Micron 90 (2016) 43–58 Contents lists available at ScienceDirect Micron j ournal 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 a b s t r a c t Article history: The typology, number and distribution pattern of antennal sensilla in two species of the genus Mecopoda Received 11 May 2016 were studied using scanning electron microscopy. The antennae of both sexes of both species attain Received in revised form 2 August 2016 a length of 10 cm. The antenna is made up of three basic segments: the scape, pedicel and flagellum, Accepted 2 August 2016 which is composed of more than 200 flagellomeres. We distinguished two types of sensilla chaetica, Available online 4 August 2016 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 Keywords: which could also have a thermo- and hygrosensitive function. The remaining types of sensilla identified Katydid Orthoptera had a purely mechanosensory function, a dual gustatory- and mechanosensory function and a thermo- Insects and/or hygrosensory function, respectively. Consistent sex specific differences in the types, numbers and Antennal sensilla distribution of antennal sensilla were not found. Interspecific differences were identified especially in Scanning electron microscopy 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 of these insect groups may even evaluate the body temperature of males as a measure for male quality; termed the “hot male hypoth- Crickets and katydids are excellent model systems that can esis” (Erregger et al., 2015). This increased thoracic temperature be used to study mating preferences based on acoustic signals could also promote the evaporation and dispersal of volatile sub- (reviewed in Gerhardt and Huber, 2002; Hedwig, 2006). During the stances that may play a role in mate attraction and final mate choice. first step in mate choice, females in these insect groups approach a Whereas long-range acoustic signals, and the information con- male by walking or flying toward the male calling song, often trav- tained therein, are received by ears in the forelegs by a well-studied elling considerable distances. They may then assess specific cues array of sensory cells in the so-called crista acustica (Schwabe, at close range before making a final mating decision. These cues 1906; Stumpner, 1996), the insect antennae are the major sen- may include courtship songs (functionally different from the calling sory organs receiving information from all other cues emitted by songs), but during courtship and the potentially subsequent copu- potential mates and from the environment. In general, an anten- lation, females may additionally assess many other cues, evaluating nal sensillum consists of a cuticular apparatus, sensory neurons, chemosensory, tactile, vibratory or visual information, which can and auxiliary cells. The outer cuticular apparatus is specialized facilitate species or kin recognition and provide important infor- according to the sensory modalities it processes and can be dis- mation about mate quality (Alexander, 1962; Balakrishnan and cerned, at least to some extent, on the basis of the morphology of Pollack, 1997; Loher and Dambach, 1989; Singer, 1998). In addi- its outer cuticular structures (Altner and Prillinger, 1980). This is tion, the considerable amount of muscular energy invested while supported by many studies where morphological examination was moving the forewings during singing is converted into heat and combined with electrophysiological methods (Altner et al., 1977, increases the temperature of the thorax that houses the muscles 1981; Schaller, 1982; Zacharuk, 1985). The presence of pores, which (Heller, 1986). Thus, researchers have hypothesized that females are the entry points for odorant molecules into the lumen of the sensillum, indicates that the sensillum has a chemosensory func- tion (Steinbrecht, 1997). Conversely, the absence of pores in the sensillum wall precludes such a function. A sensillum with a single Abbreviations: ba, sensilla basiconica; ch, sensilla chaetica; co, sensilla coelo- terminal pore can have both gustatory and mechanoreceptive func- conica; MP, multiporous; MPG, multiporous grooved; MPP, multiporous pitted; NP, tions, whereas the presence of multiple wall pores indicates that the aporous; SEM, scanning electron microscopy; TP, uniporous; tr, sensilla trichodea. ∗ Corresponding author. sensillum has an olfactory function (Altner, 1977; Zacharuk, 1985). E-mail address: [email protected] (E.S. Schneider). 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 and then washed and dehydrated in two changes of 100% ethanol body and, surprisingly, despite the apparent importance of these for 10 min each (Nagel and Kleineidam, 2015). After air-drying, the organs, the structure and distribution of antennal sensilla so far samples were mounted on aluminum stubs and sputter-coated as has only been described for one species (Neoconocephalus ensiger; described above. Slifer, 1974). This species possesses at least seven different types of To assess the general distribution pattern of the sensilla on the sensilla. Based on morphological data, which were only obtained by outer surface of the antennal segments, specimens were mounted means of light microscopy, Slifer (1974) proposed that one type is on the tips of insect pins using a small drop of liquid carbon (Leit-C, probably not innervated, and all others represent chemoreceptors, Plano GmbH, Wetzlar, Germany). After sputter coating the samples of which one may also have a tactile function. from two opposing sides, they were clamped into a custom-made ◦ In the present article, we describe the morphology, number and holder that allowed us to rotate the samples over 360 (Ditsche- distribution of sensory structures on the antennal flagella of two Kuru et al., 2011). By using this method in combination with the katydid species of the Mecopoda complex and discuss their probable cacti needles as orientation markers, we could determine the posi- sensory modality. These two closely related species were chosen tion of individual sensilla across the whole surface of different because Mecopoda sp.4 strongly increases its thorax temperature antennal segments. while singing, whereas Mecopoda elongata does not. The results of our analysis provide an essential basis for future electrophysi- 2.3. Production of sections for light and transmission electron ological and behavioral experiments that will allow researchers to microscopy evaluate the potential role of thermal and olfactory stimuli during mate choice. To gain more information about certain structural aspects of the antennal flagella and their sensilla, preliminary semi- and ultra- thin cross-sections were produced. Small pieces of the antenna 2. Materials and methods containing single to few flagellar segments were excised and imme- diately fixed overnight in iced 0.05 M cacodylate buffer containing 2.1. Animals ◦ 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 Experiments were performed with one trilling and one chirping dehydrated in a graded series of ethanol and embedded in Epon species of the katydid genus Mecopoda. The taxonomy of the genus 812 according to Luft (1961). Semi-thin sections with a thickness Mecopoda is still unresolved. Several sibling species are morpho- of 0.5–1 ␮m and ultra-thin sections with a thickness of about 70 nm logically similar, but have distinctly different calling song patterns were cut with a diamond knife using a Leica 2065 Supercut micro- (Nityananda and Balakrishnan, 2006). Insects included in this study tome and a Leica Ultracut UCT microtome, respectively. Semi-thin were taken from a laboratory breed maintained at the Institute of sections were stained with 0.1% toluidine-blue/borax solution and Zoology in Graz, which was originally established from individu- examined with an Olympus BH2 light microscope. Ultra-thin sec- als collected in a tropical rainforest in Malaysia in 2010 and 2011. tions were double-stained with 300 ppm platinum blue for 15 min Korsunovskaya (2008) described the chirping species as Mecopoda and 3% lead citrate for 7 min and examined using a FEI Tecnai G2 elongata and the trilling species as “Mecopoda sp. 4”. 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 We used the staining method invented by Slifer (1960), referred chloride. To determine the orientation of the antennae during the to here as Slifer’s staining method, to investigate the potential preparation process, the antennae were marked while still attached abundance and location of pores on the outer surface of antennal to the head capsule. Antennae were mounted on a Plexiglas (poly- sensilla. (methyl methacrylate)) holder, positioned with either the ventral After anesthetizing the animals with ethyl chloride, antennae or dorsal side facing upwards and reversibly fixed with narrow were removed and fixed in Bouin’s solution for at least 24 h. Anten- stripes of adhesive tape. Small cacti needles (Opuntia sp.) were nae were stained using a 0.5% solution of crystal violet, incubating then inserted into either the ventral or dorsal side of the antenna samples for 5–15 min.