Sensory Structures on the Antennal Flagella of Two

Micron 90 (2016) 43–58

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“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.

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

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. Otherwise, the protocol of Slifer (1960) was

using a micromanipulator (Altner et al., 1977). It proved beneﬁ- followed. Stained specimens were analyzed using an Olympus BH2

cial to punctuate the insertion side with a tungsten wire that had light microscope.

been sharpened electrolytically prior to the insertion of the needles.

In most cases, this prevented the cacti needle from snapping off. 2.5. Data acquisition and analysis

After marking the antennae, they were removed from the head cap-

sule and cut into 1.5–2.0 cm long pieces, each bearing at least one After conducting a detailed initial examination of the different

cacti needle for orientation. Samples were air dried, either directly types of sensilla found on the antennae of both sexes of the inves-

or by dehydrating them using a graded series of aqueous ethanol tigated species, we focused on the more distal ﬂagellar segments.

solutions, and then subsequently sonicated in a 1:1 mixture of chlo- We counted the ﬂagellomeres beginning from the head of the insect

roform and ethanol for several minutes. Specimens were mounted (Fig. 1a). Therefore, at least the proximal 33, and at most 44, ﬂagel-

on aluminum stubs with adhesive conductive carbon tape. They lar segments of each antenna were excluded from the quantitative

were sputter coated with gold/palladium for 60 s at 40 mA using analysis of the various sensilla parameters, because numbers and

a Bal-Tec SCD500 sputter coater. Samples were observed using a peg lengths of most types of sensilla on these segments were con-

Zeiss DSM 950 scanning electron microscope and an accelerating siderably lower than those measured on more distal segments

voltage of 15 kV. (Fig. 5). Due to the length of the antennae, sensilla density mea-

To obtain internal images of the antennal segments, antennae surements were made on every 10th ﬂagellar segment (every 5th,

were bisected along their lengths with a broken razorblade. These in case of the coeloconic sensilla, co). The density of a particular type

halves were soaked in a 1 M KOH solution for 15 min to remove of sensillum was always measured by counting its number on the

the soft tissue. Subsequently, antennae were washed twice in 70% lateral side of the segments (cf. Fig. 1), then doubling the number

ethanol for 5 min each time, sonicated in 70% ethanol for 2 min 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 distribu- of 136.2–983.6 ␮m. The trilling species Mecopoda sp.4 and the

tion pattern of sensilla on the surface of each antennal segment chirping species Mecopoda elongata displayed mean ﬂagellomere

was observed (cf. 3.3 Numbers and distribution of different sensilla lengths of 435.3 ␮m and 382.0 ␮m, respectively. The ﬂagellomeres

types on the ﬂagellar segments), the absolute sensilla density may in the Mecopoda sp.4, therefore, were signiﬁcantly longer than in

be overestimated to some degree using the methodology described Mecopoda elongata (P = 0.000). Antennae of both sexes and species

above. The surface area of the segments was determined by calcu- gradually tapered from the proximal to the distal ends with max-

lating the outer surface of a circular cylinder M = *d*h, where M is imum values of 286.2 ␮m in diameter at the base and 71.6 ␮m at

the surface area, d is the diameter and h is the length of the seg- the tip. The total antennal length ranged from 74.7 mm to 101.5 mm

ment. Sensilla density is deﬁned as the number of sensilla per mm (with 171–227 ﬂagellomeres) in Mecopoda sp.4, and from 49.7 mm

of antennal surface. The pore density (number of pores per ␮m to 75.9 mm (with 112–206 ﬂagellomeres) in Mecopoda elongata. No

in the wall of sensilla) was measured in a similar way. Measure- structural sexual dimorphism was observed for these species.

ments made on individual sensilla, such as peg length and diameter,

and inclination angle, were carried out only on sensilla for which

3.2. Morphological features of sensilla on the ﬂagellum

full proﬁle-images had been taken. To measure the peg length, a

curved line was traced from the middle of each sensillum between

By carefully examining their external shapes and dimensions,

the base and tip (Fig. 1c). Peg diameter was always measured at

two types of chaetic, one type of trichoid, ﬁve types of basiconic

the base of a sensillum, tracing a line perpendicular to the axis of

and one type of coeloconic sensilla could be distinguished in males

the peg shaft that protrudes from the antennal surface (Fig. 1c). To

and females of both species. The pores present on the sensilla were

assess the inner diameter of sockets and pore openings, we always

examined, and the sensilla were classiﬁed as aporous (NP), uni-

measured the widest possible diameter (Fig. 1c). The inclination

porous (TP) and multiporous (MP), following the terminology of

angle of a sensillum was measured distal to the antenna (i.e., in the

Altner (1977). The latter could also be differentiated into either

direction of the antennal tip, Fig. 1c). Quantitative measurements

multiporous pitted (MPP) sensilla, which have pore tubules, and

were made on a total of N = 3 animals of both sexes and species,

multiporous grooved (MPG) sensilla, which are characterized by

respectively. Measurements were carried out using ImageJ 1.46r

the presence of spoke canals, following the terminology of Zacharuk

(Rasband, National Institutes of Health, USA). Data was statistically

(1985).

analyzed using SPSS Statistics (Version 22.0.0.0, IBM Corp.). To test

Although the measured data on several aspects of the dimen-

for differences of variances, we used the Mann–Whitney U test,

sions of some sensilla types revealed signiﬁcant differences

with the level of signiﬁcance set at P < 0.05. To classify the sensilla,

between the sexes and/or species, we could not ﬁnd any differ-

we referred to the terminology of Schneider (1964), Altner (1977)

ences in terms of their general presence or typology. Data on the

and Zacharuk (1985).

measured parameters of the different types of sensilla are sum-

marized in Table 1. For a more detailed representation of the data

that also distinguishes between the sexes and species, see Table A.1

3. Results & discussion

(provided in the Appendix A). The results of the statistical analy-

ses are given in Table A.2 (provided in the Appendix A). Detailed

3.1. General structure of the antennae

morphological characterizations of the different types of sensilla

are provided in the following sections.

Antennae of both sexes of both Mecopoda species investi-

gated consisted of the scape, pedicel and many (in an intact

antennae, >200) ﬂagellomeres. The antennal surface (i.e., the out- 3.2.1. Uniporous (TP) sensilla

ermost cuticle) was covered with shallow scales, which were more Sensilla chaetica type 1 (ch1) are very long, sickle-shaped bris-

prominent on the distal segments. Cross-sections of the ﬂagellar tles with longitudinal grooves and blunt tips (Figs. 2 and 8). The

segments revealed that they are nearly round along the whole base of the bristles, which rests on the distal edge of a wide, ﬂexible

◦

length of the antenna. The ﬁrst ﬂagellomere (most proximal; cf. socket, projects from the antennal surface at an angle of 31.6–99.1

◦

Fig. 1a) differed from the others in its great length (more than (mean value of 72.5 ; Table 1; Fig. 2). The distal parts of some

1 mm). The other ﬂagellomeres were highly variable, with lengths 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 did species for which antennal sensilla have been characterized

and probably represents, at least to a certain degree, a drying arte- thus far, Slifer (1974) described a thick-walled chemoreceptor that

fact caused by the preparation procedure used. The ch1 had the highly resembled the ch1 with regard to its shape, surface structure

longest pegs, with a mean length of 97.1 ␮m (Table 1). The bris- and distribution. Five sensory neurons have been identiﬁed in this

tle length varies from 57.1 ␮m to 164.4 ␮m. At the tip of the ch1, sensillum type, one of which probably represents a mechanore-

a single terminal pore was observed (Fig. 2b). The porous charac- ceptor (Slifer, 1974). If the ch1 described here may also have a

ter was conﬁrmed by crystal violet staining, whereby the tip of the dual chemo- and mechanosensory function, requires further phys-

bristle could be stained. Due to the presence of this terminal pore, iological or ultrastructural investigation (e.g., the identiﬁcation of a

a contact-chemosensory function is likely. tubular body at the base of the sensillum). In carabid beetles of the

In general, many of the TP sensilla have been described in the genus Pterostichus, dual chemo- and mechanosensory chaetic sen-

literature as having a dual chemo- and mechanosensory function silla have been described. These house ﬁve sensory neurons, one of

and can be found on the antennae and other body appendages which has proven to be mechanosensitive (Tooming et al., 2012).

in most insect orders, e.g. Coleoptera (Daly and Ryan, 1979; The remaining four chemosensory neurons have been shown to

Jourdan et al., 1995; Tooming et al., 2012), Diptera (McIver and respond to various salts (Merivee et al., 2004), sugars (Merivee et al.,

Siemicki, 1978), Hemiptera (Steinbrecht and Müller, 1976), Lep- 2007; Merivee et al., 2008), pH (Merivee et al., 2005; Milius et al.,

idoptera (Myers, 1968), Orthoptera (Lambin, 1973) and others 2006) and phytochemicals like alkaloids and glucosides (Milius

(Zacharuk, 1985). These sensilla possess several, usually four to et al., 2011). To what compounds the chemosensory neurons in

six, chemosensory neurons with dendrites that extend through ch1 of Mecopoda may be sensitive can only be speculated until

the hair shaft towards the terminal pore and are housed together electrophysiological data are available.

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-

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58 47

3.2.2. Aporous (NP) sensilla

ch2

Sensilla chaetica type 2 ( ) are more or less straight bristles,

␮

57.5–117.9 m long, with a hair shaft that strongly tapers from 2.74

deviations

0.53 1.47 9.03 0.54

± ± ± ± about half of its length and ends in a sharp tip (Fig. 2c and d). The tip

10) 73) 168) 167) 335)

= = = = =

was often bent away from the antennal surface (Fig. 2d). The base of 3.93 2.80 N.A N.A co 20.26 7.44 N.A. 5.45 N.A. (n (n (n (n

(n ◦ ◦

± ±

standard 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 values

grooves present), but these structures were restricted to the outer 7.37

1.01 0.25 0.37

cuticular surface of the peg. We could not ﬁnd any indications of ± ± ±

17) 17) 14) 17) Mean

= = = =

the presence of pores in the sensillum wall. 6.29 1.87 2.60 72.88 N.A. N.A. ba3 N.A. N.A. N.A. (n (n (n

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 respectively.

sharp tip, grooved surface structure and are the most abundant 9.40 2.27 21.59 1.70

0.46 0.75

± ± ± ±

sensilla on the antennal surface, but lacking on the ﬁrst twenty ± ±

197) 189) 168) 153) 175) 179)

======

ﬂagellomeres (Slifer, 1974). Slifer could not ﬁnd any innervation examined, 12.50 3.07 4.90 53.71 26.46 N.A. ba2 N.A. N.A. 17.93 (n (n (n (n (n (n

of these sensilla and concluded that their function may be to pro-

were vide 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 func- 8.57 19.12 12.86 species

0.32 0.58 1.21

± ±

tion, we propose that it must be a mechanosensory function, due ± ± ±

207) 154) 130) 207) 179) 7)

and

======

to the absence of pores in the outer sensillum wall. 48.58 3.62 5.18 57.60 23.29 7.70 ba1.3 N.A. N.A. N.A. (n

(n (n

(n (n (n

Sensilla coeloconica (co) are peg-in-pit sensilla, consisting of sexes

a small peg set on the ﬂoor of a chamber sunken into the cuti-

both

␮ cle. A small central aperture with a diameter of 4.4–12.2 m (cf. of

Table 1) connects the peg inside the chamber with the surrounding 30.95 11.21 6.82

0.34 0.58 1.35

± ± ±

air. Semi-thin sections revealed that the chamber had an internal

± ± ±

295) 270) 255) 270) 179) 8)

␮ ␮ ======

diameter of about 13 m and a height of about 6 m. From out- 36.00 3.59 5.12 48.70 54.18 8.16 (n ba1.2 N.A. N.A. N.A. (n (n (n

(n (n

individuals side, 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). N

The sizes of the outer pit and aperture diameter of co were highly

from variable and especially depended on their position on the antenna.

11.77 3.48 2.15

0.62 10.68 0.36

± ± ± The diameters of both parameters gradually increased from the

± ± ±

108) 103) 102) 63) 179) 7)

======proximal to the distal ﬂagellomeres. The small peg had a mean

21.09 3.50 5.31 46.65 7.74 12.70 ba1.1 N.A. N.A. N.A. (n (n (n

(n (n (n

␮ ␮

Antennae length of 3.9 m and diameter of 2.8 m and stood perpendicu-

lar 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 available.

smooth and we could not ﬁnd any indications of the presence of 9.45 12.65 26.22

not

0.29 0.52 1.12

investigated.

± ± ± pores (Fig. 3h). The cuticle was not stained after applying Slifer’s

± ± ±

240) 213) 202) 233) 179) 7)

data ======staining method, nor could we ﬁnd any pores in ultra-thin sec-

. 54.34 3.66 5.17 63.35 31.66 tr N.A. N.A. N.A. 5.27 (n (n (n

(n (n (n

tions. However, a small apical indentation in the tip of the peg was species

N.A

visible in several preparations, which most probably represents a

clogged molting pore (Fig. 3h). Such a pore has also been described given.

in the NP sensilla coeloconica of Locusta migratoria (Altner et al., 179.13 Mecopoda

9.45 8.94

are ±

0.48 0.78

1981) and Carausius morosus (Altner et al., 1978). Peg-in-pit sen- ± ±

the

± ±

1023) 1018) 1018) 1023) 179)

in silla of insects are highly variable in their external shape (Altner and = = = = =

84.62 4.72 6.11 44.09 683.99 (n ch2 N.A. N.A. N.A. N.A. (n (n

(n (n

Loftus, 1985; Di Giulio et al., 2012; Ruchty et al., 2009) but, in most brackets

in cases, their innervation is similar. The most common type of peg-

identiﬁed in-pit sensilla is represented by a physiological triad containing

two hygro- (dry and moist) and one thermoreceptor (cold) (Altner 63.37

11.58 15.99

enclosed ±

0.53 1.12

et al., 1981; Bernard, 1974; Merivee et al., 2003, 2010; Nishikawa ) ± ±

sensilla

n ± ±

1033) 1021) 1021) 1023) 179) (

et al., 1985; Nurme et al., 2015; Tichy, 1979). On the basis of ﬁndings of = = = = =

(n ch1 97.06 72.52 N.A. N.A. N.A 264.42 N.A. (n (n (n

reported in the existing literature on NP sensilla coeloconica, which

types

include information about both the morphology and physiology of

]

these sensilla (Altner et al., 1978, 1981; Davis and Sokolove, 1975;

m ]

␮ McIver, 1973; McIver and Siemicki, 1985; Nishikawa et al., 1985; measurements m]

different

␮

Tichy, 1979; Zopf et al., 2014), we postulate that co also possess m] 7.52 [

m] ␮ the [

␮

m] [pores/ a thermo- and/or hygrosensory function in Mecopoda. Preliminary [

of ␮

[ ]

◦ electrophysiological recordings from co in Mecopoda sp.4 always [ individual

m] 5.47 [sensilla/mm aperture

pores

revealed the activity of one warm- and one cold-receptor cell (Zopf of ␮

[ m]

diameter pit

␮

diameter

angle and Römer unpublished). This physiological type is much less com-

[

wall

type

diameter

parameters

density of

mon and has so far been described especially in hematophagous

pit number socket

insects like the mosquito Aedes aegypti (Davis and Sokolove, 1975), 1 length diameter

the

the bug Rhodnius prolixus (Zopf et al., 2014) and the tropical tick Sensillum Peg Inclination Outer Sensilla Peg Inner Diameter Depression Density Table Measured and

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 49

Amblyomma variegatum (Hess and Loftus, 1984). In contrast to the the hair shaft was nearly straight, then bent to a certain degree

above mentioned triad, this physiological type that contains two and became straighter again distally. The oval-shaped wall pores

antagonistically responding thermoreceptor cells has been shown (Fig. 4f) occurred at a density of 7.70 ± 1.21 pores per ␮m (Table 1)

to respond very sensitive not only to thermal stimuli in form of which was similar to that found in ba1.2 (P = 0.463) but which was

convective heat, but also to infrared radiation (Zopf et al., 2014). signiﬁcantly lower compared to that of ba1.1 (P = 0.002) (Table 1;

Fig. 4).

3.2.3. Multiporous (MP) sensilla Sensilla basiconica type 2 (ba2) are sensilla shaped like door

Sensilla trichodea (tr) are hairs with a length of 26.6–81.3 ␮m handles and having peg lengths of 7.2–16.9 ␮m. The peg was set

and a slightly inverted, S-shaped proﬁle and a blunt tip (Fig. 4g). into an inﬂexible socket with an inner diameter of 3.1–7.4 ␮m

Like ch1, they insert into their socket at a rather steep angle of and protruded basally from the antennal surface at an angle of

◦ ◦

63.4 (Table 1). The hair shaft has its largest diameter at the base 28.3–90.0 (cf. Table 1 and Fig. 3a and b). After a few ␮m, the hair

(3.0–4.6 ␮m) and slightly tapers toward the tip (Figs. 4 g and 8). On shaft strongly curved in a distal direction to the antenna so that the

the concave surface at the base of the tr, in most cases 5–10 ␮m distal part of the peg, which was straight, was almost parallel to the

above the base, a small molting scar was visible (Fig. 4g). The antennal surface (Fig. 3a and b). A special feature of this sensillum

hair shaft distal from this structure was covered by numerous pit- type is that the socket was set proximally (referring to the direc-

ted pore openings with a density of 3.8–6.9 per ␮m (cf. Table 1; tion of the antenna) on the ﬂoor of a shallow circular depression

Fig. 4h). Tr had the lowest number of wall pores per area as com- that had a diameter of 12.3–25.0 ␮m. Furthermore, the depression

pared to the other MPP sensilla examined in the present study. In was framed, at least on its proximal half, by a cuticular rim that

tr, the pore openings appeared to be shallow depressions with a projected above the surrounding cuticle (Fig. 3b). A molting scar

rather slit-like shape. Upon applying Slifer’s staining method, the could not be identiﬁed in ba2. Several ␮m distal from the base,

distal three-quarters of the hair shaft were stained, which corre- the hair shaft was covered by narrow, longitudinal grooves (with

sponded to the location of pore openings. Based on our ﬁndings, an inter-groove distance of 80–150 nm) that extended to the tip

we propose an olfactory modality for tr. (Fig. 3b–d). This grooved part of the ba2 was stained after applying

Sensilla basiconica type 1 (ba1) are primarily slightly curved, Slifer’s staining method and this leads us to suppose that the lon-

cone-like hairs with blunted tips (Fig. 4a–f). The hair shaft inserted gitudinal grooves are multiporous, thus representing the so-called

◦ ◦

basally at an inclination angle that ranged from 23 to 83 into a spoke canals. Spoke canals are a characteristic feature of double-

socket with a diameter of 3.1–6.8 ␮m. At its base, the hair shaft had walled MPG sensilla that have been shown to be chemosensory and

a diameter that ranged from 2.7 ␮m to 4.9 ␮m. Like tr, ba1 could primarily olfactory (Altner and Prillinger, 1980; Zacharuk, 1985).

be characterized by the presence of a molting scar on the concave However, physiological combinations of thermo- and chemosen-

surface of the hair shaft in the basal region and, distal to that, by the sory (Altner et al., 1977, 1981; Schaller, 1982; Steinbrecht, 1969) as

presence of numerous wall pores (Fig. 4a–f). After applying Slifer’s well as thermo- and hygrosensitive units (Altner et al., 1977) have

staining method, the hair shaft was always stained distal to the also been described. All these modalities, as well as their combina-

molting scar, which corresponded to the region where the wall tions, might be conceivable in the ba2 of Mecopoda.

pores were located. Both the location of the molting scar and the Sensilla basiconica type 3 (ba3) have straight, short pegs with

pitted wall pores are characteristic features of MPP sensilla. MPP lengths of 4.3–9.1 ␮m that insert into their sockets at an angle of

◦

sensilla are generally considered to be olfactory (Zacharuk, 1985). 60.6–85.1 . The largest diameter of the peg was measured at its

Due to the fact that ba1 displayed variability with regard to their base ranging from 1.4 ␮m to 2.2 ␮m (Fig. 3e and f; Table 1). At the

shape, peg length and density of wall pores, we further divided proximal part of the peg, a small molting scar was identiﬁed on

this sensillum type into three subtypes described in the following its concave surface (Fig. 3f). The peg’s surface was smooth except

sections. for the distal tip, which could be characterized by the presence

Sensilla basiconica subtype 1.1 (ba1.1) have a peg length of of pitted wall-pores that were very similar in shape and dimen-

14.7–32.2 ␮m and are the shortest and stoutest sensilla within the sions to those found in ba1.1 (Fig. 3f). Applying Slifer’s staining

ba1. Their diameter is greatest at the base, and they are also char- method resulted in a dark coloration of the distal tips of these sen-

acterized by having the highest density of wall pores within the silla, which corresponded to the locations of wall pores identiﬁed

ba1 with 9.6–15.0 pores per ␮m . The wall pores appeared as very in SEM micrographs.

deep indentations with large diameters in the outer cuticular wall

(Fig. 4b). This gave the impression of a sponge-like surface, which 3.3. Numbers and distribution of different sensilla types on the

was already visible at lower magniﬁcation levels (Fig. 4a). The high ﬂagellar segments

degree of variability in the diameter of the external pits of the wall

pores along with the somewhat irregular external framing indi- The proximal 21–37 ﬂagellar segments differed from the distal

cated that the pits of at least some adjacent pores could be fused. ﬂagellomeres by 1) the complete absence of ch2 (Fig. 5a and b )

Sensilla basiconica subtype 1.2 (ba1.2) are less stout and pos- and 2) the distribution pattern of all other sensilla types, with the

sess a longer hair shaft than ba1.1 with a length of 20.8–55.0 ␮m exception of ch1, i.e., they were always grouped together in the

(Fig. 4c; Table 1). Another characteristic feature is that the largest form of sensillar ﬁelds (Fig. 5a), either on the dorsolateral and/or

diameter of the peg is not found at its base. The diameter of the ventromedial surface, on about every 2nd − 4th segment. On the

hair shaft increased above the molting scar, reaching its maximum more distal segments, all sensilla types were more or less evenly

within the ﬁrst third or quarter of the peg length and then tapering distributed in a statistical sense (Fig. 5c).

again up to the distal tip (Fig. 4c). Distal to the molting scar, oval- Common to all ﬂagellomeres was the distribution pattern of ch1.

shaped wall pores could be seen that were clearly separated from These sensilla were arranged in ﬁve longitudinal rows on each seg-

one another and evenly distributed over the hair shaft (Fig. 4d). The ment, one on the ventral, medial and dorsal surfaces and two on

density of wall pores in ba1.2 was signiﬁcantly lower (P = 0.002) the lateral surface (Fig. 5b and d). All other sensilla types, except

than that of ba1.1, with 5.9–9.9 pores per ␮m (cf. Table 1). for ch1 and ch2, were exclusively found in two longitudinal sec-

Sensilla basiconica subtype 1.3 (ba1.3) have the longest hair tors located on the dorsolateral and ventromedial surfaces of the

shaft within the ba1, from 30.7–66.0 ␮m (Fig. 8). The peg’s diam- segments as indicated in Fig. 5b) and d). In terms of the general

eter was largest at the base, ranging from 2.7 ␮m to 4.5 ␮m, then numbers of sensilla, especially of tr, ba1, ba2 and co, these were

tapering gradually up to the distal tip (Fig. 4e). The basal third of 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) sen- ch2. Antennae of Mecopoda elongata bear about 29% and 40% more

silla in Mecopoda probably represents a functional adaptation for ch1 and ch2, respectively, than Mecopoda sp.4 (Table 2; Table A.2).

the spatial localization of corresponding stimuli, which in turn is No sexual dimorphism in terms of the numbers and distribution of

also determined by the direction of antennal movements during sensilla was observed in the two species.

searching behaviors, as has been assumed e.g. in the elaterid beetle The least abundant sensillum type is represented by ba3, that

Agriotes obscurus (Merivee et al., 1997). Speciﬁc data on antennal was only observed 18–30 times per antenna (i.e., less than 1 sen-

movements during searching or resting behaviors in Mecopoda are sillum per six antennal segments were seen), most of which were

not available so far. located on the proximal 30 segments. The densities measured for

Ch2 were only observed distally from segment 27 and were the other types of sensilla are summarized in Table 2.

arranged in several alternating, longitudinal rows on each ﬂagel-

lomere as indicated in Fig. 5d. With 130 ± 34 sensilla per segment,

3.4. Sensilla-free ﬁelds

ch2 were present in the highest density of all sensilla types, fol-

lowed by ch1, which occurred at a density of 50 ± 12 sensilla per

Sensilla-free ﬁelds are areas on the ﬂagellomeres that are char-

segment (Table 2). Consistent and signiﬁcant differences between

acterized by the complete absence of sensilla (Fig. 6). The scaly

the two investigated species were only observed for the ch1 and

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 mm , 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 Mecopoda sp.4 Mecopoda elongata

Female Male Female Male

ch1 225.62 ± 51.04 (n = 45) 237.93 ± 46.61 (n = 49) 299.70 ± 53.93 (n = 42) 300.78 ± 62.75 (n = 43)

ch2 577.48 ± 150.44 (n = 45) 574.25 ± 138.87 (n = 49) 821.61 ± 155.71 (n = 42) 786.10 ± 103.71 (n = 43)

tr 37.95 ± 24.25 (n = 45) 20.87 ± 19.93 (n = 49) 33.29 ± 25.76 (n = 42) 35.77 ± 31.59 (n = 43)

ba1.1 5.69 ± 7.52 (n = 45) 9.83 ± 11.76 (n = 49) 8.42 ± 10.52 (n = 42) 6.83 ± 12.15 (n = 43)

ba1.2 49.24 ± 27.21 (n = 45) 58.35 ± 29.71 (n = 49) 55.46 ± 36.20 (n = 42) 53.36 ± 30.77 (n = 43)

ba1.3 25.56 ± 15.88 (n = 45) 25.26 ± 23.27 (n = 49) 20.92 ± 15.33 (n = 42) 21.00 ± 20.42 (n = 43)

± ± ±

ba2 21.74 16.01 (n = 45) 26.45 20.34 (n = 49) 30.79 30.07 (n = 42) 27.19 ± 17.55 (n = 43)

ba3 N.A. N.A. N.A. N.A.

co 3.80 ± 6.37 (n = 85) 6.78 ± 9.67 (n = 95) 5.66 ± 10.79 (n = 79) 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 diame-

␮

ter 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 appara-

tus 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 thermore-

ceptive function (Foelix et al., 1989).

However, because no ultrastructural data for the sensilla-free

ﬁelds in Mecopoda are available, a putative sensory function can-

not 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 anten-

Cuticular pores set with their openings on the ﬂoors of shallow

nal ﬂagellomere at different levels of magniﬁcation. a) Overview; scale bar: 40 ␮m.

depressions in the antennal surface were found on all segments of

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 the ﬂagellum (Fig. 5a and c). These had a diameter of 0.4–1.0 m and

␮ ␮

an enlarged view in the inset. Scale bar: 4 m; Inset: 1 m. 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/mm ) 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/mm ) on the most distal segments (ﬂagellomeres 111–192). about 0.4–0.5 m and a length of 1.0–4.3 m was visible on the

After removal of soft tissue by using a gentle KOH digestion method, interior (Fig. 7c and d ).This structure could clearly be differentiated

which is an approved method for the identiﬁcation of sensilla from those found in sensilla ampullaceae that have been previ-

ampullaceae (Hashimoto, 1990; Kleineidam et al., 2000; Ramirez- ously described in different species of Hymenoptera (Hashimoto,

Esquivel et al., 2014), a short tubule with a constant diameter of 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 review see Strauß and Stumpner, 2015). Thus, in terms of missing

have a secretory function and that the small tubules beneath the sex- and species-speciﬁc differences in the number of the potential

pores represent cuticular ducts, which is a characteristic for class thermosensitive sensilla, co and ba2, between the two investigated

3 epidermal glandular cells (Di Giulio et al., 2009; Giglio et al., Mecopoda species, it may well be that a preference of females for

2005; Noirot and Quennedey, 1974). These could secrete cuticu- the body temperature of singing males in Mecopoda sp.4 is built on

lar pheromones, compounds with water repellent properties like a general thermosensitivity of these sensilla, adopted through new

waxes or long-chain hydrocarbons, or protective substances. The central nervous connectivity between the thermosensory pathway

former compounds could be involved in close range intraspeciﬁc and decision making networks.

recognition, as has been already shown for several cricket species Because both convective and radiant heat determine the dis-

(Otte and Cade, 1976; Tregenza and Wedell, 1997). charge frequency, it is impossible for a single thermosensory unit

to unequivocally signal the nature of the stimulus. These individual

4. Conclusions responses are always ambiguous, not with regard to the temper-

ature change itself, but its source (Gingl and Tichy, 2001). The

Our quantitative data on the number of potential thermosen- potential presence of two different thermosensitive sensilla, pro-

sitive sensilla, co and ba2, in males and females of both Mecopoda vided they differ in their response to convective and radiant heat,

species appear not to support the “hot male hypothesis”, if only would allow an insect to distinguish between the combination of

females evaluate the body temperature of males, and only in the convective and radiant heat from the individual stimuli presented

trilling Mecopoda sp.4 males get hot during singing. It is often alone (Zopf et al., 2014). This would be a desirable property to sup-

assumed that the complexity of sensory processing may confer a port mate choice by female Mecopoda on the virtue of thermal

selection pressure favoring higher numbers of sensilla, which, how- stimuli. These females could distinguish thermal stimuli emitted

ever, may not hold for all sensory modalities. For the olfactory and by a hot male (in form of radiant heat) from stimuli arising from

visual system, the number of sensory cells and therefore the num- temperature ﬂuctuations of the surrounding air (in form of con-

ber of receptor binding proteins/photopigments is important for vective heat). Whether and to what extent the sensilla types co and

the statistical “catch” of molecules/photons to increase sensitivity ba2 respond to thermal stimuli and if female Mecopoda make use

and spatial resolution. But nevertheless, exceptions to this general of those thermal stimuli emitted by males during mate choice at

rule have also been described. In the apple maggot ﬂy Rhagoletis all, has yet to be shown through future electrophysiological and

pomonella, which contains several host-speciﬁc races that exhibit behavioral experiments.

distinct behavioral preferences for different host plants, olfactory

receptor cells are differentiated, not by their functional type or Acknowledgements

number in the antenna, but solely by their response thresholds and

temporal ﬁring patterns to volatiles emitted by their preferred host We thank Gerd Leitinger and his working group at the Institute

plants (for review see Martin et al., 2011). That sensitivity does not of Cell Biology, Histology and Embryology at the Medical University

necessarily depend on receptor number, as shown in the previ- of Graz for providing access to the scanning and transmission elec-

ous example, seems by no means the exception but rather the rule tron microscopes and for technical support. We are indebted to Sara

in other sensory modalities, such as audition and probably ther- Crockett for language editing of the ﬁrst draft of the manuscript.

mosensation. In insect ears, numbers of sensory cells vary from We thank two anonymous reviewers for their helpful and valuable

one (in species of moths) to more than 2000 (in cicadas and ancient comments. The present work was supported by a grant from the

grasshoppers), with no sex-speciﬁc difference in the number of sen- Austrian Science Fund (FWF) to H. Römer (Project: P27145-B25).

sory cells in those species where only males produce sound. Even

when hearing became non-functional in one sex, sensitivity in this Appendix A.

sex decreases, but the number of sensory cells is not affected (for

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58 55

26 33 59

) ) ta Mean

27 76

± 0. ± 0. ± 8. ) N.A. N.A. Male (n=3) (n= (n= 25 45

onga 48 70 43

19 ) ) ) ± ± ± 3. 2. 5. ta el 15

21 21 43

. 01 29

(n=9)

. . ± 0. ± 0. (n= 12

6. (n= (n= (n= 12

21 35 33 60

75 )

onga

6. ± 3. 4. 3. opoda

el . (n=1) male respectively. 66

± 0.

N.A. N.A. ) (n=9) 10 5. 74 (n=

Mec Fe 77

31 56

28 ) ) ) ) 3. ± ± 2. ±

opoda 11 52

26 26 22 42

. . co 56 03

male Male 42

± 0. ± 0. . . (n= 39 68 67

14 10 8. (n= (n= (n= (n= ) )

Mec Fe

43 33 20 52

3. 5. 3. 30 95

.1 examined, ± 0. ± 0. ± 9.

N.A. N.A. Male (n=5)

(n= (n= ) ) 34 92 78

ba1 sp.4 43 44

4. 6. 2.

) ) ) 38 13

± ± were ±

35 35 49

. 68 37

61 99

) 83

. ± 0. ± 0. . opoda

(n= (n=

11 9. (n= (n= (n= 85

20 58 46 45

84 99

sp.4 (n=1) male ± 0. ± 6. Mec 3. 5. 2. 8. N.A. N.A. (n=7) 41 (n=

Fe 66 80

species

) )

3. 3. 2. 30 50 52

opoda

23 19

) ) )

± ± ta and 21 20 45

60 26

male Male . . Mec ± 0. ± 0. ± 7. (n= (n= N.A. N.A. N.A. N.A. N.A. Male (n= (n= (n= Fe 22 46 62 75 69

onga 64 61

3. 5. 5. 3. 8. el sexes

17 30

23 38

) ) ) ) )

opoda

both ± ± ± ta 20

(n=1) (n=3)

± 0. ± 0. . 97 59

male 58 56 55 57 43

N.A. . . 91

67 63 77

(n=3) (n=2) of ± 0. ± 0. . . . 04 94

06 96 67

Mec 6. Fe 13 31 3. 4. (n= (n= (n= (n= (n=

onga 6. 1. 49 72 49 56 35

3. 4. el ba

28 14 28

24 45

) ) ) ) )

± ± ± 65

opoda

(n=5) 58 50 48 58 42

± 1. ± 0. ± 0. . . 29 30

82 31 29

male Male N.A. Male ± 0. ± 0. . . . (n=7) (n=8) (n=7) 92 9. 9.

individuals 71 97 78 70

sp.4 25 (n= (n= (n= (n= (n=

Fe 63 18 53 68 33

Mec 6. 6. 1. 2. 3

3. 5.

N 83 31 30

opoda

33 46

± ) ) ) ) ) ± ± ± 89 08 93

65 56 52 60 49

(n=9) . . . male Mec . ± 0. ± 0. ± 0. 57 71 87

± 0. ± 0. . . . N.A. (n=7) (n=7) (n=6) from 23

10 13 19 Fe 76 97 73 32

(n= (n= (n= (n= (n=

83 42 56 64 20

sp.4 7. 5. 1. 2. 3. 5.

) )

) 48 76

36 27 ) ) ) 25 44

opoda

59 ) ) ) ) ± ± ± ta ± ± ± 55

42 39 43

12 25

. 51 47 58 45

80 29 19

. . 00

61 95

. . . ± 0. ± 0. (n= (n= male Male . Antennae Mec ± 0. ± 0. . . (n= Male

17 (n= (n= (n= . 11 24

onga 11 50 27 03 82

(n= (n= (n= (n= 73 57

Fe 57 71 41 63 37

40 3. 4. 1. 8. el 3. 5. 8.

) ) available.

46 68

) ) ) 49 47

± ± ± ) ) ) ) 37 52

) ± opoda

elongata 07

± ± 44 38 42

not .

03 39 79

male . . . ± 0. ± 0. 711

(n= (n= 43

10 96

. 237 237

239 239

11 54

. 30 ± 0. ± 0. . . (n= (n= (n= Mec Fe 12 52 30 97 94

7. 5. 70 07

(n= 66 59 78 39

(n= (n= (n= (n= 2. 4.

onga 103 data 1. 9. 2 786

4. 5. =

el ) ) ba .

44 80

) ) ) 66 56

± ± ±

Mecopoda ) ) ) ) 44 64

N.A

) ± 34

60 51 49

± ± . 88 35 45

opoda

± 0. ± 0. . . . (n= (n= . 42

66 43

Male 250 250

252 252

57 76

. 20 male Male ± 0. ± 0. . . (n= (n= (n= 18 71 and 12 57 26

sp.4 58 57

7. 7.

(n= Mec 155 3. 4. Fe 76 92 81 41

1. 9. (n= (n= (n= (n= 821 given.

4. 5. ) ) sp.4

48 65

opoda

ch2 ) ) ) 46 23

are ± ± ±

) ) ) ) 52 83

) ± 01

43 40 45

± ± . 98 55 74

male 87

Mec ± 0. ± 0. . . .

(n= (n= . 32

. 293 293

294 294

62 66

. 16 ± 0. ± 0. . . (n= (n= (n= Fe 06 19 12 51 21

60 13

9. 10 (n= 138 3. 5. 68 45 87 46

1. 8. (n= (n= (n= (n= sp.4 574

4. 6. Mecopoda ) brackets

35 52

) ) ) )

in of ± ± ± ta

) ) ) ) 58 70

opoda 92 42

) 34 31 49 43

. . ± ± 91

62 00

. . . ± 0. ± 0. (n= 44

. Male 45

42 33

10 20 238 238

238 238

68 93

onga (n= (n= (n= (n= male Male Mec . . ± 0. ± 0. 46 55 21 52 64

8. 8. sexes el 3. 4. (n= 8. 150

Fe 90 47 77 40

(n= (n= (n= (n= 577

4. 6. enclosed

36 65

) ) ) ) ) )

± ± ±

both n opoda

64 33

( 46 34 28 46 42

. . 16

42 88 92

) ) ) ) 37 71

male . ± 0. ± 0. . .

) ±

in 8. ± ±

10 15 (n= (n= (n= (n= (n= Mec ta Fe 47 57 27 64 20

25 54 75

. . . .3 236 236

237 237

31 33

. ± 0. ± 0. . . 3. 5.

16 11 62 ) (n= 23 92 95 73

(n= (n= (n= (n= ba1 300

onga 27 39

5. 6. 58

) ) ) )

± ± ±

el 64 27

42 36 58 49

. . 58

59 26

. identiﬁed ± 0. ± 0. . . (n=

Male ) ) ) ) 36 72

15 23 ) ± sp.4 (n= (n= (n= (n= ± ± 50 73 44 53 25

opoda

measurements 23 13 93

3. 5. 9. . . . 245 245

252 252

97 83

. male Male ± 0. ± 0. . . ts 17 10 53

(n= Mec Fe 31 13 94 74

opoda

(n= (n= (n= (n= ) ) ) ) ) 299

± ± ±

sensilla ± ± 5. 7.

40 88

54 44 35 54 45

. . 25 29 42

96 51 56

male

63 33

. . . Mec of ch1 0. 0. 8.

10 15 3. 5. (n= (n= (n= (n= (n= surem Fe ) ) ) ) 58 31

48 57 25

) ± ± individual ± ea

11 24 61

00 93

m . . . ) ) 287 287

291 281

. . of

f ± 0. ± 1. .

types

24 40

16 13 46 66 54 ) ) )

(n= r o ± ± ± 77 05 69

(n= (n= (n= (n= ta 100 237 sp.4 77

64 61 43

5. 8. . 51 83 36

± 0. ± 0. . . . (n= (n= m Male

30 (n= (n= (n=

onga nu

38 51 34 42 53

03 64

number ) ) ) ) 59 12

opoda

3. 4.

6. 8. ) ± n= el ± ± different

69 29 04

) 45

v . . . 253 253

253 253

40 87

male Male the Mec ± 0. ± 1. . . 32 47

) ) ) ) 71

13 10 51 ± ± ± (n= the Fe 48 85 97 72

(n= (n= (n= (n= opoda

225 27 20

58 52 71 42

. . 97 61 46

5. 7. male std. . . . ± 0. ± 0. and (n= of

11 36 ± (n= (n= (n= (n= Mec Fe 35 52 55 51 93

.2 3. 4. 6. ean

) m µm] 35 47

ba1

, ) ) ) ) 88

[

± ± ± )

27 71

82 80 84 49

. . 83 09 35

± 0. ± 0. . . . deviations (n= °]

Male 10 29

[ parameters (n= (n= (n= (n= 72 47 36 47 58

sp.4 78

µm] 3. 5. [ 6.

] gle diameter rameters 2 sity sity µm]

t ) ype

[ an

30 35

Continued opoda

) ) ) ) 70

den

( ± ± ± gth

standard a/mm

um um a ti 95 21

66 62 61 45

. . 39 55 24

ill

ill ill ill male Mec ± ± 0. ± 0. . . . (n=

r socke s A.1 s s cies s A.1 measured

11 27

lina (n= (n= (n= (n= Fe 71 38 36 51 49

en en pe en nne

3. 5. 7. Sex S S [s S I Inc Peg l S Peg diameter Main Table Table values

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

were ch2 ba1.1 ba1.3 co

) ) ) ) ) ) ) ) ) ) ) )

000 000 296 834 097 286 146 344 693 354 329 575 ** ** n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. nga (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. o ale P-values el

species

) ) ) ) ) ) ) ) ) ) ) ) po

co and

000 000 826 379 589 986 196 632 888 845 127 829 ** ** n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

exact ale

(0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. Fem ty

The ) ) ) ) ) ) ) ) ) ) ) )

sexes

den

686 000 000 129 009 009 433 474 334 046 707 680 ** ** ** ** n.s. n.s. n.s. n.s. n.s. n.s. n.s. P).

silla p.4

(0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. ale

≤ Sen

M da s both

) ) ) ) ) ) ) ) ) ) ) ) co

0.05 Me

470 000 000 000 281 378 057 616 556 259 201 097 ** ** **

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. ale

(0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0.

Fem

ale Fem ale M ale Fem ale M ale M ale M ale M ale Fem ale M ale M ale Fem ale Fem ale Fem ale Fem ale Fem ale M

n.s.:

ta nga o el da po co Me Me ta nga o el da po co Me ta nga o el da po co Me Me p.4 s da po co p.4 s da po co p.4 s da po co ta nga o el da po co Me p.4 s da po co Me Me

0.05;

tr ba1.2 b a2 individuals ch1

≤

ch2 ba1.1 ba1.3 co 0.01

) ) ) ) ) ) ) ) ) from

000 000 001 001 004 000 319 234 000 ** ** ** ** ** ** ** n.s. n.s. N.A. N.A. N.A. nga (0. (0. (0. (0. (0. (0. (0. (0. (0. o ale

da 0.01;

) ) ) ) ) ) ) ) ) ) po

000 000 130 410 371 000 001 000 000 351 ** ** ** ** ** ** P n.s. n.s. n.s. n.s. N.A. N.A. ale

Antennae (0. (0. (0. (0. (0. (0. (0. (0. (0. (0.

. le Fem

(++:

) ) ) ) ) ) ) ) ) ) )

* *

190 000 002 940 156 001 079 004 025 011 005 ** ** ** ** ** n.s. n.s. n.s. n.s. N.A. naon ang p.4 Me (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. ale

cli 0.05

In M da s elongata

P ) ) ) ) ) ) ) ) ) ) ) ) co

Me * * * *

004 030 778 491 018 002 027 693 000 040 965 433 ** ** ** n.s. n.s. n.s. n.s. n.s. at ale

(0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0.

Fem

ale Fem ale Fem ale M ale M ale Fem ale M ale Fem ale M ale M ale M ale Fem ale Fem ale M ale Fem ale M ale Fem

Mecopoda

ta nga o el da po co Me ta nga o el da po co Me Me p.4 s da po co ta nga o el da po co Me p.4 s da po co p.4 s da po co p.4 s da po co Me ta nga o el da po co Me Me Me

different

ch1 a2 b ba1.2 and tr

sp.4

ch2 ba1.1 ba1.3 co

) ) ) ) ) ) ) ) ) ) ) )

ta signiﬁcantly *

000 000 000 000 000 000 000 000 000 012 640 070

** ** ** ** ** ** ** ** ** n.s. n.s. nga (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. o ale

as Mecopoda el

of ) ) ) ) ) ) ) ) ) ) ) ) po

co *

000 000 001 020 223 000 648 194 000 253 202 451 ** ** ** ** ** n.s. n.s. n.s. n.s. n.s. n.s. ale

r Me (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. ete

Fem m

sexes

regarded

d ) ) ) ) ) ) ) ) ) ) ) )

ket * *

443 000 000 048 018 000 221 000 000 059 161 654 ** ** ** ** ** n.s. n.s. n.s. n.s. n.s. p.4 (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. both ale

were er soc M da s

nn in po I

) ) ) ) ) ) ) ) ) ) ) ) co

Me *

223 000 000 609 005 000 137 000 000 001 080 018 ** ** ** ** ** ** ** n.s. n.s. n.s. n.s. ale

(0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. values

Fem

ale M ale Fem ale M ale M ale Fem ale M ale Fem ale M ale Fem ale M ale Fem ale M ale Fem ale M ale Fem ale Fem

p.4 s da po co Me Me p.4 s da po co ta nga o el da po co Me p.4 s da po co Me ta nga o el da po co Me p.4 s da po co ta nga o el da po co Me Me ta nga o el da po co identiﬁed Me

Mean

tr ba1.2 b ch1 a2 test. types

ch2 ba1.1 ba1.3 co

) ) ) ) ) ) )

sensilla ) ) ) ) ) 121 358 073 778 901 512 312

ta (0. (0. (0. (0. (0. (0. (0. * *

689 015 002 025 003 ** ** n.s. nga

(0. (0. (0. (0. (0. o ale

el n.s. n.s. n.s. n.s. n.s. n.s.

) ) ) ) ) )n.s.

da ) ) ) ) ) ) po 149 180 000 756 310

710

co (0. (0. (1. (0. (0. * * * * different (0.

017 025 021 001 009 010 ** **

ale

Mann–Whitney-

(0. (0. (0. (0. (0. (0.

n.s. n.s. n.s. n.s. n.s. Fem r

the ) ) ) ) )n.s.

the ete

) ) ) ) ) ) ) 374 059 372 095

m 091 of

(0. (0. (0. (0. (0.

000 000 001 000 000 000 005 ** ** ** ** ** ** ** p.4 Me

(0. (0. (0. (0. (0. (0. (0. ale

Peg d Peg n.s. n.s. n.s. n.s. n.s. M da s using

) ) ) )

) ) ) ) ) ) ) co ) 861 237 240 888

09 (0. (0. (0. (0. Me

000 001 000 077 000 000 000 ** ** ** ** ** ** n.s. n.s. ale

(0.

(0. (0. (0. (0. (0. (0. (0.

tested

n.s. n.s. n.s. n.s. Fem

ale Fem ale M ale M ale M ale Fem ale M ale Fem ale M ale Fem ale M ale Fem ale ale M ale Fem ale Fem ale M parameters Fem

p.4 s da po co Me ta nga o el da po co Me p.4 s da po co ta nga o el da po co Me p.4 s da po co Me ta nga o el da po co Me p.4 s da po co Me ta nga o el da po co Me Me

were

ch1 ba1.2 b a2 tr measured

ch2 ba1.1 ba1.3 co variances

main

) ) ) ) ) ) ) ) (0.8)

000 000 000 086 633 191 031 597 ** ** ** n.s. n.s. n.s. n.s. the N.A. N.A. N.A.

nga (0. (0. (0. (0. (0. (0. (0. (0. o ale

M on

) ) n.s. ) ) ) )

) ) ) ) po

000 716 422 089 234 855

co * (0. (0. (0. (0. (0. (0.

000 000 041 004 ** ** ** N.A. N.A. ale

(0. (0. (0. (0. Differences

n.s. n.s. n.s. n.s. ** Fem

) ) ) )

analyses ) ) ) ) ) ) ) 556

261 311

032

41 Peg length (0. * (0. (0. (0.

000 000 001 041 006 005 ** ** ** ** ** p.4 Me n.s. N.A.

(0.

(0. (0. (0. (0. (0. (0. ale

da s * n.s. n.s. n.s. M

po )) n.s. )

) ) ) ) ) ) ) ) ) co

683 092

(0. Me *

101 001 000 568 875 215 943 008 014 ** ** ** ale

n.s. n.s. n.s. n.s. n.s. respectively. (0. (0. (0. (0. (0. (0. (0. (0. (0. statistical

* n.s. (0. n.s. (0. Fem

ale Fem ale Fem ale M ale M ale M ale Fem ale M ale M ale Fem ale M ale ale M ale Fem ale Fem ale M ale Fem brackets. Fem

p.4 s da po co Me ta nga o el da po co p.4 s da po co Me p.4 s da po co Me Me Me Me p.4 s da po co Me ta nga o el da po co ta nga o el da po co ta nga o el da po co A.2 Me in

tr ch1 a2 b ba1.2 Table Results examined, given

E.S. Schneider, H. Römer / Micron 90 (2016) 43–58 57

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