Chem. Senses 27: 495-504,
2002
© Oxford University Press 2002
Unisex Pheromone Detectors and Pheromone-binding Proteins in Scarab Beetles
1 Laboratory of Chemical Prospecting, National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba 305-8634, Japan 2 Honorary Maeda-Duffey Laboratory, Department of Entomology, University of California at Davis, Davis, CA 95616, USA
Correspondence to be sent to: Walter S. Leal, Honorary Maeda-Duffey Laboratory, Department of Entomology, University of California at Davis, Davis, CA 95616, USA. E-mail: wsleal{at}ucdavis.edu
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Olfaction was studied in two species of scarab beetle, Anomala octiescostata and Anomala cuprea (Coleoptera: Scarabaeidae: Rutelinae), which are temporarily isolated and use the same sex pheromone compounds, (R)-buibuilactone and (R)-japonilure. Single sensillum recordings in A. octiescostata revealed highly sensitive olfactory receptor neurons (ORNs) (threshold <1 pg) that were tuned to the detection of the green leaf volatile compound (Z)-3-hexenyl acetate. As opposed to similar ORNs in another scarab species, Phyllopertha diversa, in A. octiescostata a diazo analogue elicited much lower neuronal responses than the natural ligand. Detectors for other floral and leaf compounds were also characterized. Extremely stereoselective ORNs tuned to sex pheromone were identified in male and female antennae. Biochemical investigations showed that, in A. octiescostata and A. cuprea, the pheromone-binding proteins (PBPs) isolated from male antennae were identical to PBPs obtained from female antennae. AoctPBP and AcupPBP had seven different amino acid residues. Binding of AoctPBP to (R)-japonilure is shown. PdivOBP1, which is also known to bind to (R)-japonilure, differed from AcupPBP in only two amino acid residues, one at the N-terminus and the other near the C-terminus. The structural features of the Bombyx mori PBP are compared with the sequences of eight known scarab odorant-binding proteins.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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For an insect the environment contains a myriad of chemical compounds. Most of them are physiologically irrelevant (noise), whereas others may be essential because they carry information (semiochemicals) about mate finding (sex pheromone), food sources (kairomones), oviposition sites (oviposition attractants) and many other features of the environment. While flying en route to a pheromone or other chemical source insects encounter intermittent signals with stimulus present in broken bursts of some tens of milliseconds in duration that occur with intervals of some hundreds of milliseconds (Murlis, 1997
).
This has led to the development of a remarkable selective, sensitive and
dynamic olfactory system (Kaissling,
2001).
From the perspective of molecular recognition there is another
complication. Information-carrying small hydrophobic ligands must reach the
olfactory receptors (Clyne et al.,
1999; Vosshall et
al., 1999) in order to be transduced into neuronal activities
(spikes). However, these olfactory receptors are located on islands (the
dendrites) in the sensillar lymph and many odorants cannot `swim' (they are
water insoluble). Thus, the olfactory processing starts with odorant-binding
proteins (OBPs) (Kaissling,
2001) ferrying the ligands to their receptors. Various functions
have been suggested for these proteins since their discovery
(Vogt and Riddiford, 1981).
OBPs are postulated to solubilize semiochemicals, protect the ligands from
degradation and participate in fast inactivation of chemical signals. Clearly
most of the ligands need to be solubilized. It has recently been demonstrated
that the raison d'être of these proteins is more than to
solubilize ligands. An OBP named LUSH is essential for the recognition of a
water-soluble semio-chemical (ethanol) in Drosophila melanogaster
(Kim and Smith, 2001). On the
basis of their binding abilities, OBPs have been considered to play a
significant part in the remarkable selectivity of the insect olfactory system,
but the literature is also dichotomous
(Campanacci et al.,
2001) with data suggesting that some OBPs are specific, whereas
others may be more `sloppy'. Structural biology studies with the
pheromone-binding protein (PBP) from the silkworm moth Bombyx mori
have indicated that the binding pocket forms a general hydrophobic surface for
binding (Sandler et al.,
2000). Interestingly, a C-terminal sequence, which is an extended
(unstructured) conformation in the pheromone—PBP complex, rearranges to
form a regular 
-helix that occupies the pheromone-binding pocket
(Horst et al., 2001),
thus `ejecting' the pheromone to the receptor.
We have been studying chemical communication in scarab beetles (Coleoptera:
Scarabaeidae). Identical pheromone chemistry has been characterized in species
that share the same habitat in the main land of the Japanese archipelago
(Leal, 1998), but are
temporarily isolated. For example, the sex pheromones of Anomala
octiescostata and Anomala cuprea are composed of
(R)-buibuilactone and (R)-japonilure (compounds 1 and 2
respectively) (see Figure 1) (Leal, 1991; Leal et
al., 1993,
1994a). While the sensory
physiology of the latter has been investigated in detail
(Leal and Mochizuki, 1993;
Larsson et al., 1999,
2001), little is known about
olfaction in the former (Leal,
1999). The purposes of this study were twofold. First, we aimed at
studying the sensory physiology in A. octiescostata with emphasis on
the olfactory receptor neurons (ORNs) involved in the detection of sex
pheromones, green leaf volatiles and floral compounds. Second, we isolated
PBPs from A. octiescostata and A. cuprea and cloned their
cDNAs in order to compare the primary structures of the PBPs from two species
that are involved in the detection of the same sex pheromone.
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Materials and methods |
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Insects
Eggs laid by field-captured females were collected daily and transferred to wet sand in ice cream cups, which were kept at 25°C. After hatching, grubs were individually transferred to ice cream cups filled with humus and supplied with slices of sweet potato or carrot, which were renewed weekly. Diapause termination in A. octiescostata (adults) and A. cuprea (yellow stage third-instar grubs) was achieved by cold treatment (10°C for 2-3 months) and then raising the temperature to 25°C. Adults were fed on an artificial diet for the silkworm moth B. mori (Nosan Co., Yokohama, Japan) and kept at 25°C and 50-70% relative humidity under a 16:8 h light:dark photoregime.
Semiochemicals
(R,Z)-5-(—)-(1-Octenyl)oxacyclopentan-2-one
[(R)-buibuilactone], (S)-buibuilactone,
(R,Z)-5-(—)-(1-decenyl)oxacyclopentan-2-one
[(R)-japonilure] and (S)-japonilure were obtained as
described previously (Leal,
1999). The synthesis of (Z)-3-hexenyl diazoacetate has
been reported elsewhere (Nikonov et
al., 2001). (Z)-3-Hexenol and (Z)-3-hexenyl
acetate were purchased from Wako Chemicals (Wako, Japan). Toyotama Koryo
(Tokyo, Japan) and Koei Kogyo (Yokohama, Japan) supplied anethole and geraniol
respectively.
Single sensillum recordings
Beetles were immobilized in 1.5 ml microfuge tubes that were cut at the
bottom. Their heads were sticking out of the hole and their antennae were
fixed by dental wax. The three lamellae of the antennae were separated and
held immobile with a thin metal pin. The indifferent (ground) electrode was a
thin silver wire inserted in the abdomen. Extracellular contacts with antennal
ORNs were established under a stereomicroscope with up to x300
magnification by inserting a tungsten electrode (sharpened electrolytically in
a KNO2 solution) through the cuticle adjacent to a sensillum
placodeum (Hubel, 1957). The
antennal preparation was continuously flushed by a charcoal-filtered and
moistened airstream flowing at 0.5 m/s through an 8 mm (inner diameter) glass
tube ending 10 mm before the preparation. Each test compound was applied to a
piece of filter paper (0.5 cm x 1 cm), which was dried for at least 10
min and inserted into a Pasteur pipette. Stimulus was performed by inserting
the tip of the test pipette into a hole 6 cm from the outlet of the glass tube
and air was blown through the pipette for 300 ms by a stimulus controller
(CD-02/E, Syntech, Hilversum, The Netherlands). The signal was amplified using
a Nihon Kohden MEZ-8300 amplifier (Tokyo, Japan). Antennal responses were
stored using Axotape 2.0.2 (Axon Instruments, Inc., Foster City, CA) and
visualized on a Nihon Kohden memory oscilloscope VC-11 (Tokyo, Japan). Spikes
were counted manually from the computer screen (a time bin of 250 ms). We
analyzed neuronal activity in the period 1 s before and 1 s after the onset of
stimulation. Positive responses were higher than two standard deviations of
the background activity (1 s period before stimulus onset). Net spikes were
calculated as the number of spikes during 1 s after (300 ms) stimulation minus
the number 1 s before stimulation. The results obtained were statistically
processed (Zacks, 1971) and
represented as dose—response plots.
Protein extracts
The beetles were anaesthetized on ice and their antennae and legs were
collected, frozen in liquid nitrogen and lyophilized. Homogenization was
performed in an ice-cold glass Dounce tissue grinder in 10 mM Tris—HCl,
pH 8. Homogenized samples were centrifuged twice at 12 000 r.p.m. (4°C for
5 min). The supernatants were concentrated by centrifugation under vacuum and
analyzed by native polyacrylamide gel electrophoresis (PAGE). In order to
perform sequencing, proteins were separated by native PAGE, transferred to
polyvinyl difluoride (PVDF) membranes using electroblotting and bands were cut
out and analyzed for microsequencing. For molecular mass measurements, bands
were cut out directly from gels, the proteins were electroeluted in a
microelectroeluter, concentrated by centrifugation in a Centricon 3 (Millipore
Corporation, Bedford, MA) and injected onto an on-line liquid
chromatograph—mass spectrometer (LC-MS). Binding assays followed a
previously reported protocol (Wojtasek
et al., 1998). Radiolabelled (R)-buibuilactone
and (R)-japonilure were prepared by Amersham Biosciences K. K.
(Tokyo, Japan) by catalytic reduction (with tritium gas) of the alkyne
precursors following a protocol for deuteration of these lactones
(Wojtasek et al.,
1998). Native PAGE binding assays were performed as described
previously (Wojtasek et al.,
1998).
Analytical procedures
Native PAGE (15%) analyses were performed on a Mini-PROTEAN II (Bio-Rad, Hercules, CA). Gels were stained with Coomassie blue R-250. N-terminal amino acid sequences were obtained on a Hewlett-Packard Protein Sequencer model 241 with phenylthiohydantoin (PTH) derivatives separated on a Hewlett-Packard (Agilent, Palo Alto, CA) series 1100 high-performance liquid chromatography (HPLC) system. Electrospray ionization mass spectra (ESI-MS) were obtained with a Hewlett-Packard on-line chromatograph—mass spectrometer equipped with an electrospray interface (LC/ESI-MS). HPLC separations were achieved on a Zorbax 300 CB-C8 column (150 mm x 2.1 mm and 5 µm) by using water and acetonitrile with 2% acetic acid as modifier in both solvents and a gradient from 20 to 80% acetonitrile in 15 min at a flow rate of 0.2 ml/min. The MSD parameters were as follows: skimmer collision-induced dissociation voltage (fragmentor) 120 V, drying gas flow 6 l/min, nebulizer pressure 310 KPa, nebulizer temperature 300°C and capillary voltage 3500 V. DNA sequences were obtained with an automated ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA). DNA and protein sequences were analyzed by using the MacVector software (Accelrys, Cambridge, UK).
cDNA cloning and sequencing
Total RNA was extracted (100 antennae) by using TRIzolTM Regent (Invitrogen Life Technologies, Carlsbad, CA) and mRNA was purified with the polyATract mRNA Isolation System (Promega, Madison, WI). First strand cDNA was synthesized in the presence of an oligo(dT) primer by using SuperScript IITM (Invitrogen). Two degenerate primers were designed on the basis of the first six residues of the N-terminal sequences of the isolated antenna-specific proteins from A. octiescosta and A. cuprea: ATGAG-(C/T)GA(A/G)GA(A/G)ATGGA and ATGTC(A/C/G/T)-GA(A/G)GA(A/G)ATGGA. The two primers differed only in the codons for serine (italics). Polymerase chain reactions (PCRs) were carried out in a MiniCycler (Model 150, MJ Research) by using Taq DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN) with an annealing temperature at 46°C. PCR fragments were cloned into pCR2.1-TOPO (Invitrogen) and transformed into OneShotTM Top10F' competent cells (Invitrogen). Positive insertions were identified by PCR (with M13 forward and M13 reverse primers). Sequence reactions were carried out using the Dye Terminator Cycle Sequence FS Ready Reaction Kit (Applied Biosystems) according to the instruction manual. Nucleotide sequences were determined from both strands from at least five independent clones.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Green leaf volatile detectors
Ninety-seven ORNs in the male antennae responded to the green leaf volatile
compound (Z)-3-hexenyl acetate
(Figure 1.8) (for chemical
structures, see Figure 1) in a
dose-dependent manner (Figure
2). These ORNs are the most sensitive detectors for semiochemicals
investigated thus far in scarab beetles
(Leal and Mochizuki, 1993;
Hansson et al., 1999;
Larsson et al., 1999,
2001;
Nikonov et al., 2001)
and showed a threshold lower than 1 pg
(Figure 3). They were
remarkably silent when challenged with the related alcohol
(Z)-3-hexenol (Figure
1.9) even with doses five orders of magnitude higher than the
threshold to the specific stimulus (Figure
3). Neuronal activity was always recorded from the sensilla
containing the (Z)-3-hexenyl acetate ORNs when the preparations were
stimulated with anethole (Figure
1.11). Although the threshold was almost four orders of magnitude
higher, the detectors responded in a dose-dependent manner
(Figure 2). The
(Z)-3-hexenyl acetate ORNs in A. octiescostata responded in
a dose-dependent manner to a photoaffinity-labelled compound
(Z)-3-hexenyl diazoacetate (Figure
1.10) (Figure 2).
In contrast to the same type of ORNs previously identified in another scarab
beetle Phyllopertha diversa
(Nikonov et al.,
2001), the threshold of the ORNs in A. octiescostata to
the diazo compound was three orders of magnitude higher than the threshold for
the natural compound. ORNs tuned to (Z)-3-hexenyl acetate were found
also in 61 sensilla in female antennae, but they had a 10 000-fold higher
threshold (10 ng), which is a very large difference. In addition, 38 sensilla
housing ORNs sensitive and selective to (Z)-3-hexenol were found in
both male and female antennae (data not shown).
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Detectors for floral compounds
Thirty-three sensilla in males had a combination of ORNs, with one responding to phenylethyl propionate (Figure 1.12) and geraniol (Figure 1.13). The responses to these two stimuli showed dose dependence, with a higher threshold to geraniol (Figure 4). Again, it was not possible to determine accurately whether the spikes were generated by the same or different ORNs. This type of combination neurons was also found in female antennae (26), with similar threshold and dose—response curves (data not shown).
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Sex pheromone-specific detectors in males and females
ORNs tuned to the major constituent of the pheromone system
(R)-buibuilactone were found in both male (115) and female antennae
(43). Although the pheromone detectors in males and females showed similar
dose—response curves (Figure
5), female ORNs showed a 10 times higher threshold. These
detectors were not stimulated by the antipode of the major sex pheromone
component even when challenged with 10 000 ng of (S)-buibuilactone,
in agreement with the suggested enantiomeric anosmia in A.
octiescostata (Leal,
1999). ORNs responding to the second pheromone component
(R)-japonilure (Figure
1.2) were found in both male (53) and female (25) antennae, but
they showed a higher threshold (100 ng) (data not shown). The
(R)-japonilure detectors were silent when challenged with 10 000 ng
of (S)-japonilure.
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Identification of pheromone-binding proteins
The native PAGE analyses of olfactory and control tissues from the two
species of scarab beetles A. octiescostata and A. cuprea
showed antenna-specific bands in these two species
(Figure 6). The
antenna-specific proteins showed mobility similar to the OBPs identified to
date in insects, in particular OBPs from other scarab beetles (Wojtasek et
al., 1998,
1999;
Peng and Leal, 2001;
Deyu and Leal, 2002). The
antenna-specific band in A. octiescostata showed a slightly faster
mobility than its counterpart in A. cuprea. In agreement with the
observation that males and females of A. octiescostata (this paper)
detect the female-produced sex pheromone, the antenna-specific bands were
detected in both sexes (Figure
6). This was also observed in A. cuprea (data not shown)
which also possess pheromone-detecting ORNs in the male and female antennae
(Larsson et al.,
1999).
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The native PAGE binding assays showed that AoctPBP binds to (R)-japonilure (Figure 7). However, we were not able to demonstrate association of the protein with the major pheromone constituent (R)-buibuilactone. On the other hand, assays with crude antennal extracts showed a considerable amount of radioactivity associated with slow migrating proteins in the gel (Figure 7), probably due to non-specific binding.
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The N-terminal sequences of the antenna-specific proteins in A. octiescostata males (MSEEMEELAKQL-HND-VAQT) and females (MSEEMEELAKQLHND) were indistinguishable. In addition, the amino acid sequences of the protein specific to A. cuprea antennae (male and female) gave the sequence MSEEMEELAKQLHND-VAQT.
Characterization of A. octiescostata and A. cuprea pheromone-binding proteins
Two types of PCR products were amplified from A. octiescostata and
A. cuprea with 
500 and 550 bp, yet the open reading frames for
these cDNAs were identical and both coded for proteins with 116 amino acids.
Sequences of several clones from A. octiescostata (eight from males
and seven from females) showed that the male and female PBPs are identical
(Figure 8). AoctPBP showed six
well-conserved cysteine residues at positions 16, 44, 48, 86, 95 and 114.
Based on the encoded sequence, the protein has a pI of 4.4 and a
(calculated) molecular mass of 12 940 Da (12 934 Da when considering the
formation of three disulphide bonds). The mass spectral data of the proteins
isolated from the male and female antennae were indistinguishable. The charge
envelope was deconvoluted to give a molecular mass of 12 934 Da
(Figure 9), indicating that
indeed male and female proteins are identical.
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Although the two species of scarab beetles A. octiescostata and A. cuprea use the same sex pheromone components [(R)-buibuilactone and (R)-japonilure] their PBPs are not identical. Their amino acid sequences had seven different amino acid residues (Figure 8). AcupPBP differed slightly in molecular mass (12 944 Da or 12 938 Da with disulphide bridges) and pI (calculated as 4.49). Scarab PBPs/OBPs show high amino acid identities (86-98%) (Figure 8), with the proteins from A. cuprea and P. diversa (PdivOPB1) differing in only two amino acid residues, one at the N-terminus (Met-1 versus Leu-1) and the other near the C-terminal sequence (His-101 versus Gln-101).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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An interesting characteristic of the sensory physiology of scarab beetles is the existence of highly sensitive and selective ORNs that are tuned to (Z)-3-hexenyl acetate. The threshold of these ORNs to the green leaf volatile acetate far exceeds the sensitivity of the sex pheromone detectors. The green leaf volatile ORNs in A. octiescostata showed even lower threshold responses (<1 pg) than the ORNs previously identified in P. diversa (Hansson et al., 1999
; Nikonov et al.,
2001) and A. cuprea
(Larsson et al.,
2001). The high selectivity of these neurons is demonstrated in
the lack of responses to the related alcohol (Z)-3-hexenol and other
green leaf compounds, such as (E)-2-hexenyl acetate,
(E)-2-hexenol and (E)-2-hexenal. Another feature of the
sensilla placodea housing the ORNs tuned to (Z)-3-hexenyl acetate is
the response to anethole as the second best stimulant. The threshold responses
to anethole in A. octiescostata were four orders of magnitude higher
than the response to (Z)-3-hexenyl acetate
(Figure 2). The amplitudes of
the spikes generated by the two stimuli are indistinguishable, but two lines
of evidence have suggested that different ORNs (housed in the same sensilla)
are activated. First, the (Z)-3-hexenyl acetate ORNs did not respond
even to other structurally related green leaf volatile compounds. Second,
sensilla placodea in scarab beetles in general contain more than one ORN
(Meinecke, 1975;
Kim and Leal, 2000).
In the field, adults of A. octiescostata are attracted to and
voraciously feed on dandelion (Taraxacum officinale). Field
experiments have demonstrated that the attraction is semiochemically mediated,
with (Z)-3-hexenyl acetate being one of the attractants
(Leal et al.,
1994b).
The occurrence of ORNs specific to (Z)-3-hexenyl acetate in P.
diversa and A. cuprea led us to suggest that these ORNs might
carry homologous receptor proteins in these two species of scarab beetle
(Larsson et al.,
1999). Despite the remarkable selectivity of these ORNs, the
photoaffinity-labelled (Z)-3-hexenyl diazoacetate
(Figure 1.10) mimics the
natural ligand so perfectly that the whole detecting machinery in P.
diversa was tricked by the synthetic ligand. The green leaf acetate
receptors responded to the diazo compound with even higher neural activity
than that elicited by (Z)-3-hexenyl acetate
(Nikonov et al.,
2001). Interestingly, (Z)-3-hexenyl diazoacetate could
not trick the olfactory system in A. octiescostata
(Figure 2). The responses
elicited by the diazo compound were three orders of magnitude lower than the
response to the natural acetate. These results suggest that, although tuned to
the same natural ligand, namely (Z)-3-hexenyl acetate, the specific
olfactory receptor proteins in P. diversa and A.
octiescostata are different. Olfactory receptors have been identified in
D. melanogaster (Clyne et
al., 1999; Vosshall
et al., 1999). It has been observed (L.B. Vosshall,
personal communication) that, although D. melanogaster and
Caenorhabditis elegans `smell' diacetyl (2,3-butanedione), there is
no receptor protein in flies with significant amino acid similarity to the
nematode diacetyl receptor odr10 (Zhang
et al., 1997). This is consistent with the hypothesis
that A. octiescostata and P. diversa detect
(Z)-3-hexenyl acetate with different olfactory receptor proteins.
In addition to the green leaf volatile detectors, A. octiescostata
possesses ORNs for the detection of floral compounds
(Figure 4). All sensilla
placodea housing ORNs responding to phenylethyl propionate also responded to
geraniol. These compounds are biologically relevant for A.
octiescostata as a mixture of anethole, geraniol and phenyl propionate
increases the trap catches of female beetles
(Leal et al., 1994b).
Given that the spike amplitudes in response to the two semiochemicals are
identical, it was not possible to determine accurately whether the neuronal
activities were generated by the same or different ORNs co-localized in the
same sensilla. Considering that the chemical structures of the two ligands are
unrelated (Figure 1) and taking
into consideration that each sensillum generally contains more than one ORN
(Meinecke, 1975;
Kim and Leal, 2000), it is
tempting to conclude that phenylethyl propionate and geraniol stimulate
different ORNs co-localized in the same sensilla.
As has been observed in A. cuprea
(Leal and Mochizuki, 1993;
Larsson et al.,
1999), the pheromone-detecting ORNs in A. octiescostata
also showed remarkable enantiomeric specificity. The pheromone detectors were
silent even when challenged with (S)-buibuilactone at x10 000
higher concentrations than the threshold to the major pheromone component
(R)-buibuilactone. Although the Japanese beetle Popillia
japonica and the Osaka beetle Anomala osakana detect both
enantiomers of japonilure, one as a sex pheromone and the other as a
behavioural antagonist (Wojtasek et
al., 1998), it has been suggested that these are particular
cases, with the inability to detect non-natural stereoisomers of pheromones
(enantiomeric anosmia) being a common feature in the sensory physiology of
scarab beetles (Leal,
1999).
Pheromone detectors have been found in the male and female antennae of
A. octiescostata, consistent with the observation that both male and
female beetles are attracted to the female-released sex pheromone
(Leal et al., 1994a).
As in the Japanese beetle (Kim and Leal,
2000), the number of pheromone-detecting sensilla placodea in
female antennae is lower than their counterparts in male beetles. In moths,
female antennae of most species are apparently anosmic to their own pheromone
while autodetection of female pheromones is a less observed phenomenon
(Schneider et al.,
1998) (see also references therein).
The lack of dimorphism in scarab beetles is also observed in the pheromone-detecting biochemical machinery. PBPs are found in the male and female antennae in A. octiescostata and A. cuprea (Figure 6). The identical amino acid sequence in the male and female PBPs was demonstrated by cloning the cDNAs of male and female AoctPBP (Figure 8), as well as by the measurement of the molecular masses of the native proteins (Figure 7). That A. octiescostata and A. cuprea PBPs are `unisex' is consistent with the electrophysiological data.
Despite the fact that A. octiescostata and A. cuprea use
the same sex pheromone and, consequently, their sensory systems are involved
in the detection of identical compounds, AoctPBP and AcupPBP differed in seven
residues at positions 18, 61, 64, 75, 88, 106 and 111. Even OBPs from scarab
beetles of different subfamilies can interact with the same ligand. As shown
here (Figure 7) the PBP from
A. octiescostata, which is a ruteline species, binds to
(R)-japonilure. OBP1 from P. diversa, which is a
melolonthine species, binds to the same ligand
(Wojtasek et al.,
1999). However, the amino acid sequences of the two proteins
(AoctPBP and PdivOPB1) showed only 93% identity
(Figure 8). It would be
interesting to determine whether the different amino acid residues of these
OBPs are involved in forming the binding cavities of these proteins.
We have previously observed a pH-dependent conformational change in the PBP
of the silkworm moth B. mori
(Wojtasek et al.,
1998), which leads to the formation of the acid and basic forms of
the protein, i.e. BmPBPA and BmPBPB respectively
(Damberger et al.,
2000). This conformational change was hypothesized as a mechanism
for the release of pheromone to the receptors upon interaction with negatively
charged membranes at the surface of dendrites
(Leal, 2000). Based on
structural data, it was proposed that three histidine residues at positions
69, 70 and 95, which are strictly conserved across all known lepidopteran PBPs
and OBPs, might contribute to the conformational change
(Sandler et al.,
2000). Comparison of BmPBPA with the protein
conformation in the bound complex revealed that the histidine side-chains are
indeed more widely separated in BmPBPA, which may be the result of
charge repulsion by protonation at a slightly acidic pH value
(Horst et al., 2001).
Interestingly, scarab PBPs (Figure
8) possess three strictly conserved histidine residues (His-13,
His-26 and His-102).
Of the residues involved in binding to bombykol in BmPBPB, the
most variable in other lepidopteran PBPs (Met-61, Leu-62, Ile-91 and Val-114)
are either valine, leucine, isoleucine or methionine. These variations are
suggested to be specificity determinants, with the binding cavity being most
sensitive to the length of the pheromone
(Sandler et al.,
2000). Hitherto, there is no structural data on scarab OBPs for
determining which residues are involved in pheromone binding. However, it is
worth mentioning that the four variable residues at positions 61, 64, 71 and
82 (Figure 8) are either
valine, leucine, isoleucine or methionine. Interestingly, in two proteins that
bind to (R)-japonilure, AoctPBP and PdivOBP1, these four residues are
identical. On the other hand, AcupPBP is involved in the detection of the same
pheromone system as AoctPBP, but the two proteins have a pair of different
residues at two of the positions, i.e. Val-61 and Val-64 in AoctPBP and Ile-61
and Ile-64 in AcupPBP.
In summary, research has been conducted in order to obtain a better insight into the sensory physiology of scarab beetles. ORNs specific to the detection of ecologically significant compounds have been identified in A. octiescostata. In addition to the remarkably sensitive detectors for (Z)-3-hexenyl acetate, other sensilla placodea housing ORNs tuned to floral compounds were characterized. Pheromone detectors were identified in male and female antennae. The PBPs isolated from male antennae were identical to those isolated from female antennae. The PBPs from the two species investigated, namely A. cuprea and A. octiescostata, showed only 93% amino acid similarity. Comparison of the sequences of the scarab OBPs identified to date with the structure of BmPBP shows some interesting features of scarab PBPs, which might be related to the pH-dependent conformational change and specificity binding determinants.
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Acknowledgments |
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The Japanese component of this research was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (BRAIN) granted to W.S.L. Work in the USA was made possible through direct financial support from the department, college, and chancellor's office at UCD and by USDA grant no. 01-8500-0506-GR. We appreciate the helpful discussion with members of our group, in particular Yuko Ishida and Goede Schueler and their critique of an earlier draft of the manuscript.
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Accepted February 28, 2002
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