Arthropod Structure & Development 30 (2002) 195±205
ARTHROPOD STRUCTURE & DEVELOPMENT www.elsevier.com/locate/asd
Ultrastructural characterization of antennal sensilla and immunocytochemical localization of a chemosensory protein in Carausius morosus BruÈnner (Phasmida: Phasmatidae) Gaia Monteforti, Sergio Angeli, Ruggero Petacchi, Antonio Minnocci* Scuola Superiore di Studi, Universitari e di Perfezionamento, ªS. Annaº, V. Carducci 40, I-56127 Pisa, Italy Received 6 August 2001; accepted 30 October 2001
Abstract The aim of this work was to investigate the olfactory system of the walking stick insect, Carausius morosus. Morphological, ultrastructural and immunocytochemical studies of adult female antennae were conducted by scanning and transmission electron microscopy. Extensive cross-section series were made through the last antennal segment to de®ne the cuticular apparatus, wall pore distribution and the number of innervating receptor neurons of each sensillum type. Single-walled wall pore sensilla occur in three subtypes: (i) with 27 or 28 branched receptor neurons, (ii) with two branched neurons and (iii) with one or two unbranched neurons, respectively. Double-walled wall pore sensilla were found in two subtypes with spoke channels, one with four unbranched neurons, the other with two unbranched neurons. One terminal pore sensillum was found, showing two cavities within the hair and being innervated by six sensory cells. Immunocytochemical experiments were performed to show the localization of a 19 kDa soluble protein found in the chemosensory organs of C. morosus. This protein shows an amino acid sequence homologous to the family of chemosensory proteins (CSP). The polyclonal antibody raised against the puri®ed protein (CSP-cmA) showed, for the ®rst time in CSPs, a strong labeling in olfactory sensilla, speci®cally in the sensillum lymph surrounding the dendritic branches of SW-WP sensilla and in the uninnervated lumen between the two concentric walls of DW-WP type 1 sensilla. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Antenna; Chemosensory protein; Immunocytochemistry; Olfaction; Phasmids; Scanning and transmission electron microscopy
1. Introduction The insect chemosensory system has been extensively studied in several different orders (Hansson, 1999). However, peripheral and central events of chemical transduction, and particularly the characterisation of odorant binding proteins (OBP), have been investigated mainly in holometabolous orders belonging to the Endopterygota (Vogt et al., 1999). OBPs have been described in several species of Lepidoptera (Pelosi and Maida, 1995; Robertson et al., 1999; Steinbrecht, 1999), Diptera (Hekmat-Scafe et al., 1998; Park et al., 2000), Coleoptera (Leal et al., 1998) and Hymenoptera (Danty et al., 1999). In hemimetabolous orders the chemical transduction has been less investigated. So far, in all the Exopterygota only one OBP-related protein, LAP, has been found in Lygus lineolaris (Heteroptera: Miridae) (Dickens et al., 1998). However, a new group of soluble proteins, called * Corresponding author. Tel.: 139-50-883-246; fax: 139-50-883-215. E-mail address:
[email protected] (A. Minnocci).
chemosensory proteins (CSPs), has been recently identi®ed. CSPs are thought to be involved in chemical communication, but do not show homology with the OBPs. CSPs were found not only in hemimetabolous insects, such as locusts (Orthoptera) (Angeli et al., 1999; Picimbon et al., 2000a) and cockroaches (Dictyoptera) (Picimbon and Leal, 1999), but also in holometabolous insects, such as Diptera (McKenna et al., 1994; Pikielny et al., 1994), Lepidoptera (Maleszka and Stange, 1997; Bohbot et al., 1998; Picimbon et al., 2000b) and Hymenoptera (Danty et al., 1998). The physiological function of CSPs still remains unknown, but they show a close sequence similarity (around 45±50% between any two members), the presence of signal peptides and cysteine motifs (four conserved cysteins) as well as small size (10±19 kDa) and tissue speci®city. In Schistocerca gregaria (Orthoptera) and in Mamestra brassicae (Lepidoptera), two CSPs were localized in the outer lymph of terminal pore contact sensilla (Angeli et al., 1999) and in proboscis sensilla styloconica (Nagnan-Le Meillour et al., 2000), respectively. Also in Cactoblastis cactorum
1467-8039/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 1467-803 9(01)00036-6
196
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205
(Maleszka and Stange, 1997) a CSP was found in the area of CO2 detector sensilla of the labial palp. In this context, we analyzed the antennal olfactory system of Carausius morosus (Phasmatodea), known as Indian or laboratory stick insect. It is a primitive hemimetabolous insect with a rather long life (10±12 months), found in tropical forests of southern India, and frequently used as a model for biological experiments. Males are shorter than females and appear very rarely because the species is mainly parthenogenic with sexual reproduction occurring very occasionally. The antenna of C. morosus bears several types of sensilla, but only a few studies exist, concerning light microscopy of the antennal chemosensilla (Slifer, 1966) and the ultrastructure of a thermo-hygroreceptive sensillum (Altner et al., 1978), while the ®ne structure of all the other sensillum types is unknown. Also the physiology of olfaction and chemical ecology of this species has not been studied yet. Nevertheless, in recent years several abundant proteins have been puri®ed from chemosensory organs of Phasmids (Tuccini et al., 1996; Mameli et al., 1996; Marchese et al., 2000). Speci®cally, in the parthenogenetic females of C. morosus, a 19 kDa soluble protein was found, expressed in large amounts only in their antenna and legs. Its ®rst 33 N-terminal amino acids showed signi®cant similarity (30% identity) with the OS-D protein of Drosophila, the ®rst member of the CSP family. Therefore, according to Angeli et al. (1999), we de®ne this protein as CSP-cmA, where cm stands for C. morosus and A, in lieu of a number, stands for a group of proteins. The reason for this non-standard notation is that we do not know the full sequence, but only the N-terminal part, which might correspond to more than one full sequence. In this paper we report the ®ne structure of all the different chemosensilla types present on the last segment of the antenna of C. morosus. To provide additional information, we also performed immunocytochemical essays using antisera against the CSP-cmA. We localized this protein both in the single-walled wall-pore (SW-WP) and double-walled wall-pore (DW-WP) type 1 sensilla, which are supposed to be olfactory sensilla. 2. Materials and methods 2.1. Insects Female individuals were reared on fresh Rubus spp. in winter and Tilia spp. leaves when available. They were used for the experiments at the adult stage.
microscopy at 2160 8C (Minnocci et al., 1993), coated with gold and examined using a Philips SEM 515. 2.3. Transmission electron microscopy For transmission electron microscopy, the antennae were cryo®xed by rapid immersion into super cooled liquid propane (2180 8C), according to Steinbrecht (1993), or ®xed in Karnovsky's ®xative (1965), post®xed in osmium tetroxide (1%) and dehydrated in a graded ethanol series. The specimens were embedded in Epon (Fluka) with propylene oxide as transition solvent, polymerized at 60 8C, serially sectioned using a diamond knife on a LKB ultramicrotome and mounted on Formvar-coated single hole copper grids. Finally, they were stained with uranyl acetate and lead citrate and investigated with a Jeol 100 SX electron microscope operating at 80 kV. 2.4. Puri®cation of CSP-cmA, preparation of polyclonal antibodies and Western-blot analysis The crude extract of antennal soluble proteins was prepared by homogenisation of the antennal samples in nitrogen liquid after addition of Tris buffer (20 mM Tris/ HCl, pH 7.4). The protein CSP-cmA was puri®ed by gel ®ltration and anion-exchange chromatography on Mono-Q using a Pharmacia FPLC system. Fractions of elution buffer were collected and analyzed by 13% SDS-PAGE using a Bio-Rad Mini-Protean II apparatus according to Laemmli (1970). This procedure allows to obtain an electrophoretically pure 19 kDa protein, as previously reported (Mameli et al., 1996). Antisera were obtained by injecting an adult rabbit subcutaneously with 400 mg of puri®ed CSP-mc1, followed by two additional injections of 250 mg after 18 and 30 days. The protein was emulsi®ed with an equivalent volume of Freund's complete adjuvant for the ®rst injection and with incomplete adjuvant for further injections. The animal was bled 10 days after the last injection and the serum was obtained by centrifugation at 5000 £ g for 20 min of the coagulated blood. All operations were performed according to ethical guidelines in order to minimize pain and discomfort to animals. Western-blot analyses were performed after electrophoretic separation under denaturing conditions (12% SDS-PAGE) and then the proteins samples were electroblotted on a nitrocellulose membrane. The incubations with ®rst and second peroxidase conjugate antisera were made both at dilution of 1:1000 following the procedure previously reported by Angeli et al. (1999).
2.2. Scanning electron microscopy
2.5. Immunocytochemistry
Antennae from parthenogenetic females were ®xed by immersion into 4% glutaraldehyde and dried in a CPD 030 Bal-Tech or cryo®xed in liquid nitrogen and observed frozen-hydrated by low temperature scanning electron
For immunocytochemistry, the antennae were immersed in phosphate buffered glutaraldehyde 0.1%±paraformaldehyde 3% ®xative, followed by dehydration in an ethanol series and embedded in LR White hard grade acrylic resin
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205
(Electron Microscopy Sciences, Fort Washington, USA). The sections were mounted on Formvar-coated single hole nickel grids and consecutively ¯oated on 30 ml droplets of the following solutions (for details see Steinbrecht et al. (1995)): phosphate-buffered saline (PBS) containing 50 mM glycine; PBGT (PBS containing 0.2% gelatine, 1% bovine serum albumine and 0.01% Tween 20); primary antiserum diluted with PBGT; PBGT; secondary antibody diluted 1:20 in PBGT; PBGT; PBS; water. Silver intensi®cation according to Danscher (1981) was applied to permit a visualization of the labeling at lower magni®cation. The primary antibody was used in dilutions from 1:1000 to 1:35000. The secondary antibody was anti-rabbit IgG, coupled to 10 nm colloidal gold (British BioCell, Cardiff, UK). As a control, the primary antiserum was replaced by pre-immune serum from the same rabbit from which the antiserum was obtained, at the same dilution. Finally, sections were stained with 2% uranyl acetate. 3. Results 3.1. Gross morphology The antennae are about 35 mm long with a big scape (basal diameter 800 mm, length 1700 mm), a pedicel (500 mm wide and 800 mm long) and 40±46 ¯agellar subsegments. Their number is extremely variable also between the two antennae of the same animal. The sensilla are distributed without any spatial regular pattern. Throughout the long ¯agellum there is a low number of sensilla in the proximal half (400 sensilla/mm 2 were estimated at ¯agellar segment I-XV), while sensillar density increases in the distal half (1100 sensilla/mm 2 at segment XXIV and 1700 sensilla/mm 2 at the distal segments). Anyway, the total number of sensilla/segment remains more or less the same, because of the corresponding decrease in the total surface of each ¯agellomere (mean: 196 sensilla in the ®rst four proximal ¯agellar segments and 216 in the distalmost two). Along the ¯agellum several types of chemo- and mechanosensilla, some campaniform sensilla and a small number of poreless thermo-hygrosensitive coeloconic sensilla have been described (Altner et al., 1978). We found the latter randomly on several segments of the ¯agellum, not only in the central part of the antenna, as previously reported (Weide, 1960). On the dorsal surface of the 12th ¯agellar segment a glandular bump is present, pierced by the secretory ducts of the gland cells. These glands are differentiated from the epidermis beneath the bump surface and are not provided with sensory cells (data not shown). 3.2. Fine structure The ultrastructural investigation of the different sensillum types was conducted on the last segment of the antenna, where they are present in a wide variety. After the threedimensional reconstruction of all the sensilla, three types of
197
SW-WP, two types of DW-WP, one type of terminal-pore (TP) and one type of no-pore (NP) sensilla were found. The following data are the result of three-dimensional reconstruction of eight SW-WP type 1 sensilla, 16 SW-WP type 2 sensilla, three SW-WP type 3 sensilla, 12 DW-WP type one sensilla, ®ve DW-WP type 2 sensilla, 23 TP sensilla and 29 NP sensilla. 3.2.1. SW-WP sensilla SW-WP type 1 (90 mm long with a base diameter of 4.5 mm) shows longitudinally arranged ridges (150 nm) and pores of about 50 nm diameter. The hair of the sensillum contains up to 53 branched outer dendritic segments, immersed in an extracellular electron dense ¯uid, the sensillum lymph (Fig. 1(A)). The wall is 0.5 mm thick and presents about 20±25 pores per cross section (Fig. 1(B)). Beneath the cuticle, there are 27 or 28 receptor cells and the outer dendritic segments are surrounded by a dendrite sheath, formed by the thecogen cell (Fig. 1(C)). Proximal of the dendrite sheath, the receptor cells are spatially arranged in two concentric groups, the bigger external group (Fig. 1(D), id) surrounding the smaller inner one. At this level, the receptor cells of the latter group show their ciliary segments (Fig. 1(D), cs) immersed in the inner sensillum lymph, while the receptor cells of the surrounding group show the inner dendritic segments. This pattern suggests a structural difference in the outer/ inner length ratio. The thecogen cell that segregates each neurone (Fig. 1(E)), ensheathes the inner dendritic segments and the receptor cell somata. SW-WP type 2 (70 mm long, basal diameter 3.5 mm). The wall is 0.4 mm thick, with about 40 pores per transverse section that are present between the ridges only in the distal two thirds of the hair (Fig. 2(A)), while the basal third of the cuticle is smooth. This sensillum is innervated by two sensory cells (Fig. 2(B)) extending about 26 dendritic branches into the lumen of the hair (Fig. 2(A)). The dendrite sheath surrounds the two dendritic outer segments within the sensillum-lymph cavity and ends at the hair base, the branched dendrites emerging from it. SW-WP type 3 (37 mm long, 3 mm at the base) was rarely found. The thick cuticular wall is about 0.8 mm and shows a restricted number of pores (10/section) only in the distal half of the hair. It is innervated by one or two sensory cells and their unbranched dendrites leave the dendrite sheath at the hair base (not shown). 3.2.2. DW-WP sensilla DW-WP type 1 is a `peg-like' sensillum, 15±25 mm in length and 2±3 mm in diameter at the base. The wall structure is formed by two concentric cylinders irregularly connected by spoke channels (diameters ,25 nm, Fig. 2(C)), it terminates at the tip with separate ®ngers (Fig. 2(C), inset). The outer wall, only in the distal half of sensillum, has a longitudinally grooved surface (about 30 grooves, 150 nm wide, Fig. 2(C), g), the basal part is
198
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205
Fig. 1. (A±D) Transmission electron micrographs of SW-WP type 1 sensillum in cross-section. (A) Hair cross-section showing numerous branched dendrites (d) with different size immersed in an electron dense ¯uid, the sensillum lymph (sl). Many pores (p) perforate the thin wall. (B) At higher magni®cation the pore canals (pc, 20±25 per cross section) are well visible inside the wall. (C) Below the hair base the 28 outer dendritic segments (od) are surrounded by the dendrite sheath (ds) formed by the thecogen cell (th). (D) Proximal of the dendrite sheath, the receptor cells are spatially arranged in two concentric groups, the bigger external group surrounding the smaller internal one. Receptor cells of the latter group show six ciliary segments (cs) immersed in a large inner sensillum-lymph cavity (il) while, at the same level, receptor cells of the surrounding group show the inner dendritic segments (id). Note four basal bodies (bb) and septate junctions (sj). (E) Proximally, the internal group of receptor cells shows their inner dendritic segments surrounded by the thecogen cell (th) in a mesaxonal fashion. Inside the dendrites (d), mitochondria (m) and ciliary rootlets (cr) are visible. Bar in A and D 1 mm. Bar in B, C and E 0.5 mm.
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205
smooth. The inner wall (diameter of lumen about 0.8 mm) contains four or ®ve dendrites of different size (Fig. 2(D), d), surrounded by sensillum lymph. Each dendrite corresponds to a single sensory cell. The proximal part of the outer dendritic segment is surrounded by a thick dendrite sheath. The uninnervated lumen, situated between the two cuticular cylinders, is continuous with the outer sensillumlymph cavity above the tormogen cell. Moreover, in most cases, it is full of electron dense material, probably emanating from the secretion granules located in the auxiliary cells. Immediately below the base of the sensillar hair, in the trichogen and tormogen cells, large vesicles were found, possibly representing extracted lipid droplets (not shown). At the ciliary level (Fig. 2(E)), scolopale-like structures are found and the thecogen cell forms a large labyrinth. Just below the cilia, the inner dendrites are ensheated separately by the thecogen cell (Fig. 2(F)). DW-WP type 2. This was rarely found. It presents a smaller outer cylinder (diameter less of 2 mm) with a fewer number of grooves and, consequently, of spoke channels (Fig. 3(A)). In the basal part of the peg the outer wall is completely smooth (Fig. 3(B)). The smaller inner cylinder (diameter 0.4 mm) contains a small inner sensillumlymph space and only two unbranched dendrites. Below the cuticle there are two outer dendrites surrounded by a big dendritic sheath (Fig. 3(C), ds). The apical membrane of the thecogen cell does not form a labyrinth and no vesicles are formed (Fig. 3(D)). 3.2.3. TP sensilla TP sensillum is 100±150 mm long and 5±6 mm in diameter, with longitudinally asymmetrical grooves, ¯uted aporous walls and a spatulate tip. Cross-sections of the hair show a double lumen formed by the cuticle (Fig. 3(E)), the smaller circular lumen containing ®ve dendrites (Fig. 3(F)). This sensillum is innervated by six sensory cells (Fig. 3(G)), the sixth showing a larger calibre than the others and terminating at the base of the hair with a typical tubular body. 3.2.4. NP sensilla NP sensillum is 35±37 mm long with a base diameter of 3.3 mm, shows a tip pore ®lled by electron dense material. It has a single lumen and is innervated by a single sensory cell which terminates at the base of the hair with a tubular body (not shown). 3.3. Western-blot analysis and immunocytochemistry Native CSP-cmA protein was puri®ed from about 500 antennae of adult females of C. morosus following the protocols of Mameli et al. (1996). The pure native protein was used to obtain the polyclonal antiserum against CSPcmA. By Western-blot analysis we blotted soluble protein extract patterns of C. morosus and strong labeling was associated only with the CSP-cmA. The antisera were also tested against the protein patterns of S. gregaria and Eurycantha
199
calcarata in which we previously found other proteins of the same class: CSP-sg's and CSP-ec's, respectively (Angeli et al., 1999; Marchese et al., 2000). No cross-reactivity was observed against the CSP-sg's and CSP-cm's or other soluble proteins showing a high speci®city of the antisera (Fig. 4(A)). If transverse sensilla sections from the distalmost antennal segment of C. morosus were incubated with CSP-cmA antiserum, a reproducible labeling pattern was observed, which was clearly different from that in the controls. Speci®c staining was found in 30% of the SW-WP sensilla investigated (Fig. 4(B)), exhibiting a strong labeling in the sensillum lymph surrounding the dendritic branches. In DW-WP sensilla a uniform labeling was found only in type 1, localized inside the uninnervated lumen between the two concentric walls and stronger in the basal (Fig. 4(C)) than in the apical part (Fig. 4(D)) of the peg. However, no labeling was visualized around the dendrites in the sensillum lymph. At a primary antibody dilution 1:35000, only SWWP showed speci®c labeling. Positive staining was denoted by a signal to background ratio of at least 80.
4. Discussion 4.1. Ultrastructure of antennal sensilla The absence of any regular pattern in the sensillar distribution did not allow to correlate the SEM surface morphology with the TEM reconstruction, complicating the identi®cation of sensilla and the association of sensillum labeling and sensillum type. The SW-WPs are localized principally in the distal third of the antenna (as described for the `thin-walled' sensilla in Slifer (1966)) and showed characteristics of olfactory sensilla. In SW-WP type 1, the thecogen cell segregates all the 28 neurons, as found in the `sickle-shaped sensilla' of Trissolcus basalis (Bin et al., 1989); high numbers of neurons have also been reported for basiconic sensilla of locusts (Ameismeier, 1987). Several groups of dendrites surrounded by separate dendrite sheaths were found in Cimex (Steinbrecht and MuÈller, 1976), and also in moth larvae multiple dendrite sheaths with multiple cellular envelopes are reported (Laue, 2000). The SW-WP type 2 seems to be the typical basiconic sensillum, formed by two sensory cells that upon leaving to the dendrite sheath branch into numerous dendrites. The SW-WP type 3 was rarely found in the last antennal segment. In this sensillum, the dendrites of 1 or 2 sensory cells run unbranched up to the tip of the hair as in the typical sensillum trichodeum of moths (Steinbrecht, 1987). Weide (1960) reported over 4000 trichoid sensilla on the antenna of C. morosus, but because this attribution was based only on external morphological characteristics, probably most of them were contact chemosensilla. The presence of two different DW-WP types is not so
200
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205
frequent, although three types of grooved pegs were found in Periplaneta americana (Altner and Prillinger, 1980). The ultrastructural characteristics of DW-WP sensilla of C. morosus differ in some aspects from that just described in many insect orders (Keil and Steinbrecht, 1984; Steinbrecht, 1997). In DW-WP type 1, the thecogen cell shows an apical cell membrane invaginated and folded to form a complex labyrinth, as shown also in thermo-hygroreceptive sensilla styloconica of moths (Steinbrecht et al., 1989). The vesicles found in the trichogen and tormogen cells, probably lipoid in nature and noted in the sensillum lymph of other insects, may be involved in waterproo®ng or stimulus transport (Altner et al., 1977). The DW-WP type 2 shows a different arrangement, because the apical membrane of thecogen cell does not form any extensive labyrinth. This sensillum probably correspond to the one described by Altner (1977) as `a double-walled peg with smooth outer surface'. On the other hand, two different types of DW-WP sensilla, called `coeloconica', were also found in S. gregaria (Ochieng et al., 1998). They are located in spherical cuticular pits as in Lepidoptera (Hunger and Steinbrecht, 1998), while our two types of DW-WP are both situated directly on the antennal surface, without any structural specialization, as found in type C sensilla of Cimex lectularius (Steinbrecht and MuÈller, 1976). Finally, it has to be highlighted that in C. morosus another sensillum was also found, called `coeloconic' because of its external morphology being inserted in a pit and lacks pores. This sensillum contains hygro- and thermoreceptive sensory cells (Altner et al., 1978). The TP sensillum is innervated by six sensory cells, not ®ve as Slifer (1966) described and de®ned as `thick-walled chemoreceptor peg'. Probably the sixth, mechano-receptive dendrite had been overlooked. These sensilla are typically crown-shaped on the terminal tip of the last antennal segment, as described for other contact chemoreceptors (Chapman, 1998). 4.2. CSP-cmA immunolocalization The Western-blot analyses clearly show a strong speci®city of the antiserum for CSP-cmA (Fig. 4(A)). Therefore we can assume that the immunolabeling present in the sensillar sections speci®cally marked the expression site
201
of CSP-cmA. In the immunocytochemical experiments a strong signal is visible in the sensillum lymph of SW-WP sensilla. It was not possible to assign the labelling to type 1 or type 2, because the ®xation utilized for immunocytochemistry does not allow to clearly discriminate the two types. Although there is no evidence for the function of these sensilla in C. morosus, they appear to be olfactory sensilla according to their ®ne structure. Speci®cally, the fact that CSP-cmA is abundantly expressed in the sensillar lymph of SW-WP, bathing the sensory dendrites, suggests a direct function of the protein in the chemical transduction. This is the ®rst case where a CSP is found in typical olfactory sensilla, in contrast to what was known from S. gregaria and M. brassicae (described earlier). Interestingly, DW-WP type 1 is labeled too, but with a different expression pattern. It shows labeling only in the uninnervated lumen, between the two concentric walls. The enigmatic localization of CSP-cmA in DW-WP could be associated with the presence of two different but very closely related proteins in different sensilla, that both cross-react with the polyclonal antiserum. However, it is also possible that the antiserum recognizes the same protein, which can have a different function in different sensilla. Recently Park et al. (2000) localized in Drosophila a putative odorant-binding protein, called PBPRP2, in the outer cavity of 30% of the DW coeloconic sensilla and in neither case this protein was in contact with the dendritic membranes of olfactory neurones. TP sensilla have not been labeled, although the Westernblot indicates that the antiserum for CSP-cmA labels extracts from tarsi, where numerous contact chemosensilla are present. CSP-cmA and OS-D are the only CSPs with a long amino acid chain (about 19 aa) in the ®rst part of the mature protein, as is possible to see in the table of the protein alignments (Table 1). Although we know only the ®rst 33 aminoacids of CSP-cmA, (lacking, therefore, the chance to show the homology with the other CSPs) the homology with the ®rst part of OS-D (30% identity) is evident. It could be that there are two subtypes of CSP, OS-D and CSP-cmA being the only two CSPs, known till now, with a long Nterminal region. If this is true we can expect that OS-D and CSP-cmA are expressed in similar sensilla. Indeed,
Fig. 2. (A)±(F) Transmission electron micrographs of SW-WP type 2 (A) and (B) and DW-WP type 1 (C)±(F) sensillum. C is a cryo®xed sample, all others chemically ®xed. (A) The cross-section of SW-WP type 2 hair shows more pores (p) than in type 1. They are present between the ridges only in the terminal two thirds of the peg, while the basal third of the cuticle is smooth. Dendritic branches (d) present in the lumen of the type 2 peg are less numerous than in type 1. (B) This sensillum is innervated by only two sensory cells and, distal of the dendritic ciliary constriction, the two outer dendritic segments (od) are surrounded by inner sensillum lymph and by the thecogen (th), trichogen (tr) and tormogen (to) cell. (C) The complex cuticular apparatus of DW-WP type 1 sensilla consists of an outer wall (ow) with longitudinal grooves (g) and an inner wall (iw) which surrounds the four dendrites (d). This wall structure, formed by two concentric cylinders, is irregularly connected by spoke channels (sc). In the inset the sensillum tip is visible with the typical separate cuticular ®ngers (bar 0.1 mm). (D) The peg cross-sectioned more proximally shows more regular grooves and contains dendrites (d) of different size. Each dendrite corresponds to a single sensory cell. (E) At the ciliary level the inner sensillum lymph (il) surrounds the four dendrites (d). At this level, there is no dendrite sheath and scolopale-like structures (sl) are observed in the thecogen cell. Note the large labyrinth (lb) of the thecogen cell and the abundance of mitochondria (m). (F) Inner dendritic segments (id) cross-sectioned near the cilia. The dendrites are ensheathed separately by the thecogen cell (th) that forms large invaginations containing inner-sensillum lymph. Bar in A and E 1 mm. Bar in B, C, D and F 0.5 mm.
202
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205
Fig. 3. (A)±(G) Transmission electron micrographs of DW-WP type 2 (A)±(D) and TP (E)±(G) sensillum. (A) Peg cross-section close to the tip showing the grooved outer wall (ow), the spoke channels (sc) and two unbranched dendrites (d). (B) In the basal part of the peg, the outer wall (ow) becomes completely smooth and does not present any spoke channels. (C) Below the cuticle two outer dendrites are visible surrounded by a well-developed dendrite sheath (ds). (D) At the level where the dendrite sheath ends, no droplets nor a thecogen cell labyrinth could be observed. (E) Hair cross section of TP sensillum showing a double lumen formed in the cuticular hair. (F) Detail of the smaller circular cavity containing the ®ve, chemosensory dendrites (d); microtubules (mi). (G) Proximal of the dendrite sheath the six inner dendrite segments (id) are visible, surrounded by the thecogen cell (th) and containing the ciliary rootlets (cr). The largest dendrite belongs to the sixth, mechanosensory process, which is not visible in the hair sections. Bar in A, B, C, D and E 0.5 mm, F 0.1 mm, G 1 mm.
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205
203
Fig. 4. (A, left) SDS-PAGE of crude tarsal extract of S. gregaria (S), E. calcarata (E) and C. morosus (C); (A, right): Western-blot analysis of the same extracts using antiserum raised against the CSP-cmA of C. morosus. The antiserum shows no crossreactivity with other proteins, even in case of proteins with high similarity as CSP-sg and CSP-ec. Molecular-mass markers (M) are, from the top: BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (20 kDa) and a-lactalbumin (14 kDa). (B±D): Cross-sections through different sensilla labelled with anti-CSP-cmA. (B) SW-WP oblique section speci®cally labeled in the sensillum lymph that surrounds the dendritic branches. (C)-(D) sections of DW-WP sensilla: an uniform labelling was found only in DW-WP type 1, inside the uninnervated lumen, between the two concentric walls. The signal was less strong in the apical (C) than in the basal part (D) of the peg, while no labeling was found around the dendrites in the sensillum lymph. Bar in B and D 1 mm. Bar in C 0.5 mm.
the OS-D protein on the antennae of Drosophila is expressed mainly in the region of the sensilla coeloconica, as shown by in situ hybridization (Pikielny et al., 1994; McKenna et al., 1994). These sensilla are DW-WP as the sensilla where we found the CSP-cmA. This fact could explain our observation that the CSP-cmA of C. morosus and OS-D of D. melanogaster labeled the DW-WP sensilla in contrast to the CSP-sg of S. gregaria that is expressed in the terminal pore contact sensilla (Angeli et al., 1999). In conclusion, the original characteristics of C. morosus ultrastructure and immunocytochemistry results can be explained by its very ancestral evolutionary position. Our results con®rm the hypothesis that CSPs are somehow involved in the insect chemical transduction both of odorant and taste-signals. CSPs are expressed in a speci®c association to a large range of sensilla types (including
SW-WP sensilla), suggesting that their different members can be involved in different physiological processes, as demonstrated for OBPs in Lepidoptera (Steinbrecht, 1998). Acknowledgements We gratefully acknowledge the careful and devoted technical assistance of Dr P. Lucchesi of the University of Pisa for ®lm processing and photographic printing and Prof. Paolo Pelosi for allowing us to use FPLC and his biochemical Lab. We also wish to thank Dr M. Laue, for his friendly assistance in establishing experimental protocols and Prof. R.A. Steinbrecht for all his helpful suggestions regarding cryo®xation, freeze substitution and immunocytochemistry.
204
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205
Table 1 Alignment of the N-terminal amino acid sequence of CSP-cmA and similar proteins in other insects. Conserved residues between CSP-cmA and other proteins are shaded. Bold characters indicate amino acids common among similar proteins; the four conserved cysteines are underlined. OS-D-dm (Drosophila melanogaster, McKenna et al., 1994; Pikielny et al., 1994); CSP-sg1 (Schistocerca gregaria, Angeli et al., 1999); LmigOS-D1 (Locusta migratoria, Picimbon et al., 2000a); CSP-ec1 (E. calcarata, Marchese et al., 2000); Pam8 m (Periplaneta americana, Picimbon and Leal, 1999); CSP-MbraA1 (M. brassicae, Nagnan-Le Meillour et al., 2000); CSP-bm1 (Bombyx mori, Picimbon et al., 2000b); CLP1-cc (C. cactorum, Maleszka and Stange, 1997)
References Altner, H., 1977. Insect sensillum speci®city and structure: an approach to a new typology. In: Le Magnen, J., MacLeod, P. (Eds.). Olfaction and Taste VI. Information Retrieval, London, pp. 295±303. Altner, H., Prillinger, L., 1980. Ultrastructure of invertebrate chemo-, thermo- and hygroreceptors and its functional signi®cance. International Review of Cytology 67, 69±113. Altner, H., Sass, H., Altner, I., 1977. Relationship between structure and function of antennal chemo-, hygro-, and thermoreceptive sensilla in Periplaneta americana. Cell and Tissue Research 176, 389±405. Altner, H., Tichy, H., Altner, I., 1978. Lamellated outer dendritic segments of a sensory cell within a poreless thermo- and hygroreceptive sensillum of the insect Carausius morosus. Cell and Tissue Research 191, 287± 304. Ameismeier, F., 1987. Ultrastructure of the chemosensitive basiconic single-walled wall-pore sensilla on the antennae in adults and embryonic stages of Locusta migratoria L. (Insecta, Orthoptera). Cell and Tissue Research 247, 605±612. Angeli, S., Ceron, F., Scaloni, A., Monti, M., Monteforti, G., Minnocci, A., Petacchi, R., Pelosi, P., 1999. Puri®cation, structural characterisation, cloning and immunocytochemical localisation of chemoreception proteins from Schistocerca gregaria. European Journal of Biochemistry 292, 745±754. Bin, F., Colazza, S., Isidoro, N., Solinas, M., Vinson, S.B., 1989. Antennal chemosensilla and glands, and their possible meaning in the reproductive
behaviour of Trissolcus basalis (Woll.) (Hym.: Scelionidae). Entomologica 24, 33±51. Bohbot, J., Sobrio, F., Lucas, P., Nagnan-Le Meillour, P., 1998. Functional characterization of a new class of odorant-binding proteins in the moth Mamestra brassicae. Biochemical Biophysical Research Communication 18, 489±494. Chapman, R.F., 1998. The Insects: Structure and Function. fourth ed Cambridge University Press, Cambridge. Danscher, G., 1981. Localization of gold in biological tissue. A photochemical method for light and electronmicroscopy. Histochemistry 71, 81±88. Danty, E., Arnold, G., Burmester, T., Huet, J.C., Huet, D., Pernollet, J.C., Masson, C., 1998. Identi®cation and developmental pro®les of hexamerins in antenna and hemolymph of the honeybee, Apis mellifera. Insect Biochemistry and Molecular Biology 28, 387±397. Danty, E., Briand, L., Michard-Vanhee, C., Perez, V., Arnold, G., Gaudemer, O., Huet, D., Huet, J.C., Ouali, C., Masson, C., Pernollet, J.C., 1999. Cloning and expression of a queen pheromone-binding protein in the honeybee: an olfactory-speci®c, developmentally regulated protein. Journal of Neuroscience 19, 7468±7475. Dickens, J.C., Callahan, F.E., Wergin, W.P., Murphy, C.A., Vogt, R.G., 1998. Intergeneric distribution and immunolocalization of a putative odorant-binding protein in true bugs (Hemiptera, Heteroptera). Journal of Experimental Biology 201, 33±41. Hansson, B.S., 1999. Insect Olfaction. Springer, Berlin pp. 457. Hekmat-Scafe, D.S., Steinbrecht, R.A., Carlson, J.R., 1998. Olfactory
G. Monteforti et al. / Arthropod Structure & Development 30 (2002) 195±205 coding in a compound nose. Coexpression of odorant-binding proteins in Drosophila. Annals of the New York Academic Sciences 855, 311± 315. Hunger, T., Steinbrecht, R.A., 1998. Functional morphology of a doublewalled multiporous olfactory sensillum: the sensillum coeloconicum of Bombyx mori (Insecta Lepidoptera). Tissue and Cell 30, 14±29. Karnovsky, M.J., 1965. A formaldehyde-glutaraldehyde ®xative of high osmolarity for use in electron microscopy. Journal of Cell Biology 27, 137. Keil, T.A., Steinbrecht, R.A., 1984. Mechanosensitive and olfactory sensilla of insects. In: King, R.C., Akai, H. (Eds.). Insect Ultrastructure, vol. 2. Plenum Press, New York, pp. 477±516. Laemmli, U.K., 1970. Clevage of structural proteins during the assembly of head of bacteriophage T4. Nature 227 (680), 685. Laue, M., 2000. Immunolocalization of general odorant-binding protein in antennal sensilla of moth caterpillars. Arthropod Structure and Development 29, 57±73. Leal, W.S., Wojtasek, H., Miyazawa, M., 1998. Pheromone-binding proteins of scarab beetles. Annals of the New York Academy of Sciences 855, 301±305. Maleszka, R., Stange, G., 1997. Molecular cloning, by a novel approach, of a cDNA encoding a putative olfactory protein in the labial palps of the moth Cactoblastis cactorum. Gene 20, 39±43. Mameli, M., Tuccini, A., Mazza, M., Petacchi, R., Pelosi, P., 1996. Soluble proteins in chemosensory organs of Phasmids. Insect Biochemistry and Molecular Biology 26, 875±882. Marchese, S., Angeli, S., Andolfo, A., Scaloni, A., Brandazza, A., Mazza, M., Picimbon, J.-F., Leal, W.S., Pelosi, P., 2000. Soluble proteins from chemosensory organs of Eurycantha calcarata (Insects Phasmatodea). Insect Biochemistry and Molecular Biology 30, 1091±1098. McKenna, M., Hekmat-Scafe, D., Gaines, P., Carlson, J.R., 1994. Putative Drosophila pheromone-binding proteins expressed in a subregion of the olfactory system. Journal of Biological Chemistry 269, 16340±16347. Minnocci, A., Petacchi, R., Belcari, A., 1993. Insect studies by LTSEM. Multinational Congress on Electron Microscopy, Parma 1993, 331±332. Nagnan-Le Meillour, P., Cain, A.H., Jacquin-Joly, E., FrancËois, M.C., Ramachandran, S., Maida, R., Steinbrecht, R.A., 2000. Chemosensory proteins from the proboscis of Mamestra brassicae. Chemical Senses 25, 541±553. Ochieng, S.A., Hallberg, E., Hansson, B., 1998. Fine structure and distribution of antennal sensilla of the desert locust, Schistocerca gregaria (Orthoptera: Acrididae). Cell and Tissue Research 291, 525±536. Park, S.K., Shanbhag, S.R., Wang, Q., Hasan, G., Steinbrecht, R.A., Pikielny, C.W., 2000. Expression patterns of two putative odorantbinding proteins in the olfactory organs of Drosophila melanogaster have different implications for their functions. Cell and Tissue Research 300, 181±192. Pelosi, P., Maida, R., 1995. Odorant-binding proteins in insects. Comparative Biochemistry and Physiology 111B, 503±514. Picimbon, J.F., Leal, W.S., 1999. Olfactory soluble proteins of cockroaches. Insect Biochemistry and Molecular Biology 29, 973±978. Picimbon, J.F., Dietrich, K., Breer, H., Krieger, J., 2000a. Chemosensory
205
proteins of Locusta migratoria (Orthoptera: Acrididae). Insect Biochemistry and Molecular Biology 30, 233±241. Picimbon, J.F., Dietrich, K., Angeli, S., Scaloni, A., Krieger, J., Breer, H., Pelosi, P., 2000b. Puri®cation and molecular cloning of chemosensory proteins from Bombyx mori. Archives of Insect Biochemistry and Physiology 44 (3), 120±129. Pikielny, C.W., Hasan, G., Rouyer, F., Rosbach, M., 1994. Members of a family of Drosophila putative odorant-binding proteins are expressed in different subset of olfactory hairs. Neuron 12, 35±49. Robertson, H.M., Martos, R., Sears, C.R., Todres, E.Z., Walden, K.K., Nardi, J.B., 1999. Diversity of odourant binding proteins revealed by an expressed sequence tag project on male Manduca sexta moth antennae. Insect Molecular Biology 8, 501±518. Slifer, E.H., 1966. Sense organs on the antennal ¯agellum of a walkingstick Carausius morosus Brunner (Phasmida). Journal of Morphology 120, 189±202. Steinbrecht, R.A., 1987. Functional morphology of pheromone-sensitive sensilla. In: Prestwich, G.D., Blomquist, G.J. (Eds.). Pheromone Biochemistry. Academic Press, New York, pp. 353±384. Steinbrecht, R.A., 1993. Freeze-substitution for morphological and immunocytochemical studies in insects. Microscopy Research and Technique 24, 488±504. Steinbrecht, R.A., 1997. Pore structure in insect olfactory sensilla: a review of data and concepts. International Journal of Insect Morphology and Embryology 26, 229±245. Steinbrecht, R.A., 1998. Odorant-binding proteins: expression and function. Annals of the New York Academy of Sciences 855, 323±332. Steinbrecht, R.A., 1999. Olfactory receptors. In: Eguchi, E., Tominaga, Y. (Eds.). Atlas of Arthropod Sensory ReceptorsÐDynamic Morphology in Relation to Function. Springer, Tokyo, pp. 155±176. Steinbrecht, R.A., MuÈller, B., 1976. Fine structure of the antennal receptors of the bed bug, Cimex lectularius L. Tissue and Cell 8, 615±636. Steinbrecht, R.A., Lee, J.K., Altner, H., Zimmermann, B., 1989. Volume and surface of receptor and auxiliary cells in hygro-/thermoreceptive sensilla of moths (Bombyx mori, Antheraea pernyi and A. polyphemus). Cell and Tissue Research 255, 59±67. Steinbrecht, R.A., Laue, M., Ziegelberger, G., 1995. Immunolocalization of pheromone-binding protein and general odorant-binding protein in olfactory sensilla of the silk moths Antheraea and Bombyx. Cell and Tissue Research 282, 203±217. Tuccini, A., Maida, R., Rovero, P., Mazza, M., Pelosi, P., 1996. Putative odorant-binding proteins in antennae and legs of Carausius morosus. Insect Biochemistry and Molecular Biology 26, 19±24. Vogt, R.G., Callahan, F.E., Rogers, M.E., Dickens, J.C., 1999. Odorant binding protein diversity and distribution among the insect orders, as indicated by LAP, an OBP-related protein of the True Bug Lygus lineolaris (Hemiptera, Heteroptera). Chemical Senses 24, 481±495. Weide, W., 1960. Einige Bemerkungen uÈber die antennalen Sensillen sowie uÈber das FuÈhlerwachstum der Stabheuschrecke Carausius (Dixippus) morosus Br. (Insecta: Phasmida). Wissenschaftliche Zeitschrift MartinLuther-UniversitaÈt, Halle-Wittenberg Math-Naturwiss Reihe, 9, pp. 247±250.
