Distribution and movement of Caenorhabditis elegans on a thermal gradient
Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Hakozaki, Fukuoka 812-8581, Japan
Author for correspondence (e-mail:
yohshscb{at}mbox.nc.kyushu-u.ac.jp)
Accepted 1 May 2003
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Summary |
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Key words: Caenorhabditis elegans, thermosensation, behavior, behavioral plasticity, neural integration, thermal gradient
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Introduction |
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It was previously reported that C. elegans migrates towards a
feeding temperature and stays there by moving isothermally
(Hedgecock and Russell, 1975).
Balancing of opposing neural pathways that drive worms up or down the
temperature gradient was proposed to regulate such movement
(Hedgecock and Russell, 1975
;
Mori and Ohshima, 1995
), and
genes or neurons involved in the pathways have been studied (for reviews, see
Bargmann and Mori, 1997
;
Mori and Ohshima, 1997
). When
starved, worms are said to disperse from that temperature
(Hedgecock and Russell, 1975
).
These results led to an intriguing hypothesis that worms memorize a culture
temperature in association with food condition and regulate their behavior in
reference to the memorized temperature. However, as described below, such
understanding of C. elegans behavior is not always based on very
reliable results, and therefore the basic concept of so-called `thermotaxis'
and the resulting neural model are disputable.
Previous studies on the thermal behavior (or thermotaxis) of C.
elegans were mainly performed either by observing tracks of one or a few
worms on a 9 cm agar plate carrying a radial temperature gradient or by
measuring the distribution of a worm population on a linear temperature
gradient (Hedgecock and Russell,
1975; Mori and Ohshima,
1995
; Komatsu et al.,
1996
; Hobert et al.,
1997
; Cassata et al.,
2000a
). We noticed several problems in both types of assay. First,
`isothermal movement' of a worm indicated by a circular track on a radial
gradient occurs, on average, only in a small fraction of an assay period,
although good examples have been provided. Worms more often migrate in other
temperature regions, but such behavior has not been analysed thoroughly and
understanding of the whole behavior is lacking. Second, the radial temperature
gradient changes over time, even during a 1 h assay period, and is quite
sensitive to room temperature. Therefore, even with a good thermal sensor,
precise estimation of the actual temperature of an isothermal track at the
time it is drawn is difficult. This means that although an isothermal track
was assumed to provide good evidence for memory of the growth temperature, the
relationship between the actual temperature at which it is drawn and growth
temperature is ambiguous. Third, in distribution assays of a worm population
on a linear temperature gradient, other problems exist, mainly concerning the
movement of worms. Worm motility is largely influenced by ambient temperature
(Dusenbery and Barr, 1980
),
which might bias the distribution. Detailed observation of movement is
required to assess the effect of motility as well as to determine whether
C. elegans really exhibits thermotaxis, but such studies have not
been performed until quite recently (Ryu
and Samuel, 2002
). In addition, inefficient motility of worms was
often observed when worms were handled in a buffer solution before an assay or
when they ran through a mold of Sephadex slurry, as used previously
(Hedgecock and Russell, 1975
;
Komatsu et al., 1996
;
Hobert et al., 1997
;
Cassata et al., 2000a
). Under
severe conditions, worms did not disperse well even at constant temperature,
raising the question of whether the observed accumulation on the gradient
reflects the full range of response to a temperature gradient per se.
Finally, linear temperature gradients that were used previously
(Komatsu et al., 1996
;
Hobert et al., 1997
) were
approximately 0.8 deg. cm-1, and the temperature range covered by
an assay was less than 12°C. Such gradients may not have permitted the
full range of movement of worms within the usual assay period of 60 min even
when they were fully motile. A gradient that covers a wider temperature range
should be used. The best assay period seems to be approximately 1 h, as used
before, since a longer period may lead to adaptation to the new temperature,
and a shorter period such as 30 min is clearly too short for movement over a
wide range. Given these limitations, the gradient should also be steeper.
In the present study, we improved on previous assay methods to maintain the motility of worms and examined the distribution and movement of wild-type worms on a linear, reproducible, broader and steeper thermal gradient. The results show that fed worms dispersed over a wide temperature range. The limits of distribution were modulated by feeding temperature or by starvation. However, inconsistent with previous proposals, no specific response to either feeding or starvation temperature was detected. Together with our analysis of responses in several mutants, these results suggest a fundamental change to the basic concept of thermal behavior in C. elegans.
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Materials and methods |
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Temperature gradients and preparation of thin ttx-agar plates
A reproducible and linear temperature gradient of a broad range was
produced on an aluminum slab (20 cm long x 10 cm wide x 2 mm
thick) by circulating water through 3 cm-wide chambers under each end of the
slab from two water baths regulated at 4°C and 33°C
(Fig. 1A). The room temperature
was 24-26°C. A 1.25 mm-thick ttx-agar plate containing 2% Difco Bacto agar
(Becton Dickinson, Franklin Lakes, NJ, USA) and ttx-buffer (0.3% NaCl, 25 mmol
l-1 potassium phosphate buffer, pH 6.0) was prepared. An
appropriately sized piece of the ttx-agar plate (slightly larger than the
frame wall described below) was cut out and transferred onto a thin plastic
sheet (we used a piece of OHP sheet) and was then walled with a frame to keep
worms on the plate. A frame 4 cm wide and 15 cm long was used for assays of
population distribution, while a frame 4 cm wide and 6 cm long was used for
movement analysis of individual worms. After placing the worms to be tested on
the plate, as described below, the plate was set on the aluminum slab with a
temperature gradient. Thus, the full area of the bottom of the ttx-agar plate
contacted the thin plastic sheet, which was placed on the aluminum slab. The
surface temperature of the aluminum slab and an agar plate put on the slab for
behavior tests (see below) was monitored using a thermister probe attached to
a thermometer (Anritsu 357E and HFT-58; Anritsu, Tokyo, Japan). The
temperature of the agar surface reached equilibrium within 1 min (for example,
agar surface temperatures at position 7 were 18.6±0.2°C), and a
stable gradient of approximately 1.4 deg. cm-1 in the range of
approximately 9-29°C was obtained (Fig.
1B).
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Behavior assays
Fed worms were prepared by transferring approximately 30-100 young adult
hermaphrodites to an NGM plate with plentiful E. coli and incubating
at the original temperature for several hours before assay. To starve the
worms, approximately 100 L4 larvae were transferred to a fresh plate with
E. coli and incubated overnight. Resultant adults were harvested from
the plate by flushing with ttx-buffer and collected by trapping on a 30 µm
mesh screen. Worms were further flushed with ttx-buffer to wash off E.
coli, transferred with a glass pipette onto an agar plate (2% Difco Bacto
agar in ttx-buffer) and incubated at the original temperature. To test the
effects of serotonin, worms were incubated for 6-7 h before behavioral assays
on a plate containing 2 ml of 40 mmol l-1 serotonin creatinine
sulfate complex (Sigma, St Louis, MO, USA) in M9 buffer
(Brenner, 1974) and 8 ml of 2%
agar in ttx-buffer prepared in a 6 cm-diameter dish. A control plate contained
the same amount of M9 buffer. A further 200 µl of 5 mmol l-1
serotonin solution was poured onto some of the serotonin plates 1 h prior to
the behavior assay.
For behavior assays, fed worms were allowed to briefly migrate on an agar plate to remove E. coli from their body surfaces and were then transferred to a thin ttx-agar plate prepared as described above. Starved worms were directly placed on a thin ttx-agar plate. All these procedures were performed at room temperature within approximately 10 min. Worms were manipulated by picking them up with a platinum wire to retain motility. To make sure this manipulation did not affect behavior, we also examined the fed worms collected with buffer and handled with a glass pipette. To avoid keeping the worms in a buffer for a long period, they were collected by trapping on a mesh screen instead of settling in a tube. The worms thus collected with a buffer showed good motility, and essentially the same results were obtained as for worms manipulated by picking with a wire (data not shown).
To examine population distribution on the gradient, about 30 worms were used in one experiment. The thin ttx-agar plate with worms was set on the aluminum slab with the temperature gradient and covered with an opaque lid. Preliminary experiments showed that 30 min is sometimes too short for full dispersion, whereas 2 h is too long since starvation during the assay begins to alter the behavior (data not shown); thus, assays were run for 1 h. Worms were killed using chloroform gas, the plate was sectioned into 1 cm-wide zones perpendicular to the gradient and numbers of worms in each zone were counted under a stereomicroscope.
To monitor movement of an individual worm, about five worms were put on a thin ttx-agar plate, which was placed on a thermal gradient. The apparatus was further surrounded by a wall to protect the plate from wind and to keep the temperature constant. Worms in a 2.7 cm-long and 2 cm-wide region of the gradient were observed from above through a macro lens (Nikon Micro NIKKOR 55 mm f/2.8; Nikon, Tokyo, Japan) mounted on a CCD camera (Sony DXC-C1) and recorded on videotape. Subsequent analysis was performed on a Power Macintosh computer using the Scion Image program (Scion Corporation, Frederick, MD, USA). From the recording, 120 frames were captured at roughly 5 s intervals using the built-in digitizer of Power Macintosh G3 with a magnification of 0.1 mm pixel-1. Total duration required for capturing the frames was divided by the number of intervals to obtain a mean duration between frames. XY positions of the approximate center of a worm (around the vulval position) were determined. Instantaneous velocities and their elements in directions of the thermal gradient were calculated using the displacement of the worm center in successive samples and the mean duration between frames. The values observed for worms in a 5 mm-wide zone perpendicular to the gradient were averaged. Analysis began a few minutes after the agar plate had been set on the gradient. Worms observed in a field for more than a few minutes were analysed.
Ablation of AFD neurons
A pair of AFD neurons from wild-type N2 or N2 worms carrying an
extrachromosomal array containing the gcy-8::gfp fusion gene (a kind
gift from M. Koga) as an AFD marker (Yu et
al., 1997) was eliminated by laser irradiation during the L1 stage
(Bargmann and Avery, 1995
). The
behavior of animals grown to the adult stage was examined.
Expression constructs of TAX-4 and generation of transgenic
animals
Indicated promoter regions amplified from N2 genomic DNA were fused to the
tax-4 cDNA and inserted into a pPD95.77 green fluorescent protein
(GFP) expression vector. The 1.7 kb upstream region of gcy-8
(Yu et al., 1997), the 4.6 kb
upstream region of nhr-38
(Miyabayashi et al., 1999
) and
the 4.5 kb upstream region of gpa-3
(Zwaal et al., 1997
) were
used. The construct for TAX-4 expression by the tax-4 promoter
contains the genomic region of the 13 kb promoter plus the first three exons
of the tax-4 gene and the tax-4 cDNA of the remaining part,
which is made from the tax-4::gfp fusion construct (a gift from I.
Mori). Plasmids were injected into gonads of tax-4(p678) animals at a
concentration of 100 ng µl-1 or at a concentration of 70 ng
µl-1 together with 30 ng µl-1 of an injection
marker, kin-8::gfp (a gift from M. Koga)
(Mello et al., 1991
).
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Results |
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The distribution was relatively uniform instead of peaking around the
feeding temperature (20°C), which implies that worms dispersed regardless
of the temperature gradient in a moderate temperature range but avoided warm
or cold areas. To examine the mechanisms for determining the distribution of a
worm population, we observed the movement of individual worms of the wild-type
strain freshly obtained from the CGC (all the movement analyses presented in
this paper are those of the CGC N2 strain). When worms were put inside the
distribution area, they moved both up and down the gradient
(Fig. 2C,D). The mean velocity
elements in directions of the gradient were nearly zero, indicating that both
upward and downward movement occurred at similar frequencies without strict
preference to the feeding temperature. By contrast, if worms were initially
placed at a temperature higher than the limit of the distribution
(approximately 23°C), almost all worms migrated down the temperature
gradient (Fig. 2C,D). Once
these worms reached the distribution zone, they started to move in different
directions of the gradient. Within the distribution area close to the warm
limit, worms maintained fast movement but did not enter the warmer region,
suggesting that they controlled directions of movement near the boundary to
stay inside the distribution region. We noticed some isothermal traces near
the warm boundary (Fig. 2D),
which may represent the same phenomena as those reported previously as
isothermal movement (Hedgecock and
Russell, 1975). They may occur when a worm near the boundary
migrates almost straight to prevent overrunning the boundary.
These observations indicate that wild-type worms grown at 20°C migrate independently of the temperature gradient in a moderate temperature range between approximately 23°C and 13°C or 15°C but that they sense warm temperatures above 23°C and avoid them. Worms did not go into a region colder than the distribution limit either, and therefore they may sense and avoid cold temperatures as well. Alternatively, the cold boundary may be determined by frequent turning and/or reduction of motility in a cold area: although worms near the cold boundary continued to move, their dispersion was slowed by frequent turning. We could not determine if worms placed outside the cold boundary show taxis up a temperature gradient, since they lost motility in a very short time.
When wild-type worms maintained in our laboratory and fed at 15°C or 25°C were examined, both cold and warm limits of distribution shifted by about 4°C or 2°C down or up, respectively, compared with those grown at 20°C (Fig. 2A). Wild-type worms that had been freshly obtained from the CGC and cultured at 15°C or 25°C exhibited the distribution patterns shown in Fig. 2B. The distribution limits of worms fed at 15°C shifted to lower temperatures by 3-4°C but those of worms fed at 25°C were similar to those fed at 20°C, thus showing essentially no dependence on culture temperature (Fig. 2B). The ranges of distribution of worms fed at 15°C or 25°C were 7-8°C, which were similar to those of worms fed at 20°C and narrower than those of worms maintained in our laboratory (Fig. 2A). Observation of movement of worms freshly obtained from the CGC and fed at 15°C (Fig. 2E,F) confirmed that in a region warmer than their distribution range they migrated down the temperature gradient.
Behavior of starved worms
We next examined the behavior of starved worms. In contrast to fed worms,
worms starved for several hours at either 20°C or 15°C dispersed in a
wider range of temperatures: they went into warmer temperatures and also into
colder temperatures after starved at 20°C
(Fig. 3A, upper and middle
panels). Worms starved at 25°C did not move very well hence we could not
determine the distribution of these worms. Therefore, it is likely that
starvation for several hours modulated the warm avoidance so that the worms
became less sensitive to warm temperatures. In fact, in movement analysis with
worms starved at 20°C, mean velocity elements in gradient directions
obtained were nearly zero in a warm area above 23°C
(Fig. 3B). The traces of worms
placed in a warm area did not indicate migration down a temperature gradient
either (Fig. 3C). Since these
analyses were performed during a short period of about 10 min just after
transfer from starvation culture, the results support the idea that worms
starved for several hours failed to avoid warm temperatures. If worms were
starved overnight at 20°C, the distribution range became narrow, between
approximately 17°C and 23°C (Fig.
3A, lower panel). The more important conclusion is that
distribution of worms starved either for 6 h or overnight did not indicate
avoidance of starving temperatures. Although the results with worms starved at
20°C for several hours may imply a gap around 20°C, this was not
observed with worms starved at 15°C or at 20°C for a longer period. We
confirmed this lack of avoidance of starving temperatures by observing
movement of worms starved for several hours at 20°C
(Fig. 3B,C). Worms placed at
around 20°C migrated in both directions of the temperature gradient
without any characteristic response around 20°C as judged from elements of
velocities in directions of the gradient and worm tracks.
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Exogenous serotonin is known to modulate several behaviors of C.
elegans in a manner similar to that observed with change in food levels
(Horvitz et al., 1982;
Avery and Horvitz, 1990
).
Therefore, we tested whether serotonergic signaling is involved in the
modulation of warm avoidance. Treatment of starving wild-type worms with
serotonin did not restore the behavior exhibited by fed worms
(Fig. 4). Well-fed worms of the
bas-1 mutant (Loer and Kenyon,
1993
), which has a reduced level of serotonin, avoided warm
temperatures in the same way as did wild-type worms, as shown by their
distribution (Fig. 5A) and
movement (data not shown). Another mutant with a reduced level of serotonin,
tph-1 (Sze et al.,
2000
), also avoided warm temperatures in movement analysis (data
not shown), but limits of distribution could not be determined due to slow
movement. These results indicate that serotonin is neither required for
limiting dispersal into a warm region nor sufficient to mediate food signals
for modulation of warm avoidance.
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Thermal behavior is affected in mutants with defects in some
neurons
Previous studies suggested that AFD neurons were thermosensory, based on
abnormal behavior of worms whose AFDs were either ablated or had defects
(Mori and Ohshima, 1995;
Cassata et al., 2000b
;
Satterlee et al., 2001
). Since
aberrant phenotypes were observed on a radial gradient that was thought to
cover 17-25°C, these phenotypes may suggest that AFDs are sensory neurons
for a warm temperature. However, to our surprise, AFD-killed animals clearly
avoided warm temperatures above 23°C in the same way as did wild-type
worms (Fig. 6B, right panel).
Furthermore, these animals were distributed in a temperature range similar to
the wild-type worms (Fig. 6A), and movement assays indicated that they migrated both up and down the gradient
in a region below 22°C (Fig.
6B, left panel). These results indicate that AFDs are not
essential for warm avoidance. To further examine the contribution of AFDs in
thermal behavior, we tested population distribution of ttx-1 mutant
animals, which were reported to be cryophilic
(Satterlee et al., 2001
;
Ryu and Samuel, 2002
) due to
defects in the AFD neurons. The results varied among experiments; in some
cases, the ttx-1 animals were cryophilic and accumulated in a cold
area, but in others they showed broad dispersion up to 23-24°C, as the
wild-type worms did (data not shown).
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Several mutants were reported to be aberrant in thermal behavior on a
radial gradient. We examined if they are defective in our thermal behavior
assays. Remarkably, tax-2 and tax-4 mutants
(Hedgecock and Russell, 1975;
Coburn and Bargmann, 1996
;
Komatsu et al., 1996
), which
are said to be athermotactic, dispersed into regions above 23°C
(Fig. 5A), suggesting that they
have defects in warm avoidance or its modulation. Analysis of single worm
movement of these mutants confirmed that they moved in both directions of the
gradient in a warm area at similar frequencies
(Fig. 5B). Cold limits of
distribution of both the tax-2 and tax-4 worms were warmer
than that of the wild type. tax-4 worms placed at around 15°C, a
temperature below the observed cold limit of the distribution, did not seem to
migrate up the gradient (Fig.
5B); therefore, tax-4 worms might be altered in motility
that resulted in the shift of the cold limit. The tax-4 gene is
expressed in several sensory neurons
(Komatsu et al., 1996
), among
which AFD neurons were presumed to be responsible for defects of
tax-4 mutants in thermal behavior. However, expression of
tax-4 cDNA fused to a gfp reporter gene under the control of
AFD specific promoter nhr-38 or gcy-8
(Yu et al., 1997
;
Miyabayashi et al., 1999
) in
tax-4 worms did not rescue the defects in both population
distribution tests and movement analysis
(Fig. 7), while worms
expressing tax-4 cDNA::gfp by tax-4 promoter were normal in
their behavior. We found that tax-4 cDNA::gfp expressed under the
gpa-3 promoter (Zwaal et al.,
1997
) also restored the wild-type behavior. This construct was
expressed in several neurons, including pairs of AWB, AWC, ASG, ASK and ADL,
but not in AFD, as judged by GFP fluorescence (data not shown). These results
indicate that expression of TAX-4 in neurons other than AFD is important for
warm avoidance.
|
With ttx-3 mutants (Hobert et
al., 1997), which were classified as cryophilic, the warm limit of
the distribution became much colder than those of wild-type worms but the cold
limit did not differ significantly (Fig.
5A). When ttx-3 worms were placed at around 23°C they
typically migrated down the gradient (Fig.
5B), suggesting that they avoided moderate temperatures that
wild-type worms did not avoid. Interestingly, distribution of ttx-3
mutant worms showed dependency on growth temperatures, while they were not
significantly affected by starvation (Fig.
8). Such was the case with the two putative null alleles,
ks5 (Fig. 8) and
tm268 (data not shown).
|
For mutants classified as thermophilic, we tested tax-6 and
lin-11 mutants (Hedgecock and
Russell, 1975; Mori and
Ohshima, 1995
; Hobert et al.,
1998
; Gomez et al.,
2001
). Both mutants were excluded from the warm area in the same
way as wild-type worms, and analysis of single worm movement confirmed that
both strains preferred to go down the gradient when put in a region above
23°C (Fig. 5A,B). We
confirmed previous observations (Mori and
Ohshima, 1995
) that on agar plates with a radial temperature
gradient tax-6 worms occasionally migrated around peripheral regions
(data not shown). Since their traces looked somewhat aberrant, tax-6
mutants may have defects in movement that results in altered behavior in
radial gradient tests where the temperature gradient is not linear and is less
steep in peripheral areas of the agar plates.
We also tested several other mutants with behavioral defects. Among those,
eat-4 mutant worms (Lee et al.,
1999) showed weak defects in warm avoidance, as judged by movement
analysis of individual worms (Fig.
5B). They were almost all excluded from a warm area after 1 h of
incubation (Fig. 5A), probably
because they were not completely defective in warm avoidance. Distribution of
egl-4 mutant worms (Trent et al.,
1983
; Daniels et al.,
2000
) was almost similar to that of wild-type worms, but a
fraction of worms was constantly observed in a warm area above 23°C
(Fig. 5A). Movement observation
showed that when egl-4 worms were placed at about 25°C, they at
first migrated down the temperature gradient until they reached about
23°C, as wild-type worms did. However, after several minutes, some of the
worms crossed the boundary and migrated into a warmer region
(Fig. 9). Mean velocity
elements in gradient directions indicated that warm avoidance became less
evident at that time. Therefore, the egl-4 gene is not an essential
component of warm avoidance, but its mutation results in fast loss or
modification of the avoidance.
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Discussion |
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Kinetic analyses
To examine if the worms really show directional movement to a temperature,
we analyzed movement of individual worms on a thermal gradient. Although the
initial density of worms in the movement assay to be discussed here
(approximately one worm per cm2) is lower than that in the
population distribution assay (several worms per cm2), the latter
is low enough so that interaction among worms seems negligible in both assays.
Therefore, we consider that the results obtained in the two assays can be
compared as those obtained under similar conditions. Good correlation between
both results also supports this notion (see below). The wild-type worms that
had been freshly obtained from the CGC and grown at 20°C migrated in both
directions of the gradient at similar frequencies in a wide range of moderate
temperatures (Fig. 2C). These
kinetic analyses, together with the distribution analyses discussed above,
showed absence of directional movement, or `taxis' in a strict sense
(Randall et al., 1997), and
also absence of kinetic accumulation to the growth temperature, at least for
worms cultured at 20°C. For N2 freshly obtained from the CGC and grown at
15°C, it may be possible that directional movement or kinetic accumulation
to a temperature near 15°C is responsible for the distribution pattern
shown in Fig. 2B. Avoidance of
the starvation temperature was not observed in any case
(Fig. 3). These findings are
important since they are contradictory to the previous hypothesis for
thermotaxis. Also, we observed apparent avoidance of a warm temperature (warm
avoidance) by the wild type in kinetic analyses
(Fig. 2C,D). Namely, worms
placed in a position warmer than the distribution limit migrated down the
temperature gradient until they reached the distribution area shown in
Fig. 2C,D, in which most data
points showed negative values of the velocity element in the gradient
direction. Also, worms near the warm limit of the distribution moved actively
but did not enter a warmer region (Fig.
2D). The temperature of transition from avoidance to non-avoidance
in the kinetic assay is well correlated to that of the upper limit of the
distribution range or so-called `warm boundary'. Therefore, it is strongly
suggested that the warm boundary of distribution results from this warm
avoidance, which should be based on sensation of a warm temperature. On the
other hand, mechanisms to limit dispersion into a cold area (<13°C or
15°C for worms fed at 20°C) remain unknown. Worms may sense cold
temperatures and avoid them or, alternatively, motility features near the cold
boundary, for example frequent turning, might reduce their dispersion. Based
on these results, we propose that the major thermal response in the
physiological temperature range of approximately 15-30°C, which includes
the temperature range previously used for radial temperature gradients, is
warm avoidance. We also propose that a single warm-sensing pathway is
sufficient for thermal behavior in this temperature range (see below).
Recently, Ryu and Samuel
(2002) reported evidence for
migration down thermal gradients at temperatures above the cultivation
temperature, which was obtained during behavioral analysis of worms subjected
to temporal thermal ramps. The ranges of temperature in these ramps are
similar to the warm boundaries of distribution shown in the present study, and
thus such results are roughly consistent with our finding that worms avoid
temperatures above the warm boundary. Our study also revealed that worms
disperse into a wide temperature range below the cultivation temperature in a
spatial thermal gradient instead of accumulating around the cultivation
temperature, which agrees with the result that Ryu and Samuel
(2002
) did not detect any
response to temporal thermal ramps below the cultivation temperature. Their
studies are interesting in that they suggested kinetic mechanisms for
migration down a spatial gradient and that isothermal tracking was also
analyzed. Our studies are complementary to theirs in terms of the experiments
performed and include the following points that they did not study: (1) we
studied distribution of worms in a spatial gradient in detail; (2) we also
analyzed starved worms in order to examine possible avoidance of a starving
temperature; (3) we analyzed movement of well-fed and starved worms
systematically in an entire physiological temperature range, which is much
wider than that in their analysis; and (4) we analyzed many more mutants,
eliminated the AFD neurones by laser irradiation and performed experiments to
rescue a mutant with cell-specific gene expression. On the whole, our study
suggests a fundamental change to the basic concept of thermotaxis, or
accumulation of worms around the growth temperature, whereas Ryu and Samuel
seem to assume the previous concept. Both our results and theirs are similar
in the sense that both are against the previous model in which a balance of
upward and downward drives leads to aggregation of worms around the growth
temperature.
Models for thermal responses
We discuss here possible models for thermal responses of C.
elegans in the physiological temperature range. Models 1 and 2 are based
on our present results. In model 1, the warm boundary of distribution is
determined by warm avoidance, the cold boundary results from frequent turning
or inefficient motility of worms at a cold temperature, and worms have a
single temperature sensation, which is for a warm temperature. Model 2
postulates cold avoidance based on sensation of a cold temperature as well as
warm avoidance to explain cold and warm boundaries. Logically, model 1 may
lead to a distribution pattern in which worms accumulate near the cold
boundary, giving a `cold trap'. Although clear accumulation of worms near the
cold boundary over time was not observed, we consider that both models 1 and 2
are possible at present. The previous model proposed positive migration to the
growth temperature and postulated upward and downward drives
(Hedgecock and Russell, 1975;
Mori and Ohshima, 1995
) that
may be based on cold and warm senses. This model does not require active
avoidance of warm or cold temperatures. Our present results are clearly
against the previous model per se, in the sense that directional
migration to the growth temperature was not observed, However the previous
model could be modified based on the present results into model 3, postulating
migration to a wide temperature range that is determined in relation to the
growth temperature. If model 3 is possible, it should be modified so that the
temperature range in which the postulated upward drive works is below
approximately 15°C or below the range covered by a radial gradient.
Although models 2 and 3 are conceptually different, active migration to a wide
temperature range in model 3 and avoidance of warmer and colder temperatures
outside of that temperature range in model 2, which may correspond to downward
and upward drives in model 3, could give the same distribution pattern of the
worms. The `original signals' must be different between the models: they
should be warmer and colder temperatures in model 2 and the wide range of
medium temperatures in model 3. Even our kinetic studies presented here cannot
distinguish between these two models definitively. Identification of the
original signal will give the final answer as to which model is true. However,
it is much easier to assume avoidance of warm (or cold) temperatures than to
assume attraction to a wide range of moderate temperatures, thus suggesting
avoidance. As for the mechanism of warm avoidance, we found directional
movement as shown in Fig. 2C,D,
whereas Ryu and Samuel (2002
)
found change in the duration of forward movement in response to a temporal
temperature gradient as the mechanism down a spatial temperature gradient. As
far as the temperature range of their observation fits that of our warm
avoidance, change in the duration should work at least as a part of the
mechanism of warm avoidance.
An important point is the mechanism for dependence of the thermal response
on growth temperature. In general, both warm and cold limits of the
distribution depended on a feeding temperature, although such temperature
dependence was not observed for CGC N2 worms between 20°C and 25°C
(Fig. 2B). However, since
distribution ranges were broad, it seems unlikely that worms precisely
memorize a feeding temperature and control movement by assessing the ambient
temperature in comparison with the memorized temperature. In model 1, the
change of warm boundary depending on the growth temperature is likely to be a
change in warm avoidance based on the warm sensation, and the change of cold
boundary is due to the change in motility. In model 2, change in the cold
boundary results from cold sensation. In the previous model, the change in the
distribution range was assumed to be due to the change in the balance between
upward and downward drives (Hedgecock and
Russell, 1975; Mori and
Ohshima, 1995
). In this model, both drives are controlled by
neural pathways in reference to neural memory of the growth temperature. In
models 1 and 2, it is probable that growth temperature affects warm avoidance
through neuronal plasticity. However, it could be possible that some metabolic
or physiological state, such as fatty acid composition
(Tanaka et al., 1996
),
depending on the culture temperature is the `memory' of the temperature
instead of neural memory since similar thermal avoidance is also observed in
organisms that do not have a nervous system
(Hennessey and Nelson, 1979
;
Whitaker and Poff, 1980
). In
model 1, a single neuronal pathway for thermosensation is required, whereas
two pathways are postulated in models 2 and 3.
Genes and neurons involved in the thermal response
In the previous model, athermotactic mutants such as tax-2 and
tax-4 could most simply be explained by assuming that they are
affected in the thermosensor or a thermosensory signaling pathway. Also,
cryophilic mutants such as ttx-3 are expected to be defective in
upward (thermophilic) drive or neural pathway, and thermophilic mutants such
as tax-6 are expected to be defective in downward (cryophilic) drive
or neural pathway. In our assays, the results with tax-2 or
tax-4 differed from the above prediction since they behaved as if
they were thermophilic; their cold and warm boundaries of distribution both
shifted to warmer temperatures (Fig.
5A). The upward shift of the warm boundary is correlated with an
equal frequency of downward and upward movement in the kinetic analyses above
23°C (Fig. 5B),suggesting
absence of the avoidance of warm temperatures exhibited by wild-type worms.
The upward shift of the cold boundary was suggested to be due to alteration of
motility (see Results). The results with tax-2 and tax-4
mutants are explained in this way based on models 1 or 2.
In the distribution of ttx-3 mutant worms (Figs 5A, 8), the warm boundary shifted radically to lower temperatures while cold boundaries did not change significantly as compared with those of the parental wild-type strain shown in Fig. 2A. Movement analyses detected overall downward movement in the moderate temperature region of 19-23°C, which wild-type worms do not avoid (Fig. 5B). The results with ttx-3 can be explained in models 1 or 2 by assuming that the warm sensor or its signal pathway in ttx-3 is changed to be sensitive to moderate temperatures to which wild-type worms are not sensitive. Furthermore, the dependence of ttx-3 mutant behavior on culture temperature was clearly shown for the first time in our assay (Fig. 8), which is interesting. As for the modulation by starvation, serotonin did not seem to have an important role, but the ttx-3 gene seemed to be involved since starvation did not affect distribution of ttx-3 mutant worms. These results suggest that mechanisms involved in modulation of warm avoidance by growth temperature and those by starvation are different at least in part. Interestingly, the egl-4 gene is not an essential component for warm sensation, but its mutation led to faster alteration of warm avoidance, probably due to starvation or the ambient temperature during the assay (Fig. 9). Thus, we suggest that ttx-3, tax-2, tax-4 and egl-4 genes are involved in warm avoidance or its modulation.
The neurons involved in thermal sensation and thermal signal transduction
are discussed here. Previously, AFD neurons were suggested to be major
thermosensory neurons (Mori and Ohshima,
1995). Unexpectedly, AFD-killed animals avoided warm temperatures
(Fig. 6) in the same way as did
wild type, suggesting that AFD neurons are not essential for warm avoidance.
This result may be consistent with the result of Cassata et al.
(2000b
), which suggested basic
thermotaxis without functions of AFD neurons or the ttx-1 gene. Since
AFD-killed animals showed changes in thermal responses in radial gradient
assays (Mori and Ohshima,
1995
), AFD neurons may have a role in thermal sensation, which was
not clearly shown in the present experiments. AFD was recently reported to be
involved in isothermal tracking (Ryu and
Samuel, 2002
), which was not analyzed here in detail. This was
because clear and long-distance isothermal tracking is observed only
occasionally. tax-2 and tax-4 genes have been reported to be
expressed in 10 kinds of sensory neurons including AFD
(Coburn and Bargmann, 1996
;
Komatsu et al., 1996
). These
genes are essential for normal thermal responses including warm avoidance
(Fig. 5), which is also
supported in the present study by recovery of normal behavior with
tax-4 promoter-driven expression of tax-4 cDNA in
tax-4 mutants (Fig.
7). However, AFD-specific tax-4 expression by a promoter
for nhr-38 or gcy-8 did not rescue the behavioral defects
(Fig. 7), but expression in
other neurons by the gpa-3 promoter rescued the defects. Therefore,
some of the neurons expressing tax-4, except for AFD, must be
essential for warm avoidance. Since AIY-killed animals have been reported to
be cryophilic (Mori and Ohshima,
1995
), AIY neurons may be important in warm sensation or warm
avoidance. This possibility is supported by the expression of ttx-3
in AIY, although not solely in AIY (Hobert
et al., 1997
), and by our observations showing behavioral changes
in ttx-3 mutants (Figs
5A,B,
8). We could not repeat the
thermophilic phenotype of tax-6 and lin-11 mutant worms,
which were previously proposed to have defects in the second neural pathway
that includes AIZ interneurons and opposes the defect involved in the
ttx-3 phenotype.
Role of thermal behavior in C. elegans
C. elegans is reported to avoid a harmful higher temperature such
as 33°C (Wittenburg and Baumeister,
1999). The mechanisms of warm avoidance are different from those
during escape from such higher temperatures, since tax-2 and
tax-4 mutants that showed normal responses to a high temperature
(Wittenburg and Baumeister,
1999
) were defective in warm avoidance. osm-5 and
osm-6 mutants, which respond poorly to a very high temperature
(Wittenburg and Baumeister,
1999
), clearly avoided a warm temperature above 23°C (data not
shown).
This work has led to a novel understanding of C. elegans behavior on a temperature gradient. Such behavior seems to be suitable for survival of soil microorganisms. When there is plenty of food in soil where the temperature is largely moderate, animals just disperse. Warm avoidance would prevent them from going up to the ground surface where it might be too hot or dry for survival. Worms do not disperse into a cold region either, where they cannot retain motility. However, starvation promotes worms to explore into a wider area in search of food.
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