Chemosensory tuning to a host recognition cue in the facultative specialist larvae of the moth Manduca sexta
Department of Biological Sciences, Binghamton University, State University of New York, Binghamton, NY 13902-6000, USA
* Author for correspondence (e-mail: moliva{at}binghamton.edu)
Accepted 22 July 2003
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Summary |
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Key words: indioside D, Manduca sexta, larva, sensilla, taste, receptor, host recognition
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Introduction |
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The larvae of Manduca sexta (Sphingidae) are facultative
specialists on plants in the family Solanaceae. When they feed on solanaceous
foliage, the larvae develop a strong preference for these plants, rejecting
any other potential food. In contrast, when they feed on non-solanaceous
plants or diets based on non-solanaceous foliage, they remain polyphagous
(del Campo, 1999; del Campo
and Renwick, 1999
,
2000
;
del Campo et al., 2001
;
Jermy et al., 1968
;
Rothschild et al., 1979
;
Schoonhoven, 1967
;
Yamamoto, 1974
;
Yamamoto and Fraenkel, 1960
).
In previous studies, we found that at least one of the reasons why
Manduca larvae become host-restricted when they feed on solanaceous
plants is that they develop a preference for one plant compound, indioside D,
a steroidal glycoside so far only found in Solanaceae
(del Campo, 1999
;
del Campo and Renwick, 2000
;
del Campo et al., 2001
;
Yahara et al., 1996
).
Host-restricted Manduca larvae would eat non-solanaceous food when it
was treated with indioside D or mixtures of plant compounds containing
indioside D (del Campo, 1999
;
del Campo and Renwick, 1999
,
2000
;
del Campo et al., 2001
).
Indioside D was isolated from potato foliage by bioassay guided fractionation
(del Campo, 1999
;
del Campo and Renwick, 2000
;
del Campo et al., 2001
). Of
all the potato foliage extracts and their chemical fractions containing
thousands of plant compounds, only indioside D caused host-restricted larvae
to feed on a non-host (del Campo,
1999
; del Campo and Renwick,
1999
,
2000
;
del Campo et al., 2001
). It
was therefore concluded that for potato, the recognition cue used by
host-restricted Manduca larvae was indioside D.
Host-restricted larvae choose their food based on input from taste receptor
cells located within chemosensory sensilla on their mouthparts. There are four
sets of external chemosensory sensilla. Antennal, maxillary palp and
epipharyngeal sensilla respond to chemical cues in Manduca larvae but
they do not appear to play a significant role in host-restricted feeding
behavior (de Boer,
1991a,b
,
1993
;
de Boer and Hanson, 1987
;
Glendinning et al., 1998
).
This behavior is mediated entirely by the sensilla styloconica located on the
galea, as removal of these sensilla completely eliminates food preference by
host-restricted larvae (del Campo,
1999
; del Campo et al.,
2001
; Flowers and Yamamoto,
1992
; Waldbauer and Fraenkel,
1961
). Thus, the sensory input from these sensilla is both
necessary and sufficient for host recognition by host-restricted larvae. The
bilaterally paired lateral and medial sensilla styloconica each contain four
chemoreceptor cells, which project to the subesophageal ganglion, where the
circuitry for chewing is located (Griss,
1990
; Griss et al.,
1991
; Kent and Hildebrand,
1987
; Rohrbacher,
1994a
,b
).
The responses of the sensilla styloconica to a variety of chemical compounds
have been examined (Bernays et al.,
1998
; Glendinning et al.,
2001
; Glendinning and Hills,
1997
; Glendinning et al.,
1998
,
1999a
,b
,
2000
,
2002
;
Dethier and Crnjar, 1982
;
Peterson et al., 1993
;
Schoonhoven,
1969a
,b
,
1977
;
Schoonhoven and Dethier, 1966
;
Schoonhoven and van Loon,
2002
; Städler and Hanson,
1976
). In a few cases, comparisons have been made of the responses
of these sensilla to plant or other compounds in plant-reared and wheat germ
diet-reared larvae (del Campo,
1999
; del Campo et al.,
2001
; Schoonhoven,
1969a
; Städler and
Hanson, 1976
; van Loon,
1990
). Larvae of the cabbage butterfly, Pieris, reared on
a wheat germ-based diet, showed reduced sensitivities of the sensilla
styloconica to deterrent compounds (van
Loon, 1990
). For Manduca larvae, it has been difficult to
explain the differences in food preferences of diet-reared and
solanaceous-reared larvae by differences in the responses of the sensilla
styloconica to chemical compounds. One missing factor in these studies has
been the relevant natural cue(s) for Manduca to recognize suitable
food (Bernays, 1996
;
del Campo and Renwick, 2000
;
del Campo et al., 2001
). Our
recent discovery of indioside D allows us to study the effects of dietary
experience on the responses of the sensilla to natural cues that the larvae
use to identify their normal host plants.
In a previous study we found that in plant-reared larvae, the lateral
sensilla styloconica become `tuned' to indioside D by reducing their responses
to at least three other compounds that can be found in foliage, while
maintaining their sensitivity to indioside D. We called this change in
response chemosensory tuning (del Campo et
al., 2001). Here, we present additional evidence for chemosensory
tuning in both the lateral and medial sensilla styloconica. We selected
representative compounds from four major plant chemical classes that might be
relevant to a feeding larvae: sucrose representing a generalized nutrient, KCl
representing a generalized salt, tomatine representing a solanaceous compound
that is not used as a specific recognition cue, and indioside D representing a
solanaceous compound that is a specific recognition cue for Manduca
larvae. From these data, we develop a model to describe how the
host-restricted behavior of solanaceous-reared larvae might be accomplished by
sensory tuning and its integration in the CNS.
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Materials and methods |
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Electrophysiological recordings
Satiated larvae were anesthetized by submerging them in water. They were
prepared for recording by placing them in vials of tapwater so that all
spiracles remained submerged with the head exposed for recording, using a
modification of the techniques described by Gothilf and Hanson
(1994). The head was fixed
into position with soft wax so that the medial and lateral sensilla
styloconica were accessible to recording electrodes. A ground electrode was
inserted into the mandibular musculature through a small hole in the head
capsule. Tip recordings were performed on the lateral and medial sensilla of
one side of the mouthparts of each animal using glass microelectrodes filled
with the test solutions described below. The order in which the test solutions
were applied to each sensillum was randomized. The sensilla's response was
recorded for at least 1 min per test solution. Between recordings, mouthparts
were rinsed with distilled water and dried with a tissue (Kimwipe®,
Kimberly-Clark, Roswell, WI, USA). The signals were amplified with a high
impedance amplifier (Getting Instruments, Iowa City, IA, USA) and digitally
recorded (Axon Instruments, Union City, CA, USA). Immediately after the
electrode contacted the sensilla a brief stimulus artifact obscured the
recording. This artifact (`blocking artifact') had an average duration of
13.4±0.23 ms (mean ± 95% CI; N=228 recordings). Even
for the highest firing frequencies of approx. 250 Hz, the data lost would
produce an error of only about 3.3 spikes, or 1.3% of the total.
Test solutions
All compounds tested were carried in a 50 mmol l-1 KCl
conducting solution (KCl control solution). This control solution included
0.16% polyvinylpyrrolidone-80 (PVP-80; Sigma Chemical, St Louis, MO, USA) to
increase viscosity and reduce evaporation from the electrode tip. It also
included 0.1% ethanol and 1% methanol to account for the solubility
requirements of tomatine and indioside D, respectively, as described below.
Tomatine (Sigma Chemical) was first solubilized in 10% ethanol to a
concentration of 10-2 mol l-1, and then diluted
100x in 50 mmol l-1 KCl with 0.16% PVP-80 and 1% methanol,
giving a final tomatine concentration of 100 µmol l-1. Glucose
(Sigma Chemical) was dissolved directly in the KCl conducting solution to a
concentration of 100 mmol l-1. Indioside D was obtained from fresh
potato foliage, using the methods described in del Campo and Renwick
(2000), and stored in methanol
at a concentration of 1.2x10-4 mol l-1. For
neurophysiological recordings, it was diluted 100x in 50 mmol
l-1 KCl with 0.16% PVP-80 and 0.1% ethanol, to a final
concentration of 1.2 µmol l-1. All solutions were refrigerated
between recording sessions and used within 1 week of preparation.
Data and statistical analyses
For each recording, digital traces were sampled for spike frequency at the
onset of the recording (time=0), 1, 5, 15 and 30 s. Spikes were counted for
periods of 200 ms at times 0 and 1 s, and 500 ms at all other times. Analyses
were conducted independently on lateral and medial sensilla. At analysis, the
responses of the sensilla to glucose, tomatine and indioside D were always
compared to their responses to the KCl solution. Responses were considered
`sensitive' to a solution when they were significantly higher than the
response to KCl alone. For the phasic portion of the response of each
sensillum, the first 200 ms after contact were analyzed using two-way analysis
of variance (ANOVA) tests. The factors in these fully factorial models were
diet, solution and their interaction. When a significant effect was found, a
Dunnett post-hoc test was conducted to contrast responses to
different solutions against the KCl control solution. For the tonic portion of
the responses, repeated-measure analyses were conducted on the sampled values
for each sensillum. Independent variables in the model were diet, solution,
and their interaction. Animal was included in the model as a covariant factor
to control for variability between the animals. The effect of these variables
was studied over time and independently from time. To contrast further the
responses to each solution, repeated measure analyses were conducted for each
type of dietary experience (solanaceous or wheat germ diet) to detect
different sensillar responses to the solutions. In addition, the digitized
responses of the sensilla styloconica were analyzed by individual cell spike
counts between 1 and 2 s after contact. At this time, spike shapes are quite
constant. Each sensillum contains four sensory cells. These have been
classified in a number of studies as sugar-sensitive, salt-sensitive,
inositol-sensitive and deterrent cells (for a review, see
Schoonhoven and van Loon,
2002). We did not attempt to categorize the cells by their
sensitivities to specific substances, but classified them based on their
relative amplitudes, their rise times and interspike intervals, using
commercial software (Synaptosoft, Decatur, GA, USA), and confirmed the results
by visual inspection. Four categories of cells were distinguished: a small
amplitude (S) cell, two medium amplitude units (M1 and M2), distinguished by
their rise times, and a large amplitude unit (L). For the lateral sensillum, S
cells had about half the amplitude of the M cells; L cells had approximately
twice the amplitude of the M cells. M cells were discriminated by their rise
times, which were approximately 0.3 ms for M1 and 0.8 ms for M2. For the
medial sensillum, the S cells also had about half the amplitude of the M
cells. The L cells were approximately 50% larger in amplitude than the M
cells. M cells were discriminated by their rise times of 0.4 ms for M1 and 0.9
ms for M2. Two-way ANOVA statistical tests were conducted for each individual
cell type data set. The factors in these fully factorial models were diet,
solution, and their interaction. All statistical analyses were conducted using
SPSS 10.0 (Chicago, IL, USA).
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Results |
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Phasic responses of the lateral and medial sensilla
During the first 200 ms after contact (Figs
1,
2), the responses of the
lateral sensillum (Fig. 3A) to
the tested solutions were strongly affected by the dietary experience of the
larvae (P<0.001, F=24.28, d.f.=1), and the type of
solution applied (P<0.001, F=11.74, d.f.=3). The
responses of the lateral sensilla to the different solutions were not affected
by dietary experience in the same fashion (P<0.001,
F=6.64, d.f.=3). For larvae reared on wheat germ diet, the firing
frequency of the lateral sensillum did not differ significantly from the
response to KCl control solution for glucose, indioside or tomatine (Dunnett
post-hoc tests, P>0.05, for all solution contrasts
against KCl). In contrast, for solanaceous-reared larvae, the average firing
frequency of the lateral sensillum was over twofold higher for indioside than
for KCl (Dunnett post-hoc test, contrasts against KCl:
P<0.001). However, as in wheat germ diet-reared larvae, responses
to tomatine and glucose did not differ significantly from the responses to KCl
alone (Dunnett post-hoc test, contrasts against KCl:
P>0.05 for tomatine and glucose). Moreover, in solanaceous-reared
larvae, responses to KCl, tomatine and glucose were significantly lower than
the responses to these solutions shown by diet-reared larvae. The firing
frequency in response to indioside was similar in both sets of larvae. The
substantial sensitivity to indioside shown by the lateral sensilla of the
solanaceous-reared larvae was thus primarily due to their much lower responses
to the KCl control solution.
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In contrast, the responses of the medial sensilla (Fig. 3B) to the tested solutions were weakly affected by the dietary experience of the larvae (P=0.04, F=4.58, d.f.=1), but strongly affected by the type of solution applied (P<0.01, F=4.93, d.f.=3). For wheat germ diet-reared larvae, the medial sensillum showed a significantly higher firing frequency in response to glucose or indioside when compared to its response to the KCl control solution (Dunnett post-hoc test, contrasts against KCl: P<0.001 for indioside and glucose). The response to tomatine, however, was not significantly different from the response to KCl (Dunnett post-hoc test, contrast against KCl: P>0.05, for tomatine). In contrast, while the medial sensillum of solanaceous-reared larvae showed significantly higher responses to indioside compared to KCl (Dunnett post-hoc test, contrast against KCl: P<0.001 for indioside), its response to glucose was not significantly different from its response to KCl (Dunnett post-hoc test, contrast against KCl: P>0.05, for glucose). As for the diet-reared larvae, the response to tomatine was not significantly different from KCl (Dunnett post-hoc test, contrast against KCl: P>0.05, for tomatine).
Tonic portion of the responses of the lateral and medial
sensilla
The tonic portions (1-30 s after contact) of the responses of lateral
sensilla styloconica (Fig. 4)
were modified by dietary experience (d.f.=1, F=28.34,
P<0.001) and type of solution (d.f.=3, F=16.84,
P<0.001). Moreover, there was a strong interaction between dietary
experience and type of solution tested, which suggested that not all responses
to the solutions were modified by dietary experience in the same fashion
(d.f.=3, F=11.14, P<0.001). In wheat germ diet-reared
larvae, the tonic portion of the responses of the lateral sensillum to
glucose, indioside and tomatine did not differ from the KCl control solution
(Fig. 5A, repeated-measures
analysis; P>0.05). In contrast, the tonic portion of the response
of solanaceous-reared larvae to indioside was significantly higher than the
responses to the KCl control solution (Fig.
5B). This response was sustained for at least 30 s (over time
effect, P<0.001), while there was no significant response to
glucose (P>0.05) or tomatine (P>0.05) in comparison to
the responses to KCl.
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The tonic portions of the responses of the medial sensilla styloconica (Fig. 6) were also modified by dietary experience of the larvae (d.f.=1, F=5.79, P<0.02) and type of solution (d.f.=3, F=8.98, P<0.001). In this case, however, there was no interaction between dietary experience and type of solution (d.f.=3, F=0.50, P>0.05). In wheat germ diet-reared larvae (Fig. 7A), the tonic portions of the medial sensilla responses to indioside were higher than KCl control solution for at least 5 s (P=0.001), while there were no significant responses to tomatine (P>0.05) or glucose (P>0.05) in comparison to KCl. This indicates that the sensitivity to glucose that was found in the phasic portion of the medial sensillum's response was lost during the tonic portion. For solanaceous-reared larvae (Fig. 7B) the tonic portions of the responses to indioside were significantly higher than the responses to KCl (P=0.001), while there were no significant responses to glucose (P>0.05) or tomatine (P>0.05). The response to indioside was sustained for at least 30 s after contact (over time effect, P<0.001).
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Responses of individual cells of the lateral sensillum
Responses of some individual cells in the lateral sensilla styloconica were
modified by dietary experience and type of solution at 1 s after contact
(Fig. 8). The S cell responses
(Fig. 8A) were not
significantly modified by the dietary experience of the larvae (d.f.=1,
F=1.67, P>0.05), or type of solution applied (d.f.=3,
F=2.51, P>0.05). There was no interaction of dietary
experience and solution applied (d.f.=3, F=0.29, P>0.05).
The M1 cell responses of solanaceous-reared larvae were significantly lower
than M1 cell responses (Fig.
8B) in wheat germ diet-reared larvae for all solutions except
indioside (dietary effect: d.f.=1, F=8.07, P=0.005; solution
effect: d.f.=3, F=0.21, P>0.05; interaction of dietary
effect and solution applied: d.f.=3, F=2.85, P<0.05).
This cell was sensitive to indioside in solanaceous-reared larvae (Dunnett
post-hoc contrast: P=0.001), but insensitive to all
solutions in wheat germ diet-reared larvae. The M2 cell responses
(Fig. 8C) of solanaceous-reared
larvae were lower than the responses of wheat germ diet-reared larvae for KCl
and tomatine; however, the responses to indioside and glucose were not
significantly modified by larval diet (dietary effect: d.f.=1,
F=12.80, P<0.001; solution effect: d.f.=3,
F=2.67, P=0.05; interaction of dietary effect and solution
applied: d.f.=3, F=0.44, P>0.05). For wheat germ
diet-reared larvae, no M2 responses to any of the solutions applied differed
from the KCl control solution, while in solanaceous-reared larvae this cell
was sensitive to glucose and indioside (Dunnett post-hoc contrasts
for indioside and glucose: P<0.05). The L cell responses
(Fig. 8D) of solanaceous-reared
larvae were significantly lower than responses of wheat germ diet-reared
larvae only for KCl control solution (dietary effect: d.f.=1, F=5.02,
P<0.01; solution effect: d.f.=3, F=5.02,
P<0.01; interaction of dietary effect and solution applied:
d.f.=3, F=2.26, P>0.05). This cell was sensitive only to
indioside in solanaceous-reared larvae (Dunnett post-hoc contrast:
P<0.05). For wheat germ diet-reared larvae, none of the applied
solutions resulted in a firing frequency in the L cell that was significantly
higher than its response to the KCl control solution.
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Responses of individual cells of the medial sensillum
In the medial sensillum, only the S cell responses
(Fig. 9A) were modified by
dietary experience of the larvae and type of solution at 1 s after contact.
The S cell responses of solanaceous-reared larvae were significantly lower to
glucose, while responses to KCl, indioside and tomatine were not significantly
different from the responses of wheat germ diet-reared larvae (dietary effect:
d.f.=1, F=4.01, P<0.01; solution effect: d.f.=3,
F=3.12, P<0.05; interaction of dietary effect and
solution applied: d.f.=3, F=1.12, P>0.05). In
solanaceous-reared larvae, this cell was sensitive to indioside when compared
to the KCl control solution (Dunnett post-hoc contrast:
P<0.01). The responses of the M1, M2, and L cells
(Fig. 9B-D) were not
significantly modified by dietary experience, or type of solution applied
(each analysis showed no significant dietary effect or solution effect at the
P=0.05 level). No interaction of dietary experience and type of
solution was detected in both wheat germ diet- and solanaceous-reared
larvae.
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Discussion |
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In our model, tuning the sensilla styloconica to have their strongest
responses to a host-specific recognition cue is a central component for the
induction of host specificity. There are thousands of chemical compounds found
throughout the plant kingdom, and many plant compounds have been tested on the
sensilla styloconica of Manduca larvae. Many of these plant
metabolites produce excitatory responses in the chemosensory neurons of the
sensilla styloconica (Bernays et al.,
1998; del Campo,
1999
; del Campo et al.,
2001
; Frazier and Hanson,
1986
; Glendinning and Hills,
1997
; Glendinning et al.,
1999a
,b
;
Glendinning et al., 2000
,
2001
;
Peterson et al., 1993
;
Schoonhoven,
1969a
,b
,
1972
;
Schoonhoven and Dethier,
1966
). Specific firing patterns for different compounds have been
proposed to play a key role in food selection by Manduca larvae
(Glendinning and Hills, 1997
;
Peterson et al., 1993
).
However, these studies did not address the role of dietary experience and host
recognition by means of the specific recognition cue for Manduca
larvae, indioside D (del Campo and
Renwick, 2000
; del Campo et
al., 2001
). In our study, we tested indioside as well as three
other plant compounds: KCl and glucose, which are ubiquitous among plants, and
tomatine, which is restricted to the solanaceae
(Boll, 1966
;
Schreiber et al., 1961
).
Response to KCl
We used 50 mmol l-1 KCl as our conducting solution for recording
the responses of the sensilla styloconica to different plant compounds. This
concentration was approximately 50% of the average KCl concentration in potato
foliage (Duchateau et al.,
1953; Duke and Atchley,
1986
) and wheat germ diet (Bell
and Joachim, 1976
). For the lateral sensilla styloconica,
responses to this concentration of KCl were dramatically lower in
solanaceous-reared larvae in comparison to larvae reared on wheat germ diet,
for both the phasic and tonic potions of the response. Because the
concentrations of KCl in wheat germ diet and potato foliage are essentially
the same, this difference cannot be attributed to exposure to different KCl
concentrations in the respective diets. In contrast, responses of the medial
sensilla styloconica to KCl in both types of larvae were not significantly
different, being robust for both types of animals. These data suggest that
some difference in the two types of diets besides KCl concentration leads to
the different sensory responses of the lateral sensilla to KCl in the two sets
of larvae. Furthermore, of the eight sensory neurons in the sensilla
styloconica of potato-reared larvae, only three of them showed significant
lower responses to KCl when compared to wheat germ diet-reared larvae. This
indicates that the dietary effect on neural response may be specific for only
certain sensory neurons, in this case the L, M1 and M2 neurons of the lateral
sensillum.
Response to glucose
Both medial and lateral sensilla styloconica of larvae reared on
solanaceous foliage showed lower responses to the glucose solution than
sensilla from larvae reared on wheat germ diet. However, this result was
significant only for the phasic portion of the response, and was not
maintained after 200 ms. In the lateral sensillum of both types of animals,
responses to glucose solution were not significantly different from the
response to KCl, which was a component of the glucose recording solution. This
suggests that during the phasic portion of the response, the lateral sensilla
styloconica do not show any specific sensitivity to glucose in either group of
animals, and the lower response to glucose solution shown by the
solanaceous-reared larvae is due to their lower KCl sensitivity. During the
tonic portion of the response, there is a suggestion of sensitivity to glucose
at the 1 s and 5 s time points. This may be due to responses of the M2 cell,
which shows a significant response to glucose at 1 s. The medial sensilla of
wheat germ diet-reared larvae showed significant responses to glucose during
the first 200 ms after contact, in comparison to the KCl control solution. As
was the case for the lateral sensilla, medial sensilla of solanaceous-reared
animals had lower responses to glucose than wheat germ diet-reared animals.
This lower response to glucose was not different from the response to the KCl
control solution shown by solanaceous-reared animals. Thus, the response to
glucose in the medial sensillum was dependent upon dietary experience. One of
the four taste cells in the medial sensillum of wheat germ diet-reared larvae
showed a significant response to glucose after 1 s, but we did not determine
whether the total sensillar response to glucose during the first 200 ms was
due entirely to this cell. Our findings are consistent with the results of
Glendinning et al. (2000), who
also described a taste receptor cell that responded to glucose in a distinct
phasic-tonic manner in wheat germ diet-reared larvae.
It should be noted that our study, like many earlier studies of the
responses of taste sensilla to glucose, used a much higher concentration of
glucose than is found in foliage, which is typically around 10 mmol
l-1 (Schoonhoven,
1969b). The concentration of glucose of 100 mmol l-1
was selected in this study because it was closest to the concentration used by
other researchers, and was done with the intention of comparing our findings
with those of previous studies. Concentrations of 100 mmol l-1, 150
mmol l-1, 250 mmol l-1 and 300 mmol l-1 have
all been used experimentally (e.g. Frazier
and Hanson, 1986
; Glendinning
et al., 2000
; Peterson et al.,
1993
; Schoonhoven,
1969b
). In feeding assays, even wheat germ diet-reared
Manduca larvae show only weak or no responses to glucose
(Bowdan, 1995
;
Glendinning et al., 2000
).
Thus, while a glucose response may be useful for experimental studies of the
individual properties of the taste receptor neurons, it appears unlikely to
play a direct role in food selection by the animal
(Glendinning et al.,
2000
).
Response to tomatine
We did not find a significant response to the solanaceous alkaloid
tomatine, in either the medial or lateral sensilla, of solanaceous or wheat
germ diet-reared larvae. We used a concentration of tomatine that is within
the normal range for solanaceous foliage
(Boll, 1966;
Schreiber et al., 1961
), and
similar to the concentration used in earlier experiments. The earlier studies
described a tomatine-sensitive sensory neuron in the sensilla styloconica,
which responded to this alkaloid with a delayed bursting firing pattern
starting about 30 s after contact
(Peterson et al., 1993
;
Schoonhoven, 1969b
). Although
we display data for only the first 30 s after the start of a recording, our
recordings typically lasted at least 1 min, and we expected to have recorded
the bursting response well before the recording period ended. We observed
bursting activity in the sensilla styloconica in some recordings, but this did
not appear to be a unique response to tomatine as it was found in only 50% of
all tomatine recordings, as well as in a number of animals in response to
glucose. One difference between the previous studies and our own, is the
conducting solution (Peterson et al.,
1993
; Schoonhoven,
1969b
). We used KCl in our experiments, while Peterson et al.
(1993
) used 100 mmol
l-1 NaCl. Potato foliage has a very low (<5 mmol l-1)
sodium content (Duke and Atchley,
1986
), and it is possible that a concentration of 100 mmol
l-1 in the recording pipette would influence the sensory neurons'
responses to other plant compounds, as discussed by Schoonhoven
(1969b
). Because
Manduca larvae are unlikely to ever encounter these levels of NaCl in
their natural diets, the ecological significance of this response for host
selection by Manduca larvae in a natural situation is debatable.
Response to indioside D
The clearest responses to a compound we tested on the sensilla styloconica
were to indioside D. Both the lateral and medial sensilla styloconica of
solanaceous-reared larvae had significantly higher responses to indioside D
than to any other compound we tested. This sensitivity was apparent throughout
the phasic and tonic portions of the recording.
For wheat germ diet-reared larvae, the lateral sensillum did not have a significant indioside D response. A significant phasic response to this compound was observed for the medial sensillum. This was eliminated by 5 s after contact with indioside D.
The lateral and medial sensilla styloconica of larvae reared on their
natural host plants were clearly sensitive to the host recognition cue,
indioside D. If the mean spike frequencies of indioside D at each time point
are compared with the corresponding indioside D responses in wheat germ
diet-reared larvae, they are not significantly different. The sensitivity to
indioside D that was shown by solanaceous-reared larvae was thus not due to a
higher responsiveness to this compound, but was a function of the lower
responses shown by the sensilla of these larvae to other compounds. The
sensilla had become `tuned' to indioside D. Indioside D was the only compound
of the three we tested that elicited a significantly higher response than the
KCl recording solutions and, therefore, was the only compound to which the
sensilla specifically responded. Three of the four sensory cells in the
lateral sensillum responded to indioside D, as did one of the four cells in
the medial sensillum. Thus, half of the total number of taste receptor neurons
in the sensilla styloconica responded to the chemical cue that is both
necessary and sufficient for host-restricted feeding behavior. The cellular
mechanism(s) by which sensilla exposed to potato foliage are induced to become
tuned to indioside D is not known. This could be by direct contact of
indioside D and/or other compounds in the foliage with the sensilla, or by way
of a post-ingestive feedback mechanism, as has been described for sensillar
sensitivity to amino acids in the locust
(Abisgold and Simpson,
1988).
We expect that indioside D or a structurally related compound(s) is present
in the foliage of most Solanaceae, although this remains to be determined. A
number of earlier studies have tested responses of the sensilla styloconica to
solanaceous and other types of plant saps. These recordings are comparable to
the phasic portion of our longer lasting recordings, and for saps from tomato
or Jerusalem cherry (both Solanaceae) the approximately 200 Hz spike
frequencies reported by Dethier and Crnjar
(1982) are consistent with our
values for the phasic portion of the response to indioside D. For the
phasic-tonic portion of the response (0.2-1.2 s after contact), Schoonhoven
(1969a
) reported that sensilla
styloconica of tomato-reared larvae were sensitive to solanaceous plant saps,
which resulted in spike frequencies comparable to the responses we found for
indioside D alone for this time period. Interestingly, plant saps from
non-host plants, which we expect do not contain indioside D, produced initial
spike frequencies that were substantially lower than the responses we observed
for indioside (Peterson et al.,
1993
; Schoonhoven,
1969a
).
Individual cells vs. total sensillar response
The four sensory cells in each sensillum styloconicum of Manduca
larvae have been classified by their sensitivities and patterns of response to
particular compounds or classes of compounds
(Schoonhoven, 1969a;
Peterson et al., 1993
;
Glendinning and Hills, 1997
).
Early studies recognized that chemosensory neurons in the sensilla styloconica
responded to multiple plant compounds, and that it was difficult to determine
whether the individual cell responses and/or the sum of cell responses was the
relevant information for the CNS to initiate feeding
(Dethier, 1973
;
Frazier and Hanson, 1986
;
Schoonhoven,
1969a
,b
,
1977
;
Schoonhoven and Dethier,
1966
). More recently, Bernays et al.
(2002
) also found that
relevant chemical cues for the larvae of the Arctiidae moth Estigmene
acraea, the pyrrolizidine alkaloids, stimulated three cells in the
lateral sensilla at their natural concentration. In addition, they found that
only one cell responded in a dose-dependent manner to pyrrolizidine alkaloids,
and its sensitivity was very high, although the lowest concentrations that
could stimulate this cell were probably not behaviorally significant
(Bernays et al., 2002
). Our
results showed that multiple cells respond to indioside at approximately a
fourth of its normal concentration in potato foliage. We did not conduct a
dose-dependent study with indioside D in Manduca. It is possible that
different neurons within the sensilla styloconica of Manduca have
different sensitivities to indioside, and that we may be able to identify a
cell that is particularly sensitive to it by lowering its concentration in our
recording solutions. However, the focus of the present study was to understand
how the responses of the sensilla styloconica to approximately natural
concentrations of host and non-host plant compounds might be interpreted by
the CNS to determine whether or not a feeding bout is initiated and sustained.
Thus, while an individual neuron's responses to specific compounds may be
critical for understanding different mechanisms of how taste sensilla function
and are modified by experience in Manduca, the role of the total
input from the sensilla in triggering a feeding bout cannot be ignored.
Phasic versus tonic responses
Both phasic and tonic responses of the sensilla styloconica are likely to
be important for feeding. Prior to the initiation of a feeding bout, larvae
`taste' the surface of a leaf by repeatedly touching it with their mouthparts
without biting it, producing repeated brief contacts between the taste
sensilla and the leaf surface (Devitt and
Smith, 1985; Miles and Booker,
2000
). More interestingly, Devitt and Smith
(1985
), using high speed video
recordings of feeding Euxoa messoria caterpillars, found that only
one set of chemosensilla, the lateral and medial sensilla styloconica located
on the galeae, remains in contact with foliage throughout a feeding sweep.
Each sweep is a set of bites on a leaf with no interruption, and several
sweeps comprise a full feeding bout. Only a few milliseconds before the
caterpillar's mandibles close on the foliage to complete the first bite, the
galeal chemosensilla (lateral and medial sensilla styloconica) contact the
leaf surface, remaining in contact with the foliage until just a few
milliseconds prior to the end of the feeding sweep. This would first produce a
phasic input to the CNS, probably essential for the initiation of the first
bite in a feeding bout. Once feeding has been initiated, the tonic responses
of the sensilla styloconica, still in contact with the foliage, would provide
a continuous source of excitatory inputs to the feeding circuitry. The
importance of such input for maintaining a feeding bout has been described in
a number of insects (Abisgold and Simpson,
1988
; Barton-Browne,
1975
; Bernays,
1985
; Bernays and Simpson,
1982
). Moreover, another piece of evidence for the requirement of
continous sensillar contact with food to sustain a feeding bout was provided
by Dethier and Crnjar (1982
).
They reported that Manduca larvae do not immediately stop feeding if
host foliage is quickly replaced by non-host foliage during a feeding bout;
they only began to react about 15 s after the exchange. This phenomenon may be
at least partly due to the continued excitation of the chemosensory neurons by
plant sap remaining on the mouthparts: an almost constant stimulation to the
sensilla styloconica.
Role of sensilla inputs on feeding circuitry; a model for initiating
feeding
A central pattern generator (CPG) for chewing has been localized to the
subesophageal ganglion (Griss,
1990; Rohrbacher,
1994a
,b
;
Bowden and Wyse, 2000). Elimination of thoracic input to the subesophageal
ganglion causes continuous chewing movements of the mandibles, leading to the
conclusion that there is an inhibitory input to this circuit of thoracic
origin (Griss et al., 1991
;
Rowell and Simpson, 1992
).
Excitatory sensory input resulting from the application of solanaceous plant
sap to the mouthparts increases the frequency of the chewing rhythm in larvae
with intact or cut connectives (Griss et
al., 1991
; Rowell and Simpson,
1992
). These observations led Rowell and Simpson
(1992
) to propose that feeding
was triggered when the level of excitation from chemo- and mechano-sensilla on
the mouthparts surpassed a threshold of inhibition to the chewing CPG that was
set in the thoracic ganglia. The source of this inhibition is not known, but
it was hypothesized to be related to the hunger status of the larva. An
additional source of inhibition to the chewing CPG could be from taste
receptor cells that respond to deterrent compounds in plant saps
(Frazier and Hanson, 1986
;
Glendinning and Hills, 1997
;
Glendinning et al.,
1999a
,b
,
2000
,
2001
;
Peterson et al., 1993
;
Schoonhoven,
1969a
,b
,
1972
). Because host-restricted
feeding behavior in Manduca is dependent only on the sensilla
styloconica (Waldbauer and Fraenkel,
1961
; del Campo et al.,
2001
), the dramatically different food choices shown by wheat germ
diet-reared and solanaceous foliage-reared larvae must be based on differences
in the contributions of these sensilla to the total excitatory and inhibitory
input to the feeding circuitry. We propose that in order to activate the
chewing circuit and initiate feeding, the total excitatory input from all
taste sensilla on the mouthparts must be sufficient to surpass a threshold
level of inhibition to this circuitry that is determined by thoracic
inhibition and any inputs from deterrent sensory cells. For wheat germ
diet-reared larvae, the excitatory inputs from the sensilla styloconica help
exceed this threshold by responding robustly to many different plant
compounds, and should therefore initiate feeding bouts on many different
plants, as has been described (de Boer,
1992
; Schoonhoven,
1967
; Yamamoto,
1974
; del Campo and Renwick,
1999
). In contrast, the sensilla styloconica of larvae reared on
solanaceous foliage are tuned to indioside D, with relatively low responses to
other plant compounds, and thus would not contribute sufficient excitatory
input to initiate a feeding bout unless the host-specific indioside D is part
of the stimulus. Our study tested representatives of four types of potentially
relevant chemical stimuli; a salt, a sugar, a solanaceous specific compound
that is not used as a recognition cue and the specific host recognition cue
for these larvae. Expanding the variety of tested compounds to include more
types of salts, sugars and deterrents at different concentrations would be a
useful way to further test our model.
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