The analysis of the mechanosensory origin of the infrared sensilla in Melanophila acuminata (Coeloptera; Buprestidae) adduces new insight into the transduction mechanism

Arthropod Structure & Development 36 (2007) 291e303 www.elsevier.com/locate/asd

The analysis of the mechanosensory origin of the infrared sensilla in Melanophila acuminata (Coeloptera; Buprestidae) adduces new insight into the transduction mechanism Anke Schmitz a, Angelika Sehrbrock b, Helmut Schmitz a,* a

Institute of Zoology, University of Bonn, Poppelsdorfer Schloss, D-53115 Bonn, Germany b Forschungszentrum caesar, Ludwig-Erhardt-Allee 2, D-53175 Bonn, Germany Received 14 December 2006; accepted 1 February 2007

Abstract The thoracic infrared (IR) sensilla of the pyrophilous jewel beetle Melanophila acuminata most likely have evolved from hair mechanoreceptors (sensilla trichodea). To further elucidate the sensory transduction mechanism, the morphology of IR sensilla and of neighbouring hair mechanoreceptors was investigated by using conventional electron microscopical techniques (SEM, TEM) in combination with focused ion beam milling (FIB). It was assumed that any deviation from the bauplan of a sensillum trichodeum is of particular concern for the transduction of IR radiation into a mechanical stimulus. Thus, the structures supposed to be relevant for stimulus uptake and transduction were homologized. Compared to a hair mechanoreceptor, an IR sensillum shows the following special features: (i) the formation of a complex cuticular sphere instead of the bristle; the sphere consists of an outer exocuticular shell as well as of an inner porous mesocuticular part. (ii) The enclosure of the dendritic tip of the mechanosensitive neuron inside the sphere in a fluid-filled inner pressure chamber which is connected with a system of microcavities and nanocanals in the mesocuticular part. Hence we propose that an IR sensillum most probably acts as a microfluidic converter of infrared radiation into an increase in internal pressure inside the sphere which is measured by the mechanosensitive neuron. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Infrared receptor; Mechanoreceptor; Transduction mechanism; Forest fire; Phylogenetic development; FIB

1. Introduction Buprestid beetles of the genus Melanophila inhabit the palaearctic as well as the nearctic parts of the world. As a special behavioural feature, beetles of both sexes approach forest fires because their brood depends on burnt wood as larval food (Apel, 1989; Champion, 1909; Evans, 1962; Linsley, 1943). Initially, the freshly burnt area serves as a meeting place where the males vigorously search for females. After mating, the females deposit the eggs under the bark of the burnt trees. However, the outbreak of a forest fire is highly unpredictable. In the northern boreal forests, where most Melanophila species can be

* Corresponding author. Tel.: þ49 0228 732 071; fax: þ49 0228 735 458. E-mail address: [email protected] (H. Schmitz). 1467-8039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.asd.2007.02.002

found, a fire statistically occurs only every 50e200 years (Engelmark, 1984; Heinselmann, 1973; Zackrisson, 1977). Because finding a fire is crucial for the survival of all Melanophila species, Melanophila beetles should be able to detect fires from distances as large as possible. Therefore, it is reasonable to suppose that the sensory organs which are used for fire detection have been subjected to a strong evolutionary pressure especially with regard to sensitivity. For the detection of fires, Melanophila beetles are equipped with special antennal smoke detectors (Schu¨tz et al., 1999) and thoracic IR receptors (Evans, 1964, 1966a,b; Schmitz and Bleckmann, 1997). The IR receptors are situated in two pit organs which are located on the metathorax. Each IR organ houses about 70 IR sensilla which are closely packed together at the bottom of the pit (Evans, 1966b; Vondran et al., 1995). From the outside, a single sensillum can be recognized by a hemispherical

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dome with a diameter of about 12e15 mm. The dome is built by a thin cuticle which represents the outer boundary of a spherical internal cavity. The cavity is almost completely filled out by a tiny cuticular sphere with a diameter of about 10 mm. Based on transmission electron microscopical observations, Vondran et al. (1995) described that the sphere consists of three different zones: (i) an outer lamellated mantle; (ii) an intermediate layer of unstructured cuticle revealing many irregularly arranged microcavities; and (iii) an innermost zone where the cuticle appears uniform except for some spots of higher electron density. The sphere is connected to the vertex of the outer cuticular dome by a small cuticular stalk and the narrow gap surrounding the sphere is filled out by leaflike extensions of at least two enveloping cells (Vondran et al., 1995). In contrast to these findings, it was reported recently that a single large air-filled cavity is situated in the centre of the sphere (Evans, 2005). From below, the sphere is innervated by a single sensory cell. All morphological as well as all physiological data available so far have demonstrated that this cell is a ciliary mechanoreceptor (Schmitz and Bleckmann, 1998; Schmitz et al., 1997; Vondran et al., 1995). Based on the differing morphological results, two models of sensillum function exist. However, these models are inconsistent with each other. (i) In the first one a so called photomechanic principle was established (Schmitz and Bleckmann, 1998; Schmitz et al., 1997). The authors proposed that the biomolecules (i.e. proteins and chitin) of the cuticular sphere strongly absorb mid-IR radiation. In a way not described in detail, the resulting thermal expansion of the cuticular sphere is measured by the mechanoreceptor. (ii) Assuming the presence of an air-filled cavity inside the sphere, Evans (2005) proposed in the second model that IR radiation enters this cavity by a small apical waveguide with a diameter of about 1.5 mm. Due to the absorption of IR photons at the inner cuticular walls of the cavity; the enclosed air is heated up and expands. In a way not further specified the resulting increase in gas pressure should stimulate the mechanoreceptor. In this paper an attempt was made to conclusively resolve the details of the transduction mechanism of the Melanophila IR sensillum. For this purpose, we tried to understand the phylogenetic origin of the IR sensilla, which most probably have evolved from hair mechanoreceptors (sensilla trichodea) of the lateral body wall. This approach seemed very promising in Melanophila species because in the vicinity of the field of IR sensilla at the bottom of the pit organ, incompletely developed IR sensilla can be found (Vondran et al., 1995). The comparative study of common hair mechanoreceptors, of incompletely developed sensilla, and of fully developed IR sensilla should help to retrace the gradual transition from a well-known insect hair mechanoreceptor to an IR sensillum. For this purpose, the morphology and ultrastructure of the sensilla in and around the IR pit organs were investigated with a variety of methods. To get comprehensive insight into the three dimensional construction of the cuticular apparatus the technique of focused ion beam (FIB) milling was used. The FIB device used in our study is based on a conventional scanning electron microscope (SEM), extended by an additional

Gallium-ion gun. Similar to the SEM electron beam, the beam of the ion gun is focused and deflected by an electro optical lens system. Both beams are headed towards the same region of the specimen but are tilted by 54 with respect to each other (Fig. 1). Because of the large mass of the Gallium-ions they are able to ablate the specimen in the focus with an adequate rate. The deflection of the ion-beam is controlled by a central unit, allowing the beam to scan a wide variety of different patterns. Therefore, it is possible to simply drill holes, cut trenches or mil away complete areas. 2. Materials and methods 2.1. Animals Adult beetles hatched from burnt logs which were stored in the animal house of the Zoological Institute of Bonn University. The wood originated form forest fires in Brandenburg happening in summer 2002 and 2003, respectively. Animals were kept for several weeks in plastic boxes and fed with raisins, peanuts and walnuts. Water was given ad libidum. 2.2. Scanning electron microscopy Beetles fixed in 70% ethanol were cleaned by sonication in a mixture of chloroform/ethanol (2:1) for 2 min. After drying in air, specimens were glued on holders with carbon glue (Leit-C, Fa. Neubauer), sputtered with gold, and examined in a LEO 440i (Leica, Bensheim, Germany) scanning electron microscope (SEM). 2.3. Focused ion beam (FIB) technique To prevent drying artefacts, specimens fixed in 70% ethanol were dehydrated through an ascending series of ethanol and finally immersed in HMDS (Nation, 1983). After air-drying, defined slices were prepared with the focused ion beam (FIB)

Fig. 1. Schematic diagram of the focused ion beam setup. (1) Axis of the ion gun. (2) Axis of the electron gun. (3) Scanning scheme of the ion-beam used for the preparation (line scans). (4) Projected field of view of the electron beam. (5) Specimen laid open by the successive ablation of previous layers by the ion beam.

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machine LEO 1540XB. This device allows the ablation of probe material by a focused ion beam with high resolution. Simultaneously the probe can be imaged by a scanning electron microscope to control the milling process. The electron beam of the SEM is tilted by 54 with respect to the ion beam (cf. Fig. 1). The diameter and the current of the ion-beam can be adjusted. This allows the user to choose between fast ablation (e.g. to cut away obstacles in the view path) and precise preparations with sub-mm resolution. The minimal beam diameter is about 50 nm, the scanning resolution is 8 nm. All preparations with the FIB were done in the ‘‘milling for depth’’ mode with the milling depth parameter set to 25 nm in Silicon. Prior to the cross sections a coarse milling step was performed in front of the region of interest to remove material that would otherwise block the sight of the SEM. For that purpose the beam parameters were set to 10 nA and 30 kV. The coarse milling was stopped 2 mm in front of the region of interest and the current was reduced to 1 nA for the following fine milling. The milling was interrupted every few nanometres to take an inlense secondary electron picture by the SEM with 5 kV acceleration voltage, an aperture of 120 mm and activated high current option. 2.4. Light and transmission electron microscopy Sensory IR pit organs of 6 beetles together with about 0.5 mm of surrounding cuticle were excised in iced glutaraldehyde fixative (3% glutaraldehyde in 0.05 mol l1 cacodylate buffer, pH 7.1; osmolarity 380e400 mosmol l1) and fixed overnight. The organs were then washed in buffer, postfixed with 1.5% OsO4 in the same buffer, dehydrated through an ascending ethanol series and embedded in Epon 812. Semithin and ultrathin sections were cut with a Reichert Ultracut Microtome using glass- or diamond knives. Semithin sections (0.5 mm) were stained with a 0.05% toluidine-blue/borax solution and examined with a Leitz DM RBE light microscope. To demonstrate the different types of cuticle, the Epon resin was removed in some sections (2 mm thick) by immersion in an alcoholic KOH solution (Weyda, 1982). After washing in 96% ethanol, sections were stained with a Mallory trichrome solution for 5 min. Non-melanized exocuticle, which does not really stain, appears in a faint yellow or amber, mesocuticle appears red and endocuticle blue (Weyda, 1982). Digital images of the sections were taken with a Nikon Coolpix 5000. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM 109 TEM. Sections were taken from 10 IR organs. Longitudinal as well as transverse sections were cut through IR sensilla, incompletely developed IR sensilla and hair mechanoreceptors. 3. Results 3.1. Outer morphology of the sensilla in and around the IR pit organ The paired IR pit organs (Fig. 2A) are located ventrolaterally at the anterior border of the metathorax adjoining the

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coxae of the middle legs. Towards the mesothorax, the pits are demarcated by an edge from where the surface rapidly lowers about 0.1 mm down to the field of IR sensilla. From the level of the metathorax encompassing an IR organ, the surface slopes less abruptly towards the bottom of the pit. Fullydeveloped IR sensilla associated with wax glands are densely packed at the bottom of each pit organ (Fig. 2AeC). From the outside, a single IR sensillum is characterized by a domeshaped elevation with a small apical depression. Furthermore, the state of development of the IR sensilla within a given organ is not uniform. Looking at the size and the symmetry of a sensillum we found that sensilla in the dorsal region are larger showing a better state of development than sensilla located in the ventral half. This can be recognized also in the organ shown in Fig. 2A. At the top edge of the posterior slope of the IR organ some hair mechanoreceptors (sensilla trichodea), which are also associated with wax glands, can be found (Fig. 2A,C). Such contact mechanoreceptors are distributed all over the body of Melanophila beetles. Additionally, sensilla can be found which represent incompletely developed IR sensilla. We classified those sensilla into: (i) ‘‘intermediate sensilla’’ which show features of hair mechanoreceptors as well as features of IR sensilla (Fig. 2B); and (ii) ‘‘hidden sensilla’’ where the spheres are sunken to a variable degree into the surrounding cuticle (Fig. 2A,C). In some beetles, the intermediate sensilla can be found at the ventral edge of the IR sensilla field. Here, a small group of more or less well developed IR sensilla also shows a bristle of different length. The bristle arises from the small apical depression on top of the dome typical for an IR sensillum (Fig. 2B). Hidden sensilla can mainly be found on the ventral half of the posterior slope (Fig. 2A,C). They represent underdeveloped IR sensilla, whose spheres are partially or completely sunken into the cuticle (see also Fig. 6). The extent of sinking of the sphere is positively correlated with the distance of the sensillum to the bottom of the pit (cf. Fig. 2A,C). 3.2. Inner structure of an IR-sensillum Semithin sections stained with Mallory trichrome stain showed that the composition of the body wall cuticle outside the pit organ corresponds to the common scheme well-known in insects: below a layer of melanized (i.e. dark pigmented) exocuticle, a layer of red mesocuticle can be found. The outer surface of the exocuticle is covered by a thin epicuticle (<1 mm) which can only be discerned with the TEM at higher magnifications. However, even with the TEM, an epicuticular wax layer could not be identified. Most probably the wax layer was removed during the fixation procedure. Finally, an inner purple to blue layer of unsclerotized endocuticle can be found (Fig. 3A). However, at the bottom of the pit organ, cuticular composition is different. The most striking feature is that dark pigmented exocuticle is missing. Close to the field of IR sensilla, the exocuticle gradually loses its dark colour and gets more and more amber (see arrow in Fig. 3A). So it stains like the shaft of the bristle of a hair mechanoreceptor (Fig. 3B,C). The bristles

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Fig. 2. (A) SEM image of a pit organ and its posterior surroundings. Anterior is up and ventral is on the right. At the bottom of the pit measuring about 300  120 mm, more than 70 dome-shaped IR sensilla and associated wax glands are located. Arrowheads indicate some of the hidden sensilla with partly sunken spheres; arrows indicate some hidden sensilla with totally sunken spheres. Hair mechanoreceptors are noticeable by their bristles (br; bar 30 mm). (B) Three intermediate sensilla located at the ventral edge of the field of fully-developed IR sensilla (br: mechanoreceptive bristle; wa: wax gland; bar 10 mm). (C) Detail from the ventrally located area of the posterior slope of the pit organ shown in (A). Rectangles indicate the two hidden sensilla which were opened with the focused ion beam (cf. Fig. 6; bar 10 mm).

are stiff and look whitish like the IR sensilla when viewed under a binocular microscope. Therefore, we conclude that the amber staining material represents hard but unpigmented exocuticle. At the bottom of the pit organ, this kind of achromatic exocuticle forms the outermost thin covering of the hemispherical domes of the IR sensilla and the outer mantle of the internal spheres (Fig. 3A,D,E). The inner thin layer of the domes covering the spheres consists of reddish mesocuticle. Mesocuticle also fills out the inner lumen of the spheres (Fig. 3A,D,E). However, inside the innermost core of the sphere a small portion of amber exocuticle can be found (see asterisk in Fig. 3D). This exocuticular area builds a pear-shaped mass with its largest part situated about in the middle of the sphere. The slender part continues into the apical depression of the outer dome.

Hence, light microscopical results demonstrate that the sphere in principle consists of solid exo- and mesocuticular parts. However, our ultrastructural examinations revealed a lot of submicroscopic hollow spaces inside the sphere. Micro- or even nanocavities and canals can especially be found in the middle region (Figs. 4AeD and 5A,B) which coincides with the part of the sphere staining red with the Mallory stain thereby indicating mesocuticle. Because the cavities in this mesocuticular layer are obviously interconnected to each other, the intermediate layer shows a porous appearance. A larger cavity was always found in the lower part of the sphere at the border between the exocuticular lamellated mantle and the intermediate layer (Figs. 4C,D and 5CeF). We called this conspicuous cavity the ‘‘inner pressure chamber’’ as will be explained below.

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Fig. 3. Semithin sections through IR sensilla, hair mechanoreceptors, and a hidden sensillum stained with Mallory trichrome stain. Melanized exocuticule (exo1): dark brown to black, non-melanized exocuticle (exo2): faint yellow, mesocuticle (meso): red, unsclerotized endocuticle (endo): blue. (A) Two neighbouring IR sensilla. Both spheres are cut peripherally through the outer lamellated mantle (lm, yellowish) and the intermediate layer (il, red). On the left, the cuticle of the anterior wall of the pit organ is shown. Note that the outer melanized exocuticle (upper left corner) gradually looses its dark colour becoming more and more yellowish towards the bottom of the pit. Arrow indicates transition region between both types of exocuticle. wa: wax gland (bar 10 mm). (B) Section through the basal region of a hair mechanoreceptor. The base of the bristle (br) is sunken about 13 mm in the mesocuticle. The wall of the canal enclosing the bristle consists of dark exocuticle. Near the knobbed base of the bristle, the exocuticle looses the dark coloration and the encasement around the bristle base (arrow) appears yellowish (bar 3 mm). (C) Section through a hair shaft (br: bristle, bar 3 mm). (D) Semithin section through the centre of an IR sensillum. Note the yellowish innermost region inside the sphere (asterisk). il: intermediate layer, lm: lamellated mantle, pc: pressure chamber, wa: wax gland (bar 5 mm). (E) Section through a hidden sensillum concealed in the cuticle. A small stalk of dark exocuticle extends towards the sphere. Arrow points to the inner region of the sphere where a faint dark coloration can still be discerned, lm: lamellated mantle (bar 5 mm).

Looking at the ultrastructure of the inner core of the sphere which appeared amber in the Mallory stained sections, it turned out that this portion of cuticle is homogenous (Figs. 4D and 5A). No cavities or indications for the existence of chitin fibres could be found. In contrast to this, chitin fibres are a striking feature of the outer mantle of the sphere. The prominent lamellation is caused by helicoidally deposited chitin fibres. As a striking feature, layers become gradually thicker towards the outside of the sphere (Fig. 5A,B). A few extremely small canals run centrifugally through the outer mantle (see arrows in Fig. 5B). By means of that, a fluid-filled connection between the cavity system of the mesocuticular layer inside the sphere and the cleft around the sphere is established. Furthermore, the thickness of the outer mantle increases significantly from the top to the bottom of the sphere (Figs. 3D,E, 4BeD and 5B). From below, a canal with a diameter of about 2 mm penetrates the bottom of the outer lamellated mantle (Figs. 3D, 4D and 5D). The dendritic outer segment (DOS) of the sensory cell runs through this canal. About 1 mm below the bottom of

the inner pressure chamber, the lumen of the canal abruptly narrows and opens into the inner pressure chamber by a small funnel-shaped pore. Initially, this pore has a diameter of about 1 mm which narrows down to 0.3 mm. The tip of the DOS is squeezed through this pore (Fig. 5E). The space between the dendritic membrane and the conical pore is filled out with an electron dense matter which most probably represents material of the dendritic sheath. Thus, the olive-shaped tip of the DOS is situated inside the inner pressure chamber (Figs. 5E,F and 7). The flattened upper portion of the tubular body can be found inside the tip which is embedded in a mass of chitin fibres (Fig. 5C,E,F). When taking a closer look at these fibres, it became apparent, that two kinds of fibre-like material can be identified: (i) a mass of lighter fibres directly surrounding the dendritic tip (Fig. 5E,F); and (ii) a weak layer of darker material which builds a thin covering on top of the mass of the lighter fibrous material (see arrowheads in Fig. 5E,F). Results are summarized in the schematic drawing provided in Fig. 7.

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Fig. 4. SEM images of an IR sensillum successively opened by a focused ion beam (FIB). (A) Sensillum opened in the region of the outer lamellated mantle (lm) of the internal sphere. wa: wax gland. (B) A few mm deeper inside the sphere, the porous intermediate layer (il) becomes visible. (C) In the region where the small cuticular stalk extends from the apical depression to the sphere, the inner pressure chamber (pc) becomes visible. Note that some of the small canals are connected to the pressure chamber. (D) In the centre of the sphere, an area of solid cuticle can be discerned (asterisk). A small dendritic canal (arrow) extends towards the inner pressure chamber (pc), (all bars 2 mm).

3.3. Inner structure of hair mechanoreceptors and hidden sensilla The bauplan of the hair mechanoreceptors (sensilla trichodea) close to the pit organ is corresponding to the well-known scheme of cuticular insect mechanoreceptors (Keil, 1997; McIver, 1975; Thurm, 1969). As a special feature of the

mechanoreceptors investigated in our study, the base of the hair is sunken about 12 mm below the outer surface of the cuticle (Fig. 5G). Consequently, a small canal runs from the exterior down to the base of the bristle. As revealed by the Mallory stained sections, the wall of the canal consists of melanized exocuticle (Fig. 3B). However, at the level of the base of the bristle, the cuticle of the wall looses its dark colour,

Fig. 5. (A) TEM image of a horizontal section through an IR sensillum at the level where the sphere shows its maximal diameter. The gap around the sphere is filled out by extensions of enveloping cells. Asterisk indicates the innermost cuticular core (il: intermediate layer, lm: lamellated mantle, bar 5 mm). (B) TEM image showing details of the outer lamellated mantle (lm). The mantle is partly penetrated by centrifugally oriented nanocanals (arrows). pc: pressure chamber (bar 2 mm). (C) TEM section through an IR sensillum at the level of the inner pressure chamber (pc). The tip of the dendrite (arrow) inside the pressure chamber is surrounded by a dense meshwork of chitin fibres (bar 3 mm). (D) Inner pressure chamber (pc) opened by FIB. Note bundles of dried microfibres (arrow) inside. Asterisk indicates the dendritic canal (bar 2 mm). (E) Longitudinal section through the inner pressure chamber (pc). The olive-shaped tip of the dendrite (arrow) is embedded in a mass of chitin fibres filling out the pressure chamber. Double arrowhead points to pieces of darker fibrous material overlaying the lighter fibres. Large arrow indicates electron dense material plugged between the dendritic membrane and the small conical pore in the lamellated mantle (lm). tb: tubular body (bar 1 mm). (F) Detail of the inner pressure chamber from (C). The tip of the dendrite (arrow) is completely enclosed by fibrous material. A thin irregular layer of dark fibrous material (arrowhead) demarcates the pressure chamber distally. The irregular appearance of the cuticle is caused by the spongy structure of the intermediate layer (il, bar 1 mm). (G) Basal region of a hair mechanoreceptor exposed by FIB. The hair base is located about 12 mm below the cuticular surface. Arrow indicates the fibres of the dried joint membrane connecting the bristle (br) to the surrounding cuticle (bar 3 mm). (H) Corresponding TEM image of the basal region of a hair mechanoreceptor. The tip of the dendrite (arrow) is directly connected to the cuticle of the bristle and supported by a socket septum (ss). The joint membrane (jm) contains numerous chitin fibres and is covered by a thin layer of (dark) cuticle. Asterisk indicates the bell-shaped encasement of the bristle base. wa: wax gland (bar 1 mm). (I) TEM section through the inner pressure chamber of a hidden sensillum situated at the upper edge of the pit organ. The tip of the dendrite (arrow) is already totally enclosed by the fibre-rich former joint membrane; some fragments of the dark cuticular material formerly connecting the base of the hair to the surrounding cuticle are also visible (arrowhead). Note that the round cuticular enclosure shows no lamellation and is not yet separated from the surrounding cuticle by a gap (bar 2 mm).

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becomes yellowish, and builds a bell-shaped encasement around the base of the bristle (Figs. 3B and 5H). The base of the bristle is connected to the cuticle of the encasement by a fibrous joint membrane which is covered by an extremely thin layer of electron-dense cuticle (Fig. 5H). The tip of the DOS, which is directly connected to the cuticle of the bristle, is supported by a socket septum. The socket septum arises from the lower circular opening of the encasement around the bristle base (Fig. 5H).

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Because intermediate sensilla (cf. Fig. 2B) cannot be found in each beetle, we obtained sections through an intermediate sensillum only once. Here, the base of the bristle was developed and innervated in the same way as in an ordinary hair mechanoreceptor. Despite the fact that a more or less well pronounced hemispherical bulge existed around the base of the hair, no internal sphere was found. On the other hand, the hidden sensilla which can be found in the posterior wall of the IR organ are already more

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Fig. 6. Hidden sensilla partly exposed by focused ion beam (FIB). (A) Upper edge of the posterior wall of the IR organ. Arrow indicates the place where a totally hidden sensillum was uncovered by FIB (sensillum is marked in Fig. 2C by the lower rectangle; bar 10 mm). (B) Detail of the sensillum from (A). The inner sphere is situated about 6 mm under the surface and only incompletely separated from the surrounding cuticle. The inner pressure chamber (pc) as well as the spongy material of the intermediate layer (il) can be seen. Asterisk indicates dendritic canal (bar 2 mm). (C) After some additional mm of material removal, the spongy material of the intermediate layer (il) becomes fully visible (bar 2 mm). (D) Detail of a hidden sensillum marked in Fig. 2C by the upper rectangle. The cuticle covering the sphere is already slightly bulged out; however, the inner sphere is still incompletely separated from the surrounding cuticle. The inner pressure chamber (pc) contains a tangled mass of shrunken fibres (il: intermediate layer, bar 2 mm).

developed like IR sensilla: distinct spheres were found which show well-developed inner pressure chambers and the spongy nature of the intermediate layers is evident (Fig. 6BeD). However, one important difference to a fully developed IR sensillum exists: the hidden spheres are only incompletely separated from the surrounding cuticle by narrow clefts (Figs. 3E, 5I, and 6BeD). As already mentioned above, the sunken spheres become more and more lifted out of the cuticle as the distance to the bottom of the pit decreases. As a consequence, the cuticle which covers the sphere becomes thinner (cf. Fig. 6B,D). Because the upper cuticular layer of the posterior wall consists of melanized exocuticle, the hidden spheres are always covered by black exocuticle (Fig. 3E). 4. Discussion 4.1. Benefits of the FIB technique in arthropod cuticle research Originally the FIB-technology was developed to prepare anorganic samples like silicon-chips.

The results presented here demonstrate that this technique is also suitable for handling rigid organic materials like insect cuticle. The FIB images can be directly compared to TEM images and e due to the threedimensional impression e can provide additional insight into complex spatial structures. No thermal damage has been observed at the dissected surfaces (see also Marko et al., 2006). In conclusion, the FIB-System may be used like a scalpel or microtome with nm-resolution; however, it has the big advantage that no mechanical forces act on the sample and that it is not necessary to embed the sample into a resin. Both the preparative ion-beam and the imaging electronbeam may be used simultaneously. Therefore, it is possible to follow the automated preparation on-line and to intervene manually if necessary. The SEM picture is also used to plan the next preparation steps. The dedicated software allows the user to draw lines or areas as an overlay onto the SEM-picture. These structures will then be successively scanned by the ionbeam. The system may also be configured to continuously mill away surfaces and to automatically take a series of SEM-pictures in equidistant layers. Therefore, a dataset can

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Fig. 7. Schematic reconstruction of an IR sensillum. Asterisk indicates innermost portion of unmelanized exocuticle. The tip of the dendrite enclosed in the pressure chamber (pc) is embedded in a mass of fibrous material. exo: exocuticle, meso: mesocuticle, endo: endocuticle, il: spongy intermediate layer.

be produced that contains the complete 3D-structure of the object. 4.2. Cuticular composition and ultrastructure of an IR sensillum In principle, the findings of the previous work of Vondran et al. (1995) as briefly described in the introduction could be corroborated. Basically, the internal sphere of an IR sensillum consists of solid cuticle. Only the middle (i.e. intermediate) layer shows an irregular network of small cavities and nanocanals. On the other hand, the existence of a large airfilled space in the centre of the sphere, which has been postulated recently, (Evans, 2005) could not be verified. However, by our recent investigations we also could show some new structural features of the spheres. By the staining experiments with Mallory triple stain we could identify the different types of cuticle building up the cuticular apparatus of an IR sensillum. It turned out that the outer dome covering the sphere as well as the lamellated outer mantle of the sphere consists of colourless b-sclerotized exocuticle (Andersen, 1971; Andersen and Barrett, 1971). Because the mantle is reinforced by intersecting layers of chitin fibres, the sphere has an outer hard shell of high mechanical stability. In contrast to this, the intermediate layer consists of less sclerotized and, therefore, softer mesocuticle. No evidence was found for the existence of chitin fibres. Finally, a small portion of b-sclerotized cuticle fills out the centre of the upper half of the sphere (cf. Fig. 7).

The ultrastructural investigation revealed the existence of very small nanocanals extending through the outer mantle (Fig. 5B). Most probably all cavities in the intermediate layer are interconnected. Furthermore, a distinct pressure chamber connected with the mesocuticular cavity system was identified in the lower part of the sphere. Inside the pressure chamber, the tip of the mechanosensory dendrite is located which is enclosed by a dense meshwork of chitin fibres. The authors are convinced that all cavities inside the sphere are filled with a liquid in living beetles. Reasons are that: (i) we never found an air-filled cavity which indicates that buffer solution as well as resin could get into all cavities inside the sphere. (ii) There is no evidence, that any watertight epicuticular and/or wax layer surrounds the outer lamellated mantle. It is not known that the apical extensions of the enveloping cells surrounding the sphere (Vondran et al., 1995) are able to secrete a wax layer. Therefore, water can get into the cavities by cuticular diffusion and through the nanocanals. (iii) We frequently found diffuse spots of electron-dense material in the cavity system. This indicates the existence of molecules of higher molecular weight dissolved in the intracavital water which became agglutinated during fixation. 4.3. Retracing the way of evolution: conversion of a hair-mechanoreceptor into an IR receptor At a first glance, the IR sensilla, which have been described so far only in pyrophilous beetles of the genus Melanophila, seem to be quite different from contact hair

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mechanoreceptors. However, incompletely developed IR sensilla, which can be found close to the field of fully-developed IR sensilla, provide first evidence that the IR sensilla have evolved from common mechanoreceptors of the bristle type (i.e. sensilla trichodea). Such mechanoreceptors, which can be found in any insect, serve as near-field receptors for the perception of mechanical forces (e.g. touch). The mechanical stimulus deflects a bristle of different length which stimulates a bipolar mechanosensory cell (McIver, 1985; Thurm, 1964). The dendritic tip, which is mostly supported by the subcuticular socket septum (Gaffal, 1975), is attached to the hair base via the dendritic sheath. In order to transmit the mechanical stimulus to the mechanosensory dendrite efficiently, no energy has to be lost by the deformation of the bristle. Therefore, the bristle consists of an amorphous, stiff exocuticle. The bristle is connected to a surrounding cuticular socket by an elastic joint membrane which allows a directed deflection of the bristle and a subsequent return into its original position. For this purpose, the joint membrane is composed of chitinous fibres embedded in an amorphous matrix. Most probably, the matrix contains the elastic protein resilin (Keil, 1997; WeisFogh, 1960). Most mechanoreceptor hairs act as first-order levers. By deflection of the outer part of the bristle, the tip of the DOS is deformed. This deformation immediately results in an opening of stretch-activated ion channels located in the cell membrane (French, 1992; Gillespie and Walker, 2001). The intermediate sensilla (Fig. 2B) clearly show features of hair mechanoreceptors (a bristle) as well as of IR sensilla (a dome-shaped elevation). It appears that a rough negative correlation exists between the length of the bristle and the visibility of a hemispherical elevation. In the few intermediate sensilla we investigated, no internal sphere was found and the base of the bristle was developed as in a normal hair mechanoreceptor (cf. Fig. 5H). This may point to a disruption of the gradient of a morphogenetic factor controlling the development of the sphere and/or the bristle. When analysing the positions of the hidden spheres it became evident that hidden spheres are located at spots where hair mechanoreceptors would be expected (cf. Figs. 2A,C and 6A). Also the association of the hidden spheres with wax glands corroborates the hypothesis that hidden spheres represent transformed sensilla trichodea. We consider the hidden sensilla to be incompletely developed IR sensilla because their spheres are less exposed than the spheres of fully-developed IR sensilla. The greater the distance to the bottom of the pit, the deeper the sensilla are sunken into the surrounding cuticle. Consequently, at the upper edge of the pit, hidden sensilla are only detectable by a small depression next to the openings of the accompanying wax gland. In those totally hidden sensilla, the outermost tip of the mechanoreceptive sensory cell which is already located in a pressure chamber (Figs. 5I and 6B) is situated about 10e12 mm below the outer surface of the cuticle. This corresponds well to the conditions in a sensillum trichodeum (cf. Fig. 5G). In contrast to this, dendritic tips of fully developed IR sensilla are situated only 1e 2 mm below the cuticular surface (Fig. 4C,D). Thus, a tendency can be postulated to lift the spheres out of the cuticle and to bring the site of transduction closer to the surface.

Hidden spheres are covered by darkly pigmented exocuticle. As in fully-developed IR sensilla, the spheres are connected to the outer layer of exocuticle by a small cuticular stalk. In the hidden spheres, this stalk consists of dark exocuticle (Fig. 3E). By comparison with normal hair mechanoreceptors, the origin of the stalk becomes clear: it obviously consists of the fused exocuticular walls of the canal through which the bristle runs in a hair mechanoreceptor (cf. Fig. 3B). Therefore, the small depressions which can be seen from the outside represent the funnel-shaped relics of this fusion. That the stalk stains yellowish in fully developed IR sensilla is due to b-sclerotization of the exocuticle at the bottom of the pit organ. In some of the Mallory stained sections it appeared that some dark staining took place in the upper centre of the hidden spheres (see arrow in Fig. 3E). This provides additional evidence that the innermost pear-shaped core of the spheres consists of exocuticle. The narrow gap which surrounds the sphere in fully-developed IR sensilla with the exception of the stalk (cf. Figs. 3D, 4AeD, 5AeC and 7) is only incompletely developed in the hidden spheres (cf. Figs. 3E, 5I and 6BeD). Hence, it seems not very likely that the enveloping cells which extend inside the cleft (Vondran et al., 1995) may be involved in the secretion of the sphere. It has to be postulated that the gap around the sphere increases, the more the sphere bulges out of the cuticle. Therefore, the cleft filled out by apical extensions of enveloping cells most probably has a functional significance (see below). Consequently, a morphogenetic gradient has to be postulated which is responsible: (i) for the development of the cleft around the spheres; and (ii) e at the same time e for lifting the spheres out of the cuticle. Obviously, this gradient decreases with increasing distance from the bottom of the pit. 4.4. Hypothetical model about the conversion of a hair mechanoreceptor into an IR sensillum Fig. 8 depicts our current hypothesis about the evolutionary steps which deem necessary to convert a hair mechanoreceptor into an IR sensillum. As already mentioned, the exocuticular walls of the canal through which the bristle runs have to fuse (see arrows in Fig. 8A). Additionally, the bell-shaped enclosure of the bristle base, which most probably consists of colourless b-sclerotized exocuticle, has to increase considerably. By the additional inclusion of chitin fibres, the outer lamellated mantle of the sphere could be produced. The next important step is that the fibres of the joint membrane lose the contact to the base of the bristle and become connected to the tip of the mechanosensory dendrite. We speculate that during this reorganisation process mesocuticular material can get into the growing outer mantle. The inner core of exocuticular material (marked by an asterisk in Fig. 7) which can be found in the upper part of the sphere, most probably represents remains of the merged wall material which becomes to lie inside the sphere. It has to be proposed that the circular connection of the joint membrane to the cuticle of the former bell-shaped enclosure around the bristle base persists. This region becomes incorporated into the lamellated sphere and

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Fig. 8. Proposed evolutionary pathway from a hair mechanoreceptor towards an IR sensillum: (A) a fusion of the walls of the exocuticular canal through which the bristle runs in a hair mechanoreceptor has to be proposed (arrows 1). In which way the lamellated outer mantle is formed and how mesocuticular material gets into the mantle is still unclear. exo1: melanized (black) exocuticle. (B) After the formation of an internal sphere which is totally hidden in the cuticle, a cleft begins to grow around the sphere (arrows 2). (C) The next step is the ascending of the sphere so that the covering cuticle builds a thin-walled hemispherical dome (arrow 3). exo2: b-sclerotized (colourless) exocuticle.

builds the lateral wall and the bottom of the inner pressure chamber of the sphere. From below, a gap begins to develop around the sphere which allows the outgrowing apical extensions of the enveloping cells to enwrap the sphere (see arrows in Fig. 8B). By these steps, the formation of a hidden sphere can be explained (Fig. 8B). The next steps which finally result in the formation of a fully-developed IR sensillum are straightforward to postulate: spheres must be lifted out of the cuticle (arrow in Fig. 8C) and the gap has to be extended around the sphere up to the apical stalk. Nevertheless, the fate of the bristle remains enigmatic in our model. In principle, two possibilities exist: (i) the bristle became totally reduced; or (ii) the material of the bristle may (partly?) persist in the exocuticular core in the upper half of the sphere and e sclerotized to a lesser degree e in the mesocuticular layer. Future investigations have to show which hypothesis is correct. 4.5. Present model of the transduction mechanism There is a strong relationship between structure and function in insect sensilla (Altner, 1977; Gnatzy and Tautz, 1980; Hunger and Steinbrecht, 1998). Thus it is promising to infer the function of an IR sensillum by means of its composition and ultrastructure. Our data strongly imply that IR sensilla can be regarded as derivatives of hair mechanoreceptors. This is consistent with the hypothesis that hair mechanoreceptors exhibit an important preadaptation with regard to the sensing of IR radiation: the biopolymers of the cuticular apparatus (i.e. proteins and chitin) as well as the water strongly absorb in the mid-IR region (Bunker and Jensen, 1998; Herzberg and Huber, 1950; Sowards et al., 2001). Therefore, it can be speculated that in hypothetical ancestral forms of Melanophila beetles which began to take advantage of the food resources created by a fire, hair mechanoreceptors may have been stimulated by strong IR radiation. Thermal expansion of the cuticle

close to the dendritic tip and of the water, e.g. inside the outer receptor lymph space, could have unspecifically stimulated the dendritic tip. According to this developmental scenario, an evolutionary pressure was exerted onto ventrolaterally located mechanoreceptors which finally led to the formation of the IR sensilla in their present form. For the further enhancement of sensitivity, it was of great importance to ensure that a maximal amount of energy is directed to the mechanosensitive ion channels. Therefore, the IR absorbing cuticular apparatus has to fulfil three important requirements: (i) the number of absorbing molecules has to be as high as possible; (ii) all molecules have to be involved in absorption (i.e. the existence of non-absorbing ‘‘futile’’ molecules have to be avoided); and (iii) the overall expansion of the absorbing matter has to be ‘‘focused’’ onto the dendritic tip. Consequently, the optimal dimensions of the cuticular apparatus of an IR sensillum can be determined by considering the penetration depth of mid-IR radiation into the cuticle. Because no data dealing with the penetration depth of mid-IR radiation into insect cuticle were available, we calculated the penetration depth of 3 mm IR radiation into water, bone and collagen from the respective absorption coefficients (Forrer et al., 1993). Results showed that penetration depth for water is 1 mm, for bone 4.4 mm, and for collagen 7.5 mm. Because of the structural similarities of collagen and cuticle, we estimated a penetration depth of approximately 6 mm for cuticle. Considering that the outer dome of an IR sensillum has a radius of about 6 mm, 3 mm IR radiation could penetrate up to the mid point of an IR sensillum. The water inside the enveloping cells surrounding the sphere should not significantly restrict IR penetration into the sphere because the thickness of the cellular layer is far below 1 mm. So the first two of the above requirements are met, because each water or cuticle molecule theoretically can serve as a primary absorber and any further increase in sensillum size would result in non-absorbing molecules inside. The outer lamellated mantle and especially the fluid in the spongy intermediate layer can serve to fulfil the third

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requirement: bringing about a maximal mechanical impact onto the dendritic tip. We propose that, first of all, the exocuticle of the outer mantle partly absorbs IR and thereby helps to heat the core of the sphere. However, due to sclerotization of the proteins and incrustation of helicoidally arranged chitin fibres (Andersen, 1979; Hazel et al., 2001; Vincent, 1980), thermal expansion of the mantle will be considerably (maybe two or three orders of magnitude) lower than that of the spongy mesocuticle inside. Although there is no direct correlation between the hardness and the coefficient of thermal expansion of a material, a general tendency exists that harder materials show a lower coefficient of thermal expansion than softer materials. In our model we propose that this is also true for the different types of insect cuticle. It has been measured that hardness and modulus (i.e. elasticity) of the different types of cuticle can cover a range of six orders of magnitude (Vincent and Wegst, 2004). Especially the hardness of chitin fibres is extraordinary and can be more than 150 GPa which corresponds to the hardness of gold (Peter, 1993; Vincent and Wegst, 2004). Due to the assumed larger thermal expansion coefficient of the apparently chitin fibre-free mesocuticular material of the intermediate layer we propose that the expansion of the intermediate layer is impaired by the outer mantle. As a consequence, the lumina of the microcavities will faintly decrease and the water which also strongly absorbs at 2.9 mm (Bunker and Jensen, 1998), thereby expanding, will be squeezed out of the cavities. It can be assumed that the softer mesocuticle of the intermediate layer contains a much larger amount of water than the harder exocuticle (Barbakadze et al., 2006; Vincent and Wegst, 2004), which will enhance IR absorption at 2.9 mm caused by the water molecules. Because the water is incompressible, expansion of the entire intermediate layer will immediately result in an increase in pressure in the pressure chamber. The resulting excess pressure can only be compensated by a pliable structure. Because the membrane of the dendritic tip is the only compliant structure inside the sphere, the resulting deformation of the membrane will open stretchactivated ion channels. We propose that the spongy intermediate layer fulfils two other important tasks: (i) The bandwidth of absorption is considerably increased because the cuticle shows a much broader absorption maximum in the mid-IR region than the water (Herzberg and Huber, 1950). (ii) The partitioning of the water-filled lumen into microcavities and nanocanals causes a considerable surface enlargement. Consequently, more heat energy can be conducted from the cuticle to the water. The remaining constituents of the former fibrous joint membrane inside the pressure chamber most probably are also parts of the transduction machinery. We suppose that, as in joint membranes of hair mechanoreceptors, a compliant protein matrix (resilin?) surrounds the chitin fibres. The membranous material sustains the tip of the dendrite in its initial position and probably is involved in transmitting pressure to the dendritic membrane. Because it has been described that IR sensilla adapt rapidly (Hammer et al., 2001; Schmitz and Bleckmann, 1998; Schmitz et al., 2000), it may be possible

that the compliant matrix also manages a fast reset of the system when the pressure decreases. In this respect, the nanocanals in the lamellated mantle are of interest (Fig. 4B). We suppose that these canals may allow a slow shifting of fluid. By this means a suddenly increasing excess pressure inside the sphere can be diminished and also low-frequency changes in ambient temperature will probably not induce the generation of action potentials. If this is true, the IR sensillum would have an inbuilt compensation mechanism for low-frequency temperature changes. Our results strongly suggest that a microfluidic component inside the intermediate layer is of crucial importance for the function of a photomechanic IR sensillum. In this respect, the role of the thin layer of enveloping cells around the sphere becomes clear: because any air inside the microcavities would be fatal, evaporated water can be substituted immediately. Acknowledgements We are grateful to Karl-Heinz Apel1 from the Landesforstanstalt Eberswalde (LFE) who supplied us with burnt wood infested with larvae of Melanophila acuminata. We are grateful to the Nees-Institut fu¨r Biodiversita¨t der Pflanzen of Bonn University for the use of the SEM. Manfred Mu¨rtz (Institute for Laser Medicine, University of Du¨sseldorf) calculated the penetration depth of IR radiation into biological materials and Michael Tewes (Center of Advanced European Studies and Research, Bonn) provided Fig. 1 and gave valuable information about the FIB technique. Supported by a grant of the American Defence Advanced Research Projects Agency (DARPA; Grant No. FA 9550-05-1-0422). References Altner, H., 1977. Insektensensillen: Bau- und Funktionsprinzipien. Verhandlungen der Deutschen Zoologischen Gesellschaft 70, 139e153. Andersen, S.O., 1971. Phenolic compounds isolated from insect hard cuticle and their relationship to the sclerotization process. Insect Biochemistry 1, 157e170. Andersen, S.O., 1979. Insect cuticle. Annual Review of Entomology 24, 29e 61. Andersen, S.O., Barrett, F.M., 1971. The isolation of ketocatechols from insect cuticle and their possible role in sclerotization. Journal of Insect Physiology 17, 69e83. Apel, K.-H., 1989. Zur Verbreitung von Melanophila acuminata DEG. (Col., Buprestidae). Entomologische Nachrichten und Berichte 33, 278e280. Barbakadze, N., Enders, S., Gorb, S., Arzt, E., 2006. Local mechanical properties of the head articulation cuticle in the beetle Pachnoda marginata (Coleoptera, Scarabaeidae). Journal of Experimental Biology 209, 722e 730. Bunker, P.R., Jensen, P., 1998. Molecular Symmetry and Spectroscopy, second ed. NRC Research Press, Ottawa. Champion, G.C., 1909. A buprestid and other Coleoptera on pine injured by ‘‘heath fires’’ in N.W. Surrey. Entomologist’s Monthly Magazine 45, 247e250.

1

This paper is dedicated to our colleague and friend Karl-Heinz Apel, one of the world-leading experts in the biology and the behaviour of buprestid beetles, who passed away much too early in April 2006.

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