“Sensory structures on the antennal flagella of two katydid species of the genus Mecopoda (Orthoptera, Tettigonidae)”

Micron 90 (2016) 43–58

Contents lists available at ScienceDirect

Micron journal homepage: www.elsevier.com/locate/micron

“Sensory structures on the antennal flagella of two katydid species of the genus Mecopoda (Orthoptera, Tettigonidae)” Erik S. Schneider ∗ , Heinrich Römer Institute of Zoology, Karl-Franzens-University of Graz, Universitätsplatz 2/1, 8010 Graz, Austria

a r t i c l e

i n f o

Article history: Received 11 May 2016 Received in revised form 2 August 2016 Accepted 2 August 2016 Available online 4 August 2016 Keywords: Katydid Orthoptera Insects Antennal sensilla Scanning electron microscopy

a b s t r a c t The typology, number and distribution pattern of antennal sensilla in two species of the genus Mecopoda were studied using scanning electron microscopy. The antennae of both sexes of both species attain a length of 10 cm. The antenna is made up of three basic segments: the scape, pedicel and flagellum, which is composed of more than 200 flagellomeres. We distinguished two types of sensilla chaetica, one type of sensilla trichodea, five types of sensilla basiconica and one type of sensilla coeloconica. The possible function of the sensilla was discussed. Six types of sensilla were considered as olfactory, one of which could also have a thermo- and hygrosensitive function. The remaining types of sensilla identified had a purely mechanosensory function, a dual gustatory- and mechanosensory function and a thermoand/or hygrosensory function, respectively. Consistent sex specific differences in the types, numbers and distribution of antennal sensilla were not found. Interspecific differences were identified especially in terms of the numbers of sensilla chaetica. © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Crickets and katydids are excellent model systems that can be used to study mating preferences based on acoustic signals (reviewed in Gerhardt and Huber, 2002; Hedwig, 2006). During the first step in mate choice, females in these insect groups approach a male by walking or flying toward the male calling song, often travelling considerable distances. They may then assess specific cues at close range before making a final mating decision. These cues may include courtship songs (functionally different from the calling songs), but during courtship and the potentially subsequent copulation, females may additionally assess many other cues, evaluating chemosensory, tactile, vibratory or visual information, which can facilitate species or kin recognition and provide important information about mate quality (Alexander, 1962; Balakrishnan and Pollack, 1997; Loher and Dambach, 1989; Singer, 1998). In addition, the considerable amount of muscular energy invested while moving the forewings during singing is converted into heat and increases the temperature of the thorax that houses the muscles (Heller, 1986). Thus, researchers have hypothesized that females

Abbreviations: ba, sensilla basiconica; ch, sensilla chaetica; co, sensilla coeloconica; MP, multiporous; MPG, multiporous grooved; MPP, multiporous pitted; NP, aporous; SEM, scanning electron microscopy; TP, uniporous; tr, sensilla trichodea. ∗ Corresponding author. E-mail address: [email protected] (E.S. Schneider).

of these insect groups may even evaluate the body temperature of males as a measure for male quality; termed the “hot male hypothesis” (Erregger et al., 2015). This increased thoracic temperature could also promote the evaporation and dispersal of volatile substances that may play a role in mate attraction and final mate choice. Whereas long-range acoustic signals, and the information contained therein, are received by ears in the forelegs by a well-studied array of sensory cells in the so-called crista acustica (Schwabe, 1906; Stumpner, 1996), the insect antennae are the major sensory organs receiving information from all other cues emitted by potential mates and from the environment. In general, an antennal sensillum consists of a cuticular apparatus, sensory neurons, and auxiliary cells. The outer cuticular apparatus is specialized according to the sensory modalities it processes and can be discerned, at least to some extent, on the basis of the morphology of its outer cuticular structures (Altner and Prillinger, 1980). This is supported by many studies where morphological examination was combined with electrophysiological methods (Altner et al., 1977, 1981; Schaller, 1982; Zacharuk, 1985). The presence of pores, which are the entry points for odorant molecules into the lumen of the sensillum, indicates that the sensillum has a chemosensory function (Steinbrecht, 1997). Conversely, the absence of pores in the sensillum wall precludes such a function. A sensillum with a single terminal pore can have both gustatory and mechanoreceptive functions, whereas the presence of multiple wall pores indicates that the sensillum has an olfactory function (Altner, 1977; Zacharuk, 1985).

http://dx.doi.org/10.1016/j.micron.2016.08.001 0968-4328/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).

44

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

The length of a katydid antenna can be five times greater than its body and, surprisingly, despite the apparent importance of these organs, the structure and distribution of antennal sensilla so far has only been described for one species (Neoconocephalus ensiger; Slifer, 1974). This species possesses at least seven different types of sensilla. Based on morphological data, which were only obtained by means of light microscopy, Slifer (1974) proposed that one type is probably not innervated, and all others represent chemoreceptors, of which one may also have a tactile function. In the present article, we describe the morphology, number and distribution of sensory structures on the antennal flagella of two katydid species of the Mecopoda complex and discuss their probable sensory modality. These two closely related species were chosen because Mecopoda sp.4 strongly increases its thorax temperature while singing, whereas Mecopoda elongata does not. The results of our analysis provide an essential basis for future electrophysiological and behavioral experiments that will allow researchers to evaluate the potential role of thermal and olfactory stimuli during mate choice. 2. Materials and methods 2.1. Animals Experiments were performed with one trilling and one chirping species of the katydid genus Mecopoda. The taxonomy of the genus Mecopoda is still unresolved. Several sibling species are morphologically similar, but have distinctly different calling song patterns (Nityananda and Balakrishnan, 2006). Insects included in this study were taken from a laboratory breed maintained at the Institute of Zoology in Graz, which was originally established from individuals collected in a tropical rainforest in Malaysia in 2010 and 2011. Korsunovskaya (2008) described the chirping species as Mecopoda elongata and the trilling species as “Mecopoda sp. 4”.

and then washed and dehydrated in two changes of 100% ethanol for 10 min each (Nagel and Kleineidam, 2015). After air-drying, the samples were mounted on aluminum stubs and sputter-coated as described above. To assess the general distribution pattern of the sensilla on the outer surface of the antennal segments, specimens were mounted on the tips of insect pins using a small drop of liquid carbon (Leit-C, Plano GmbH, Wetzlar, Germany). After sputter coating the samples from two opposing sides, they were clamped into a custom-made holder that allowed us to rotate the samples over 360◦ (DitscheKuru et al., 2011). By using this method in combination with the cacti needles as orientation markers, we could determine the position of individual sensilla across the whole surface of different antennal segments. 2.3. Production of sections for light and transmission electron microscopy To gain more information about certain structural aspects of the antennal flagella and their sensilla, preliminary semi- and ultrathin cross-sections were produced. Small pieces of the antenna containing single to few flagellar segments were excised and immediately fixed overnight in iced 0.05 M cacodylate buffer containing 3% glutardialdehyde at 4 ◦ C. After 2 h post-fixation with 1.5% OsO4 in the same buffer and rinsing in buffer solution, specimens were dehydrated in a graded series of ethanol and embedded in Epon 812 according to Luft (1961). Semi-thin sections with a thickness of 0.5–1 ␮m and ultra-thin sections with a thickness of about 70 nm were cut with a diamond knife using a Leica 2065 Supercut microtome and a Leica Ultracut UCT microtome, respectively. Semi-thin sections were stained with 0.1% toluidine-blue/borax solution and examined with an Olympus BH2 light microscope. Ultra-thin sections were double-stained with 300 ppm platinum blue for 15 min and 3% lead citrate for 7 min and examined using a FEI Tecnai G2 transmission electron microscope.

2.2. Scanning electron microscopy (SEM) specimen preparation

2.4. Crystal violet staining of whole antennae

Animals were decapitated after anesthetizing them with ethyl chloride. To determine the orientation of the antennae during the preparation process, the antennae were marked while still attached to the head capsule. Antennae were mounted on a Plexiglas (poly(methyl methacrylate)) holder, positioned with either the ventral or dorsal side facing upwards and reversibly fixed with narrow stripes of adhesive tape. Small cacti needles (Opuntia sp.) were then inserted into either the ventral or dorsal side of the antenna using a micromanipulator (Altner et al., 1977). It proved beneficial to punctuate the insertion side with a tungsten wire that had been sharpened electrolytically prior to the insertion of the needles. In most cases, this prevented the cacti needle from snapping off. After marking the antennae, they were removed from the head capsule and cut into 1.5–2.0 cm long pieces, each bearing at least one cacti needle for orientation. Samples were air dried, either directly or by dehydrating them using a graded series of aqueous ethanol solutions, and then subsequently sonicated in a 1:1 mixture of chloroform and ethanol for several minutes. Specimens were mounted on aluminum stubs with adhesive conductive carbon tape. They were sputter coated with gold/palladium for 60 s at 40 mA using a Bal-Tec SCD500 sputter coater. Samples were observed using a Zeiss DSM 950 scanning electron microscope and an accelerating voltage of 15 kV. To obtain internal images of the antennal segments, antennae were bisected along their lengths with a broken razorblade. These halves were soaked in a 1 M KOH solution for 15 min to remove the soft tissue. Subsequently, antennae were washed twice in 70% ethanol for 5 min each time, sonicated in 70% ethanol for 2 min

We used the staining method invented by Slifer (1960), referred to here as Slifer’s staining method, to investigate the potential abundance and location of pores on the outer surface of antennal sensilla. After anesthetizing the animals with ethyl chloride, antennae were removed and fixed in Bouin’s solution for at least 24 h. Antennae were stained using a 0.5% solution of crystal violet, incubating samples for 5–15 min. Otherwise, the protocol of Slifer (1960) was followed. Stained specimens were analyzed using an Olympus BH2 light microscope. 2.5. Data acquisition and analysis After conducting a detailed initial examination of the different types of sensilla found on the antennae of both sexes of the investigated species, we focused on the more distal flagellar segments. We counted the flagellomeres beginning from the head of the insect (Fig. 1a). Therefore, at least the proximal 33, and at most 44, flagellar segments of each antenna were excluded from the quantitative analysis of the various sensilla parameters, because numbers and peg lengths of most types of sensilla on these segments were considerably lower than those measured on more distal segments (Fig. 5). Due to the length of the antennae, sensilla density measurements were made on every 10th flagellar segment (every 5th, in case of the coeloconic sensilla, co). The density of a particular type of sensillum was always measured by counting its number on the lateral side of the segments (cf. Fig. 1), then doubling the number to approximate the number present on the entire segment surface.

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

45

Fig. 1. Schematic drawings of Mecopoda showing a right antenna (a) and a single flagellar segment (b) and their respective orientation with respect to the rest of the body (indicated by the small arrows). (c) Schematic drawing of a single sensillum displaying the convex and concave surfaces, as well as proximal and distal ends of a sensillum. Colored lines indicate how different measurements were taken. Abbreviations: s = scape; p = pedicel; f = flagellar sub-segments. Drawings are not to scale.

Due to the fact that a certain level of asymmetry in the distribution pattern of sensilla on the surface of each antennal segment was observed (cf. 3.3 Numbers and distribution of different sensilla types on the flagellar segments), the absolute sensilla density may be overestimated to some degree using the methodology described above. The surface area of the segments was determined by calculating the outer surface of a circular cylinder M = *d*h, where M is the surface area, d is the diameter and h is the length of the segment. Sensilla density is defined as the number of sensilla per mm2 of antennal surface. The pore density (number of pores per ␮m2 in the wall of sensilla) was measured in a similar way. Measurements made on individual sensilla, such as peg length and diameter, and inclination angle, were carried out only on sensilla for which full profile-images had been taken. To measure the peg length, a curved line was traced from the middle of each sensillum between the base and tip (Fig. 1c). Peg diameter was always measured at the base of a sensillum, tracing a line perpendicular to the axis of the peg shaft that protrudes from the antennal surface (Fig. 1c). To assess the inner diameter of sockets and pore openings, we always measured the widest possible diameter (Fig. 1c). The inclination angle of a sensillum was measured distal to the antenna (i.e., in the direction of the antennal tip, Fig. 1c). Quantitative measurements were made on a total of N = 3 animals of both sexes and species, respectively. Measurements were carried out using ImageJ 1.46r (Rasband, National Institutes of Health, USA). Data was statistically analyzed using SPSS Statistics (Version 22.0.0.0, IBM Corp.). To test for differences of variances, we used the Mann–Whitney U test, with the level of significance set at P < 0.05. To classify the sensilla, we referred to the terminology of Schneider (1964), Altner (1977) and Zacharuk (1985).

3. Results & discussion 3.1. General structure of the antennae Antennae of both sexes of both Mecopoda species investigated consisted of the scape, pedicel and many (in an intact antennae, >200) flagellomeres. The antennal surface (i.e., the outermost cuticle) was covered with shallow scales, which were more prominent on the distal segments. Cross-sections of the flagellar segments revealed that they are nearly round along the whole length of the antenna. The first flagellomere (most proximal; cf. Fig. 1a) differed from the others in its great length (more than 1 mm). The other flagellomeres were highly variable, with lengths

of 136.2–983.6 ␮m. The trilling species Mecopoda sp.4 and the chirping species Mecopoda elongata displayed mean flagellomere lengths of 435.3 ␮m and 382.0 ␮m, respectively. The flagellomeres in the Mecopoda sp.4, therefore, were significantly longer than in Mecopoda elongata (P = 0.000). Antennae of both sexes and species gradually tapered from the proximal to the distal ends with maximum values of 286.2 ␮m in diameter at the base and 71.6 ␮m at the tip. The total antennal length ranged from 74.7 mm to 101.5 mm (with 171–227 flagellomeres) in Mecopoda sp.4, and from 49.7 mm to 75.9 mm (with 112–206 flagellomeres) in Mecopoda elongata. No structural sexual dimorphism was observed for these species. 3.2. Morphological features of sensilla on the flagellum By carefully examining their external shapes and dimensions, two types of chaetic, one type of trichoid, five types of basiconic and one type of coeloconic sensilla could be distinguished in males and females of both species. The pores present on the sensilla were examined, and the sensilla were classified as aporous (NP), uniporous (TP) and multiporous (MP), following the terminology of Altner (1977). The latter could also be differentiated into either multiporous pitted (MPP) sensilla, which have pore tubules, and multiporous grooved (MPG) sensilla, which are characterized by the presence of spoke canals, following the terminology of Zacharuk (1985). Although the measured data on several aspects of the dimensions of some sensilla types revealed significant differences between the sexes and/or species, we could not find any differences in terms of their general presence or typology. Data on the measured parameters of the different types of sensilla are summarized in Table 1. For a more detailed representation of the data that also distinguishes between the sexes and species, see Table A.1 (provided in the Appendix A). The results of the statistical analyses are given in Table A.2 (provided in the Appendix A). Detailed morphological characterizations of the different types of sensilla are provided in the following sections. 3.2.1. Uniporous (TP) sensilla Sensilla chaetica type 1 (ch1) are very long, sickle-shaped bristles with longitudinal grooves and blunt tips (Figs. 2 and 8). The base of the bristles, which rests on the distal edge of a wide, flexible socket, projects from the antennal surface at an angle of 31.6–99.1◦ (mean value of 72.5◦ ; Table 1; Fig. 2). The distal parts of some bristles point towards the antennal surface (Fig. 5). This extreme

46

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

Fig. 2. SEM-micrographs of ch1 (a and b) and ch2 (c and d). (a) Proximal portion of ch1. The hair shaft is inserted basally into a wide flexible socket and protrudes perpendicular to the cuticle. Scale bar: 10 ␮m. (b) Distal portion of ch1. As the sickle-shaped hair shaft arcs towards the tip, its distal part is almost parallel to the antennal surface. Scale bar: 3 ␮m. Inset shows the tip of the sensillum with a view of its terminal pore (TP). Scale bar: 2 ␮m. (c) Proximal portion of ch2. The base of the hair shaft is inserted at an acute angle into a tight socket, resting on its concave surface. Scale bar: 10 ␮m. (d) Distal portion of ch2. The pore-less hair shaft strongly tapers throughout its length, ending in a sharp tip. Scale bar: 10 ␮m.

bending, however, may not reflect the condition in the living insect and probably represents, at least to a certain degree, a drying artefact caused by the preparation procedure used. The ch1 had the longest pegs, with a mean length of 97.1 ␮m (Table 1). The bristle length varies from 57.1 ␮m to 164.4 ␮m. At the tip of the ch1, a single terminal pore was observed (Fig. 2b). The porous character was confirmed by crystal violet staining, whereby the tip of the bristle could be stained. Due to the presence of this terminal pore, a contact-chemosensory function is likely. In general, many of the TP sensilla have been described in the literature as having a dual chemo- and mechanosensory function and can be found on the antennae and other body appendages in most insect orders, e.g. Coleoptera (Daly and Ryan, 1979; Jourdan et al., 1995; Tooming et al., 2012), Diptera (McIver and Siemicki, 1978), Hemiptera (Steinbrecht and Müller, 1976), Lepidoptera (Myers, 1968), Orthoptera (Lambin, 1973) and others (Zacharuk, 1985). These sensilla possess several, usually four to six, chemosensory neurons with dendrites that extend through the hair shaft towards the terminal pore and are housed together with a single mechanoreceptor whose dendrite terminates below the base in a typical mechanosensory tubular body (Steinbrecht and Müller, 1976; Zacharuk, 1985). In N. ensiger, the only katy-

did species for which antennal sensilla have been characterized thus far, Slifer (1974) described a thick-walled chemoreceptor that highly resembled the ch1 with regard to its shape, surface structure and distribution. Five sensory neurons have been identified in this sensillum type, one of which probably represents a mechanoreceptor (Slifer, 1974). If the ch1 described here may also have a dual chemo- and mechanosensory function, requires further physiological or ultrastructural investigation (e.g., the identification of a tubular body at the base of the sensillum). In carabid beetles of the genus Pterostichus, dual chemo- and mechanosensory chaetic sensilla have been described. These house five sensory neurons, one of which has proven to be mechanosensitive (Tooming et al., 2012). The remaining four chemosensory neurons have been shown to respond to various salts (Merivee et al., 2004), sugars (Merivee et al., 2007; Merivee et al., 2008), pH (Merivee et al., 2005; Milius et al., 2006) and phytochemicals like alkaloids and glucosides (Milius et al., 2011). To what compounds the chemosensory neurons in ch1 of Mecopoda may be sensitive can only be speculated until electrophysiological data are available.

5.45 ± 9.03 (n = 335) N.A. N.A. Density of wall pores [pores/␮m2 ]

683.99 ± 179.13 (n = 179) N.A. 2

Sensilla density [sensilla/mm ]

264.42 ± 63.37 (n = 179) N.A.

31.66 ± 26.22 (n = 179) 5.27 ± 1.12 (n = 7)

7.74 ± 10.68 (n = 179) 12.70 ± 2.15 (n = 7)

54.18 ± 30.95 (n = 179) 8.16 ± 1.35 (n = 8)

23.29 ± 19.12 (n = 179) 7.70 ± 1.21 (n = 7)

N.A.

N.A.

17.93 ± 2.27 (n = 175) 26.46 ± 21.59 (n = 179) N.A. N.A. N.A Depression diameter [␮m]

N.A.

N.A.

N.A.

N.A.

N.A. N.A. N.A. Diameter of pit aperture [␮m]

Outer pit diameter [␮m]

Inclination angle [◦ ]

Peg diameter [␮m]

Inner socket diameter [␮m]

N.A.

N.A.

N.A.

N.A.

N.A.

20.26 ± 2.74 (n = 168) 7.44 ± 1.47 (n = 167) N.A.

N.A

3.93 ± 0.53 (n = 10) 2.80 ± 0.54 (n = 73) N.A

co ba3

6.29 ± 1.01 (n = 17) 1.87 ± 0.25 (n = 17) 2.60 ± 0.37 (n = 14) 72.88 ± 7.37 (n = 17) N.A. 12.50 ± 1.70 (n = 197) 3.07 ± 0.46 (n = 189) 4.90 ± 0.75 (n = 168) 53.71 ± 9.40 (n = 153) N.A.

ba2 ba1.3

48.58 ± 8.57 (n = 207) 3.62 ± 0.32 (n = 154) 5.18 ± 0.58 (n = 130) 57.60 ± 12.86 (n = 207) N.A. 36.00 ± 6.82 (n = 295) 3.59 ± 0.34 (n = 270) 5.12 ± 0.58 (n = 255) 48.70 ± 11.21 (n = 270) N.A.

ba1.2 ba1.1

21.09 ± 3.48 (n = 108) 3.50 ± 0.36 (n = 103) 5.31 ± 0.62 (n = 102) 46.65 ± 11.77 (n = 63) N.A. 54.34 ± 9.45 (n = 240) 3.66 ± 0.29 (n = 213) 5.17 ± 0.52 (n = 202) 63.35 ± 12.65 (n = 233) N.A. 84.62 ± 9.45 (n = 1023) 4.72 ± 0.48 (n = 1018) 6.11 ± 0.78 (n = 1018) 44.09 ± 8.94 (n = 1023) N.A. 97.06 ± 15.99 (n = 1033) 5.47 ± 0.53 (n = 1021) 7.52 ± 1.12 (n = 1021) 72.52 ± 11.58 (n = 1023) N.A. Peg length [␮m]

tr ch2 ch1 Sensillum type

Table 1 Measured parameters of the different types of sensilla identified in the Mecopoda species investigated. Antennae from N = 3 individuals of both sexes and species were examined, respectively. Mean values ± standard deviations and the number of individual measurements (n) enclosed in brackets are given. N.A. = data not available.

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

47

3.2.2. Aporous (NP) sensilla Sensilla chaetica type 2 (ch2) are more or less straight bristles, 57.5–117.9 ␮m long, with a hair shaft that strongly tapers from about half of its length and ends in a sharp tip (Fig. 2c and d). The tip was often bent away from the antennal surface (Fig. 2d). The base of the hair shaft was inserted at an angle of 44.1◦ ± 8.9◦ (mean ± std. dev.) into a very tight socket and rested on its concave surface. The surface structure was similar to that of ch1 (i.e., longitudinal grooves present), but these structures were restricted to the outer cuticular surface of the peg. We could not find any indications of the presence of pores in the sensillum wall. Ch2 seemed to share characteristics with the sharp-tipped hairs described by Slifer (1974) on the antennal flagellum of the katydid N. ensiger. These sensilla also exhibit the same shape with a very sharp tip, grooved surface structure and are the most abundant sensilla on the antennal surface, but lacking on the first twenty flagellomeres (Slifer, 1974). Slifer could not find any innervation of these sensilla and concluded that their function may be to provide protection for the other, more delicate sensory sensilla on the antenna. This is an interesting hypothesis that could also prove true for ch2 in Mecopoda. However, if ch2 possesses any sensory function, we propose that it must be a mechanosensory function, due to the absence of pores in the outer sensillum wall. Sensilla coeloconica (co) are peg-in-pit sensilla, consisting of a small peg set on the floor of a chamber sunken into the cuticle. A small central aperture with a diameter of 4.4–12.2 ␮m (cf. Table 1) connects the peg inside the chamber with the surrounding air. Semi-thin sections revealed that the chamber had an internal diameter of about 13 ␮m and a height of about 6 ␮m. From outside, the co can be recognized as roundish elevations of the cuticle with diameters ranging from 13.6 ␮m to 27.2 ␮m (Fig. 3g; Table 1). The sizes of the outer pit and aperture diameter of co were highly variable and especially depended on their position on the antenna. The diameters of both parameters gradually increased from the proximal to the distal flagellomeres. The small peg had a mean length of 3.9 ␮m and diameter of 2.8 ␮m and stood perpendicular to the center of the floor, whereas the tip of the peg directly faced the aperture (Fig. 3g). The cuticular surface of the peg was smooth and we could not find any indications of the presence of pores (Fig. 3h). The cuticle was not stained after applying Slifer’s staining method, nor could we find any pores in ultra-thin sections. However, a small apical indentation in the tip of the peg was visible in several preparations, which most probably represents a clogged molting pore (Fig. 3h). Such a pore has also been described in the NP sensilla coeloconica of Locusta migratoria (Altner et al., 1981) and Carausius morosus (Altner et al., 1978). Peg-in-pit sensilla of insects are highly variable in their external shape (Altner and Loftus, 1985; Di Giulio et al., 2012; Ruchty et al., 2009) but, in most cases, their innervation is similar. The most common type of pegin-pit sensilla is represented by a physiological triad containing two hygro- (dry and moist) and one thermoreceptor (cold) (Altner et al., 1981; Bernard, 1974; Merivee et al., 2003, 2010; Nishikawa et al., 1985; Nurme et al., 2015; Tichy, 1979). On the basis of findings reported in the existing literature on NP sensilla coeloconica, which include information about both the morphology and physiology of these sensilla (Altner et al., 1978, 1981; Davis and Sokolove, 1975; McIver, 1973; McIver and Siemicki, 1985; Nishikawa et al., 1985; Tichy, 1979; Zopf et al., 2014), we postulate that co also possess a thermo- and/or hygrosensory function in Mecopoda. Preliminary electrophysiological recordings from co in Mecopoda sp.4 always revealed the activity of one warm- and one cold-receptor cell (Zopf and Römer unpublished). This physiological type is much less common and has so far been described especially in hematophagous insects like the mosquito Aedes aegypti (Davis and Sokolove, 1975), the bug Rhodnius prolixus (Zopf et al., 2014) and the tropical tick

48

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

Fig. 3. SEM-micrographs of ba2 (a–d), ba3 (e and f) and co (g and h). (a) Top-view (scale bar: 10 ␮m) and (b) profile view of multiporous-grooved ba2 (scale bar: 3 ␮m). High magnification images of the distal (c) and proximal parts (d) of the hair shaft showing longitudinal grooves (indicated by black arrows) that represent spoke canals. Positions of images c) and d) are indicated by lettered rectangles given in b). Scale bars in c) and d): 500 nm. (e) Overview image of ba3 illustrating its relatively small dimensions (scale bar: 20 ␮m). (f) High magnification image of ba3 reveals the presence of several pitted wall pores that are located exclusively at the tip of the hair shaft (black arrow) and a molting scar on the concave surface at the base (white arrowhead). Scale bar: 2 ␮m. (g) Overview of co, consisting of a small, straight peg that is perpendicular to the floor of a pit sunken in the cuticle. A small aperture connects the peg inside the pit to the surrounding air. Scale bar: 10 ␮m. (h) High magnification image of co with partially damaged pit wall offers a free view of the smooth peg inside the pit. White arrowhead indicates the location of a clogged molting pore. Scale bar: 2 ␮m.

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

Amblyomma variegatum (Hess and Loftus, 1984). In contrast to the above mentioned triad, this physiological type that contains two antagonistically responding thermoreceptor cells has been shown to respond very sensitive not only to thermal stimuli in form of convective heat, but also to infrared radiation (Zopf et al., 2014). 3.2.3. Multiporous (MP) sensilla Sensilla trichodea (tr) are hairs with a length of 26.6–81.3 ␮m and a slightly inverted, S-shaped profile and a blunt tip (Fig. 4g). Like ch1, they insert into their socket at a rather steep angle of 63.4◦ (Table 1). The hair shaft has its largest diameter at the base (3.0–4.6 ␮m) and slightly tapers toward the tip (Figs. 4 g and 8). On the concave surface at the base of the tr, in most cases 5–10 ␮m above the base, a small molting scar was visible (Fig. 4g). The hair shaft distal from this structure was covered by numerous pitted pore openings with a density of 3.8–6.9 per ␮m2 (cf. Table 1; Fig. 4h). Tr had the lowest number of wall pores per area as compared to the other MPP sensilla examined in the present study. In tr, the pore openings appeared to be shallow depressions with a rather slit-like shape. Upon applying Slifer’s staining method, the distal three-quarters of the hair shaft were stained, which corresponded to the location of pore openings. Based on our findings, we propose an olfactory modality for tr. Sensilla basiconica type 1 (ba1) are primarily slightly curved, cone-like hairs with blunted tips (Fig. 4a–f). The hair shaft inserted basally at an inclination angle that ranged from 23◦ to 83◦ into a socket with a diameter of 3.1–6.8 ␮m. At its base, the hair shaft had a diameter that ranged from 2.7 ␮m to 4.9 ␮m. Like tr, ba1 could be characterized by the presence of a molting scar on the concave surface of the hair shaft in the basal region and, distal to that, by the presence of numerous wall pores (Fig. 4a–f). After applying Slifer’s staining method, the hair shaft was always stained distal to the molting scar, which corresponded to the region where the wall pores were located. Both the location of the molting scar and the pitted wall pores are characteristic features of MPP sensilla. MPP sensilla are generally considered to be olfactory (Zacharuk, 1985). Due to the fact that ba1 displayed variability with regard to their shape, peg length and density of wall pores, we further divided this sensillum type into three subtypes described in the following sections. Sensilla basiconica subtype 1.1 (ba1.1) have a peg length of 14.7–32.2 ␮m and are the shortest and stoutest sensilla within the ba1. Their diameter is greatest at the base, and they are also characterized by having the highest density of wall pores within the ba1 with 9.6–15.0 pores per ␮m2 . The wall pores appeared as very deep indentations with large diameters in the outer cuticular wall (Fig. 4b). This gave the impression of a sponge-like surface, which was already visible at lower magnification levels (Fig. 4a). The high degree of variability in the diameter of the external pits of the wall pores along with the somewhat irregular external framing indicated that the pits of at least some adjacent pores could be fused. Sensilla basiconica subtype 1.2 (ba1.2) are less stout and possess a longer hair shaft than ba1.1 with a length of 20.8–55.0 ␮m (Fig. 4c; Table 1). Another characteristic feature is that the largest diameter of the peg is not found at its base. The diameter of the hair shaft increased above the molting scar, reaching its maximum within the first third or quarter of the peg length and then tapering again up to the distal tip (Fig. 4c). Distal to the molting scar, ovalshaped wall pores could be seen that were clearly separated from one another and evenly distributed over the hair shaft (Fig. 4d). The density of wall pores in ba1.2 was significantly lower (P = 0.002) than that of ba1.1, with 5.9–9.9 pores per ␮m2 (cf. Table 1). Sensilla basiconica subtype 1.3 (ba1.3) have the longest hair shaft within the ba1, from 30.7–66.0 ␮m (Fig. 8). The peg’s diameter was largest at the base, ranging from 2.7 ␮m to 4.5 ␮m, then tapering gradually up to the distal tip (Fig. 4e). The basal third of

49

the hair shaft was nearly straight, then bent to a certain degree and became straighter again distally. The oval-shaped wall pores (Fig. 4f) occurred at a density of 7.70 ± 1.21 pores per ␮m2 (Table 1) which was similar to that found in ba1.2 (P = 0.463) but which was significantly lower compared to that of ba1.1 (P = 0.002) (Table 1; Fig. 4). Sensilla basiconica type 2 (ba2) are sensilla shaped like door handles and having peg lengths of 7.2–16.9 ␮m. The peg was set into an inflexible socket with an inner diameter of 3.1–7.4 ␮m and protruded basally from the antennal surface at an angle of 28.3–90.0◦ (cf. Table 1 and Fig. 3a and b). After a few ␮m, the hair shaft strongly curved in a distal direction to the antenna so that the distal part of the peg, which was straight, was almost parallel to the antennal surface (Fig. 3a and b). A special feature of this sensillum type is that the socket was set proximally (referring to the direction of the antenna) on the floor of a shallow circular depression that had a diameter of 12.3–25.0 ␮m. Furthermore, the depression was framed, at least on its proximal half, by a cuticular rim that projected above the surrounding cuticle (Fig. 3b). A molting scar could not be identified in ba2. Several ␮m distal from the base, the hair shaft was covered by narrow, longitudinal grooves (with an inter-groove distance of 80–150 nm) that extended to the tip (Fig. 3b–d). This grooved part of the ba2 was stained after applying Slifer’s staining method and this leads us to suppose that the longitudinal grooves are multiporous, thus representing the so-called spoke canals. Spoke canals are a characteristic feature of doublewalled MPG sensilla that have been shown to be chemosensory and primarily olfactory (Altner and Prillinger, 1980; Zacharuk, 1985). However, physiological combinations of thermo- and chemosensory (Altner et al., 1977, 1981; Schaller, 1982; Steinbrecht, 1969) as well as thermo- and hygrosensitive units (Altner et al., 1977) have also been described. All these modalities, as well as their combinations, might be conceivable in the ba2 of Mecopoda. Sensilla basiconica type 3 (ba3) have straight, short pegs with lengths of 4.3–9.1 ␮m that insert into their sockets at an angle of 60.6–85.1◦ . The largest diameter of the peg was measured at its base ranging from 1.4 ␮m to 2.2 ␮m (Fig. 3e and f; Table 1). At the proximal part of the peg, a small molting scar was identified on its concave surface (Fig. 3f). The peg’s surface was smooth except for the distal tip, which could be characterized by the presence of pitted wall-pores that were very similar in shape and dimensions to those found in ba1.1 (Fig. 3f). Applying Slifer’s staining method resulted in a dark coloration of the distal tips of these sensilla, which corresponded to the locations of wall pores identified in SEM micrographs. 3.3. Numbers and distribution of different sensilla types on the flagellar segments The proximal 21–37 flagellar segments differed from the distal flagellomeres by 1) the complete absence of ch2 (Fig. 5a and b ) and 2) the distribution pattern of all other sensilla types, with the exception of ch1, i.e., they were always grouped together in the form of sensillar fields (Fig. 5a), either on the dorsolateral and/or ventromedial surface, on about every 2nd − 4th segment. On the more distal segments, all sensilla types were more or less evenly distributed in a statistical sense (Fig. 5c). Common to all flagellomeres was the distribution pattern of ch1. These sensilla were arranged in five longitudinal rows on each segment, one on the ventral, medial and dorsal surfaces and two on the lateral surface (Fig. 5b and d). All other sensilla types, except for ch1 and ch2, were exclusively found in two longitudinal sectors located on the dorsolateral and ventromedial surfaces of the segments as indicated in Fig. 5b) and d). In terms of the general numbers of sensilla, especially of tr, ba1, ba2 and co, these were greater in the dorsolateral sector than in the ventromedial one. The

50

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

Fig. 4. SEM-micrographs of multiporous pitted (MPP) sensilla ba1.1 (a and b), ba1.2 (c and d), ba1.3 (e and f) and tr (g and h). White arrowheads mark the position of a molting scar located on the concave surface at the base of each MPP sensillum. A close-up of a molting scar of ba1.1 is shown in the inset of subfigure a). Black and white arrows indicate individual pore openings located in the sensillum wall. Scale bars: a) 5 ␮m, Inset: 2 ␮m; c), e) and g) 10 ␮m; b), d), f) and h) 2 ␮m.

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

51

Fig. 5. General distributional pattern of sensilla on the antennal flagellum. a) SEM-micrograph of the 22nd flagellomere of a female Mecopoda sp.4 showing the typical distribution pattern of sensilla on the most proximal flagellomeres. Most sensilla types are grouped together in the form of sensillar fields (indicated by dotted circle). Scale bar: 100 ␮m. b) Schematic cross-section through a proximal flagellomere as shown in subfigure a). Ch1 are arranged in five longitudinal rows along the antennal segments. Other sensilla types, except for ch1 and ch2, can only be found within two longitudinal sectors located on the dorsolateral and ventromedial surface of a segment, respectively (indicated by red areas). c) SEM-micrograph of the 110th flagellomere of a female Mecopoda sp.4 showing the typical distribution pattern of sensilla on the more distal flagellomeres. Scale bar: 100 ␮m. d) Schematic cross-section through a typical distal flagellomere as shown in subfigure c). In addition to the above-mentioned distributional pattern of proximal flagellomeres (cf. subfigure b), the distal segments are further characterized by the presence of ch2 that are arranged in several alternating, longitudinal rows. Abbreviations: ba1.1–ba1.3 = basiconic sensilla subtype1.1–subtype1.3; ba2 = basiconic sensilla type2; ch1, ch2 = chaetic sensilla type1 and type2; co = coeloconic sensilla; cp = cuticular pores; tr = trichoid sensilla. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

asymmetric distribution of the mentioned (mainly olfactory) sensilla in Mecopoda probably represents a functional adaptation for the spatial localization of corresponding stimuli, which in turn is also determined by the direction of antennal movements during searching behaviors, as has been assumed e.g. in the elaterid beetle Agriotes obscurus (Merivee et al., 1997). Specific data on antennal movements during searching or resting behaviors in Mecopoda are not available so far. Ch2 were only observed distally from segment 27 and were arranged in several alternating, longitudinal rows on each flagellomere as indicated in Fig. 5d. With 130 ± 34 sensilla per segment, ch2 were present in the highest density of all sensilla types, followed by ch1, which occurred at a density of 50 ± 12 sensilla per segment (Table 2). Consistent and significant differences between the two investigated species were only observed for the ch1 and

ch2. Antennae of Mecopoda elongata bear about 29% and 40% more ch1 and ch2, respectively, than Mecopoda sp.4 (Table 2; Table A.2). No sexual dimorphism in terms of the numbers and distribution of sensilla was observed in the two species. The least abundant sensillum type is represented by ba3, that was only observed 18–30 times per antenna (i.e., less than 1 sensillum per six antennal segments were seen), most of which were located on the proximal 30 segments. The densities measured for the other types of sensilla are summarized in Table 2. 3.4. Sensilla-free fields Sensilla-free fields are areas on the flagellomeres that are characterized by the complete absence of sensilla (Fig. 6). The scaly surface structure, abundant everywhere else on the antennal cuti-

52

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

Table 2 Sensilla density, given as numbers of sensilla per mm2 , of different types of sensilla identified in both sexes of both Mecopoda species investigated. Antennae of N = 3 individuals of both sexes and species were examined, respectively. Mean values ± standard deviations and the number of individual measurements (n) enclosed in brackets are given. N.A. = data not available. Sensillum type

ch1 ch2 tr ba1.1 ba1.2 ba1.3 ba2 ba3 co

Mecopoda sp.4

Mecopoda elongata

Female

Male

Female

Male

225.62 ± 51.04 (n = 45) 577.48 ± 150.44 (n = 45) 37.95 ± 24.25 (n = 45) 5.69 ± 7.52 (n = 45) 49.24 ± 27.21 (n = 45) 25.56 ± 15.88 (n = 45) 21.74 ± 16.01 (n = 45) N.A. 3.80 ± 6.37 (n = 85)

237.93 ± 46.61 (n = 49) 574.25 ± 138.87 (n = 49) 20.87 ± 19.93 (n = 49) 9.83 ± 11.76 (n = 49) 58.35 ± 29.71 (n = 49) 25.26 ± 23.27 (n = 49) 26.45 ± 20.34 (n = 49) N.A. 6.78 ± 9.67 (n = 95)

299.70 ± 53.93 (n = 42) 821.61 ± 155.71 (n = 42) 33.29 ± 25.76 (n = 42) 8.42 ± 10.52 (n = 42) 55.46 ± 36.20 (n = 42) 20.92 ± 15.33 (n = 42) 30.79 ± 30.07 (n = 42) N.A. 5.66 ± 10.79 (n = 79)

300.78 ± 62.75 (n = 43) 786.10 ± 103.71 (n = 43) 35.77 ± 31.59 (n = 43) 6.83 ± 12.15 (n = 43) 53.36 ± 30.77 (n = 43) 21.00 ± 20.42 (n = 43) 27.19 ± 17.55 (n = 43) N.A. 5.43 ± 8.59 (n = 76)

cle, was lacking in these specific regions (Fig. 6) as were regions of dark cuticular pigmentation (only visible in light microscopical images). Sensilla-free fields occurred only 2–3 times per antenna (depending on its total length). They were always located on the most proximal part of the medial surface of a segment, had a diameter of about 150 ␮m and covered roughly one-third of the segment’s circumference (Fig. 6a). At higher levels of magnification, several small pore openings in the outer cuticle were visible (Fig. 6b). These exhibited an inner diameter of less than about 60 nm and therefore could clearly be differentiated from cuticular pores found in other parts of the antenna (cf. 3.5 Cuticular pores on the antennal surface). Semi-thin cross-sections through these sensilla-free fields revealed no significant differences in the cuticle with regard to its thickness. A reduction in cuticular thickness of about 80% is a characteristic feature of the olfactory pore plates that have been identified on the antennae of root-feeding Melolontha melolontha larvae (Eilers et al., 2012). These pore plates, however, are similar in their outer appearance to the sensilla free-fields in Mecopoda. The infrared organs of the Australian buprestid beetle Merimna atrata are another example of cuticular areas that lack sensilla with an outer cuticular apparatus and which are further characterized by the loss of dark pigments and a considerable reduction of cuticular thickness (Schmitz et al., 2000; Schneider and Schmitz, 2014). Each of the infrared organs in these pyrophilous beetles is innervated by a single large multipolar neuron and two scolopidia (Schmitz et al., 2001; Schneider and Schmitz, 2013). These infrared organs are intended to help the beetle detect and approach forest fires and navigate safely through freshly burnt areas using infrared radiation as adequate stimulus (Schmitz et al., 2015). Another interesting example for internal sensilla that lack an outer cuticular apparatus was found in the aristal sense organ of Drosophila and other dipterans (Foelix et al., 1989). These sensilla comprise two sensory neurons, one of which shows several ultrastructural similarities to insect thermoreceptors and most probably possesses a thermoreceptive function (Foelix et al., 1989). However, because no ultrastructural data for the sensilla-free fields in Mecopoda are available, a putative sensory function cannot yet be ruled out. We hypothesize that these specific regions have either a secretory or, if innervated, most probably an olfactory function, which has to be tested in future experiments. 3.5. Cuticular pores on the antennal surface Fig. 6. SEM-micrographs of a sensilla-free field on the medial surface of an antennal flagellomere at different levels of magnification. a) Overview; scale bar: 40 ␮m. b) Higher magnification of the cuticular surface within a sensilla-free field. White arrowheads mark the position of small pore openings, one of which is depicted in an enlarged view in the inset. Scale bar: 4 ␮m; Inset: 1 ␮m.

Cuticular pores set with their openings on the floors of shallow depressions in the antennal surface were found on all segments of the flagellum (Fig. 5a and c). These had a diameter of 0.4–1.0 ␮m and variable appearances (Fig. 7a and b). They were most abundant on the proximal 21–37 flagellomeres (i.e., on average about 130 pores per segment or ↔ 700 pores/mm2 ) and their numbers declined towards the antennal tip to only about 20 pores per segment (130

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

53

Fig. 7. SEM-micrographs of cuticular pores on the antennal surface. Exterior view (a and b): cuticular pores, variable in their appearance and slightly sunken in the antennal surface, can be identified. Interior view (c and d): after removal of soft tissue, slender cuticular tubules became visible beneath these structures. These were variable in length and probably represent cuticular ducts of class 3 epidermal glandular cells. Scale bars: 2 ␮m.

Fig. 8. Schematic drawings of the different types of sensilla found in the two Mecopoda species investigated. Drawings show the characteristic shape and relative proportion of these sensilla. All drawings were made at the same scale (scale bar: 10 ␮m). Underneath the drawings of the respective types of sensilla, their abbreviated names, a short description of their pore system according to the classification scheme of Altner (1977) and Zacharuk (1985) and their proposed modality based on the morphological data presented in this study are listed. Abbreviations: ba = basiconic; ch = chaetic; co = coeloconic; MPG = multiporous grooved; MPP = multiporous pitted; NP = aporous; TP = uniporous (with a terminal pore); tr = trichoid.

pores/mm2 ) on the most distal segments (flagellomeres 111–192). After removal of soft tissue by using a gentle KOH digestion method, which is an approved method for the identification of sensilla ampullaceae (Hashimoto, 1990; Kleineidam et al., 2000; RamirezEsquivel et al., 2014), a short tubule with a constant diameter of

about 0.4–0.5 ␮m and a length of 1.0–4.3 ␮m was visible on the interior (Fig. 7c and d ).This structure could clearly be differentiated from those found in sensilla ampullaceae that have been previously described in different species of Hymenoptera (Hashimoto, 1990; Slifer and Sekhon, 1961) and Diptera (Boo and McIver, 1975;

54

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

Felippe-Bauer and Bauer, 1990). We assume that these structures have a secretory function and that the small tubules beneath the pores represent cuticular ducts, which is a characteristic for class 3 epidermal glandular cells (Di Giulio et al., 2009; Giglio et al., 2005; Noirot and Quennedey, 1974). These could secrete cuticular pheromones, compounds with water repellent properties like waxes or long-chain hydrocarbons, or protective substances. The former compounds could be involved in close range intraspecific recognition, as has been already shown for several cricket species (Otte and Cade, 1976; Tregenza and Wedell, 1997). 4. Conclusions Our quantitative data on the number of potential thermosensitive sensilla, co and ba2, in males and females of both Mecopoda species appear not to support the “hot male hypothesis”, if only females evaluate the body temperature of males, and only in the trilling Mecopoda sp.4 males get hot during singing. It is often assumed that the complexity of sensory processing may confer a selection pressure favoring higher numbers of sensilla, which, however, may not hold for all sensory modalities. For the olfactory and visual system, the number of sensory cells and therefore the number of receptor binding proteins/photopigments is important for the statistical “catch” of molecules/photons to increase sensitivity and spatial resolution. But nevertheless, exceptions to this general rule have also been described. In the apple maggot fly Rhagoletis pomonella, which contains several host-specific races that exhibit distinct behavioral preferences for different host plants, olfactory receptor cells are differentiated, not by their functional type or number in the antenna, but solely by their response thresholds and temporal firing patterns to volatiles emitted by their preferred host plants (for review see Martin et al., 2011). That sensitivity does not necessarily depend on receptor number, as shown in the previous example, seems by no means the exception but rather the rule in other sensory modalities, such as audition and probably thermosensation. In insect ears, numbers of sensory cells vary from one (in species of moths) to more than 2000 (in cicadas and ancient grasshoppers), with no sex-specific difference in the number of sensory cells in those species where only males produce sound. Even when hearing became non-functional in one sex, sensitivity in this sex decreases, but the number of sensory cells is not affected (for

review see Strauß and Stumpner, 2015). Thus, in terms of missing sex- and species-specific differences in the number of the potential thermosensitive sensilla, co and ba2, between the two investigated Mecopoda species, it may well be that a preference of females for the body temperature of singing males in Mecopoda sp.4 is built on a general thermosensitivity of these sensilla, adopted through new central nervous connectivity between the thermosensory pathway and decision making networks. Because both convective and radiant heat determine the discharge frequency, it is impossible for a single thermosensory unit to unequivocally signal the nature of the stimulus. These individual responses are always ambiguous, not with regard to the temperature change itself, but its source (Gingl and Tichy, 2001). The potential presence of two different thermosensitive sensilla, provided they differ in their response to convective and radiant heat, would allow an insect to distinguish between the combination of convective and radiant heat from the individual stimuli presented alone (Zopf et al., 2014). This would be a desirable property to support mate choice by female Mecopoda on the virtue of thermal stimuli. These females could distinguish thermal stimuli emitted by a hot male (in form of radiant heat) from stimuli arising from temperature fluctuations of the surrounding air (in form of convective heat). Whether and to what extent the sensilla types co and ba2 respond to thermal stimuli and if female Mecopoda make use of those thermal stimuli emitted by males during mate choice at all, has yet to be shown through future electrophysiological and behavioral experiments. Acknowledgements We thank Gerd Leitinger and his working group at the Institute of Cell Biology, Histology and Embryology at the Medical University of Graz for providing access to the scanning and transmission electron microscopes and for technical support. We are indebted to Sara Crockett for language editing of the first draft of the manuscript. We thank two anonymous reviewers for their helpful and valuable comments. The present work was supported by a grant from the Austrian Science Fund (FWF) to H. Römer (Project: P27145-B25). Appendix A.

Table A.1 Main measured parameters of the different types of sensilla identified in both sexes of Mecopoda sp.4 and Mecopoda elongata. Antennae from N = 3 individuals of both sexes and species were examined, respectively. Mean values ± standard deviations and the number of individual measurements (n) enclosed in brackets are given. N.A. = data not available. Sensillum parameters, mean ± std.dev., n=number of measurements ch1

Sensillum type Species Sex

Mecopoda sp.4 Female Male 97.40 ± 100.00 ± 13.69 16.11 (n=253) (n=291)

Peg length [µm]

ch2 Mecopoda elongata Female Male 94.97 ± 95.31 ± 17.23 16.25 (n=252) (n=237)

Mecopoda sp.4 Female Male 90.68 ± 87.62 ± 8.42 9.02 (n=238) (n=294)

tr Mecopoda elongata Female Male 81.57 ± 78.11 ± 7.66 7.10 (n=252) (n=239)

Mecopoda sp.4 Female Male 56.57 ± 57.00 ± 10.89 8.40 (n=59) (n=65)

ba1.1 Mecopoda elongata Female Male 53.82 ± 49.67 ± 9.29 6.91 (n=58) (n=58)

Mecopoda sp.4 Female Male

Mecopoda elongata Female Male

22.60 ± 20.61 ± 3.64 (n=23) 2.84 (n=38)

20.56 ± 3.78 (n=28)

21.01 ± 3.75 (n=19)

Peg diameter [µm]

5.48 ± 0.59 (n=253)

5.77 ± 0.58 5.31 ± 0.36 (n=287) (n=245)

5.23 ± 0.37 (n=236)

4.77 ± 0.58 4.68 ± 0.52 4.76 ± 0.44 4.66 ± 0.37 (n=238) (n=293) (n=250) (n=237)

3.71 ± 0.25 3.83 ± 0.33 3.63 ± 0.24 3.49 ± 0.23 (n=50) (n=56) (n=51) (n=56)

3.62 ± 0.30 (n=21)

3.58 ± 0.43 (n=35)

3.43 ± 0.31 (n=26)

3.33 ± 0.25 (n=21)

Inner socket diameter [µm]

7.85 ± 1.12 (n=253)

8.05 ± 1.31 7.13 ± 0.72 (n=287) (n=245)

6.92 ± 0.71 (n=236)

6.40 ± 0.70 6.45 ± 0.83 5.92 ± 0.64 5.59 ± 0.52 (n=238) (n=293) (n=250) (n=237)

5.41 ± 0.44 5.42 ± 0.46 5.18 ± 0.45 4.72 ± 0.38 (n=47) (n=52) (n=48) (n=55)

5.75 ± 0.50 (n=20)

5.46 ± 0.44 (n=35)

5.33 ± 0.56 (n=26)

4.60 ± 0.45 (n=21)

Sensilla density 2 [sensilla/mm ]

69.45 ± 13.24 (n=281) 237.93 ± 46.61 (n=49)

74.83 ± 10.13 (n=252) 299.70 ± 53.93 (n=42)

73.33 ± 11.54 (n=237) 300.78 ± 62.75 (n=43)

47.93 ± 8.33 (n=238) 577.48 ± 150.44 (n=45)

46.66 ± 10.32 (n=294) 574.25 ± 138.87 (n=49)

41.76 ± 7.43 (n=252) 821.61 ± 155.71 (n=42)

39.54 ± 5.96 (n=239) 786.10 ± 103.711 (n=43)

63.61 ± 11.12 (n=58) 37.95 ± 24.25 (n=45)

64.71 ± 13.08 (n=60) 20.87 ± 19.93 (n=49)

68.31 ± 9.30 (n=58) 33.29 ± 25.76 (n=42)

56.63 ± 13.97 (n=57) 35.77 ± 31.59 (n=43)

46.26 ± 45.99 ± 8.61 (n=19) 8.99 (n=13) 5.69 ± 7.52 (n=45)

9.83 ± 11.76 (n=49)

52.03 ± 14.11 (n=22) 8.42 ± 10.52 (n=42)

35.29 ± 6.12 (n=9) 6.83 ± 12.15 (n=43)

Table A.1 (Continued)

ba1.2 Mecopoda sp.4 Female Male

Mecopoda elongata Female Male

36.39 ± 36.83 ± 35.97 ± 34.51 ± 7.51 (n=70) 6.78 (n=88) 6.78 (n=71) 6.03 (n=66) 3.71 ± 0.30 3.72 ± 0.35 3.51 ± 0.32 3.38 ± 0.24 (n=66) (n=82) (n=58) (n=64) 5.38 ± 0.35 5.47 ± 0.47 4.93 ± 0.47 4.51 ± 0.40 (n=62) (n=80) (n=52) (n=61) 51.55 ± 11.95 (n=61) 49.24 ± 27.21 (n=45)

47.09 ± 10.27 (n=84) 58.35 ± 29.71 (n=49)

52.61 ± 11.27 (n=71) 55.46 ± 36.20 (n=42)

42.83 ± 8.64 (n=54) 53.36 ± 30.77 (n=43)

ba1.3 Mecopoda sp.4 Mecopoda elongata Female Male Female Male 48.96 ± 47.42 ± 50.58 ± 46.91 ± 8.25 8.16 9.28 (n=58) 8.12 (n=49) (n=54) (n=46) 3.63 ± 3.73 ± 0.27 3.57 ± 0.36 3.52 ± 0.35 0.29 (n=42) (n=34) (n=34) (n=44) 5.33 ± 5.44 ± 0.39 5.27 ± 0.65 4.64 ± 0.52 0.42 (n=28) (n=36) (n=31) (n=35) 57.51 ± 53.59 ± 64.88 ± 55.62 ± 10.40 15.64 10.64 10.92 (n=54) (n=58) (n=46) (n=49) 25.56 ± 25.26 ± 20.92 ± 21.00 ± 15.88 23.27 15.33 20.42 (n=45) (n=49) (n=42) (n=43)

ba2 Mecopoda sp.4 Female Male

ba3 Mecopoda elongata Female Male

Mecopoda sp.4 Female Male

co Mecopoda elongata Female Male

Mecopoda sp.4 Female Male

12.98 ± 12.88 ± 12.03 ± 11.80 ± 5.97 ± 0.83 6.71 ± 1.28 6.06 ± 0.17 1.60 (n=46) 1.58 (n=66) 1.70 (n=49) 1.73 (n=36) (n=7) (n=7) (n=3)

N.A.

3.41 (n=1)

3.06 ± 0.48 3.18 ± 0.44 2.97 ± 0.46 3.03 ± 0.48 1.73 ± 0.31 1.97 ± 0.14 1.96 ± 0.30 (n=43) (n=60) (n=44) (n=42) (n=7) (n=8) (n=2)

N.A.

2.66 ± 0.68 2.92 ± 0.68 2.77 ± 0.34 2.70 ± 0.33 (n=7) (n=30) (n=9) (n=27)

5.19 ± 0.65 4.71 ± 0.80 4.94 ± 0.68 4.82 ± 0.76 2.32 ± 0.30 2.78 ± 0.28 (n=40) (n=51) (n=38) (n=39) (n=6) (n=7)

3.04 (n=1)

N.A.

N.A.

N.A.

N.A.

N.A.

51.55 ± 57.35 ± 52.39 ± 50.29 ± 76.01 ± 8.13 (n=23) 9.57 (n=56) 9.07 (n=47) 8.57 (n=27) 7.23 (n=9)

70.65 ± 6.92 (n=5)

67.20 ± 4.94 (n=3)

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

5.66 ± 10.79 (n=79)

5.43 ± 8.59 (n=76)

21.74 ± 16.01 (n=45)

26.45 ± 20.34 (n=49)

30.79 ± 30.07 (n=42)

27.19 ± 17.55 (n=43)

N.A.

4.34 ± 0.39 (n=5)

Mecopoda elongata Female Male

3.80 ± 6.37 6.78 ± 9.67 (n=85) (n=95)

3.74 (n=1)

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

72.87 ± 10.29 (n=253) 225.62 ± 51.04 (n=45)

Inclination angle [°]

3.48 ± 0.26 (n=3)

55

56 Table A.2 Results of statistical analyses on the main measured parameters of the different sensilla types identified in both sexes of Mecopoda sp.4 and Mecopoda elongata. Antennae from N = 3 individuals of both sexes and species were examined, respectively. Differences of variances were tested using the Mann–Whitney-U test. Mean values were regarded as significantly different at P < 0.05 (++: P < 0.01; +: 0.01 ≤ P < 0.05; n.s.: 0.05 ≤ P). The exact P-values are given in brackets.

(0.310)

n.s. (0.202)

n.s. (0.640)

n.s. (0.080)

n.s. (0.161)

* (0.018)

n.s. (0.654)

n.s. (0.070)

n.s. (0.451)

** (0.001)

n.s. (0.319)

** (0.000)

n.s. (0.234)

n.s. (0.693)

** (0.004)

** (0.000)

* (0.025)

** (0.000)

N.A.

N.A.

N.A.

N.A.

N.A.

* (0.040)

** (0.000)

n.s. (0.965)

* (0.011)

n.s. (0.433)

** (0.005)

N.A.

n.s. (0.351)

Female

Mecopoda sp.4

Male Female Male

Male

** (0.000)

** (0.000)

** (0.000)

** (0.000)

n.s. (0.296)

** (0.000)

** (0.000)

** (0.000)

** (0.000)

n.s. (0.826)

n.s. (0.129)

n.s. (0.379)

n.s. (0.834)

n.s. (0.589)

n.s. (0.097)

Female Male

ch1 Mecopoda elongata Mecopoda sp.4

Female

** (0.000)

tr

Female

n.s. (0.079)

Male

** (0.000)

n.s. (0.470)

n.s. (0.281)

** (0.009)

n.s. (0.378)

** (0.009)

n.s. (0.986)

n.s. (0.433)

n.s. (0.196)

n.s. (0.146)

n.s. (0.632)

n.s. (0.344)

Female

** (0.001)

Male

** (0.002)

* (0.027)

** (0.000)

Mecopoda sp.4

Female Male

n.s. (0.156)

Female

* (0.018)

Mecopoda elongata

Female

Mecopoda sp.4

Male Female Male

** (0.004)

Female Male

ch1 Mecopoda elongata

n.s. (0.371)

tr

Mecopoda sp.4

** (0.001)

Female

* (0.012)

n.s. (0.410)

Male

n.s. (0.253)

n.s. (0.940)

Male n.s. (0.686)

n.s. (0.057)

n.s. (0.286)

n.s. (0.616)

n.s. (0.474)

n.s. (0.556)

n.s. (0.334)

n.s. (0.888)

* (0.046)

n.s. (0.845)

n.s. (0.354)

n.s. (0.127)

n.s. (0.329)

Female

n.s. (0.059)

n.s. (0.130)

Male

** (0.000)

** (0.002)

Female

** (0.000)

n.s. (0.778)

n.s. (0.491)

** (0.001)

ba1.2

** (0.000)

** (0.001)

** (0.000)

** (0.000)

Mecopoda elongata

** (0.000)

** (0.000)

Mecopoda sp.4

Male Female

** (0.000)

Male

** (0.000)

Female

n.s. (0.194)

Male

** (0.000)

Mecopoda elongata

n.s. (0.648)

Mecopoda sp.4

n.s. (0.221)

Mecopoda elongata

Female Male

** (0.000)

Female

** (0.000)

ba1.2

Female

Mecopoda sp.4

Male Female Male Female Male

ch1 Mecopoda elongata Mecopoda sp.4 tr Mecopoda elongata

** (0.000)

n.s. (0.137)

** (0.000)

** (0.000)

Female

Male

(0.095) n.s.

* (0.018)

** (0.000)

ba2

(0.312)

** (0.005)

Male

** (0.000)

* (0.030)

Female

n.s.

** (0.005)

** (0.000)

** (0.004)

Female

Mecopoda elongata

(0.888) n.s.

(0.512)

n.s. (0.223)

Male

n.s.

(0.756) n.s.

** (0.000)

Female

(0.240)

n.s.

* (0.020)

Male

n.s.

(0.901)

* (0.048)

Mecopoda sp.4

(0.237)

(1.000) n.s.

** (0.001)

ba2

n.s.

* (0.010)

(0.372) n.s.

** (0.000)

n.s. (0.609)

** (0.000)

Mecopoda elongata

n.s.

(0.778)

Male

** (0.000)

** (0.000)

Female

** (0.000)

n.s.

Male

** (0.000)

** (0.003)

Female

** (0.000)

Mecopoda sp.4

Female Male Female Male

** (0.009)

(0.861)

(0.073)

ba1.2

Female

Mecopoda sp.4

Male Female Male Female Male

Mecopoda elongata Mecopoda sp.4 tr Mecopoda elongata

(0.180) n.s.

Male

Female Male

Mecopoda elongata

n.s. (0.855)

n.s.

(0.358)

** (0.001)

(0.059) n.s.

** (0.000)

n.s. (0.190)

n.s. (0.259)

n.s. (0.693)

co

** (0.005)

n.s.

** (0.000)

Male

co

* (0.014)

N.A.

** (0.000)

** (0.000)

co

** (0.006)

** (0.000)

n.s.

co

co

** (0.008)

** (0.001)

Mecopoda elongata

N.A.

* (0.025)

Mecopoda sp.4

N.A.

(0.149)

** (0.002)

ba2

N.A.

Female

N.A.

Male

N.A.

n.s.

n.s. (0.09)

Female

n.s. (0.234)

Male

* (0.041)

Female

n.s. (0.215)

n.s. (0.943)

n.s. (0.597)

Male

n.s. (0.41)

ba1.2

n.s. (0.875)

Mecopoda elongata

Male Female Female Male Female Male

* (0.031)

n.s. (0.077)

* (0.021)

** (0.000)

Female

ba1.3

Male

Mecopoda sp.4

n.s. (0.089)

n.s. (0.568)

(0.374)

** (0.000)

ba1.3

ba1.2

n.s. (0.191)

n.s.

* (0.025)

Male

** (0.000)

ba1.3

Mecopoda elongata

** (0.004)

n.s. (0.311) n.s. (0.422)

** (0.000)

ba1.3

Mecopoda sp.4

** (0.001)

ba1.3

ba2

** (0.000)

n.s. (0.633)

Mecopoda sp.4

Female Female Male

(0.8)

tr

n.s. (0.716) n.s.

** (0.000)

* (0.015)

n.s. (0.223)

Female

ba1.1

Female

n.s. (0.086)

** (0.000)

n.s. (0.443)

ba1.1

Mecopoda elongata

* (0.041)

** (0.001)

Male

ba1.1

Mecopoda sp.4

(0.032)

n.s. (0.689)

ba1.1

Mecopoda elongata

** (0.000)

* (0.017)

** (0.000)

Female (0.121)

n.s. (0.201)

n.s. (0.707)

n.s. (0.097)

n.s. (0.680)

n.s. (0.575)

n.s. (0.829)

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

*

(0.556)

Male

ch2

(0.01) n.s.

ba1.1

n.s. (0.092) n.s. (0.261)

*

** (0.000)

Female

n.s. (0.091) n.s. (0.710) n.s.

Sensilla density Mecopoda sp.4 Mecopoda elongata

ch2

** (0.000)

ch1

** (0.000)

Male

Inclinaon angle Mecopoda sp.4 Mecopoda elongata

ch2

** (0.001)

n.s. (0.683)

Male

(0.000)

Female

** (0.000)

ba2

Female

**

Male

Inner socket diameter Mecopoda sp.4 Mecopoda elongata

Mecopoda elongata

ch2

Male

n.s. (0.101)

** (0.000)

Mecopoda sp.4

Female

** (0.000)

Mecopoda elongata

Male

ch2

ch1

Mecopoda sp.4

Female

Peg diameter Mecopoda sp.4

Mecopoda elongata

Peg length Mecopoda sp.4 Mecopoda elongata

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

References Alexander, R.D., 1962. Evolutionary change in cricket acoustical communication. Evolution 16 (4), 443–467. Altner, H., Loftus, R., 1985. Ultrastructure and function of insect thermo-and hygroreceptors. Annu. Rev. Entomol. 30 (1), 273–295. Altner, H., Prillinger, L., 1980. Ultrastructure of invertebrate chemo-, thermo-, and hygroreceptors and its functional significance. In: Bourne, G.H., Danielli, J.F. (Eds.), International Review of Cytology, vol. 67. Academic Press, pp. 69–139. Altner, H., Sass, H., Altner, I., 1977. Relationship between structure and function of antennal chemo-, hygro-, and thermoreceptive sensilla in Periplaneta americana. Cell Tissue Res. 176 (3), 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 Tissue Res. 191 (2), 287–304. Altner, H., Routil, C., Loftus, R., 1981. The structure of bimodal chemo-, thermo-, and hygroreceptive sensilla on the antenna of Locusta migratoria. Cell Tissue Res. 215 (2), 289–308. Altner, H., 1977. nsect sensillum specificity and structure: an approach to a new typology. In: Lemagnen, MacLeod (Eds.), Olfaction and Taste. Information Retrieval Ltd., London, pp. 295–303. Balakrishnan, R., Pollack, G., 1997. The role of antennal sensory cues in female responses to courting males in the cricket Teleogryllus oceanicus. J. Exp. Biol. 200 (3), 511–522. Bernard, J., 1974. Étude électrophysiologique de récepteurs impliqués dans l’orientation vers l’hôte et dans l’acte hématophage chez un Hémiptère, Triatoma infestans. Thesis, Rennes, France. Boo, K.S., McIver, S.B., 1975. Fine structure of sunken thick-walled pegs (sensilla ampullacea and coeloconica) on the antennae of mosquitoes. Can. J. Zool. 53 (3), 262–266. Daly, P.J., Ryan, M.F., 1979. Ultrastructure of antennal sensilla of Nebria brevicollis (Fab.) (Coleoptera: Carabidae). Int. J. Insect Morphol. Embryol. 8 (3), 169–181. Davis, E.E., Sokolove, P.G., 1975. Temperature responses of antennal receptors of the mosquito, Aedes aegypti. J. Comp. Physiol. 96 (3), 223–236. Di Giulio, A., Rossi Stacconi, Marco Valeri, Romani, R., 2009. Fine structure of the antennal glands of the ant nest beetle Paussus favieri (Coleoptera, Carabidae, Paussini). Arthropod Struct. Dev. 38 (4), 293–302. Di Giulio, A., Maurizi, E., Rossi Stacconi Marco Valerio Romani, R., 2012. Functional structure of antennal sensilla in the myrmecophilous beetle Paussus favieri (Coleoptera, Carabidae, Paussini). Micron 43 (6), 705–719. Ditsche-Kuru, P., Schneider, E.S., Melskotte, J.E., Brede, M., Leder, A., Barthlott, W., 2011. Superhydrophobic surfaces of the water bug Notonecta glauca: a model for friction reduction and air retention. Beilstein J. Nanotechnol. 2 (1), 137–144. Eilers, E.J., Talarico, G., Hansson, B.S., Hilker, M., Reinecke, A., 2012. Sensing the underground: ultrastructure and function of sensory organs in root-feeding Melolontha melolontha (Coleoptera: Scarabaeinae) larvae. PLoS One 7 (7), e41357. Erregger, B., Hartbauer, M., Kovac, H., Stabentheiner, A., Römer, H., 2015. Multimodale Kommunikation: Komplexe Gesänge und Substratvibrationen einer tropischen Laubheuschreckenart der Gattung Mecopoda, in: Nachrichtenblatt der Bayerischen Entomologen, vol. 53. Entomologentag, München. 07.03.2015, pp. 94–97. Felippe-Bauer, M.L., Bauer, P.G., 1990. Sensilla ampullacea on the antennae of Culicoides paraensis (Goeldi, 1905) with notes on other Culicoides (Diptera: Ceratopogonidae). Memórias do Instituto Oswaldo Cruz 85 (2), 235–237. Foelix, R.F., Stocker, R.F., Steinbrecht, R.A., 1989. Fine structure of a sensory organ in the arista of Drosophila melanogaster and some other dipterans. Cell Tissue Res. 258 (2), 277–287. Gerhardt, H.C., Huber, F., 2002. Acoustic Communication in Insects and Anurans: Common Problems and Diverse Solutions. University of Chicago Press, Chicago, USA. Giglio, A., Ferrero, E.A., Brandmayr, T.Z., 2005. Ultrastructural identification of the antennal gland complement in Siagona europaea Dejean 1826, a myrmecophagous carabid beetle. Acta Zool. (Stockholm) 86 (3), 195–203. Gingl, E., Tichy, H., 2001. Infrared sensitivity of thermoreceptors. J. Comp. Physiol. A 187 (6), 467–475. Hashimoto, Y., 1990. Unique features of sensilla on the antennae of Formicidae (Hymenoptera). Appl. Entomol. Zool. 25 (4), 491–501. Hedwig, B., 2006. Pulses, patterns and paths: neurobiology of acoustic behaviour in crickets. J. Comp. Physiol. A 192 (7), 677–689. Heller, K.-G., 1986. Warm-up and stridulation in the buschcricket, Hexacentrus unicolor Serville (Orthoptera, Conocephalidae, Listroscelidinae). J. Exp. Biol. 126 (1), 97–109. Hess, E., Loftus, R., 1984. Warm and cold receptors of two sensilla on the foreleg tarsi of the tropical bont tick Amblyomma variegatum. J. Comp. Physiol. A 155 (2), 187–195. Jourdan, H., Barbier, R., Bernard, J., Ferran, A., 1995. Antennal sensilla and sexual dimorphism of the adult ladybird beetle Semiadalia undecimnotata Schn. (Coleoptera: Coccinellidae). Int. J. Insect Morphol. Embryol. 24 (3), 307–322. Kleineidam, C., Romani, R., Tautz, J., Isidoro, N., 2000. Ultrastructure and physiology of the CO2 sensitive sensillum ampullaceum in the leaf-cutting ant Atta sexdens. Arthropod Struct. Dev. 29 (1), 43–55. Korsunovskaya, O.S., 2008. Acoustic signals in katydids (Orthoptera, Tettigonidae): communication I. Entomol. Rev. 88 (9), 1032–1050.

57

Lambin, M., 1973. The antennal sensilla of some cockroaches with special reference to Blaberus craniifer (Burm.). Zeitschrift für Zellforschung und Mikroskopische Anatomie 143 (2), 183–206. Loher, W., Dambach, M., 1989. Reproductive behavior. In: Huber, F., Moore, T.E., Loher, W. (Eds.), Cricket Behavior and Neurobiology. Cornell University Press, Ithaca, pp. 43–83. Luft, J.H., 1961. Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9 (2), 409–414. Martin, J.P., Beyerlein, A., Dacks, A.M., Reisenman, C.E., Riffell, J.A., Lei, H., Hildebrand, J.G., 2011. The neurobiology of insect olfaction: sensory processing in a comparative context. Prog. Neurobiol. 95 (3), 427–447. McIver, S., Siemicki, R., 1978. Fine structure of tarsal sensilla of Aedes aegypti (L.) (Diptera: Culicidae). J. Morphol. 155 (2), 137–155. McIver, S., Siemicki, R., 1985. Fine structure of antennal putative thermo-/hygrosensilla of adult Rhodnius prolixus Stål (Hemiptera: Reduviidae). J. Morphol. 183 (1), 15–23. McIver, S.B., 1973. Fine structure of antennal sensilla coeloconica of culicine mosquitoes. Tissue Cell 5 (1), 105–112. Merivee, E., Rahi, M., Luik, A., 1997. Distribution of olfactory and some other antennal sensilla in the male click beetle Agriotes obscurus L. (Coleoptera: Elateridae). Int. J. Insect Morphol. Embryol. 26 (2), 75–83. Merivee, E., Vanatoa, A., Luik, A., Rahi, M., Sammelselg, V., Ploomi, A., 2003. Electrophysiological identification of cold receptors on the antennae of the ground beetle Pterostichus aethiops. Physiol. Entomol. 28 (2), 88–96. Merivee, E., Renou, M., Mänd, M., Luik, A., Heidemaa, M., Ploomi, A., 2004. Electrophysiological responses to salts from antennal chaetoid taste sensilla of the ground beetle Pterostichus aethiops. J. Insect Physiol. 50 (11), 1001–1013. Merivee, E., Ploomi, A., Milius, M., Luik, A., Heidemaa, M., 2005. Electrophysiological identification of antennal pH receptors in the ground beetle Pterostichus oblongopunctatus. Physiol. Entomol. 30 (2), 122–133. Merivee, E., Must, A., Milius, M., Luik, A., 2007. Electrophysiological identification of the sugar cell in antennal taste sensilla of the predatory ground beetle Pterostichus aethiops. J. Insect Physiol. 53 (4), 377–384. Merivee, E., Märtmann, H., Must, A., Milius, M., Williams, I., Mänd, M., 2008. Electrophysiological responses from neurons of antennal taste sensilla in the polyphagous predatory ground beetle Pterostichus oblongopunctatus (Fabricius 1787) to plant sugars and amino acids. J. Insect Physiol. 54 (8), 1213–1219. Merivee, E., Must, A., Luik, A., Williams, I., 2010. Electrophysiological identification of hygroreceptor neurons from the antennal dome-shaped sensilla in the ground beetle Pterostichus oblongopunctatus. J. Insect Physiol. 56 (11), 1671–1678. Milius, M., Merivee, E., Williams, I., Luik, A., Mänd, M., Must, A., 2006. A new method for electrophysiological identification of antennal pH receptor cells in ground beetles: the example of Pterostichus aethiops (Panzer, 1796) (Coleoptera, Carabidae). J. Insect Physiol. 52 (9), 960–967. Milius, M., Merivee, E., Must, A., Tooming, E., Williams, I., Luik, A., 2011. Electrophysiological responses of the chemoreceptor neurones in the antennal taste sensilla to plant alkaloids and glucosides in a granivorous ground beetle. Physiol. Entomol. 36 (4), 368–378. Myers, J., 1968. The structure of the antennae of the Florida Queen butterfly, Danaus gilippus berenice (Cramer). J. Morphol. 125 (3), 315–328. Nagel, M., Kleineidam, C.J., 2015. Two cold-sensitive neurons within one sensillum code for different parameters of the thermal environment in the ant Camponotus rufipes. Front. Behav. Neurosci. 9, 240. Nishikawa, M., Yokohari, F., Ishibashi, T., 1985. The antennal thermoreceptor of the camel cricket, Tachycines asynamorus. J. Insect Physiol. 31 (7), 517–524. Nityananda, V., Balakrishnan, R., 2006. A diversity of songs among morphologically indistinguishable katydids of the genus Mecopoda (Orthoptera: Tettigoniidae) from Southern India. Bioacoustics 15 (3), 223–250. Noirot, C., Quennedey, A., 1974. Fine structure of insect epidermal glands. Annu. Rev. Entomol. 19 (1), 61–80. Nurme, K., Merivee, E., Must, A., Sibul, I., Muzzi, M., Di Giulio, A., Williams, I., Tooming, E., 2015. Responses of the antennal bimodal hygroreceptor neurons to innocuous and noxious high temperatures in the carabid beetle, Pterostichus oblongopunctatus. J. Insect Physiol. 81, 1–13. Otte, D., Cade, W., 1976. On the role of olfaction in sexual and interspecies recognition in crickets (Acheta and Gryllus). Anim. Behav. 24 (1), 1–6. Ramirez-Esquivel, F., Zeil, J., Narendra, A., 2014. The antennal sensory array of the nocturnal bull ant Myrmecia pyriformis. Arthropod Struct. Dev. 43 (6), 543–558. Ruchty, M., Romani, R., Kuebler, L.S., Ruschioni, S., Roces, F., Isidoro, N., Kleineidam, C.J., 2009. The thermo-sensitive sensilla coeloconica of leaf-cutting ants (Atta vollenweideri). Arthropod Struct. Dev. 38 (3), 195–205. Schaller, L., 1982. Structural and functional classification of antennal sensilla of the cockroach, Leucophaea maderae. Cell Tissue Res. 225 (1), 129–142. Schmitz, H., Schmitz, A., Bleckmann, H., 2000. A new type of infrared organ in the Australian fire-beetle Merimna atrata (Coleoptera: Buprestidae). Naturwissenschaften 87 (12), 542–545. Schmitz, H., Schmitz, A., Bleckmann, H., 2001. Morphology of a thermosensitive multipolar neuron in the infrared organ of Merimna atrata (Coleoptera, Buprestidae). Arthropod Struct. Dev. 30 (2), 99–111. Schmitz, A., Schneider, E.S., Schmitz, H., 2015. Behaviour of the Australian ‘fire-beetle’ Merimna atrata (Coleoptera: Buprestidae) on burnt areas after bushfires. Rec. West. Aust. Mus. 30 (1), 1. Schneider, E.S., Schmitz, H., 2013. Bimodal innervation of the infrared organ of Merimna atrata (Coleoptera, Buprestidae) by thermo- and mechanosensory units. Arthropod Struct. Dev. 42 (2), 135–142.

58

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58

Schneider, E.S., Schmitz, H., 2014. Thermomechanical properties of the stimulus transducing cuticle in the infrared organ of Merimna atrata (Coleoptera, Buprestidae). J. Morphol. 275 (9), 991–1003. Schneider, D., 1964. Insect antennae. Annu. Rev. Entomol. 9 (1), 103–122. Schwabe, J., 1906. Beiträge zur Morphologie und Histologie der typanalen Sinnesapparate der Orthopteren. In: Chun, C. (Ed.), Zoologica. Original-Abhandlungen aus dem Gesamtgebiet der Zoologie, vol. 20. E. Schweizerbartsche Verlagsbuchhandlung (E. Nägele), Stuttgart, pp. 1–154. Singer, T.L., 1998. Roles of hydrocarbons in the recognition systems of insects. Am. Zool. 38 (2), 394–405. Slifer, E.H., Sekhon, S.S., 1961. Fine structure of the sense organs on the antennal flagellum of the honey bee, Apis mellifera Linnaeus. J. Morphol. 109 (3), 351–381. Slifer, E.H., 1960. A rapid and sensitive method for identifying permable areas in the body wall of insects. Entomol. News 71. Slifer, E.H., 1974. Structures on the antennal flagellum of a katydid, Neoconocephalus ensiger (Orthoptera, Tettigoniidae). J. Morphol. 143 (4), 435–443. Steinbrecht, R.A., Müller, B., 1976. Fine structure of the antennal receptors of the bed bug, Cimex lectularius L. Tissue Cell 8 (4), 615–636. Steinbrecht, R.A., 1969. Comparative morphology of olfactory receptors. In: Pfaffmann, C. (Ed.), Olfaction and Taste, vol. 3. Rockefeller University Press, New York, pp. 3–21.

Steinbrecht, R.A., 1997. Pore structures in insect olfactory sensilla: a review of data and concepts. Int. J. Insect Morphol. Embryol. 26 (3–4), 229–245. Strauß, J., Stumpner, A., 2015. Selective forces on origin, adaptation and reduction of tympanal ears in insects. J. Comp. Physiol. A 201 (1), 155–169. Stumpner, A., 1996. Tonotopic organization of the hearing organ in a bushcricket. Naturwissenschaften 83 (2), 81–84. Tichy, H., 1979. Hygro- and thermoreceptive triad in antennal sensillum of the stick insect, Carausius morosus. J. Comp. Physiol. 132 (2), 149–152. Tooming, E., Merivee, E., Must, A., Luik, A., Williams, I.H., 2012. Antennal sugar sensitivity in the click beetle Agriotes obscurus. Physiol. Entomol. 37 (4), 345–353. Tregenza, T., Wedell, N., 1997. Definitive evidence for cuticular pheromones in a cricket. Anim. Behav. 54 (4), 979–984. Zacharuk, R.Y., 1985. Antennae and sensilla. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon Press, Oxford. Zopf, L.M., Lazzari, C.R., Tichy, H., 2014. Differential effects of ambient temperature on warm cell responses to infrared radiation in the bloodsucking bug Rhodnius prolixus. J. Neurophysiol. 111 (6).