Chem. Senses 26: 885-896,
2001
© Oxford University Press 2001
Immunolocalization of Odorant-binding Proteins in Noctuid Moths (Insecta, Lepidoptera)
Max-Planck-Institut für Verhaltensphysiologie, D-82319 Seewiesen, Germany 1 Present address: Institute of Zoology, Academia Sinica, Beijing, China
Correspondence to be sent to: R. A. Steinbrecht, Max-Planck-Institut für Verhaltensphysiologie, D-82319 Seewiesen, Germany. e-mail: steinbrecht{at}mpi-seewiesen.mpg.de
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Odorant-binding proteins were studied in the noctuid moths Agrotis segetum, Autographa gamma, Helicoverpa armigera, Heliothis virescens and Spodoptera littoralis using antisera raised against the pheromone-binding protein (PBP) and general odorant-binding protein 2 (GOBP2) of Antheraea polyphemus (Saturniidae). Proteins immunoreacting with these antisera were only found on the antennae and PBP and GOBP2 could be identified on western blots of males and females of all five species. PBPs were predominantly localized in sensilla trichodea and GOBP2 in sensilla basiconica, in good correlation with the stimulus specificity of the receptor cells in these sensilla. In H. armigera and H. virescens the majority of the s. trichodea immunoreacted with the antiserum against PBP of A. polyphemus; in A. segetum, A. gamma and S. littoralis, on the other hand, a high percentage of s. trichodea remained unlabelled. Probably, the PBP expressed in these sensilla is so different that it does not immunoreact with the antiserum used. Such a protein was found by native PAGE of antennal extracts of A. segetum and S. littoralis. These data correlate with the fact that the two heliothine species use pheromones with the same alkyl chain length as A. polyphemus, while the other three species use pheromones with shorter chains. In H. armigera, H. virescens, A. gamma and S. littoralis female antennae were also immunolabelled and a large number of PBP-expressing s. trichodea was consistently found. In S.littoralis this fits with the electrophysiologically recorded high pheromone sensitivity of female s. trichodea, whereas in females of H. armigera and H. virescens no or only weak responses to pheromone stimulation have been reported. Therefore, PBP expression in a sensillum does not necessarily imply pheromone sensitivity of its receptor cells.
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Odorant-binding proteins (OBPs) have now been characterized for a great number of insect species. Immunolocalization studies have shown that they are secreted into the sensillum lymph that surrounds the peripheral sensory processes of olfactory receptor neurons. Thus, they are present in the regions where the first steps of olfactory signal transduction take place and functions in odorant solubilization and transport, odorant deactivation and general detoxification have been proposed [for reviews see Pelosi, Pelosi and Maida, Steinbrecht and Vogt et al. (Pelosi and Maida, 1995
; Pelosi, 1998; Steinbrecht, 1998; Vogt et al., 1999)].
Because insect sensilla house only a limited number of olfactory receptor neurons (ORNs) (in pheromone-sensitive sensilla trichodea usually two or three), different groups of ORNs may have a different microenvironment and different OBPs may be associated with ORNs of different specificity. Thus, a more specific role of OBPs is possible, e.g. to preselect from the universe of odorants only certain classes of molecules and to bind and transport only these to the receptor proteins, which then ‘see’ only this preselection of odorants, including their specific stimulus (Vogt et al., 1991; Prestwich et al., 1995).
In the first immunocytochemical study using an antiserum against the PBP of Antheraea polyphemus it was shown that this antiserum not only labels sensilla on the antennae of this species, but also sensilla of other moth species, e.g. Bombyx mori and Autographa gamma. The latter, a noctuid moth, proved interesting in so far as only a fraction of the long s. trichodea was labelled, whereas the rest, although morphologically indistinguishable, remained unlabelled. This was in sharp contrast to the two silkmoth species, where the pheromone-sensitive long s. trichodea were labelled without exception (Steinbrecht et al., 1992).
Also in contrast to the silkmoth, we observed positive PBP labelling in long s. trichodea of female noctuid moths (Steinbrecht et al., 1994). In female silkmoths long s. trichodea are either absent, as in A. polyphemus and Antheraea pernyi, or express GOBP2, as in B.mori, fitting with their response to general (non-pheromone) odours (Steinbrecht et al., 1995).
These observations, and the fact that many noctuids are important pest insects, encouraged us to systematically study the expression of OBPs immunoreacting with our antisera against PBP and GOBP2 of A. polyphemus in several noctuid species. We choose Autographa gamma, Helicoverpa armigera, Heliothis virescens and Spodoptera littoralis and also performed immunoblotting and preliminary immunolabelling with Agrotis segetum. The results clearly demonstrate a complex pattern of OBP expression in noctuids which in a way reflects their complex pheromone signalling system.
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Animals
Spodoptera littoralis Boisd. and A. segetum Schiff. were obtained as pupae from Dr Bill Hansson (University of Lund), H. armigera Fabr. and H. virescens Hb. from Dr Elke Hartlieb (Seewiesen). Autographa gamma L. were caught with a light trap during the summer months in Seewiesen. Except for A. gamma, where the age of moths was unknown, moths of the other species were used 2–7 days after emergence.
Preparation of tissue extracts
Male and female antennae, heads, proboscides and legs of H. virescens were homogenized on ice for 20 min with a home-made motor driven homogenizer in 20 mM Tris–HCl buffer (pH 7.2) in the presence of 1 mM phenylmethylsulphonyl fluoride as protease inhibitor. Homogenates were centrifuged at 10 000 g for 15 min and the supernatants used in further experiments.
Native PAGE
Electrophoresis under non-denaturating conditions (native PAGE) was performed in polyacrylamide gels using a BioRad Mini-Protean II apparatus and a discontinuous buffer system according to Laemmli (Laemmli, 1970). Proteins were stained with Coomassie Brilliant Blue R250.
Antisera
The anti-PBP(Apol) and anti-GOBP2(Apol) antisera were raised by immunizing rabbits with purified native PBP or GOBP2 of A. polyphemus as described (Steinbrecht et al., 1992, 1995).
Immunoblotting
After electrophoretic separation, proteins were electrotransferred onto nitrocellulose membranes by the semi-dry blotting procedure of Kyhse-Anderson (Kyhse-Anderson, 1984). First, nitrocellulose membranes were blocked with 1% bovine serum albumin in phosphate-buffered saline and 0.05% Tween 20 for 1 h at room temperature and then incubated overnight with the anti-PBP(Apol) and anti-GOBP2(Apol) antisera at a dilution of 1:500. Bound antibodies were detected with goat anti-rabbit IgG coupled to horseradish peroxidase (Bio-Rad; dilution 1:3000) and 4-chloro-1-naphtol as substrate.
Immunocytochemistry
Antennae were cryofixed and freeze-substituted as described in detail by Steinbrecht (Steinbrecht, 1993), embedded in LR White resin (The London Resin Co., UK) and sectioned with a diamond knife. The section plane was oriented parallel to the long axis of the antenna so that the maximum number of sensilla was cross-sectioned (Figure 4A). Anti-PBP(Apol) and anti-GOBP2(Apol) were used as primary antibodies at dilutions of 1:3000–1:9000. Goat-anti-rabbit IgG conjugated with 10 nm colloidal gold (Amersham) served as secondary antibody at a dilution of 1:20. Optional silver intensification according to Danscher (Danscher, 1981) enlarged the grains to some 40 nm. Sections were stained with uranyl acetate. Details of the immunocytochemical protocols are given in Steinbrecht et al. (Steinbrecht et al., 1992, 1995).
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For qualitative comparison at least 100 sensilla from one or two animals were observed for each species and sex and labelled with anti-PBP(Apol) and anti-GOBP2(Apol) on consecutive sections. Most sensilla were checked twice with each antiserum at different levels. For quantitative evaluation of labelling density at least 12 labelled sensilla of a given type were chosen at random, photographed at 20 000:1 and the negatives scanned with a flatbed scanner (UMAX Powerlook II) connected to an Apple Power PC (8500/120). Image analysis (i.e. measurement of area and number of particles) was done with a customized version of NIH Image (v.1.59 and v.1.62, developed at the US National Institutes of Health, customized by Steve Barrett, Surface Science Research Centre, University of Liverpool) and self-made macros.
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Immunoblots
In a first set of experiments we tested whether the two antisera raised against purified native PBP and GOBP2 of A. polyphemus cross-reacted with proteins from antennal homogenates of male and female A. segetum and S. littoralis. Equal homogenates of male and female antennae of A. polyphemus were used as controls (Figure 1). In all three species tested and in both male and female antennae the two classes of proteins were clearly labelled by the antisera. The anti-PBP immunoreactive bands showed a different electrophoretic migration as compared with the anti-GOBP2 labelled bands and the proteins appeared to be present in different concentrations in the three species, as well as in males and females. A positive immunoreaction for anti-PBP(Apol) and anti-GOBP2(Apol) was also observed with antennal proteins from H. virescens, H. armigera and A. gamma [Figure 2 and data not shown; see also figure 1 in Steinbrecht et al. (Steinbrecht et al., 1992)]. It is noteworthy that in S. littoralis the bands stained by anti-PBP(Apol) were stronger in females than in males, while in males (and also in A. segetum) there were highly expressed proteins (marked * in Figure 1A) that had similar electrophoretic properties but did not immunoreact with anti-PBP(Apol).
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Further, we tested the tissue specificity of the two antisera used in this study by comparing immunoblots from crude extracts of male and female antennae with those of heads, proboscides and legs of H. virescens. Both anti-PBP(Apol) and anti-GOBP2(Apol) revealed immunoreactive proteins only in homogenates from male and female antennae, and not from the other tissues (Figure 2).
Sensillum types and immunolabelling
Noctuid antennae, like those of all Lepidoptera, are multimodal sensory organs carrying not only olfactory sensilla but also thermo-/hygrosensitive s. styloconica, mechanosensitive s. chaetica and s. campaniformia and s. chaetica with mechanosensitive and gustatory neurons. All these sensilla in our experiments did not show more than background labelling.
Olfactory sensilla either belong to the single-walled category (s. trichodea and s. basiconica) or to the double-walled s. coeloconica. The latter in all five species were not labelled by the two antisera (n > 100 s. coeloconica). Positive labelling was only encountered with the single-walled s. trichodea and s. basiconica; it was found in the sensillum lymph in the hair lumen and the sensillum lymph cavity below the hair base (Figure 3). In addition, we found labelled secretory granules in the auxiliary cells of labelled sensilla, as described in detail previously (Steinbrecht et al., 1992).
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Sensilla trichodea are characterized by a thick, tapering cuticular wall penetrated by relatively few pores and largely unbranched sensory dendrites, whereas s. basiconica have branched dendrites and a thin cuticular wall with densely packed, conspicuous pores. Occasionally sensilla with intermediate characteristics are also found, e.g. ‘Zwischentyp’ (Steinbrecht, 1973
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When considering the size of the cuticular hair or peg and the number of innervating ORNs, morphological subtypes can be defined for both s. trichodea and s. basiconica [see, for example, Koh et al., Meng et al. and Steinbrecht (Steinbrecht 1973; Meng et al., 1989; Koh et al., 1995)]. In this study we did not, however, attempt a rigorous classification of subtypes, as this cannot be definitively achieved without complete 3-dimensional reconstruction. Taking into account the fact that short s. trichodea have a smaller diameter at a given level compared with large s. trichodea, we tentatively classified these subtypes in male and female S. littoralis and in male H. virescens. The short s. trichodea are usually located closer to the medial plane of the antenna and are abundant in both sexes, while the long s. trichodea are more abundant laterally on both sides and are predominant in males (Figure 4) [for a more detailed morphology of various noctuid antennae see Hallberg, Jefferson et al., Koh et al., Lavoie-Dornik and McNeil and Ljungberg et al. (Jefferson et al., 1970; Hallberg, 1981; Lavoie-Dornik and McNeil, 1987; Ljungberg et al., 1993; Koh et al., 1995)].
Complementary expression of PBP and GOBP2
Altogether, in the four species studied 1569 sensilla (1150 s. trichodea and 419 s. basiconica) were mapped, identified as to their type and position and labelled with the two antisera against PBP and GOBP2. Sensilla that were treated only with one antiserum are not included in this number and nor are those sensilla that could not unequivocally be ascribed to a certain sensillum type. We did not find any s. trichodeum labelled with both antisera; there was either PBP or GOBP2 labelling or no labelling (Figure 3). The labelling characteristics of the different species, sexes and sensillum types are shown as pie charts giving the percentage of PBP-positive, GOBP2-positive and non-labelling sensilla for each group (Figures 5, 6).
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Even at first glance it is obvious that s. basiconica predominantly express GOBP2 while s. trichodea predominantly express PBP. The labelling pattern of s. basiconica appears relatively uniform; that of s. trichodea, however, shows remarkable differences between the sexes of each species investigated, as well as between the different species. The percentage of PBP(Apol)-positive s. trichodea varied from only 31% in male A. gamma to 99% in H. armigera males. In females of the two heliothine species the percentage of s. trichodea expressing a cross-reacting PBP was significantly lower than in the males. On the other hand, in A. gamma and S. littoralis we found more PBP-labelled s. trichodea in females than in males (in S. littoralis twice as many). In A. segetum the majority of male s. trichodea were not labelled with anti-PBP(Apol) (data not shown).
Only very few s. basiconica (0–6%) were shown to immunoreact with anti-PBP(Apol). In contrast, s. trichodea were only rarely (0–19%) labelled with anti-GOBP2(Apol). This ‘opposite labelling’ of s. trichodea was particularly prominent in S. littoralis and female H. virescens. It was almost exclusively found for short s. trichodea located close to the medial plane of the antenna (Figure 4). Sensilla with opposite labelling tended to be found in clusters of two or even three sensory hairs in close proximity.
In those cases where two individuals were studied (H. virescens and S. littoralis males) the relative distribution of OBP expression was similar, except that the rare cases of opposite labelling were sometimes missing in the smaller samples (Figure 7).
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Labelling of subtypes
As mentioned above, we attempted to discriminate long and short s. trichodea of both sexes of S. littoralis and of male H. virescens according to their hair diameter at a given level. Only clearly identified subtypes are plotted as to their labelling characteristics in Figure 6. Evidently short s. trichodea show a different labelling pattern to long s. trichodea. This is particularly true for male and female S. littoralis.
As shown in Figure 4, short s. trichodea are generally more common close to the medial plane of the antenna and this is also the region where we find the greatest labelling variability of s. trichodea in the other species.
Labelling intensity
In this study we did not intend to evaluate all labelling quantitatively, although there were substantial differences in labelling intensity. Labelling with anti-PBP(Apol) usually resulted in high grain densities (200–700 grains/µm2). In contrast, labelling with anti-GOBP2(Apo) was generally rather weak in all noctuid species studied, rarely exceeding grain densities of 70 grains/µm2 at a serum concentration of 1:3000 (in s. basiconica of A. polyphemus we observed 200 grains/µm2 at 1:6000).
The long s. trichodea in male S. littoralis that were counted as not labelled with anti-PBP(Apol) in Figures 4B, 5 and 6 still showed a low number of grains (on average 7 grains/µm2), which might be more than background. A particularly clear grouping of s. trichodea according to labelling density with anti-PBP(Apol) was observed in A. gamma (Figures 8, 9). As we had arbitrarily set the threshold for positive PBP labelling at 100 grains/µm2 the population of s. trichodea marked by an asterisk in Figure 9 is included among the unlabelled sensilla in the graph in Figure 5, thus, our statements about immunoreactivity disregard very weakly labelling cases.
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Sensillum types and function
Noctuid moths are well known for fairly complex multicomponent pheromone signals emitted by the conspecific female, each pheromone component being received by a different set of receptor cells on the male antennae. There are usually one or two main components which, when used in the correct ratio, may be fully sufficient for attraction in behaviour experiments; in other cases the addition of minor pheromone components is essential to make the blend as effective as a virgin female [for reviews see Hansson and Priesner (Priesner, 1979; Hansson, 1995)]. In addition to receptor cells tuned to the various conspecific pheromone components, male antennae often house receptor cells responding to pheromone components of sympatric species, which when excited inhibit upwind flight and thus help in avoiding interbreeding [for a review see Mustaparta (Mustaparta, 1997)].
As a general rule, pheromone receptor cells in noctuids are found in the long s. trichodea standing out in distinct rows laterally on the antenna. Electrophysiological experiments have further shown that the male-specific long s. trichodea are not a uniform population. This is in contrast to saturniid, bombycid and sphingid moths, where the long s. trichodea are not only morphologically but also functionally uniform with identical sets of receptor cells for the different pheromone components in almost every sensillum (Kaissling, 1979; Kaissling et al., 1989; Meng et al., 1989). Noctuid long s. trichodea, although morphologically indiscriminatable, house different combinations of receptor cells, owing to the fact that there are no more than two or three receptor cells in any single hair [see Koh et al. (Koh et al., 1995) and references therein], whilst a much higher number of pheromone components (sometimes more than seven) and interspecific signals are received (Priesner, 1979, 1980; Löfstedt et al., 1982).
The function of short s. trichodea and s. basiconica is less well characterized in Noctuidae. Recording from these sensilla is more difficult and so is discrimination of the different sensillum types under the light microscope and even the scanning electron microscope. Many investigators, therefore, have classified three or four types of trichoid sensilla according to size, shape of hair and hair tip and the angle at which these sensilla stand out from the antennal surface (Jefferson et al., 1970; Lavoie-Dornik and McNeil, 1987; Mochizuki et al., 1992). Intermediate types, which combine features of s. trichodea and s. basiconica, further complicate the morphological classification of olfactory sensilla and it has been proposed that all single-walled olfactory sensilla are a continuum of structural features with the long s. trichodea at one end and the short s. basiconica at the other end of the spectrum (Steinbrecht, 1996a). These problems have to be considered, in particular when comparing data from different authors.
Electrophysiological experiments with female moths have usually concentrated on the reception of plant odours, which may apparently be received by s. trichodea as well as by s. basiconica (Almaas and Mustaparta, 1991; Todd and Baker, 1993; Anderson et al., 1995) and s. coeloconica (Pophof, 1997). The dogma that female moths do not smell their own pheromone may have suppressed systematic investigation of female pheromone sensitivity in the past. However, female pheromone autodetection has been demonstrated for several moth species other than silkmoths [see Schneider et al. and Todd and Baker (Todd and Baker, 1993; Schneider et al., 1998) and references therein]. Spodoptera littoralis, as described below, is a particularly good example (Ljungberg et al., 1993).
Cross-reactivity of antisera
This study clearly shows that the two antisera raised against purified native PBP and GOBP2 of A. polyphemus cross-react with OBPs of other moth species but retain their specificity for the two OBP subclasses, PBP and GOBP2, as also shown by immunoblots. As in Antheraea, only antennal proteins are labelled with these antisera, in contrast to the so-called ‘OS-D-related proteins’ (Vogt et al., 1999), which may also be expressed on mouthparts, legs and elsewhere in the body. The general expression pattern of PBP in male s. trichodea and of GOBP2 in s. basiconica of both sexes, predicted by Vogt et al. (Vogt et al., 1991) and first observed in A. polyphemus and B.mori (Laue et al., 1994; Steinbrecht et al., 1995), was also observed in the noctuid species studied here. A novel finding is the heterogeneous labelling pattern of male s. trichodea and the surprisingly high expression rate of PBP in female s. trichodea in noctuid moths.
The weak labelling of some sensilla may either indicate that the OBP in these sensilla has a weak affinity for the antisera used or that the concentration of the cross-reacting OBP is low; perhaps a non-labelling OBP, such as GOBP1 or ABPX, forms the main constituent in the sensillum lymph of these hairs (see below). That there may be more than one OBP expressed in the same sensillum was first been demonstrated in Drosophila (Hekmat-Scafe et al., 1997) and is also true for moths (Maida et al., 1999).
In two of the investigated noctuid species PBPs have been fully sequenced. Hel-1 is a PBP of H. virescens (Krieger et al., 1993) and has 73% amino acid identity to the main PBP of A. polyphemus (APO-1) (Raming et al., 1989). Aseg-1, a PBP of A. segetum, was characterized and sequenced by LaForest et al. (LaForest et al., 1999); this protein shows only 58% sequence homology to APO-1, as determined by a BLAST search on the SwissProt database. The more similar PBP of H. virescens strongly immunoreacts with the anti-PBP(Apol) antiserum, as shown in western blots. In A. segetum, however, only a minor protein band is labelled with this serum, whereas a major band with PBP-like electrophoretic properties, probably representing the major male PBP, remains unlabelled. This fits well with the immunocytochemical results: almost all s. trichodea are labelled with anti-PBP(Apol) in male H. virescens, while only a small fraction of these sensilla is labelled with this antiserum in male A. segetum. PBP labelling in H. armigera very much resembles that in H. virescens. Autographa gamma and S. littoralis, on the other hand, are more like A. segetum in their labelling properties. Thus, the main PBP of these species may be less similar to the main PBP of A. polyphemus than the PBPs of the two heliothine species.
If we compare the compounds used by the species studied as intra- and interspecific signals (Table 1) it becomes apparent that a major difference between the two heliothine and the other three noctuid species is the length of the alkyl chain (mainly C16 in H. virescens and H. armigera, C14 and shorter in S. littoralis, A. segetum and A. gamma). Antheraea polyphemus uses C16 alkyl compounds as pheromone components. The question now arises: is the structure of PBPs directly related to the structure of the bound pheromones? Attempts to answer this question by comparing amino acid sequences have so far not led to conclusive answers (Merritt et al., 1998; Willett, 2000), however, as long as the binding site is not known, such comparisons may be too vague. Although the properties of a given PBP to react with a given antibody are probably not the same that define its binding properties with pheromones, it appears worthwhile to seek such a correlation. Indeed, in the present study we observed a correlation between antiserum cross-reactivity of the PBPs and chain length of the pheromones of the different species. A similar correlation has earlier been shown for several other moth species: of nine lepidopteran species from three different superfamilies, those that use pheromones of the same alkyl chain length as A. polyphemus show strong labelling of s. trichodea with anti-PBP(Apol), while others with a shorter chain length do not, independent of their taxonomic relatedness to Antheraea (Steinbrecht, 1996b).
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The GOBP2 of H. virescens (Hel-10) has been fully sequenced (Krieger et al., 1993
). It has 79.4% amino acid identity to the GOBP2 of A. pernyi (the full sequence of the GOBP2 of A. polyphemus is not yet known). Despite this high value we found a rather low immunoreactivity of anti-GOBP2(Apol) with the GOBPs of all noctuid moths examined, showing that immunoreactivity of antisera is not strictly correlated with amino acid identity of the antigens. OBP expression and functional specificity
The heterogeneous labelling pattern of male s. trichodea reflects the heterogeneous functional specificity of the sensilla in the five noctuid species studied. Most probably there is a complex distribution mosaic of several PBPs and GOBPs in every species. Three different PBPs were purified from male antennae of another noctuid, Mamestra brassicae (Nagnan-Le Meillour et al., 1996), which were found to have different binding affinities for the major pheromone component (Maibèche-Coisne et al., 1997). Three different PBPs have now also been found in male A. polyphemus and A. pernyi (Maida et al., 2000).
Noctuids do feed as adults, in contrast to, for example, saturniids and bombycids, and orientation to complex host odour signals must be expected. In addition to receptor cells for intra- and interspecific signalling substances, s. trichodea may also contain olfactory neurons tuned to green leaf volatiles, flower odours and oviposition repellents. These were mainly found in short s. trichodea of both sexes [S. littoralis [Anderson et al., 1993, 1995]; H. virescens (Almaas and Mustaparta, 1991; Røstelien et al., 2000a,b)]. It remains to be shown, however, whether in noctuid moths expression of GOBP2 correlates with plant odour specificity of the sensilla in the same way as in female silkmoths (Steinbrecht et al., 1995). Unfortunately, there are so far no in vitro binding data for GOBPs.
Whether the few observed cases of ‘opposite labelling’ (long s. trichodea expressing GOBP2, s. basiconica expressing PBP) also represent functionally exceptional sensilla remains unclear. The fact that such sensilla were often found in clusters might hint at an early developmental mistake in the cell lineage of these sensilla.
PBP expression in female noctuid moths
The high percentage of positive PBP labelling in female S. littoralis fits well with the electrophysiological data of Ljungberg et al., who observed receptor cells responding with a low threshold to the pheromone component Z9,E11-14:Ac (Table 1) in female long and short s. trichodea (Ljungberg et al., 1993). Concomitantly, a PBP immunoreactive with the anti-PBP(Apol) serum is expressed in most female long and short s. trichodea; in the male this expression is restricted to the majority of short s. trichodea. In the male long s. trichodea receptor cells tuned to the other pheromone component Z9,E12-14:Ac and to the interspecific signal Z9-14:OH were found; the majority of these sensilla in the male were not labelled and obviously express a different PBP. Thus, different PBPs might be involved in the reception of compounds that differ by as little as the position of one double bond.
Does expression of a PBP in a sensillum cogently imply pheromone sensitivity of the receptor cells in this sensillum? Thus far the evidence from silkmoths and Spodoptera appears conclusive. Our findings in Heliothinae, however, cast some doubt on this notion. The overwhelmingly high expression of PBP in females of both H. armigera and H. virescens shows unequivocally that PBPs are common in s. trichodea of female heliothine moths. Recently a PBP has even been cloned from female Heliothis zea (Callahan et al., 2000). This stands in sharp contrast to the presently available electrophysiological data. Almaas and Mustaparta only occasionally found receptor neurons in female s. trichodea of H. virescens which responded better to pheromone components than to ‘green odours’ and these had a much lower sensitivity than the pheromone-sensitive neurons in the male (Almaas and Mustaparta, 1991). Pheromone stimuli were also tried during single cell recordings from female H. armigera, but no responses have been obtained (M. Stranden and H. Mustaparta, personal communication). Thus we should not assume that PBP expression in a sensillum necessarily means pheromone sensitivity of this sensillum. PBPs may also serve other functions than contributing to stimulus specificity, e.g. play a role in general stimulus solubilization, detoxification of the sensory apparatus or rapid stimulus deactivation (Ziegelberger, 1996; Kaissling, 1998, 2001).
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Acknowledgments |
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We gratefully acknowledge the expert technical assistance of Barbara Müller and the critical comments of K.-E. Kaissling and Gunde Ziegelberger. We are grateful to Hanna Mustaparta, T.-J. Almaas and their collaborators in Trondheim for stimulating discussions and the permission to quote unpublished results. Shan-gan Zhang was supported by the K.C. Wang Education Foundation, State Key Laboratory of Integrated Management of Pest Insects and Rodents and the National Nature Science Foundation (39570115) during his stay in Seewiesen.
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References |
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Accepted April 9, 2001
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