28::snb-1::gfp, a transgene in which the Pceh-28
promoter drives M4 specific expression (WormBase 2006;
http://www.wormbase.org/db/gene/gene?name=ceh-28)
of the synaptic vesicle marker SNB-1::GFP
(Nonet, 1999). In these
transgenic animals, we found punctate clusters of SNB-1::GFP along M4 axons in
both the isthmus and terminal bulb (Fig.
6A), indicating that M4 did in fact innervate the terminal bulb in
C. elegans. To confirm that the observed punctae truly corresponded
to synaptic structures, we also expressed Pceh-28::snb-1::gfp in
unc-104 mutants, in which synaptically localized proteins such as
SNB-1::GFP should mislocalize to neuronal cell bodies
(Nonet, 1999;
Nonet et al., 1998) due to
synaptic vesicle transport defects (Hall
and Hedgecock, 1991; Otsuka et
al., 1991). Indeed, in unc-104 mutants carrying
Pceh-28::snb-1::gfp, we found SNB-1::GFP mislocalized to the M4 cell
body (data not shown).
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Therefore, M4 innervates the TB in C. elegans. Why then, despite
the presence of synapses, is TB behavior unaffected by M4? The gene
slo-1 encodes a Ca2+ activated K+ channel that
negatively modulates neurotransmission
(Wang et al., 2001), and we
observed a suggestive phenotype on EPGs (electropharyngeograms) from
slo-1 mutants.
The EPG is an extracellular current recording of pharyngeal muscles during
pumping (Avery et al., 1995;
Raizen and Avery, 1994).
Peristalsis does not generate EPG currents. MC stimulation of pharyngeal
muscles causes small positive spikes on EPGs, which either (1) remain as
single positive spikes if MC fails to trigger full muscle action potentials
(i.e. they represent MC EPSPs) or (2) are followed by large positive and
negative spikes representing full muscle depolarizations and repolarizations,
if MC successfully elicits muscle action potentials
(Raizen and Avery, 1994;
Raizen et al., 1995). In
addition to MC activity, activity from the M3 neurons to repolarize pharyngeal
muscles during action potentials also appears on EPGs. M3 IPSPs are visible as
trains of small negative spikes during the muscle action potential
(Raizen and Avery, 1994). MC
and M3 are the only neurons whose functions are observed in EPGs (see
Fig. 6B), and inactivation of
MC and M3 by laser ablations or mutations such as eat-2
(McKay et al., 2004;
Raizen et al., 1995) and
eat-4 (Lee et al.,
1999) abolishes their corresponding EPG signals. A schematic of
typical EPG signals, including MC and M3 spikes, is shown in
Fig. 6B.
Consistent with slo-1's known function, EPGs of slo-1
mutants included phenotypes indicative of increased MC and M3
neurotransmission, such as larger MC and M3 spikes
(Wang et al., 2001; J.-T. A.
Chiang and L. Avery, unpublished observations). However, even when MC and M3
functions were removed, slo-1 EPGs were still different from wild
type. Specifically, even though single positive spikes are normally absent
from EPGs from MC–M3– wild-type and eat-2; eat-4 animals
(Raizen et al., 1995), we
found that single positive spikes were still present in MC–M3–
slo-1 and eat-2; eat-4; slo-1 animals
(Fig. 6D-F). Thus, in a
slo-1 mutant, some neuron other than MC can activate pharyngeal
muscles.
We conducted additional laser ablations in eat-2; eat-4; slo-1
animals to identify the source of these novel single positive spikes. We found
that when we ablated M4 in eat-2; eat-4; slo-1 animals, single
positive spikes were largely eliminated
(Fig. 6G). Thus, mutating
slo-1 activated the M4 neuron, and the novel single positive spikes
in slo-1 mutants corresponded to M4 EPSPs in pharyngeal muscles. We
then wished to test if activation of M4 caused increased pumping as well.
Based on M4's innervation pattern, slo-1 should cause increased
pumping in the TB, but not in the corpus. To test this, we examined the
effects of slo-1 in the eat-5 background. The eat-5
mutation causes gap junction defects and uncouples the TB from the corpus,
which allows TB pumping to be assessed independently of corpus pumping
(Starich et al., 1996).
Indeed, we found that eat-5; slo-1 animals had over twofold greater
TB pumping than eat-5 animals
(Fig. 6H), consistent with the
idea that slo-1 activated M4 caused increased TB pumping. [We were
unable to test if M4 ablation abolished the increased TB pumping in eat-5;
slo-1 animals, because M4 ablation causes starvation
(Avery and Horvitz, 1987) with
confounding effects of increased and irregular pumping
(Avery and Horvitz, 1990;
Raizen et al., 1995).]
Finally, if slo-1 acted directly in the M4 neuron, M4 should also
express slo-1. To determine the expression pattern of slo-1,
we examined the expression of Pslo-1::SLO-1::GFP, a GFP fusion
transgene containing a 5.2 kb Pslo-1 promoter. Indeed, expression was
seen in M4 (Fig. 6I),
consistent with slo-1 acting directly in the M4 neuron. Expression
was also detected in most other pharyngeal neurons (data not shown),
consistent with slo-1's aforementioned effects on other pharyngeal
neurons, such as MC and M3.
Together, the novel M4 dependent single positive spikes and increased TB
pumping suggested that loss of slo-1 activated M4 to stimulate TB
pumping. Since slo-1 encodes a negative modulator of
neurotransmission (Wang et al.,
2001), these results suggested that C. elegans
M4–TB synapses can function, but their synaptic activity is normally
reduced in wild-type C. elegans to a level where M4 has no effect on
TB behavior, i.e. M4–TB synapses are silenced in C. elegans.
Based on this, we suggest that silencing of M4–TB synapses may account
for the evolutionary differences in M4–TB stimulation between the
Rhabditidae and other free-living soil nematode families.
Another gene that is required for MC to stimulate pharyngeal pumping is
eat-18 (McKay et al.,
2004; Raizen et al.,
1995). However, whereas eat-2 encodes a nicotinic channel
subunit specifically localized to the MC-corpus neuromuscular junction,
eat-18 is a gene that is required for the surface expression of
nicotinic receptors throughout the pharynx
(McKay et al., 2004).
Interestingly, in contrast to eat-2; eat-4; slo-1 animals, we found
that eat-18; eat-4; slo-1 animals lacked single positive spikes
(Fig. 6J). We then also tested
whether the nicotinic antagonist d-tubocurarine
(Raizen et al., 1995) can
affect single positive spikes, and indeed, 100 µmol l–1
d-tubocurarine drastically reduced the number of single positive spikes in
eat-2; eat-4; slo-1 EPGs (Fig.
6K). Together, the eat-18 and d-tubocurarine experiments
indicated that M4-TB synapses are cholinergic, and stimulate the TB muscles
via nicotinic receptors.
Isthmus/TB pumping/peristalsis rates respond to food density changes
With regard to pumping and peristalsis patterns of free-living soil
nematode families, we described above that corpus pumping generally occurred
independently of isthmus and TB motions
(Fig. 2A–D) (except
coupled corpus/anterior isthmus/TB pumping in the Rhabditidae). We found that
when we diluted the food in the animals' surroundings (see Materials and
methods for experimental setup), corpus pumping rates were increased or
similar to those in normal food conditions, but isthmus/TB pumping/peristalsis
rates were generally reduced (Fig.
7). This suggested that independent regulation of corpus and
isthmus/TB contraction rates may be useful for responding to changes in food
density. When food is scarce (i.e. the dilute condition), the corpus still
pumps rapidly to maximize the amount of food ingested; yet, since less food is
ingested overall, isthmus and TB contraction rates are down-regulated for
energy efficiency as less food needs to be passed posteriorly and chewed by
the grinder. Additionally, since reducing food density has effects similar to
ablating M4 (compare the effects of M4 ablation in
Fig. 4 with decreasing food
density in Fig. 7), we
hypothesize that M4 may be important for mediating the food density response
of isthmus/TB contractions.
|
|
Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
|---|
Altogether, these observations form a broad overview of how pharyngeal
behaviors and neuronal stimulation evolved in free-living soil nematodes, and
provide a framework for investigating the underlying cellular and genetic
mechanisms of change. Some details, however, will require future
clarification. With respect to pharyngeal behaviors, for example, it is
unclear which evolved first in the Rhabditidae family: (1) switch of anterior
isthmus from peristalsis to pumping, or (2) coupling of anterior isthmus/TB
pumping to the corpus. In Poikilolaimus regenfussi, a basal
Rhabditidae species (Kiontke and Fitch,
2005), we observed anterior isthmus pumping, but TB pumps were
often weakly coupled to the corpus, suggesting incomplete gap junction
coupling. This observation is potentially informative, but since the
phylogenetic relationships between P. regenfussi and other
Rhabditidae species are still under investigation
(Kiontke and Fitch, 2005), it
is unclear whether P. regenfussi signifies that gap junction coupling
evolved after anterior isthmus pumping, or if gap junctions were secondarily
weakened in the P. regenfussi lineage. This can be resolved with
future clarification of the Rhabditidae phylogeny.
With respect to how neuronal stimulation of the pharynx evolved, the major
gap in our understanding is the ancestral functions of the M4 and M2 neurons.
We attempted laser ablations in T. lirellus, but were unsuccessful
due to technical problems (see Materials and methods). Further, other species
that are closely related to the free-living soil nematodes, such as the
Plectidae (Blaxter et al.,
1998), do not have peristalsis and do not have three-part
pharynxes (von Lieven, 2003;
J.-T. A. Chiang, unpublished), and were therefore not useful as outgroups.
Thus, determination of the ancestral functions of pharyngeal neurons await the
availability of more favorable outgroup species.
Having characterized how pharyngeal behaviors and their neuronal
stimulation evolved, we then focused on understanding the mechanisms by which
M4–TB stimulations are absent specifically in the Rhabditidae family. In
C. elegans, we found that M4 actually innervates the TB, even though
it has no detectable function there in wild type. Further, slo-1
encodes a BK type Ca2+-activated K+ channel that
negatively modulates neurotransmission
(Wang et al., 2001), and novel
EPG spikes and increased TB pumping suggested that M4–TB stimulations
occurred in slo-1 mutants. Thus, M4–TB synapses in C.
elegans apparently have the capability for function, but
neurotransmission at these synapses is reduced to undetectable or irrelevant
levels. Based on this, we propose that M4–TB synapses have been silenced
during the evolution of the Rhabditidae family, leading to the lack of
M4–TB stimulations. Interestingly, using the eat-18 mutation
and the drug d-tubocurarine, we also demonstrated the cholinergic nature of
M4–TB synapses. TB muscles in C. elegans had been shown to
express surface nicotinic channels (McKay
et al., 2004), but their functional significance was unclear. An
admittedly speculative, but potentially interesting, possibility is that these
channels represent nonfunctional remnants from previously active M4–TB
synapses.
Although the slo-1 phenotypes suggest that modulation of
neurotransmission at M4–TB synapses may be important for the absence of
M4–TB stimulation in the Rhabditidae, we emphasize that there are
clearly alternative possibilities – for example, changes in the number
of M4–TB synapses, or changes in the way TB muscles respond to M4
stimulation. Additional future studies, including characterization of
M4–TB synapses in the non-Rhabditidae families, will help distinguish
between the different possibilities. Nonetheless, we have also identified
several additional C. elegans genes that can activate M4–TB
synapses when mutated, and they also encode presynaptic ion channel subunits
that modulate neurotransmission, such as unc-2 and unc-36,
which encode subunits of a voltage-gated Ca2+ channel (M. Steciuk,
unpublished observations). Thus, modulation of neurotransmission is an
attractive hypothesis, and we hope to identify evolutionary differences in
these genes that correlate with the activity/inactivity of M4–TB
synapses in each family. In particular, differential expression of
alternatively spliced BK type Ca2+-activated K+ channels
are known to be of physiological importance (i.e.
Atkinson et al., 2000;
Rosenblatt et al., 1997), and
an interesting hypothesis is that differential expression of a specific SLO-1
isoform in M4, in the Rhabditidae family, is critical for inactivating
M4–TB synapses.
Given the observed evolutionary differences in pharyngeal behaviors, what
functional implications might they have? That is, are they advantageous or
adaptive in any way? As shown above (Fig.
7), the general lack of corpus and isthmus/TB coupling appeared to
be useful for responding to changes in food density. Further, in the
Rhabditidae family, the switch in the anterior isthmus from peristalsis to
pumping was likely important, as computational modeling shows that anterior
isthmus pumping drastically enhances the efficiency of food transport by the
corpus (Avery and Shtonda,
2003).
On the other hand, other evolutionary differences in pharyngeal behaviors
did not have obvious functional consequences. In several cases, they even
appeared paradoxical. For example, why TB pumping became coupled to corpus
pumping in the Rhabditidae family is somewhat perplexing, since this prevented
the ability to independently regulate TB pumping rates in response to food
density changes. In the Diplogasteridae, the loss of the TB grinder and TB
pumping is also perplexing, since the ability to chew was lost. Other
researchers have also been intrigued by this
(von Lieven and Sudhaus, 2000;
Zhang and Baldwin, 1999), and
while the reason is not obvious, it may be related to the diversified diet of
the Diplogasteridae – it is the only free-living soil nematode family
known to feed on non-bacterial food sources
(von Lieven and Sudhaus,
2000).
Our results demonstrate the utility of comparative studies between C.
elegans and other nematodes for studying behavioral evolution. One major
advantage of studying C. elegans is its relative simplicity, a
feature shared by nematodes in general. Thus, as in our study of the pharynx,
the evolution of nematode behaviors and neuronal functions can be
characterized in terms of very precise and discrete changes. In particular,
the use of laser ablations in comparative studies may be a powerful way to
examine how neuronal functions evolve at the level of single neurons and small
neuronal circuits. To complement these advantages, the wealth of techniques
and resources in C. elegans can be helpful for understanding the
underlying cellular and genetic mechanisms of change, as with our study on
M4–TB stimulation. Currently, molecular and genetic techniques are being
actively developed for non-C. elegans nematodes as well, such as in
Pristionchus pacificus (Kenning
et al., 2004; Srinivasan et
al., 2003; Srinivasan et al.,
2002), which will be of value. For similar reasons to these, there
has already been much interest in using C. elegans and related
nematodes as models for studying the evolution of developmental mechanisms
(i.e. Dichtel et al., 2001;
Felix et al., 2000;
Sommer, 1997), and we think
that comparative studies in C. elegans and related nematodes can also
provide useful models for investigating the mechanisms that underlie
behavioral and neuronal evolution.
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List of abbreviations |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
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Acknowledgments |
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Footnotes |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionList of abbreviationsReferences |
|---|
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P<0.05).