Chem. Senses 28: 349-359,
2003
© Oxford University Press 2003
Perception of Noxious Compounds by Contact Chemoreceptors of the Blowfly, Phormia regina: Putative Role of an Odorant-binding Protein
1 Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan 2 Bio-oriented Technology Research Advancement Institution, Saitama 331-8537, Japan 3 Faculty of Human Development, Kobe University, Kobe 657-8501, Japan 4 Department of Applied Physics and Chemistry, University of Electro-Communications, Chofu, Tokyo 182-8585, Japan
Correspondence to be sent to: Dr Mamiko Ozaki, Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Kyoto 606-8585, Japan. e-mail mamiko{at}ipc.kit.ac.jp
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Abstract |
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The blowfly, Phormia regina, has sensilla with four contact-chemoreceptor cells and one mechanoreceptor cell on its labellum. Three of the four chemoreceptor cells are called the sugar, the salt and the water receptor cells, respectively. However, the specificity of the remaining chemoreceptor cell, traditionally called the `fifth cell', has not yet been clarified. Referring to behavioral evaluation of the oral toxicity of monoterpenes, we measured the electrophysiological response of the `fifth cell' to these compounds. Of all the monoterpenes examined, D-limonene exhibited the strongest oral toxicity and induced the severest aversive behavior with vomiting and/or excretion in the fly. D-Limonene, when dispersed in an aqueous stimulus solution including dimethyl sulfoxide or an odorant-binding protein (OBP) found in the contact-chemoreceptor sensillum, the chemical sense-related lipophilic ligand-binding protein (CRLBP), evoked impulses from the `fifth cell'. Considering the relationship between the aversive effects of monoterpenes and the response of the `fifth cell' to these effects, we propose that the `fifth cell' is a warning cell that has been differentiated as a taste system for detecting and avoiding dangerous foods. Here we suggest that in the insect contact-chemoreceptor sensillum, CRLBP carries lipophilic members of the noxious taste substances to the `fifth cell' through the aqueous sensillum lymph. This insect OBP may functionally be analogous to the von Ebner's grand protein in taste organs of mammals.
Key words: monoterpene, oral toxicity, aversive behavior, bitter taste reception, odorant-binding protein
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Introduction |
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The contact-chemoreceptor sensilla on the labellum of the fly are in the form of a hair housing five sensory neurons, i.e. four contact-chemoreceptors and one mechanoreceptor neuron (Ozaki and Tominaga, 1999
). Although three of the four chemoreceptor neurons
are termed the sugar, the salt and the water receptor cells after their
adequate stimuli, respectively, the fourth neuron has simply been called the
`fifth cell'. Dethier and Hanson reported the responsiveness of the `fifth
cell' to fatty acids (Dethier and Hanson,
1968), but these authors did not change this terminology, because
its function had not then been clarified conclusively. Furthermore, Gillary
had previously reported that Cs+ or Rb+ ions evoke
impulses not only of the salt receptor cell but also of the `fifth cell'
(Gillary, 1966). Hence, this
cell is sometimes called the `second salt receptor cell'. However, the `fifth
cell' could be expected to have another function. Dethier showed that the
response to some vapors induced vigorous impulses of the `fifth cell'
(Dethier, 1972), suggesting
that the `fifth cell' is sensitive to odorants as well. Ozaki et al.
showed that the response of the `fifth cell' to fragrant components of apple
juice is depressed in the presence of antibodies raised against
chemical-sense-related lipophilic-ligand-binding protein (CRLBP), which is an
odorant-binding protein (OBP) commonly found in the olfactory and the
contact-chemoreceptor sensilla of the blowfly, Phormia regina
(Ozaki et al., 1995).
OBPs are small, water-soluble proteins and widely found in olfactory systems
of different animals (Vogt,
1995; Pelosi,
1996; Vogt et al.,
1999). The OBPs are involved in the first specific biochemical
step in odor reception and are thought to carry lipophilic odorants to the
olfactory receptor cells through hydrophilic surroundings
(Vogt et al., 1985;
Prestwich et al.,
1995; Vogt, 1995;
Pelosi, 1996; Steinbrecht,
1996,
1999;
Ziegelberger, 1996;
Kaissling, 1998). Therefore,
Ozaki et al. suggested that the `fifth cell', with the help of CRLBP,
could be involved in the recognition of volatile, more lipophilic plant
compounds.
Phytophagous lepidopterous caterpillars have been shown to have
contact-chemoreceptor neurons in the maxillae that are sensitive to deterrent
or bitter-tasting compounds (Dethier,
1973; Frazier and Hanson,
1986; Schoonhoven,
1987; Blaney and Simmonds,
1988; Chapman et al.,
1991; Glendinning and Hills,
1997). The importance of deterrent neurons had been recognized in
phytophagous lepidopterous caterpillars by studying the relationship between
food-plant selection behavior of these insects and toxicity of secondary
metabolites of plants (Harley and
Thoresteinson, 1967;
Schoonhoven, 1972;
Schoonhoven et al.,
1992; Peterson et
al., 1993; Bernays and
Chapman, 1994; Bernays et
al., 2000).
Generally, P. regina, has saprophagous larvae and the adults
insects visit plants to feed on nectar or sap, among many other potential food
sources. During the search for feeding or oviposition sites, the flies will
come in contact also with potentially toxic compounds
(Dethier, 1993), thus the
activation of neurons sensitive to such compounds leading to the avoidance of
really toxic compounds could elicit aversive behavior. Recently, Liscia and
Solari (Liscia and Solari,
2000) reported for Protophormia terraenovae that the
`fifth cell' responds to substances bitter for humans, such as quinine,
amiloride, nicotine and caffeine. They also showed that these flies avoided
drinking sucrose solution mixed with quinine or amiloride.
In addition to the study of Liscia and Solari
(Liscia and Solari, 2000),
which indicates a possible function of the `fifth cell' as a deterrent or a
bitter taste receptor cell, here we show that the `fifth cell' of P.
regina actually perceives non-polar monoterpenes. We also suggest that
the CRLBP might function as a carrier protein for the monoterpenes to the
`fifth cell', which is an indispensable receptor neuron for the `toxin'
detection system in the fly.
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Materials and Methods |
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Flies
The blowflies, P. regina, originally donated from Professor Morita's laboratory in Kyushu University, were reared in our laboratory at 22 ± 2°C using the method of Morita (personal communication), and after emergence, fed with 0.1 M sucrose and water.
Preparation of stimuli
The monoterpenes (D-limonene, L-limonene, cineole, citral, ß-myrcene) and toluene were purchased from Nakarai Tesuque Corporation, Kyoto, Japan. We directly applied these pure chemicals to several sites of the fly body for the toxicity test.
Monoterpenes are mostly non-polar and cannot be easily dissolved in aqueous solutions. Therefore, we dispersed these chemicals in 10 mM NaCl with 0.25, 1 or 4% dimethyl sulfoxide (DMSO) for the aversive proboscis extension reflex tests (aversive PER tests) and the electrophysiological tip-recording experiments. Ten millimolar NaCl was added to every stimulus solution, because the electrophysiological tip-recording method needs an electrolyte in stimulus solutions. We also used 1, 1.25, 5 or 20 µM CRLBP, 20 µM ß-lactoglobulin (BLG) or 20 µM bovine serum albumin (BSA) instead of DMSO. CRLBP was purified from the P. regina labellar tissue as mentioned below. BLG, BSA and DMSO were purchased from Nakarai Tesuque Corporation, Kyoto, Japan.
Equal volumes (
0.5 ml) of a lipophilic substance
(D-limonene, L-limonene, cineole, citral, ß-myrcene
or toluene) and 10 mM NaCl solution containing a certain concentration of
DMSO, CRLBP BLG or BSA were mixed in a glass vial under oxygen-free conditions
and centrifuged to separate into aqueous and oil layers. The aqueous layer was
recovered and used as a stimulus solution for the aversive PER tests or for
electrophysiological experiments after measuring the concentration of the
contained lipophilic substance. The resulting concentrations of
D-limonene, L-limonene, cineole, citral, ß-myrcene
or toluene in the stimulus solutions were determined by gas chromatography
(GC). This was accomplished by mixing the stimulus solution with n-hexane (v/v
1:3) and separation of the mixture into aqueous and hexane layers. As
quantification standard for the GC, dodecane was added to the recovered hexane
layer at the concentration of 10 µg/ml. In the chromatograms, the peaks of
D-limonene, L-limonene, cineole, citral, ß-myrcene
and toluene were identified, and their concentrations were calculated by
referring to the concentration of dodecane.
Isolation of CRLBP
Lipophilic-ligand-binding protein (CRLBP) was purified from the blowfly
labella according to Ozaki's method (Ozaki
et al., 1995). The labella of the blowflies were isolated
from heads and homogenized in a hand mortar with a small amount of liquid
nitrogen for 20 min. The homogenate was mixed with the sample buffer (4.75 mM
sodium barbiturate–HCl, 10% glycerol, pH 6.8) and centrifuged at 15 000
r.p.m. for 10 min at 4°C, and the supernatant was applied to the native
polyacrylamide gel electrophoresis.
The gel systems of Ornstein (Ornstein,
1964) and Davis (Davis,
1964) were used with some modifications: instead of Tris buffers,
we used barbiturate buffers (stacking gel buffer: 9.3 mM sodium
barbiturate–HCl pH 6.7; running gel buffer: 91.1 mM sodium
barbiturate–HCl buffer pH 8.9; electrode buffer: 41.1 mM sodium
barbiturate–glycine buffer pH 8.3); the stacking and the running gels
were made of 4.5 and 7.5% acrylamide, respectively. Electrophoresis was
carried out at a current of 40 mA for 1.5 h in a cold chamber, so that the
temperature of the gel could be adjusted to 20–24°C.
Since the CRLBP is a highly acidic protein (pI = 3.6)
(Ozaki et al., 1995),
it migrated faster than other proteins. Thus, we could cut the gel, including
CRLBP, after electrophoresis without any contamination. The gel piece with
CRLBP was put in a dialysis bag with a small amount of electrode buffer and
set for horizontal submarine electrophoresis. CRLBP was then
electrophoretically eluted out from the gel overnight and dialyzed against
distilled water overnight in a cold chamber at 5°C. The concentration of
CRLBP in the resulting solution was determined by the CBB protein quantifying
method. For storage, we lyophilized the solution to get a dry powder that was
kept at–80°C. For experimental usage, we made appropriate
concentrations of CRLBP solutions by dissolving the protein in distilled water
or buffers.
Toxicity test
The flies, 5–6 days after emergence from a single batch, were immobilized by securing their wings with washing pins. For toxicity test, a 0.3 µl drop of pure D-limonene, L-limonene, cineole, citral, ß-myrcene or toluene was directly applied with a glass capillary to the labellum and other body parts (see Figure 1) of 10 flies. Thirty minutes after this treatment (the applied chemicals were allowed to evaporate spontaneously), the flies were classified into three groups of conditions and counted as: killed, handicapped and recovered. The same test was repeated six times for each chemical, and the toxicity was evaluated.
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Aversive proboscis extension (PER) test
Flies from a single batch were immobilized 5–6 days after emergence by securing their wings with washing pins. We first gave distilled water to the flies until they were satiated with water, and then subjected them to the test.
We carefully touched the labellar chemosensilla with the stimulus solution
in a micropipette tip, so that the flies could only drink sparingly from it.
Twenty flies were tested with the same stimulus, and the number of flies
showing full extension of a strained proboscis followed by vomiting and/or
faecal excretion (see inset of Figure
2A) was counted and expressed as a percentage. This test was
repeated six times. It seems likely that the fly can vomit exhaustively by
using the full extension of a strained proboscis. Dethier reviewed feeding and
aversive behaviors of the blowfly in detail, but did not mention much about
this type of PER (Dethier,
1976). However, in order to evaluate a positive aversive behavior
against toxins for the fly counting, we selected this type of PER and called
it `aversive PER', discriminating it from a passive aversive-touching response
like retracting the proboscis.
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Electrophysiological procedure
In electrophysiological experiments, adult flies, 5–6 days after
emergence, were used. The electrophysiological response was recorded from the
tip of an LL type chemosensillum located on the outer margin of the labellum,
using the tip-recording method described first by Hodgson et al.
(Hodgson et al.,
1955). If the tip-recording method, which has been widely used for
water-soluble stimulants, could also be applied for lipophilic, less
conductive substances, it can be expected that the chemoreceptor neurons
sensitive to D-limonene would be determined. In order to make the
tip-recording method applicable for recording responses to
D-limonene, we added DMSO or CRLBP to the aqueous stimulus solution
and dispersed D-limonene in it. We stimulated the sensillar tip
with the D-limonene-dispersing 10 mM NaCl solution with DMSO or
CRLBP in a glass capillary recording electrode. We also examined BLG and BSA
instead of CRLBP. The stimulus duration was 30 s and the interval between
stimuli was at least 10 min. Evoked impulses were recorded through a band-pass
filter (100–2000 Hz). The receptor neurons from which the recorded
impulses originate can be determined, because the impulses of the four
receptor cells in a sensillum have different amplitudes. The ambient
temperature was 20–24°C throughout the experiments.
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Results |
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Toxicity of D-limonene
The contact toxicity of D-limonene, L-limonene, cineole, citral, ß-myrcene and toluene, when applied to different sites on the fly's body, is summarized in Figure 1, which shows the percentage of flies killed, handicapped and recovered 30 min after application of 0.3 µl of test substance. Application of any of these chemicals produced more or less convulsive shaking and paralysis of wings and legs. However, the strength of the locomotive disorder depended on the individual chemical. After application of toluene, for example, 100% of flies were alive and more than 87% of them recovered normal locomotive activity within 30 min, regardless of the application site. Citral and ß-myrcene evoked paralysis of legs lasting 30 min in a significant percentage of flies, but these substances seemed less toxic than D-limonene, L-limonene or cineole. The strength of the locomotive disorders also depended on the application sites. Cineole, when applied to the antennae, and D-limonene and L-limonene, when applied to the labellum, showed the strongest toxicity. After application of D-limonene, L-limonene and cineole to the labellum, the death rates were 87, 40 and 30% respectively. These death rates were regarded as indicators for relative oral toxicities of the chemicals applied to the labellum.
It can be concluded that D-limonene produced the severest oral toxicity of all examined chemicals. The toxicity of the other compounds was in the following order: D-limonene > L-limonene > cineole > citral > ß-myrcene > toluene.
Aversive PER to monoterpenes
When we touched the labellar sensilla with D-limonene-dispersing solution, the fly showed aversive PER (inset of Figure 2A), in which the proboscis was extended between the prothorathic legs in a protective movement. An irritated movement of labellum and abdomen were usually observed prior to the aversive PER, and vomiting and excretion of faeces were often induced as well. These behaviors can help the fly to avoid intake of toxic substances.
The inset of Figure 2B shows the feeding PER to stimulation of the labellar sensilla with sucrose solution. The fly extends the proboscis towards the pipette tip filled with sucrose solution and opens the labellar lobes ready to suck. Thus, the aversive and the feeding PERs can be discriminated from each other by careful observation. The difference in the starvation effect between the aversive and the feeding PERs strongly suggested that these PERs are totally different in their behavioral meanings. Forty flies hatched from the same batch were divided into two groups of 20 flies each: one group was fed with 0.1 M sucrose for 5 days after emergence until the PER test, and the other group was fed with 0.1 M sucrose until 36 h prior to the test and starved after that (water was continuously supplied to both groups). Five days after emergence, the PER test to D-limonene dispersed in 10 mM NaCl solutions with 0, 0.25, 1 or 4% DMSO was carried out in both groups at the same time. The ratio of flies showing the aversive PER increased as the concentration of DMSO was increased (72% of flies showed aversive PER at 4% DMSO), but no significant difference was found between the fed (open columns) and the starved groups (closed columns) (Figure 2A). On the other hand, the ratio of flies showing feeding PER increased as the concentration of sucrose was increased, and it was obviously higher in the starved group (closed columns) than that in the fed flies (open columns) at every sucrose concentration (Figure 2B). Significant differences in the ratio of flies showing feeding PER between the starved and the fed groups were confirmed either at 0.0625, 0.25 or 1 M sucrose by unpaired t-test (P < 0.01).
We carried out the PER test to D-limonene, L-limonene, cineole, citral, ß-myrcene or toluene-containing solutions, respectively, and the ratios of the flies showing aversive PER are indicated in Figure 3 (open columns).
The concentrations (µg/ml) of examined chemicals (indicated above the columns in Figure 3) varied with the concentration of DMSO. These concentrations might be underestimated if the chemicals could not completely be transferred to the hexane layer for GC measurement (see Materials and methods). ß-Myrcene was difficult to dissolve in 10 mM NaCl either with or without DMSO; cineole, citral, and toluene were, however, easily dissolved in 10 mM NaCl even in the absence of DMSO. Figure 3 indicates that D- and L-limonene are hardly dissolved without DMSO, but when dispersed with DMSO, could induce aversive PER more effectively than cineole, citral and toluene. In order to exclude purely olfactory repellent effects, we carried out also aversive PER tests to D-limonene with flies whose olfactory input was eliminated by removing their antennae and maxillary palps (closed columns). We found no significant differences as compared to intact flies (open columns). We conclude that olfactory cues are not as important as gustatory cues for the aversive PER in P. regina.
Thus, the order of gustatory effectiveness of the examined chemicals in the aversive PER was as follows: D-limonene > L-limonene > cineole, citral or toluene (the effect of ß-myrcene was not evaluated due to the weak dispersion of this compound).
In subsequent tests we used, instead of DMSO, CRLBP, which is an intrinsic lipophilic-ligand-binding protein purified from the labellar tissue of P. regina, to disperse D-limonene in aqueous solutions. As with DMSO, the ratio of flies showing the aversive PER to the D-limonene-dispersing solution with CRLBP was increased as the concentration of CRLBP was increased (Figure 4). In the presence of 20 µM CRLBP, 74.7% of flies showed aversive PER. Dispersion of D-limonene with BLG or BSA was far less effective.
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Electrophysiological response of the `fifth cell' to D-limonene
Since D-limonene is highly toxic and effectively induced aversive behavior of P. regina as mentioned above, we carried out electrophysiological experiments to find which taste receptor neuron responded. We mainly used D-limonene for stimulation, because the other monoterpenes did not induce such sensitive responses from the `fifth cell' (Data not shown).
The impulses of the four functionally different chemoreceptor neurons in a labellar sensillum were discriminated from each other by their amplitudes; the `fifth cell' stimulated with 0.5 M CsCl, 0.74 ± 0.08 mV (n = 20); the water receptor cell with 10 mM NaCl, 0.49 ± 0.08 mV (n = 20); the sugar receptor cell with 0.5 M sucrose, 1.04 ± 0.04 mV (n = 20); the salt receptor cell with 0.5 M NaCl, 1.29 ± 0.11 mV (n = 20).
In the labellar sensillum of P. regina, up to 4% pure DMSO did not
induce any obvious impulses except for the impulses from the water receptor
cell (see Figure 5A bottom).
Figure 5A (top) shows a
recording from the LL type of labellar sensillum to
D-limonene-dispersing 10 mM NaCl solutions with 4% DMSO. The small
impulses appeared immediately after the beginning of the stimulation and
gradually diminished by adaptation, while the larger impulses appeared after a
significant latency. Referring to the standard amplitudes of impulses
(mentioned above), it is tentatively defined that the small (0.53 ±
0.03 mV, n = 10) and the large impulses (0.76 ± 0.11 mV,
n = 10) are derived from the water receptor cell and the `fifth
cell', respectively. In this record, 8 s latency is seen until the first
appearance of the `fifth cell' impulse, probably because it takes time for
D-limonene molecule to be transferred from the DMSO-containing
environment in the stimulus solution to an intrinsic carrier protein, CRLBP in
the receptor lymph. Figure 5A
(bottom) is the control record in the absence of D-limonene. The
impulses of the `fifth cell' diminish, and only the impulses of the water
receptor cell are seen. It is therefore concluded that the `fifth cell'
responds to D-limonene. Among the examined sensilla, we found some
(2 in 56 trials) in which, for unknown reasons, the water receptor cell
generated no impulses. Figure
5B (top) shows the impulse caused by
D-limonene-dispersing 10 mM NaCl solution with 4% DMSO in one such
sensilla. Only the impulses of the `fifth cell' (0.74 ± 0.05 mV,
n = 10) can be clearly seen. Here we estimated responding neurons
based only on the impulse amplitudes, hence we use the traditional term `fifth
cell' as a tentative name for the D-limonene-sensitive neuron.
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Figure 6A shows the impulses generated by D-limonene dispersed in 10 mM NaCl solution with 1 µM CRLBP. The impulses of the water receptor cell (0.53 ± 0.04 mV, n = 13) and the `fifth cell' (0.79 ± 0.10 mV, n = 25) can be seen. These impulses are qualitatively similar to those in Figure 5A, in which DMSO was used to disperse D-limonene in the stimulus solution. This implies that CRLBP can help D-limonene to be dissolved in an aqueous environment. In the control record from the same sensillum stimulated with 10 mM NaCl solution containing 1 µM CRLBP (Figure 6B), only the impulses of the water receptor cell did appear. Figure 6C shows four types of nerve impulses recorded in the LL type of labellar sensillum: (a) the impulses of the `fifth cell' (to 0.5 M CsCl); (b) the water receptor cell (to 10 mM NaCl); (c) the sugar receptor cell (to 0.1 M sucrose); and (d) the salt receptor cell (to 0.5 M NaCl). D-Limonene, when dispersed with BLG or BSA instead of CRLBP, evoked fewer impulses from the `fifth cell', at least for 30 s after the beginning of stimulation (data not shown). This suggests that BLG or BSA are acting differently to CRLBP in the taste sensillum of P. regina (see Discussion).
Except for the cases as shown in Figure 5B, we usually observed that the `fifth cell' and the water receptor cell impulses were generated by stimulation with D-limonene-containing solutions. However, the time course of impulse generation in the `fifth cell' was not always constant. The duration of the latency varied, and the peak of the `fifth cell' activity, at which the highest impulse frequency was observed, appeared at different times after the beginning of stimulation. At the present time, it is unknown why such differences appeared among the sensilla. In fact, these differences made it difficult to investigate the concentration–response relationship of the `fifth cell' activity, and so we selected the number of impulses generated for 5 s at the peak of the cell activity and plotted these in Figure 7A. Figure 7A shows such concentration-response relationships of D-limonene dissolved with DMSO (closed circles) and with CRLBP (open circles), respectively. The D-limonene concentration dependency of the aversive behavior is also indicated in Figure 7B.
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Discussion |
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The `fifth cell' and its role in the taste system of the fly
We can hypothesize that the sense of taste in insects is also categorized
into some fundamental tastes, four functionally differentiated taste receptor
cells in a sensillum may primarily be responsible for such fundamental tastes.
The electrophysiological action of the sugar receptor cell, which responds not
only to sugars but also to nucleotides
(Amakawa et al., 1992)
or amino acids (Shiraishi and Kuwabara,
1970), induces feeding PER in the hungry flies
(Dethier, 1976). This implies
that the sugar receptor cell of the fly is responsible for a more general sign
of food intake rather than a sign of sugars. Both water and salts are
indispensable supplements. The fly chooses which one to take in order to
optimize its internal water–salt balance. The feeding PER induction to
water or salt may be regulated by the central nervous system referring to the
internal requirement (Dethier,
1976; Moss and Dethier,
1983).
As for the `fifth cell', how does its activity reflect on the behavior of
the fly? Prior to the present paper, Liscia and Solari reported that the
`fifth cell' of P. terraenovae responded to some substances that
humans find bitter (Liscia and Solari,
2000). In addition to the electrophysiological results, Liscia and
Solari showed that the fly preferred pure sucrose solution to mixtures of
sucrose plus quinine or amiloride. These results might easily lead to the
conclusion that the `fifth cell' of the fly has a functional analogy to the
bitter taste receptor cell of humans. In P. regina, however, it has
been reported that alkaloids inhibit the responsiveness of the sugar receptor
cell (Dethier and Bowdan,
1989,
1992). Recently, Sadakata
et al. discussed the inhibitory effects of amiloride on the sugar
receptor cell of the fleshfly, Boettcherisca peregria, but did not
show any response to amiloride itself
(Sadakata et al.,
2002). If substances that taste bitter to humans have inhibitory
effects on the sugar receptor cell in P. terraenovae, such inhibitory
effects could explain the behavioral results of Liscia and Solari
(Liscia and Solari, 2000),
independently of the `fifth cell' activity. In order to indicate a direct
contribution of the `fifth cell' activity to insect behavior, it is necessary
to evaluate the behavior induced by the electrophysiological activity of the
`fifth cell'.
In our study, the toxicity of D-limonene and other monoterpenes (Figure 1) and their gustatory effectiveness estimated by the aversive PER test (Figure 3) showed the same order. Therefore, it seemed convenient to use D-limonene and other monoterpenes as warning taste stimuli, which would activate the `fifth cell'. However, monoterpenes purified as essential oils were not directly applicable for the electrophysiological tip-recording method. The sidewall recording method (Morita and Yamashita, 1955) would have been an alternative, but the aversive movement of the labellum, which often occurrs even in the isolated head preparation, prevented us from inserting an electrode into the sidewall of a fine sensillum and from keeping it stable during impulse recording. Thus, we attempted the tip-recording method, in which monoterpene-containing electrolytes were used as the stimuli, and carried out the behavioral tests with the same stimuli. Such stimuli evoked the impulses of the water receptor cell as well (Figures 5A top and 6A). However, it is thought that it is not the impulses of the water receptor cell, but rather those of the `fifth cell' that trigger the aversive reactions, because the impulses of the water receptor cell by themselves did not induce any aversive reactions from the fly.
If the activation of the `fifth cell' triggers the aversive PER, the
electrophysiological latency should be shorter than the behavioral latency
(Dethier, 1968). Inspecting the
impulse recordings to D-limonene-containing solutions in detail,
2–15 s latencies are seen. Sometimes, the aversive PER occurred later
than 20 s after the beginning of stimulation. However, there was some overlap
and we could not measure the electrophysiological and the behavioral latencies
in the same animal at the same time. As for concentration dependency, there is
little inconsistency between the electrophysiological activity of the `fifth
cell' and the aversive reaction of the fly
(Figure 7). Thus, all of our
results tend to suggest that the `fifth cell' is a warning cell to induce
aversive reaction from the fly.
Nevertheless, aversive behavior is not always induced when the `fifth cell'
generates impulses. Dethier (Dethier,
1976) and Ozaki et al.
(Ozaki et al., 1995)
observed that apple juice stimulation of the taste sensillum of P.
regina elicited the impulses of both the sugar and the `fifth cell'. In
the case of apple juice, the fly did not show the aversive PER but the feeding
PER. Unknown components of apple juice, which stimulate the `fifth cell', may
have weak toxicity. As has been discussed in the mammalian bitter taste
receptor cell (Garcia and Hankins,
1975), the `fifth cell' of P. regina might also respond
to potentially toxic substances, even if they are not really toxic. This seems
reasonable, considering the relationship between deterrence and toxicity of
plant secondary compounds for phytophagous insects
(Bernays, 1991). Monoterpenes,
some of which were used in the present work, belong to one of the biggest
groups of secondary metabolites of plants. Plants use the toxicities of these
compound to defend themselves against herbivores, but some insects counter
such toxins by evolving detoxification systems to protect themselves. Thus,
the sensitivity of a contact-chemoreceptor neuron to phagostimulants may be
diverse (Bernays and Chapman,
2000).
In fact, the `fifth cell' responds to quite different structures of
molecules, the toxicities of which have not been investigated in detail. This
is one of the main reasons why the `fifth cell' has not been given a name
according to its function. Considering the study of Liscia and Solari
(Liscia and Solari, 2000) and
the present work together, however, we can now define this insect taste
receptor cell as a deterrent cell, or more precisely a warning cell for
gustatory suspicion of toxicity. It is noticed that this cell is functionally
analogous to the mammalian bitter taste receptor cell, although all substances
bitter for humans or other mammals may not be recognized as deterrents in
insects.
CRLBP, an odorant-binding protein in the taste system and its role
In 1995, a unique type of OBP, called CRLBP, was found by Ozaki et
al. to be widespread in the olfactory and taste organs of P.
regina. Their findings were reminiscent of Dethier's observation
(Dethier, 1972) that the
`fifth cell' of P. regina responds to some vapors, and that the
`fifth cell' could be involved in the recognition of volatile, more generally
lipophilic substances. Odor perception by contact-chemoreceptor neurons
reported in other insects (Städler
and Hanson, 1975;
Städler, 1984) may also
involve lipophilic substance transport by CRLBP or its relatives.
Prior to the discovery of CRLBP, Schmale et al.
(Schmale et al.,
1990) and Bläker et al.
(Bläker et al.,
1993) reported a gustatory homolog of mammalian OBP, von Ebner's
gland protein (VEGP), and predicted that VEGP could be a carrier protein for
some lipophilic, probably bitter substances. Considering the role of the
`fifth cell' discussed in the present paper, CRLBP seems to be functionally
analogous to VEGP, although mammalian and insect OBPs have quite different
structures. VEGP has a lipophilic inside pocket of ß-barrel type but
CRLBP does not. Because VEGP could not be obtained commercially, we examined
BLG, which is also a mammalian lipophilic ligand-binding protein that has a
ß-barrel type inside pocket like VEGP. However, we found that BLG, which
hardly solubilized D-limonene, did not replace CRLBP
(Figure 4). The ligand-binding
spectrum of BLG may be different from that of CRLBP, probably because of their
different structures. Presumably, mammalian and insect OBP families
convergently appeared in different phylogenetic branches of animal evolution,
so that they have different ligand-binding spectra and are not functionally
compatible with each other.
As shown in Figure 4, 20
µM BSA solubilized 30.1 ± 1.5 µg/ml D-limonene, while
1.25 µM CRLBP solubilized 23.5 ± 3.4 µg/ml. Nevertheless,
D-limonene with 20 µM BSA did not elicit aversive PER as
effectively as with 1.25 µM CRLBP. Possibly BSA would not pass
D-limonene to the receptor molecule on the `fifth cell' as
perfectly as CRLBP, the lipophilic ligand carrier in the taste and the
olfactory organs of P. regina
(Ozaki et al., 1995).
When comparing the data of Figure
4 and
7, we find that 4% DMSO
consistently solubilized 90 µg/ml of D-limonene and
elicited 70% aversive PER. However, there is a quantitative inconsistency
on the effect of CRLBP; Figure
4 shows that 5 µM CRLBP solubilized 158 ± 19
µg/ml of D-limonene to elicit 45% aversive PER;
Figure 7 shows that 100
µg/ml D-limonene solubilized with the same amount of CRLBP
elicited 85% aversive PER. The most likely explanation for this inconsistency
is structural and/or functional differences in the purified CRLBP, as we used
different batches in the two experiments.
It was reported that the CRLBP is a major protein component in the labellar
contact-chemosensilla in P. regina
(Ozaki et al., 1995),
but we do not exactly know its concentration in the sensilla. Considering that
the concentration of the PBP of Antheraea polyphemus was measured to
be 10 mM in the olfactory sensilla (Klein
et al., 1986;
Kaissling, 1998), we can
roughly estimate the intrinsic concentration of the CRLBP to be in the same
range. The CRLBP concentrations used in this study were 1000 times lower,
because of technical difficulties for large-scale purification of CRLBP from
the fly tissue. If we could use the intrinsic concentration of CRLBP, the
gustatory sensitivity of the `fifth cell' and the aversive PER sensitivity of
the fly might be increased and/or the electrophysiological and the behavioral
latencies might be shortened.
The results of the present study suggest that the taste receptor molecules
of the `fifth cell' to which CRLBP passes its ligands might resemble olfactory
receptors. In insect olfactory sensilla, the ligand-solubilizing effect of
OBPs has been electrophysiologically demonstrated by Van den Berg and
Ziegelberger (Van den Berg and
Ziegelberger, 1991), and recently a contribution of OBPs to
receptor specificity has been shown in addition
(Pophof, 2002). Our study in
Phormia is the first electrophysiological study on the role of an OBP
in an insect taste sensillum.
Which receptor molecules on the `fifth cell' can receive a ligand from CRLBP? How does the CRLBP bind or release the ligand for the receptor molecule? In order to answer these questions and understand the warning mechanism of the `fifth cell', especially for lipophilic toxins, the ligand-binding spectrum of CRLBP and its carrier-receptor relation should precisely be investigated.
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Acknowledgments |
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We thank Ms Maki Takehara and Mr Atsushi Seto for their technical assistance in the PER test and Ms Kaori Tagashira for her help in electrophysiological experiment. This study was supported by grants from the HFSP Organization and the PROBRAIN to M.O.
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References |
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Accepted April 10, 2003
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