Expression of odorant-binding and chemosensory proteins and spatial map of chemosensilla on labial palps of Locusta migratoria (Orthoptera: Acrididae)

Arthropod Structure & Development 35 (2006) 47–56 www.elsevier.com/locate/asd

Expression of odorant-binding and chemosensory proteins and spatial map of chemosensilla on labial palps of Locusta migratoria (Orthoptera: Acrididae) Xin Jin, Shan-gan Zhang, Long Zhang * Key Lab for Biological Control of Ministry of Agriculture, Department of Entomology, China Agricultural University, Beijing 100094, China Received 5 July 2005; accepted 9 November 2005

Abstract The sensilla on labial palps in Locusta migratoria were observed and mapped using light microscopy, scanning and transmission electron microscopy. A dome region on the tip of the fourth segment (distal segment) of labial palps is mainly covered with sensilla chaetica (about 98%), and few sensilla basiconica (2%). The total number of both types of sensilla is significantly higher in females than in males. Sensilla chaetica can be further subdivided into three groups containing 6, 7 or 10 neurons. Immunocytochemical localization of odorant-binding protein (OBP) and chemosensory proteins (CSPs) was performed on ultrathin sections of sensilla on labial palps. The antiserum against odorant-binding protein from Locusta migratoria (LmigOBP) only labelled sensilla basiconica, with gold granules only found in the sensillum lymph. Chemosensory protein instead was specifically present in the outer sensillum lymph of all three subgroups of sensilla chaetica with antiserum against CSP-I from Schistocerca gregaria (SgreCSP-I). In contrast these three subgroups were never labelled with antiserum against CSP-II from Locusta migratoria (LmigCSP-II). In addition, a few sensilla chaetica could not be stained with any of the antisera used. q 2006 Elsevier Ltd. All rights reserved. Keywords: Ultrastructure; Type; Chemoreception; Immunocytochemistry; Locust; Palp

1. Introduction It is well known that locusts perceive environmental stimuli through their chemosensory organs, including those on the maxillary and labial palps. Information on the physiological function of the palps of insects is still limited. In Drosophila, the maxillary palps have been suggested to be accessory olfactory organs (de Bruyne et al., 1999; Shanbhag et al., 1999). In locusts, the maxillary and labial palps are essentially dedicated to contact chemoreception and play a major role in food selection, on the basis of the anatomy and movements of palps (Blaney and Chapman, 1969a, 1970; Klein, 1981), as well as of electrophysiological data (Blaney, 1974). Most of the palpal chemosensory sensilla are concentrated on the tip dome of palps. These terminal sensilla include contact chemoreceptors with a single relatively large pore at the tip, and a few multi-wall-pored olfactory sensilla as well. On two closely related locust species, Locusta migratoria and * Corresponding author. Tel.: C86 10 62731303; fax: C86 10 62731048. E-mail address: [email protected] (L. Zhang).

1467-8039/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2005.11.001

Schistocerca gregaria, there are two subtypes of terminal contact chemoreceptors (taste receptors), containing 6 and 10 sensory neurons, respectively (Blaney and Chapman, 1969b; Blaney et al., 1971), and one type of olfactory receptors’, sensilla basiconica (Blaney, 1977) on maxillary palps. In the cricket, Gryllus bimaculatus, nine types of sensilla, including three types of tip-pored sensilla and three types of olfactory sensilla, have been identified on the tip globe of maxillary palps (Klein, 1981). There are no detailed studies on labial palp sensilla of other orthopterans so far. Various types of chemosensilla may contain different soluble binding proteins in their lymph, belonging to two major families, odorant-binding proteins (OBPs: Vogt and Riddiford, 1981; McKenna et al., 1994; Pikielny et al., 1994; Pelosi and Maida, 1995; Pelosi, 1998; Steinbrecht, 1998) and chemosensory proteins (CSPs: Angeli et al., 1999; Nagnan-Le Meillour et al., 2000; Picimbon et al., 2000; Jacquin-Joly et al., 2001; Wanner et al., 2004). OBPs and CSPs are small polypeptides, of 120–130 and 100–110 amino acids, respectively, but completely unrelated in their amino acid sequences. OBPs contain six conserved cysteines, while CSPs only present four. Both OBPs and CSPs are folded in compact structures, rich in a-helical domains, but again exhibiting completely

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distinct and unique architectures (Tegoni et al., 2004). The function of these proteins was proposed to be involved in chemoreception on the basis of their binding ability with chemical molecules and the specific existence of the proteins in sensilla with a suggested chemosensory function (Steinbrecht, 1998). Consequently, OBPs of Lepidoptera have been subdivided into PBPs (pheromone-binding proteins), GOBPs (general odorant-binding proteins) and ABPX (antennal binding protein X) (Pelosi, 1998). The sequences of two closely related OBPs, called LmigOBPs, have been reported in L. migratoria (Ban et al., 2003a) and three sub-classes of CSPs are expressed in both L. migratoria and S. gregaria (Angeli et al., 1999; Picimbon et al., 2000; Ban et al., 2003b; Jin et al., 2005). Immunocytochemical localization of OBP and CSPs was performed in chemosensilla of the locusts. CSP-I is present in chaetic sensilla of antennae, maxillary palps and tarsi of S. gregaria (Angeli et al., 1999). In L. migratoria, OBP is expressed in the wall-porous sensilla basiconica and trichodea, and CSPs are present in s. chaetica on the antennae, palp, and tarsi (Jin et al., 2005). So far immunocytochemistry, applied to insects’ OBPs and CSPs, has shown that: (i) OBPs are generally present in olfactory sensilla, but have occasionally also been found in taste sensilla, as in the case of PBPRP2, one OBP of Drosophila (Shanbhag et al., 2001a); (ii) in Lepidoptera PBPs are typically associated with male sensilla trichodea, GOBPs with sensilla basiconica of both sexes, although often the picture is more complex (Steinbrecht et al., 1994; Steinbrecht, 1996; Zhang et al., 2001); (iii) generally a single type of OBP or CSP is expressed in each sensillum, although examples of co-expression have been described (Hekmat-Scafe et al., 1997). With more antibodies available, co-localization of several OBPs and/or CSPs may possibly be the rule rather than the exception (Steinbrecht, pers. comm). (iv) CSPs have been most often detected in unipore contact chemosensilla, but sometimes are also found in olfactory sensilla (Monteforti et al., 2002).

2. Materials and methods 2.1. Insects Locusts (L. migratoria) were raised in our department at 28–30 8C, relative humidity 60%, and photoperiod of 18 h:6 h light:dark. Fresh wheat shoots were provided daily. The distal segments of labial palps from adult locusts were dissected for the experiments soon after emergence. 2.2. Light microscopy (LM) The palps were treated with 10% sodium hydroxide overnight and dehydrated by 100% ethanol followed by 1:1 ethanol/xylene and 100% xylene. The tip region of each palp was dissected and spread on a slide, then mounted in Canadian gum. Based on LM observation, the numbers of terminal chemosensilla on palps of both sexes were statistically compared using Student’s t-test. 2.3. Scanning electron microscopy (SEM) For SEM, the distal segments of palps were cut into two parts with a knife along the longitudinal direction and fixed in 70% ethanol for 2 h, then cleaned in ultrasonic bath (250 W) for 1 min in the same solution. After treatment with 100% ethanol for 30 min, the samples were dried in air. The palpal parts were mounted on holders with the outer surface up and after gold-coating examined in a HITACHI S570 or FEI Quanta 200 SEM. 2.4. Transmission electron microscopy (TEM) For TEM, labial palps were cut and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer solution, PBS (pH 7.4), then post-fixed with 2% OsO4 in 0.1 M PBS (pH 7.4), followed by dehydration in an ethanol series and 100% acetone. Embedding was done via propylene oxide in Epon 812. Ultrathin sections were cut with a glass knife on a LKB V Ultramicrotome and mounted on Formvar-coated grids. The specimens were observed in a HITACHI H-7500 TEM. 2.5. Immunocytochemistry

To our knowledge, this paper for the first time investigates the spatial map and the fine structure of chemosensillum types on labial palps of L. migratoria and the exact immunolocalization of OBP and CSPs in different subtypes of palpal sensilla. It is believable that the immunolabelling results, combined with electrophysiological information can indicate interesting correlations between soluble proteins expressed in a particular type of sensillum and the stimuli to which the same sensilla respond. Moreover, a spatial and functional map of the various sensilla types could provide useful information towards understanding the role of OBPs and CSPs in chemoreception, as well as about odour and taste coding (Steinbrecht et al., 1994; Shanbhag et al., 1999; Shanbhag et al., 2001b; de Bruyne et al., 2001;).

The chemical fixation was done by immersion of palps into a mixture of paraformaldehyde (4%) and glutaraldehyde (2%) in 0.1 M PBS (pH 7.4) followed by dehydration in an ethanol series. The samples were embedded in LR White resin (Taab, Aldermaston, Berks, UK) by polymerization at 60 8C in tightly closed gelatine capsules. Ultrathin sections were cut with a glass knife on a LKB V Ultramicrotome and mounted on Formvar-coated grids. For immunocytochemistry, the grids were subsequently floated on 30 ml droplets of the following solutions, mainly adapted from Steinbrecht et al. (1992): in brief, PBS containing 50 mM glycine, PBGT (PBS containing 0.2% gelatine, 1% bovine serum albumine and 0.02% Tween-20), primary

X. Jin et al. / Arthropod Structure & Development 35 (2006) 47–56

antiserum diluted with PBGT, 6 washings with PBGT, secondary antibody in PBGT, 2 washings on each PBGT, PBS glycine, PBS and water. Optional silver intensification (Danscher, 1981) increased the size of the gold granules from 10 to about 40 nm; 2% uranyl acetate increased the tissue contrast for observation in the transmission electron microscope (HITACHI H-7500). The following antisera were used in this study, all raised against recombinant proteins: anti-SgreCSP-I (CSP-I of S. gregaria, Angeli et al., 1999; Acc. No. AF070964); antiLmigOBP (OBP of L. migratoria, Ban et al., 2003a, Acc. No. AY542076) and anti-LmigCSP-II (CSP-II of L. migratoria, Ban et al., 2003b, Acc. No. AY149658). The preparation of antisera can be checked in Jin et al. (2005). The primary antisera were used at dilutions of 1:1000 for anti-OBP and 1:10000 for anti-CSPs and incubated at 4 8C overnight. As a control, the primary antiserum was replaced by serum from a healthy rabbit at the same dilution. Secondary antibody was anti-rabbit IgG, coupled to 10 nm colloidal gold (AuroProbee EM, GAR G10, Amersham) diluted 1:20 and incubated at room temperature for 60–120 min. Immunocytochemical labelling was done on sections of two male and two female adults of L. migratoria.

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(P!0.001). Basiconic sensilla, instead, revealed no difference in numbers between sexes (PO0.1). 3.2. Sensilla basiconica Sensilla basiconica are stubby, short pegs (about 15 mm in length), and are equipped with a broad socket, about 13.5 mm in outer width. At half length of the hair, the diameter of the shaft is about 2.5 mm. The cuticular wall is about 260 nm thick and is perforated by numerous pores with a density of about 31 pores/mm2 of cuticle. These pores of up to 65 nm in diameter are connected to pore tubules (Fig. 2(A) and (B)). This sensillum type is innervated by about 15 sensory neurons associated with auxiliary cells (Fig. 2(C)). The inner dendritic segments are unbranched but distally, the dendrites are divided into many branches (about 40) almost filling the outer sensillum lymph cavity of the hair shaft. The largest dendrite branch reaches 0.75 mm in diameter, while the smallest one is only about 0.2 mm (Fig. 2(B)). The fine structural details are very similar to s. basiconica on the maxillary palps of L. migratoria (Blaney, 1977). 3.3. Sensilla chaetica

3. Results 3.1. Spatial map of chemosensilla on sensory dome of labial palps Each four-segmented labial palp of L. migratoria presents a sensory dome on the distal segment (Fig. 1(A) and (B)). Most of the palpal cuticle is sclerotized and sculptured into hexagonal plates, studded with pegs and giant hairs, which probably represent mechanoreceptor hairs type I and type III, respectively (Blaney and Chapman, 1969a). However, the cuticle of the sensory dome is soft and membranous, it displays some gland openings and is densely covered with papillae and sensilla (Fig. 1(C) and (D)), named terminal sensilla by Blaney and Chapman (1969a). The boundary between the dome and other parts of the forth segment runs more proximally at the inner face, and several giant sensory hairs type III are located along this boundary (Fig. 1(E)). Based on external morphology, three types of terminal sensilla, viz. s. chaetica, s. basiconica and s. campaniformia, can be observed by LM and SEM. The first two are hairshaped, s. chaetica being longer than s. basiconica with thicker cuticular wall and a small socket (Fig. 1(C) and (D)). S. campaniformia display a pore in the center, show an oval cuticular plate encircled by a socket and are very rare in this region (Fig. 1(D)). While s. chaetica are uniformly distributed on the dome, s. basiconica are scattered in a medial zone of the dome (Fig. 1(D) and (E)). The numbers of both s. chaetica and basiconica have been counted on 20 labial palps from five adult males and five females. The values are reported in Table 1. Females present a significantly higher total number of chaetic sensilla than males

Sensilla chaetica are 20–25 mm in length, and present longitudinal grooves on the wall, a crest and an elliptic tip-pore system, as can be observed by SEM. The peg is 3.5–4.8 mm in diameter at the base and tapers gently to 1.7 mm just under the crest. Each peg has a socket of 9–11 mm outer diameter, 7 mm inner diameter and 3 mm depth (Fig. 3(A)). S. chaetica on labial palps are subdivided into three subtypes: Ch6 is innervated by six neurons, Ch7 by seven neurons and Ch10 by ten neurons (Fig. 3(B)). It has to be noted that on the maxillary palps of locusts only two subtypes of s. chaetica containing 6 and 10 neurons, respectively, have been observed (Blaney and Chapman, 1969b; Blaney et al., 1971). Apart from the different neuron numbers, all three subtypes are similar in fine structure of neurons and auxiliary cells (for details see Blaney and Chapman, 1969b; Blaney et al., 1971). In the inner lymph cavity of the hair shaft, there are 5, 6 and 9 unbranched dendrites, respectively, of chemosensory neurons in the three subtypes (Fig. 3(C)–(E)). In addition, each sub-type contains one mechanosensory dendrite terminating at the base of the hair shaft (not shown). The dendrites are enclosed in a dendrite sheath, which separates the inner and outer sensillum lymph cavity. The diameters of the dendrite sheaths of subtypes Ch6 and Ch7 are about 0.65–0.7 mm (Fig. 3(C) and (E)), whereas that of subtype Ch10 is 1 mm at the base (Fig. 3(D)). Some mechanoreceptor hairs type III close to the dome region were also observed on the ultrathin sections. These giant hairs are equipped with a thicker cuticular wall without wall pores and show lymph cavities without dendrites. Only one dendrite teminates at the base of the shaft (see also Blaney and Chapman, 1969a).

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Fig. 1. The light micrograph (A) and scanning electron micrograph (B) show an obvious sensory dome on the distal segment of the labial palp of Locusta migratoria. The dome is densely covered with terminal sensilla. (C) The hair-shaped chemosensilla on the sensory dome consist of sensilla chaetica (ch) and s. basiconica (ba). Compared with the former one, s. basiconica have a wider socket and a thinner cuticular wall as seen by light microscopy. (D) The gentle, undulating surface of sensory dome forming many papillae (pa) is covered with numerous sensilla chaetica and a few sensilla basiconica; some gland openings (gl) are found as well. Along the boundary of the dome, there are a few s. campaniformia (ca) with one sunken pore in the center and several mechanoreceptors of type III (III). The rigid cuticle of the distal segment is sculptured into hexagonal plates and equipped with some sensory hairs type I (I). (E) The schematic distribution of various types of sensilla shows the boundary of the dome running more proximally at the inner face. S. chaetica are largely represented and uniformly distributed on the dome, while s. basiconica are scattered in a medial zone of the dome. Scale bars 40 mm (5 mm for inset in D).

3.4. Labelling with anti-CSPs and OBP Antiserum against SgreCSP-I heavily labelled sensilla chaetica of subtypes Ch6 (Fig. 4(A)) and Ch10 (Fig. 4(B)), while subtype Ch7 was only weakly stained (Fig. 4(C)). The

gold granules were concentrated in the outer sensillum lymph surrounding the dendrite sheath, while the inner sensillum lymph containing dendrite branches was never labelled. In addition, the sensillum lymph of mechanoreceptor type III was also heavily labelled with anti-SgreCSP-I (Fig. 4(E)), as were

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Table 1 Chemosensilla on sensory dome of labial palps of L. migratoria

Males Females P

Number

Total sensilla

S. chaetica

S. basiconica

Proportion of s. basiconica

10 10

294G16 337G30 !0.001

289G16 330G29 !0.001

5G2 7G2 O0.1

1.77% 1.99%

Numbers are meansGSE; significance of sex differences (P) was calculated by Student’s t-test.

the suspension fibers at the shaft base (not shown). However, none of the three subtypes of s. chaetica was labelled with antiLmigCSP-II (not shown). Only few chaetic sensilla remained unlabelled with either antiserum against CSPs (Fig. 4(D)). As in these experiments the number of innervating neurons and details of the fine structure remained unknown, we named this non-labelling subgroup subtype Chx. The antiserum against LmigOBP only labelled s. basiconica. The grains were very concentrated in the sensillum lymph bathing the numerous dendrites as well as in the wall-pores (Fig. 4(F)). The cuticle and the dendrites of all sensory hairs were not labelled by any antiserum (Fig. 4(A)–(F)). The controls show no labelling or weak labelling not significantly higher than background (not shown).

The labelling patterns of adult males and females were similar. Table 2 summarizes the distribution of OBP and CSPs in labial palp sensilla, as determined with the corresponding polyclonal antisera. 4. Discussion Chemoreception, both taste and olfaction, is essential in locusts as well as in all insects, as it may initiate very important behaviour responses, such as mating, oviposition and feeding (Gillott, 1980; Chapman, 2003). In accordance with the general functional morphology of insect sensilla (for a review see Steinbrecht, 1984) and with single-sensillum electrophysiological recording in locusts, the tip-pored chaetic sensilla on maxillary palps are involved in contact chemoreception of

Fig. 2. (A) Sensilla basiconica display a wide socket (s) and numerous wall pores (31 pores/mm2) as seen by scanning electron microscopy. (B) The cross section through the shaft of a s. basiconicum reveals that the cuticular wall (cw) is perforated by many wall pores (p) connected to pore tubules (pt). The sensillum lymph cavity is almost filled with numerous dendritic branches (d). (C) Beneath the cuticle, 15 inner dendritic segments originating from 15 sensory neurons are surrounded by auxiliary cells, viz. thecogen cell (th), trichogen cells (tr) and tomogen cells (to). Scale bars 2 mm in (A, C), 1 mm in (B).

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Fig. 3. (A) Scanning electron micrograph displays longitudinal ridges, a single terminal pore (tp) and a socket (s) at the base of sensillum chaeticum. (B) A transverse section beneath the cuticle indicates three subtypes Ch6, Ch7 and Ch10 of s. chaetica, containing 6, 7 and 10 inner dendritic segments (arrowheads around Ids), respectively. (C–E) Transverse sections at about the base level of the hairs from subtype Ch6 (C), Ch7 (E) and Ch10 (D): The inner sensillum lymph cavities (isl) contain 5 (C), 6 (E), 9 (D) unbranched dendrites (d), respectively, while one mechanosensory dendrite is terminating just beneath the base of sensilla (not shown). The dendrite sheaths (sh) envelop the dendrites, separating inner (isl) and outer sensillum lymph (osl) cavities. cw, cuticular wall of sensilla. Scale bars 5 mm in (A and B), 1 mm in (C–E).

hydrophilic compounds (Blaney, 1974), while wall-pored sensilla, s. basiconica and s. trichodea on the antennae respond to olfactory stimuli, such as aggregation and sex pheromones (Ochieng and Hansson, 1999). The tip sensory field of the cricket maxillary palp comprises approximately 5000 sensilla of nine morphologically distinguishable types. These sensilla include mechanoreceptors, single-walled and double-walled olfactory sensilla and various types of contact chemosensilla differing not only in external morphology such as length, shape and cuticular grooves, but also in neuron numbers (Klein, 1981). The sensilla on the sensory dome of locust maxillary (Blaney, 1977) and labial palps (this study, Fig. 1(E); Table 1) are mostly (more than 95%) sensilla chaetica, but their number is much smaller than that observed in the cricket. Furthermore, all the s. chaetica in

the locust have almost identical external morphology. Such differences could be related to the different habitats of locusts and crickets. Crickets live underground and may need a more complex chemosensory system, particularly for contact chemoreception. Our observations showed that there are more terminal s. chaetica on female palps than on male palps. Possibly females need a more complex chemosensory system to trigger specific female functions, such as oviposition. It has already been stated that the smaller number of antennal chemoreceptors on gregarious locusts as compared to solitary locusts might be related to a less sensitive olfactory system in the former phase (Ochieng et al., 1998), a notion corroborated by single-sensillum electrophysiology (Ochieng and Hansson, 1999).

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Fig. 4. Subtypes Ch6 (A) and Ch10 (B) of sensilla chaetica display strong labelling with anti-SgreCSP-I specifically in the outer sensillum lymph (osl) cavity without dendrites, but not in the inner sensillum lymph (isl) cavity. Subtype Ch7 (C), however, was labelled very weakly. In addition, one unknown subtype Chx of s. chaetica (D) was not labelled with the antibodies tested. The sensillum lymph (sl) of mechanoreceptor hair type III (E) was also heavily labelled with anti-SgreCSP-I. (F) S. basiconicum (ba) labelled in sensillum lymph (sl) and wall pores (p) with anti-LmigOBP. The cuticlar walls (cw), dendrites (d), and inner sensillum lymph cavities of all tested sensilla never show more than unspecific labelling. s, socket. Scale bars 1 mm in (A–C), 2 mm in (D and E), 500 nm in (F).

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Table 2 The labelling in various sensilla types with anti-OBP and anti-CSPs Antisera Types of sensilla

SgreCSP-I

LmigCSP-II

LmigOBP

S. chaetica subtype Ch6 S. chaetica subtype Ch7 S. chaetica subtype Ch10 S. chaetica subtype Chx S. basiconica Mechanoreceptor type III

C

B B B B B B

B B B B C B

C Strong labeling,

C B B C

, weak labeling, B no labeling.

The olfactory sensilla of locusts are mainly distributed on the antennae and are classified as single-walled sensilla basiconica and s. trichodea, and double-walled s. coeloconica (Steinbrecht, 1969, 1984, Ameismeier, 1987; Ochieng et al., 1998; Shao et al., 2002; Jin et al., 2004). Apart from the antennae, a few s. basiconica have been observed on maxillary (Blaney, 1977) and labial palps (this study, Fig. 1(C)–(E)). The external morphology of the palpal s. basiconica is very similar to the antennal ones, except for a prominent socket in connection with a membranous cuticle (Fig. 2(A)). The number of sensory neurons in s. basiconica seems lower on the labial palp (15 for one s. basiconicum observed, Fig. 2(C)) than on the antennae (20–50 sensory neurons reported by Ochieng et al., 1998). Whether the s. basiconica on both olfactory organs have different physiological functions, still has to be experimentally verified. So far only the responses of antennal olfactory receptor neurons of S. gregaria to odours have been studied by electrophysiological recording (Ochieng and Hansson, 1999). Western blotting showed that OBP is expressed only in the antennae of both species of locusts (Jin et al., 2005), however, our much more sensitive immunolabelling results showed that OBP is not only expressed on the antennae but also on the labial palps (Fig. 4(F)). In both cases this protein is present in the lymph of single-walled wall-pore olfactory sensilla. In contrast to the great diversification of OBPs in Lepidoptera (Zhang et al., 2001) and Diptera (Shanbhag et al., 2001b), the widespread presence of a single type of OBP in locusts seems to support a non-specific function of this protein in odour perception, although we cannot exclude the presence of other proteins of the OBP family, yet to be identified. On locust antennae, only one type of contact chemoreceptors (sensilla chaetica) containing 6 sensory neurons, has been observed (Ochieng et al., 1998; Jin et al., 2004). Previous reports on locust maxillary palps indicated two subtypes of terminal contact chemosensilla containing 6 and 10 sensory neurons (Blaney and Chapman, 1969b; Blaney et al., 1971). Here, we report a third subtype, Ch7, morphologically similar to the other two, but containing the dendrites of 7 sensory neurons (Fig. 3(B) and (E)). The occurrence of three subtypes of s. chaetica on the palps indicates complex gustatory functions. Functional classification of these different subtypes of palpal chaetic sensilla, however, requires electrophysiological recordings of identified single sensilla.

The existence of different functional subtypes of s. chaetica on the labial palps is supported by our immunolabelling data (Fig. 4(A)–(D)). While the antennal chaetic sensilla were labelled both with antibodies against recombinant SgreCSP-I and LmigCSP-II (Angeli et al., 1999; Jin et al., 2005), none of the chaetic sensilla on labial palps was ever labelled with antiLmigCSP-II (this study). (However, crude extracts of labial palps weakly reacted with anti-LmigCSP-II in western blots (Jin et al., 2005)). The small subgroup Chx doesn’t even express CSP-I. The general role of CSPs remains undefined so far and probably there is not a unique function for all the proteins of this class. In fact, CSPs are present in various organs of insects (Angeli et al., 1999; Paesen and Happ, 1995; Jacquin-Joly et al., 2001; Ban et al., 2003b) and may be involved in different physiological processes, such as lipid carrier, production, release or reception of pheromones and even leg regeneration (Maleszka and Stange, 1997; Bohbot et al., 1998; Kitabayashi et al., 1998; Nagnan-Le Meillour et al., 2000; Jacquin-Joly et al., 2001; Picone et al., 2001; Monteforti et al., 2002; Lartigue et al., 2002; Briand et al., 2002; Ban et al., 2003b). The first immunolocalization experiment with a protein of this family showed that locust CSP-I was strictly limited to contact chemoreceptors. It was presumed that this CSP could play a role similar to that of OBPs by carrying contact chemical stimuli to the receptor sites (Angeli et al., 1999). However, further immunolabelling studies showed that these proteins are only concentrated in the outer receptor lymph of taste sensilla (Jin et al., 2005; this study, Fig. 4(A)–(C)). Similar results have also been found on taste bristles labelled with anti-PBPRP2 of Drosophila; this protein, therefore, was not considered to function as stimulus carrier, but to be involved in general detoxification (Shanbhag et al., 2001a). Moreover, CSP-I in the locust was also found in the wings (Ban et al., 2003b), in the subcuticular space between normal epidermal cells and the antennal cuticle (Jin et al., 2005) and in the lymph cavity of purely mechanosensitve bristles (this study, Fig. 4(E)). Therefore, CSP-I probably has multiple still unknown functions that challenge further experiments. Acknowledgements We thank Drs Paolo Pelosi and Li-ping Ban for supplying all the antisera tested. We are grateful to Dr P. Pelosi and two anonymous reviewers for critical comments and revisions on the manuscript. We thank Ms Hong-jing Hao for skillful technical assistance on electron microscopy. The research was supported by the grants from Ministry of Science and Technology of China (2004BA528B-3) and China Agricultural University. References Ameismeier, F., 1987. Ultrastructure of the chemosensitve basiconic singlewalled wall-pore sensilla on the antennae in adults and embryonic stage of Locusta migratoria L. (Insecta, Orthoptera). Cell and Tissue Research 247, 605–612.

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