Micron 106 (2018) 7–20

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Micron

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Species- and sex-specific distribution of antennal olfactory sensilla in two T tortricid moths, Epiphyas postvittana and Planotortrix octo ⁎ Gwang Hyun Roha, Kye Chung Parkb, Hyun-Woo Ohc, Chung Gyoo Parka,d, a Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea b New Zealand Institute for Plant and Food Research, Christchurch, New Zealand c Korea Research Institute of Bioscience & Biotechnology, Daejeon 34141, Republic of Korea d Institute of Life Science (BK21+ Program), Gyeongsang National University, Jinju 52828, Republic of Korea

ARTICLE INFO ABSTRACT

Keywords: We investigated the morphology and distribution of antennal sensilla in males and females of two tortricid Antenna moths, Epiphyas postvittana and Planotortrix octo, by scanning electron microscopy. The number and overall Morphology length of flagellomeres were significantly greater in females than in males in both species. The antennae of each Olfaction species bearing six morphological types of sensilla (trichodea, basiconica, coeloconica, auricillica, chaetica, and Scanning electron microscopy styloconica), with different numbers and distributions along the antennae. Among these sensilla, four types Sensilla (trichodea, basiconica, coeloconica, and auricillica) displayed multi-porous cuticular surfaces, indicating that Trichodea their primary sensory function is olfactory. Each of these four types of sensilla could be further classified into subtypes according to their size, shape, and surface structure. Both E. postvittana and P. octo exhibited sexual dimorphism of the profiles of antennal olfactory sensilla. Trichoid sensilla were the most abundant type in both species. Subtype I trichoid sensilla were male-specific in both species, indicating that they are responsible for the perception of conspecific female sex pheromone. By contrast, subtype II trichoid sensilla were more abundant in female antennae in both species, suggesting that some subtype II trichoid sensilla are involved in female-specific behaviors, such as oviposition. Chaetic and styloconic sensilla displayed relatively even distributions along the antennae. Our results indicate that the antennae of E. postvittana and P. octo have species-specific and sex-specific profiles of olfactory sensilla. The morphological information obtained in our study provides a basis for elec- trophysiological and behavioral studies of the olfactory sensory function of each morphological type of sensilla.

1. Introduction sensory modalities are often related to the purpose of sensory reception, such as finding host plants and mates of a particular species (Cossé Antennae are the major sensory organs in insects, bearing various et al., 1998; Baker et al., 2004; Ansebo et al., 2005; Pophof et al., 2005; types of sensilla for the detection of chemical and physical sensory cues Sun et al., 2011). The morphology and distribution of antennal sensilla from the surrounding environment. Each sensillum is an independent are often characteristic features of a specific group of insects and can be sensory unit containing sensory neurons, auxiliary cells, and various used for taxonomic identification (Notaro-Muñoz et al., 1997; Molero- extracellular components, and these components are spatially and Baltanás et al., 2000). The morphological identification of sensilla with physiologically isolated from the outside of the sensillum (Steinbrecht, specific sensory modalities can provide valuable information for sub- 1997). sequent electrophysiological or behavioral studies of the functions of These antennal sensilla display diverse morphological features, and sensilla (Baker et al., 2004; Malo et al., 2004; Pophof et al., 2005; Sun the specific external shape and morphological characters are typically et al., 2011). related to the sensory functions of sensilla. For example, thick and long Olfactory sensilla are the most abundant type of sensilla in the an- setae with basal sockets and no surface pores are typical features of tennae of many groups of insects, such as flies (Stocker, 2001; mechanosensilla, the presence of one or a few pores at the tip indicates Sukontason et al., 2004), beetles (Merivee et al., 2002; Ploomi et al., a gustatory function, and the presence of numerous nanoscale pores 2003), bees (Frasnelli et al., 2010), and moths (Gómez et al., 2003; throughout the cuticular surface suggests an olfactory function Gómez and Carrasco, 2008; Sun et al., 2011). These antennal olfactory (Zacharuk, 1980). The presence and abundance of sensilla with specific sensilla display distinct shapes, such as trichoid (long hair with a sharp

⁎ Corresponding author at: Institute of Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea. E-mail address: [email protected] (C.G. Park). https://doi.org/10.1016/j.micron.2017.12.006 Received 12 September 2017; Received in revised form 15 December 2017; Accepted 18 December 2017 Available online 21 December 2017 0968-4328/ © 2017 Elsevier Ltd. All rights reserved. G.H. Roh et al. Micron 106 (2018) 7–20 tip), basiconic (short hair with a blunt tip), auricillic (rabbit ear shape), 2.2. Scanning electron microscopy placoid (round dome shape), and coeloconic (short peg with long- itudinal grooves). Despite the diverse shapes, all types of olfactory For scanning electron microscopy, antennae of adult moths were sensilla, except coeloconic sensilla, have numerous nanoscale pores excised at the base around the scape and fixed in 70% ethanol for ap- (approximately 20–50 nm in diameter) on their cuticular surface proximately 24 h. The antennae were then dried in closed plastic con- (Onagbola and Fadamiro, 2008; Kim et al., 2016; Roh et al., 2016; Wee tainers with desiccants and mounted on a piece of double-sided sticky et al., 2016). Instead, coeloconic sensilla possess longitudinal grooves tape attached to the top of aluminum specimen stubs. The samples were running along the sensory peg (Faucheux et al., 2006; Diongue et al., then gold-coated in a sputter coater (Q15ORS; Quorum, Lewes, UK). 2013; Roh et al., 2016). These pores and grooves appear to be the en- The antennal preparations were observed using two different scanning trance for odor molecules into the olfactory sensillum before reaching electron microscopes (JCM-5000, JEOL, Tokyo, Japan; FEI Quanta 250 their corresponding receptors on dendritic membranes through the FEG, FEI, Hillsboro, OR, USA) at 10 kV. sensillum lymph (Steinbrecht, 1997; Shields and Hildebrand, 1999). Different insect species belonging to the same group appear to share 2.3. Identification of olfactory sensilla and classification into morphological similar types of olfactory sensilla, although the detailed morphological types properties of sensilla can be species-specific. For example, elongated placoid sensilla are typical olfactory sensilla in hymenopteran species After counting the flagellomeres in male and female antennae of E. (Gao et al., 2007; Onagbola and Fadamiro, 2008), round placoid sen- postvittana and P. octo, the sensilla present in the 1st, 6th, 11th, 21th, silla are typical in homopteran species (Broeckling and Salom, 2003), 31th, and 41th flagellomeres were examined at low magnifi cations of short basiconic and auricillic sensilla are typical in dipteran species under × 5000. These sensilla were then classified into distinct groups, (Ilango et al., 1994; Sukontason et al., 2004, 2007), and long trichoid such as trichodea, basiconica, coeloconica, auricillica, chaetica, and sensilla are typical in moths (Diongue et al., 2013; Roh et al., 2016). styloconica, according to their gross morphology at low magnification. The light brown apple moth Epiphyas postvittana and the green- The sensilla belonging to each group were randomly sampled and ex- headed leafroller moth Planotortrix octo are polyphagous tortricid amined at high resolution, up to × 50,000, in which the detailed moths endemic to Australia and New Zealand (Danthanarayana, 1975; structures of the cuticular surfaces of sensilla were carefully examined. Suckling and Brockerhoff, 2010). Although they are polyphagous, fe- When numerous pores with diameters of 20–50 nm were present on the male moths of each species demonstrate preferences and aversions to- sensillum surface, the sensilla were regarded as olfactory. Peg-shaped wards distinct groups of plants for oviposition (Wearing et al., 1991; sensilla (coeloconica) with narrow longitudinal grooves were also re- Mclaren and Suckling, 1993; Suckling et al., 1998; Suckling and garded as olfactory. Then, the sensilla in each group were further Brockerhoff, 2010; Brockerhoff et al., 2011), indicating that female classified into subtypes based on their size, the presence of a basal moths process plant volatile cues via the olfactory sensory system. Both socket, and surface morphology. The sizes (length and width) of fla- E. postvittana and P. octo use multi-component female sex pheromones, gellomeres and sensilla were measured using ImageJ (Rasband, suggesting the presence of male-specific ORNs (olfactory receptor 1997–2006). The overall length and total number of flagellomeres on a neurons) for the detection of conspecific female sex pheromone com- flagellum were measured for five male and five female antennae in the ponents. Recent molecular studies have revealed the sex-biased ex- two species. pression of odorant receptors in the antennae of E. postvittana (Corcoran et al., 2015) and P. octo (Steinwender et al., 2016), which suggests the 2.4. Distribution of olfactory sensilla presence of male-specific and female-specific ORNs in each species. The morphological types of sensilla and their distribution often The total number of olfactory sensilla on each flagellomere was exhibit sexual dimorphism, and the sex-specific olfactory sensilla con- estimated using the formula (n/a)×A(Roh et al., 2016), where n is the tain ORNs responsible for the detection of volatile compounds related number of sensilla observed in a defined surface area (a) on each fla- to sex-specific behaviors. The male-specific long trichoid sensilla in gellomere. A is the total surface area of a flagellomere, calculated by moths, for example, typically contain ORNs responsive to conspecific multiplying the basal width by the longitudinal length, assuming that female sex pheromones and related compounds (Hansson et al., 1995; the flagellomere is cylindrical, and subtracting the area occupied by Cossé et al., 1998; Baker et al., 2004). In this context, information on scales. Then, the distribution of each type of olfactory sensilla was the morphology and distribution of olfactory sensilla can be very useful analyzed and compared between the two species and between the sexes. for inferring and studying the function of each type of olfactory sensilla Six flagellomeres (1st, 6th, 11th, 21st, 31st, and 41 st flagellomeres) and, subsequently, for understanding the chemical communication from five female and five male adults of two species were subjected to a systems of given species. In this study, the morphological types of an- quantitative analysis of olfactory sensilla. tennal olfactory sensilla and their distributions in E. postvittana and P. octo were investigated by scanning electron microscopy. The profiles of 3. Statistical analysis different morphological types of olfactory sensilla were then compared between two species and between males and females. The implications The length, basal width, and number of the four olfactory sensilla of the species-specific and sex-specific profiles of olfactory sensilla for subtypes were compared between sexes using the Student’s t-test and the chemical communication system of these species are discussed. generalized linear model (GLM), and means were separated by the least significant difference (LSD) test. Difference in length and width among sensilla types and the number of sensilla and subtypes within each 2. Materials and methods sensilla type were analyzed in each sex and species using GLM, and the means were separated by LSD tests. All statistical analyses were con- 2.1. Insects ducted using SAS 9.4 (SAS Institute, Cary, NC, USA).

Pupae of E. postvittana and P. octo were obtained from the lab co- 4. Results lonies maintained at the Mt. Albert Research Centre, Auckland, New Zealand. Male and female pupae of each species were placed in separate 4.1. Gross antennal morphology containers with 8% sugar water and kept at room temperature. One- to two-day-old moths were used in our experiments. The lengths of male and female antennae were 3.8 ± 0.1 mm and 4.6 ± 0.2 mm in E. postvittana and 5.2 ± 0.1 mm and 5.8 ± 0.1 mm

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Fig. 1. External gross morphology and array of antennal sensilla in male (A and C) and female (B and D) adults of E. postvittana and in male (G and I) and female (H and J) adults of P.octo. The ventral part of the antenna was covered with dense sensilla and the dorsal part was covered with scales. Arrays of male-specific s. trichodea subtype I (arrow) were present in male antennae (C and I) but absent in female antennae (D and J) in E. postvittana and P. octo. Distributions of six different types of sensilla on antennal flagellomeres in male and female adults of E. postvittana (E and F) and P. octo (K and L). Length and width of each flagellomere in both sexes of E. postvittana (M) and P. octo (N). Sensilla trichodea (Tr), s. basiconica (Ba), s. auricillica (Au), s. coeloconica (Co), s. chaetica (Ch), and s. styloconica (St). in P. octo, respectively. The numbers of flagellomeres in each male and species (Fig. 1). By contrast, the width of each flagellomere gradually female antenna were 43.4 ± 0.9 and 48.2 ± 0.6, respectively, in E. decreased towards the distal end of antennae in both species (Fig. 1M, postvittana, and 48.2 ± 0.2 and 50.0 ± 0.5 in P. octo. Antennae were N). significantly longer and the number of flagellomeres was significantly greater in females than in males in the two species (Length: E. post- vittana, df =8, t = 4.45, P = 0.002; P. octo, df =8, t = 3.64, 4.2. Morphological analysis of sensilla P = 0.007, Number of flagellomeres: E. postvittana, df =8, t = 4.38, P = 0.002; P. octo, df =8, t = 3.09, P = 0.015). The length of each The antennal sensilla of both E. postvittana and P. octo could be flagellomere increased towards the center of flagellomeres and de- classified into six types based on their gross morphology (Figs. 1–6). creased towards the proximal and distal ends of antennae in both These six types were trichodea, basiconica, auricillica, coeloconica, chaetica, and styloconica. Three types of sensilla (trichodea, basiconica,

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Fig. 2. Distribution and fine morphology of different subtypes of sensilla trichodea on antennal flagellomeres in both sexes of E. postvittana and P. octo. Three different subtypes of s. trichodea (Tr I, II, and III) in males (A and I) and two different subtypes of s. trichodea (Tr II and III) in females of E. postvittana and P. octo (B and J, respectively). Basal parts of subtypes of s. trichodea (Tr I, II, and III) in E. postvittana (C, D, and E) and P. octo (K, L, and M). Pores on the surface (arrows) in s. trichodea subtype I, II, and III in E. postvittana (F, G, and H) and those in s. trichodea subtype I and II in P. octo (N and O). Length and width of each subtype of s. trichodea in both sexes of E. postvittana (P and Q) and P. octo (R and S).

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Fig. 3. Distribution and fine morphology of different subtypes of sensilla basiconica on antennal flagellomeres in E. postvittana and P. octo. Three different subtypes of s. basiconica (Ba I, II, and III) in E. postvittana (A and B) and one subtype of s. basiconica (Ba I) in P. octo (H). Subtypes of s. basiconica (Ba I, II, and III) in E. postvittana (C, D, and E). Pores on surface (arrow) in s. basiconica subtype I and II in E. postvittana (F and G) and in s. basiconica subtype I in P. octo (I). Length and width of each subtype of s. basiconica in both sexes of E. postvittana (J and K). and auricillica) displayed numerous pores with diameters of approxi- as olfactory. By contrast, two other types of sensilla (chaetica and sty- mately 20–50 nm on their cuticular surface (Figs. Figure 2F, G, H, N, O, loconica) did not display any nanoscale pores on their cuticular surface. Figure 3F, G, I; and Figure 4G, H), and therefore the main sensory Fewer olfactory sensilla were present in the proximal and distal function of these sensilla was regarded as olfactory. The coeloconic flagellomeres than in the central flagellomeres in both species (Fig. 7) sensilla exhibited longitudinal grooves with widths of approximately (E. postvittana, male: df = 5, 24, F = 20.60, P<0.001, female: df =5, 50–100 nm along the cuticular surface of the central peg (Fig. 5), and 24, F = 14.02, P<0.001; P. octo, male: df = 5, 24, F = 27.75, therefore the main sensory function of these sensilla was also regarded P<0.001, female: df = 5, 24, F = 10.34, P<0.001). However, the

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Fig. 4. Distribution and fine morphology of different subtypes of sensilla auricillica on antennal flagellomeres in E. postvittana and P. octo. Three different subtypes of s. auricillica (Au I, II, and III) in E. postvittana (A, B, and C) and two different subtypes of s. auricillica (Au I and II) in P. octo (I and J). Subtypes of s. auricillica (Au I, II, and III) in E. postvittana (D, E, and F) and s. auricillica subtype I (Au I) in P. octo (K). Pores on the surface (arrow) in s. auricillica subtype I and III in E. postvittana (G and H). density of olfactory sensilla (the number of sensilla per unit area) was basiconic and coeloconic sensilla in most antennal segments in both relatively higher in the distal flagellomeres (Fig. 7)(E. postvittana, male: sexes of E. postvittana, and the opposite pattern was observed in P. octo df = 5, 24, F = 8.42, P<0.001, female: df = 5, 24, F = 14.02, (Fig. 8)(E. postvittana, male: df = 17, 72, F = 6.50, P<0.001, female: P<0.001; P. octo, male: df = 5, 24, F = 30.18, P<0.001, female: df = 17, 72, F = 5.12, P<0.001; P.octo, male: df = 17, 72, F = 11.20, df = 5, 24, F = 10.84, P<0.001). Similar trends were observed in P<0.001, female: df = 17, 72, F = 15.38, P<0.001). males and females. Trichoid sensilla were the most abundant among the four types of olfactory sensilla in both sexes of the two species (Table 2) 4.3. Sensilla trichodea (E. postvittana, male: df = 3, 16, F = 999.13, P<0.001, female: df = 3, 16, F = 186.77, P<0.001; P.octo, male: df = 3, 16, Trichoid sensilla could be characterized as having a thin, hair-like F = 569.13, P<0.001, female: df = 3, 16, F = 497.36, P<0.001). shape with a pointed tip (Fig. 2). All trichoid sensilla examined at high The three other types of sensilla exhibited slight differences in abun- resolution in our study exhibited numerous nanoscale pores on their dance in both sexes in E. postvittana, in the decreasing order of aur- cuticular surface. In both E. postvittana and P. octo, all trichoid sensilla icillica, basiconica, and coeloconica (Table 2). The auricillic sensilla displayed regularly spaced and angled lateral ridges along the sensilla, were more abundant in E. postvittana than in P. octo (Table 2)(df =3, and the nanoscale pores were located between the ridges (Fig. 2F, G, H, 16, F = 25.67, P< 0.001). But, the coeloconic sensilla showed the N, and O). opposite pattern (df = 3, 16, F = 31.35, P<0.001). The relative The trichoid sensilla could be further classified into three subtypes abundance of auricillic sensilla was slightly greater than those of in each species based on their length, width, and shape (Table 1 and

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Fig. 5. Distribution and fine morphology of different subtypes of sensilla coeloconica on antennal flagellomeres in E. postvittana and P. octo. Two different subtypes of s. coeloconica (Co I and II) in E. postvittana (A) and in P. octo (E and F). S. coeloconica subtypes I and II in E. postvittana (B and C). Surface (arrow) of the peg in s. coeloconica in E. postvittana (D). S. coeloconica subtypes I and II in P. octo with or without the fence (G and H, respectively).

Fig. 2). The subtype I trichoid sensilla, the longest and thickest among length: df = 1, 44, F = 3.67, P = 0.062, width: df = 1, 44, F = 1.09, the three subtypes of trichoid sensilla, were present only in male an- P = 0.302; P.octo, subtype II, length: df = 1, 56, F = 0.17, P = 0.683, tennae in both species (Table 1 and Figs. 1 and 2). Subtype I trichoid width: df = 1, 56, F = 7.76, P = 0.007, subtype III, length: df = 1, 43, sensilla in male P. octo were longer (96.1 ± 2.5 μm) than those of male F = 3.74, P = 0.059, width: df = 1, 43, F = 15.10, P<0.001). Sub- E. postvittana (53.1 ± 1.1 μm) (male: df = 11, 181, F = 380.48, type II and subtype III trichoid sensilla were longer in P. octo than in E. P<0.001), whereas the width of subtype I trichoid sensilla did not postvittana (Table 1). differ between the two species (4.4 ± 0.1 μm in male P. octo and Subtype I and II trichoid sensilla were the most abundant subtypes 4.3 ± 0.1 μm in male E. postvittana)(Table 1) (male: df = 11, 181, in males and females of both species (Table 2)(E. postivittana, male: F = 100.52, P < 0.001). In E. postvittana, subtype II trichoid sensilla df = 10, 44, F = 181.26, P< 0.001; female: df = 9, 40, F = 172.42, were significantly longer in females than in males, but were wider in P< 0.001; P. octo, male: df = 7, 32, F = 150.97, P< 0.001; female: males than in females. The length and width of subtype III trichoid df = 6, 28, F = 233.38, P< 0.001). Subtype II in both species and sensilla were not different between sexes. In P. octo, the lengths of subtype III trichoid sensilla were significantly more abundant in fe- subtype II and subtype III trichoid sensilla were not significantly dif- males than in males in E. postvittana (Table 2)(E. postvittana, subtype II: ferent between sexes. The width of subtype II was wider in males than df =8, t = −8.59, P< 0.001, subtype III: df =8, t = −3.26, in females, but the subtype III was wider in females than in males P = 0.011; P. octo, subtype II: df =8,t = −3.94, P = 0.004). However, (Table 1)(E. postvittana, subtype II, length: df = 1, 44, F = 40.10, the number of subtype III was similar in the two sexes in P. octo P<0.001, width: df = 1, 44, F = 15.39, P<0.001, subtype III, (Table 2)(P. octo, subtype III: df =8,t = 0.00, P = 1.000). The overall

Fig. 6. Sensilla styloconica (St) and s. chaetica (Ch) on antennal flagellomeres in E. postvittana (A and C) and P. octo (F and H). S. styloconica on the terminal flagellomere (D), the surface of s. styloconica (B), and the surface of s. chaetica (E) in E. postvittana. S. styloconica on the terminal flagellomere (I), the surface of s. styloconica (G), and the surface of s. chaetica (J) in P. octo.

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Fig. 7. Total number of olfactory sensilla (s. tri- chodea, s. basiconica, s. auricillica, and s. coeloco- nica) (lines) and the number of sensilla per 10,000 μm2 (bars) on the six antennal flagellomeres of E. postvittana (A) and P. octo (B) (means + SE, N = 5). The number of sensilla on 10,000 μm2 of each flagellomere was counted to compare the den- sity of sensilla among flagellomeres. Numbers of four sensilla types (s. trichodea, s. basiconica, s. coelo- conica, and s. auricillica) were pooled. Total number of olfactory sensilla and density of sensilla on the six antennal flagellomeres were compared by general- ized linear model (GLM) and the means were sepa- rated by LSD at P = 0.05. Means with different let- ters are significantly different among the six flagellomeres of each sex.

number of antennal trichoid sensilla of E. postvittana was greater than P. octo (Table 1 and Fig. 3). Subtype I basiconic sensilla were sig- that of P. octo (Table 2)(df = 3, 16, F = 21.23, P<0.001). In both nificantly longer in males than in females, but the width of subtype I did species, the subtype I trichoid sensilla displayed a relatively uniform not differ between the sexes of E. postvittana. The lengths and widths of distribution in each flagellomere, aligning along the circumferential subtype II and III of basiconic sensilla were similar between males and lines close to both ends of the flagellomere (Figs. 1 and 2). By contrast, females of the E. postvittana (Table 1)(E. postvittana, subtype I, length: subtype II and subtype III trichoid sensilla showed random distributions df = 1, 12, F = 6.34, P = 0.027, width: df = 1, 12, F = 0.00, in each flagellomere (Fig. 2). P = 0.956; subtype II, length: df = 1, 15, F = 3.09, P = 0.099, width: df = 1, 15, F = 0.00, P = 0.957; subtype III, length: df = 1, 11, F = 0.00, P = 0.970, width: df = 1, 11, F = 0.59, P = 0.459). The ba- 4.4. Sensilla basiconica siconic sensilla of P. octo also exhibited similar lengths and widths between males and females (Table 1) (subtype I, length: df = 1, 24, Basiconic sensilla could be characterized as a cone-shaped hair with F = 2.00, P = 0.169, width: df = 1, 24, F = 3.96, P = 0.058). a blunt tip (Fig. 3). All basiconic sensilla examined at high resolution in The numbers of each basiconic sensilla subtype of E. postvittana and our study exhibited numerous nanoscale pores on their cuticular sur- P. octo were not significantly different between the sexes (Table 2)(E. face (Fig. 3F, G, and I). In E. postvittana, many of the nanoscale pores on postvittana, subtype I: df =8,t = −1.59, P = 0.151; subtype II: df =8, basiconic sensilla displayed somewhat elongated shapes, and a series of t = −1.44, P = 0.187; subtype III: df =8,t = 0.29, P = 0.782, P. octo, such pores formed a number of short longitudinal grooves along the subtype I: df =8, t = −1.85, P = 0.102). It appeared that antennal sensilla (Fig. 3F, G). In P. octo, the cuticular surface of basiconic sensilla basiconic sensilla of P. octo were more abundant than those of E. post- showed regularly spaced and acutely edged longitudinal ridges along vittana (Table 2)(df = 3, 16, F = 5.18, P = 0.010). In P. octo, basiconic which nanoscale pores were aligned (Fig. 3I). sensilla were more abundant throughout the distal region from the 11th The basiconic sensilla could be further classified into three subtypes flagellomere, regardless of sex ( Fig. 8). A somewhat similar trend was in each sex of E. postvittana based on their length and shape (Table 1 observed in female E. postvittana. By contrast, however, basiconic and Fig. 3). By contrast, only one type of basiconic sensilla was found in

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Fig. 8. Abundance of three sensillum types (s. basiconica, s. coeloconica, and s. auricillica) relative to s. trichodea (100%) and their distribution on the six antennal flagellomeres of each sex of E. postvittana (A, B) and P. octo (C, D) (means ± SE, N = 5). The relative abundance was compared by generalized linear model (GLM) and the means were separated by LSD at P = 0.05. Different letters indicate significant differences in the relative abundance among the six flagellomeres.

Table 1 Size and shape of each subtype of antennal olfactory sensilla identified in E. postvittana and P. octo (mean ± SE, N = 3–30).

Species Sensillum type Subtype Length (μm) Basal width (μm)

Male a Female a Male a Female a Shape

E. postvittana Trichodea Tr I 53.1 ± 1.1 b – 4.3 ± 0.1 a – Curved Tr II 36.0 ± 0.5 d, Bb 40.8 ± 0.6 c, A 3.3 ± 0.0 b, A 3.0 ± 0.0 a, B Curved Tr III 25.9 ± 0.8 e 27.7 ± 0.6 d 2.1 ± 0.1 def 2.2 ± 0.1 b Straight Basiconica Ba I 25.9 ± 1.3 e, A 22.0 ± 0.9 e, B 2.4 ± 0.2 cd 2.3 ± 0.2 b Curved Ba II 10.1 ± 0.6 g 11.5 ± 0.4 h 2.3 ± 0.3 cde 2.3 ± 0.1 b Curved Ba III 14.0 ± 0.8 gf 13.9 ± 1.0 g 2.4 ± 0.1 cd 2.2 ± 0.2 b Straight Auricillica Au I 16.7 ± 0.8 f 17.9 ± 0.7 f 2.5 ± 0.2cc 2.5 ± 0.2c b Narrow rabbit ear Au II 13.8 ± 0.9 gf NA 3.5 ± 0.3 b NA Wide rabbit ear Au III NA NA NA NA Flat Coeloconica Co I NA NA 7.7 ± 0.2d 8.0 ± 0.1d With fence Co II NA NA NA 7.8 ± 0.2d Without fence

P. octo Trichodea Tr I 96.4 ± 2.5 a – 4.4 ± 0.1 a – Curved Tr II 57.0 ± 0.6 b 57.4 ± 0.5 a 3.2 ± 0.1 b, A 3.0 ± 0.1 a, B Curved Tr III 42.4 ± 0.9 c 44.7 ± 0.8 b 2.0 ± 0.1 ef, B 2.4 ± 0.1 b, A Mixed Basiconica Ba I 16.1 ± 0.4 f 17.2 ± 0.7 f 1.9 ± 0.1 f 2.2 ± 0.1 b Curved Auricillica Au I NA NA NA NA Narrow rabbit ear Au II NA NA NA NA Flat Coeloconica Co I NA NA 9.3 ± 0.3d 9.1 ± 0.2d With fence Co II NA NA NA NA Without fence

NA: Lengths or widths could not be measured because the subtypes were tangled with many other subtypes. – Not found in female E. postvittana and P. octo. a Lengths and widths of subtypes, except s. coeloconica, in the same column are significantly different when marked with the same small letters (LSD at P = 0.05). b Lengths and widths of each sensilla subtype marked with the same capital letters are significantly different between the sexes within a species (LSD at P = 0.05). c The width of s. auricillica subtype I was measured at its middle part. d The width of s. coeloconica was defined as the diameter of the fence.

15 G.H. Roh et al. Micron 106 (2018) 7–20

Table 2 The number of each subtype of sensilla on male and female antennal flagellomeres of E. postvittana and P. octo (mean ± SE; N = 5).

Sensilla type Subtype E. postvittana P. octo

Male a Female a Male a Female a

Trichodea Tr I 130.0 ± 4.4a – 114.1 ± 5.8 a – Tr II 82.5 ± 6.7 b 164.2 ± 6.8 a*d 91.4 ± 4.3 b 117.8 ± 5.0 a * Tr III 26.2 ± 5.1 d 52.8 ± 6.5 b* 55.8 ± 4.6 c 55.9 ± 1.5 b Total 238.7 ± 4.9 ABb,ac 217.0 ± 11.4 B, a 261.2 ± 8.7 A, a 173.8 ± 5.4C, a

Basiconica Ba I 8.7 ± 1.3 ef 12.8 ± 2.3 d 24.9 ± 2.0 e 32.7 ± 3.5 c Ba II 5.9 ± 1.9 ef 9.0 ± 1.2 de –– Ba III 2.0 ± 0.8 ef 1.6 ± 1.2 e –– Total 16.6 ± 3.0 B, c 23.4 ± 3.0 B, c 24.9 ± 2.0 AB, bc 32.7 ± 3.5 A, b

Auricillica Au I 37.9 ± 2.4 c 38.8 ± 6.7 c 3.1 ± 1.1 fg 12.8 ± 1.6 e* Au II 8.1 ± 0.5 ef* 2.0 ± 0.5 de 10.0 ± 1.1 f 5.5 ± 1.9 ef Au III 8.5 ± 1.3 ef 9.4 ± 1.1 de –– Total 54.4 ± 3.5 A, b 50.2 ± 7.3 A, b 13.1 ± 2.1 B, c 18.3 ± 0.9 B, c

Coeloconica Co I 9.6 ± 0.8 e 12.8 ± 1.1 d* 37.4 ± 3.7 d * 21.1 ± 1.8 d Co IIe 0.6 ± 0.4 f 0.2 ± 0.2 e 0.0 ± 0.0 g 0.5 ± 0.3 f Total 10.2 ± 1.1 C, c 13.0 ± 1.1C, c 37.4 ± 3.7 A, b 21.6 ± 1.7 B, c

– Not found on the examined antennal flagellomeres. a Mean numbers of sensilla subtypes marked with different small letters in the same column are significantly different (LSD at P = 0.05). b Total numbers of sensilla subtypes marked with different capital letters in the same row are significantly different (LSD at P = 0.05). c Total numbers of sensilla subtypes marked with different small letters in the same column are significantly different (LSD at P = 0.05). d Asterisks (*) indicate a significant difference in the number of sensilla subtypes between males and females in the same species (t-test at P = 0.05). e Coeloconica subtype II was not found on the six flagellomeres examined but was found on some other flagellomeres in small numbers. sensilla became less abundant towards distal flagellomeres in male E. (Table 2)(df =8,t = 2.13, P = 0.06). Auricillic sensilla of E. postvittana postvittana (Fig. 8). were more abundant than those of P. octo (Table 2)(df = 3, 16, F = 25.67, P<0.001).

4.5. Sensilla auricillica 4.6. Sensilla coeloconica Auricillic sensilla could be characterized as flat, rabbit ear, or shoe- horn-like elongated ovoid in shape (Fig. 4). The main difference be- Coeloconic sensilla could be characterized as short pegs (Fig. 5). tween auricillic sensilla and three other types (trichoid, basiconic, or Unlike the three other types of olfactory sensilla, the cuticular surface coeloconic) of olfactory sensilla was that the base was narrower than of coeloconic sensilla did not contain any nanoscale pores. Instead, – the middle region in auricillic sensilla, whereas the base of the three linear grooves with widths of 50 100 nm were present along the peg fi other types of olfactory sensilla was wider than the rest of the structure. (Fig. 5B, D, and G). The coeloconic sensilla could be further classi ed All auricillic sensilla examined at high resolution in our study exhibited into two subtypes in each species based on the presence of long- numerous nanoscale pores on their cuticular surface. In many cases, itudinally bent pointed microtrichia towards the central peg (Table 1 these pores were aligned in lines, forming longitudinal grooves in both and Fig. 5). fi species (Fig. 4G, H). Most coeloconic sensilla identi ed in both species were subtype I, The auricillic sensilla were further classified into three subtypes in and subtype II coeloconic sensilla with no surrounding microtrichia E. postvittana and two subtypes in P. octo, mainly based on their shapes were extremely rare (Table 2). fi (Table 1 and Fig. 2). Subtype I and II auricillic sensilla of E. postvittana There were signi cantly more subtype I coeloconic sensilla than displayed narrow or wide rabbit ear-like shapes, respectively, with a subtype II coeloconic sensilla in both sexes of the two species (Table 2). ff fi semicircular cross section (Fig. 4). Subtype I auricillic sensilla of P. octo Additionally, the number of subtype I sensilla di ered signi cantly exhibited narrow rabbit ear-like shapes. By contrast, subtype III aur- between the sexes in both species, but the number of subtype II did not ff − icillic sensilla could be distinguished from subtype I or II by their flat di er (Table 2)(E. postvittana, subtype I: df =8,t = 2.39, P = 0.044; shape in both species (Table 1 and Fig. 4C, F, and J). In male E. post- subtype II: df =8, t = 0.89, P = 0.397 and P. octo, subtype I: df =8, − vittana, the length of subtype I auricillic sensilla was similar to that of t = 3.84, P<0.001, subtype II: df =8, t = 1.63, P = 0.141). The subtype II auricillic sensilla, but the basal width of subtype I auricillic coeloconic sensilla were more abundant in P. octo than in E. postvittana sensilla was significantly smaller than that of subtype II auricillic sen- (Table 2)(df = 3, 16, F = 31.35, P<0.001). The coeloconic sensilla silla (Table 1). were clustered at the distal part of segments in both sexes of the two The number of subtype I auricillic sensilla was significantly greater species, and their abundance increased towards the distal end of an- than that of other subtypes in both sexes of E. postvittana (male: df = 10, tennae in both sexes of the two species, most markedly in male P. octo 44, F = 181.26, P<0.001; female: df = 9, 40, F = 172.42, (Fig. 8). P<0.001), but was not different in both sexes of P. octo (Table 2). The subtype I and III auricillic sensilla were similar in number in the two 4.7. Sensilla chaetica and sensilla styloconica sexes in E. postvittana (subtype I: df =8,t = −0.14, P = 0.891; subtype ,III: df =8 t = −0.57, P = 0.586), but subtype II auricillic sensilla in E. Chaetic sensilla were thick (width: 2.7 ± 0.1 μminE. postvittana postvittana were significantly more abundant in males than in females and 3.0 ± 0.1 μminP. octo) and long (length: 55.6 ± 3.1 μminE. (df =8, t = 8.49, P<0.001). Additionally, the subtype I auricillic postvittana and 60.4 ± 3.3 μminP. octo) with no nanoscale pores on sensilla were more abundant in females than in males in P. octo the cuticular surface (Fig. 6C, E, H, and J). Styloconic sensilla were (Table 2)(df =8, t = −5.13, P<0.001). However, the number of thick columns (width and length: 4.0 ± 0.2 μm and 16.8 ± 1.4 μmin subtype II auricillic sensilla did not differ between the sexes in P. octo E. postvittana, and 4.1 ± 0.2 and 16.1 ± 0.8 μminP. octo,

16 G.H. Roh et al. Micron 106 (2018) 7–20 respectively) with cone-shaped distal ends and no nanoscale pores on function (Zacharuk, 1980; Steinbrecht, 1997). Various types of olfac- the cuticular surface (Fig. 6A, B, D, F, G, and I). These two types of tory sensilla, such as trichoid, basiconica, and auricillica, display a sensilla were regarded as non-olfactory based on the absence of na- number of nanoscale pores on the cuticular surface (Roh et al., 2016; noscale surface pores. In both species, one styloconic sensillum was Wee et al., 2016). Similarly, narrow longitudinal grooves are present present at the distal end of each flagellomere, except on the terminal along the central peg of another type of olfactory sensilla, coeloconic flagellomere, where several styloconic sensilla were located (Fig. 6D, I). sensilla (Pophof, 1997; Gómez et al., 2003; Roh et al., 2016; Wee et al., 2016). The ultrastructure of the pores and grooves indicate a lack of 5. Discussion endo- and exo-cuticular layers under these structures (Koh et al., 1995; Steinbrecht, 1997; Sun et al., 2011) through which airborne odor mo- 5.1. Gross morphology lecules are suggested to enter (Steinbrecht, 1997). Electrophysiological studies have indicated that the trichoid, basi- The gross external morphologies of the antennae of E. postvittana conic, auricillic, and coeloconic sensilla bearing nanoscale pores or and P. octo were similar to those in other tortricid (Lepidoptera) spe- longitudinal grooves are olfactory (Pophof, 1997; Baker et al., 2004; cies, such as Grapholita molesta (George and Nagy, 1984), Adoxophyes Ansebo et al., 2005; Pophof et al., 2005). Our observations indicated orana (Cuperus, 1985), Talponia batesi (Gómez and Carrasco, 2008), that nanoscale pores are uniformly distributed in sensilla trichoid, ba- and Cydia pomonella and C. succedana (Roh et al., 2016), and were also siconica, and auricillica. Similar examples have been found in other similar to those of other moth species (Sun et al., 2011; Diongue et al., tortricid moths, such as C. pomonella and C. succedana (Roh et al., 2013). The trend of longer flagellomeres in the mid-antennal region and 2016), as well as in P. xylostella (Wee et al., 2016). It is unclear if the slender flagellomeres towards the distal end of antennae found in E. size of pores is related to the molecular receptive range of the sensilla. postvittana and P. octo was similar to trends described in other tortricid An atomic force microscopy study of moth sensilla has indicated that moths, such as C. pomonella and C. succedana (Roh et al., 2016), as well the chemical composition differs between pores and other areas as other moth species, e.g., Plutella xylostella (Lepidoptera: Plutellidae) (Maitani et al., 2010). (Wee et al., 2016). Our results showed that female antennae have more flagellomeres 5.3. Trichodea than male antennae in E. postvittana and P. octo, similar to other moths, such as Zamagiria dixolophella (Lepidoptera: Pyralidae) (Gómez et al., The predominance of trichoid sensilla in the antennae of E. post- 2003), but unlike some other tortricid moths, such as C. pomonella, C. vittana and P. octo appears to be common in moths (Faucheux, 1990a; succedana (Roh et al., 2016), and T. batesi (Gómez and Carrasco, 2008), Faucheux, 1991; Jung et al., 1999; Gómez and Carrasco, 2008; Sun in which the number of flagellomeres appears to be larger in males or et al., 2011; Diongue et al., 2013 , Roh et al., 2016). Their detailed similar between the sexes. surface morphology, displaying regularly spaced and angled lateral ridges and numerous nanoscale pores between the ridges, is also a 5.2. Olfactory sensilla common feature of trichoid sensilla in moths (Roh et al., 2016; Wee et al., 2016). Our results indicate that the morphological types of olfactory sen- The olfactory sensory function of trichoid sensilla has been well silla and their distribution on the antennae are species-specific, and characterized by electrophysiological studies in several moth species, auricillic sensilla are more abundant than basiconic or coeloconic and these analyses have indicated the presence of functional ORNs in- sensilla in E. postvittana. However, the abundance of the three sensilla side the sensilla (Hansson et al., 1995; Cossé et al., 1998; Baker et al., types differed in P. octo. In other tortricid moths, like C. pomonella and 2004). Similarly, it has been suggested that the major sensory function C. succedana, auricillic sensilla are more abundant than basiconic or of the trichoid sensilla in the antennae of E. postvittana and P. octo is coeloconic sensilla (Roh et al., 2016). However, basiconic sensilla are olfactory. It has also been suggested that the ridges on the cuticular more abundant than auricillic or coeloconic sensilla in Homoeosoma surface enhance the contact between the trichoid sensilla by main- nebulella (Lepidoptera: Pyralidae) (Faucheux, 1991). taining air flow close to the sensillum surface (Maitani et al., 2010), and The six different morphological types of antennal sensilla (tri- the differences in chemical composition between nanoscale pores and chodea, basiconica, auricillica, coeloconica, chaetica, and styloconica) other areas of sensillum surface contribute to the selective entry of identified in E. postvittana and P. octo appear to be shared by a number specific groups of odor molecules through the pores (Steinbrecht, 1997; of tortricid moth species (Razowski and Wojtusiak, 2004; Roh et al., Maitani et al., 2010). 2016). By contrast, butterflies and other groups of insects display spe- Our results indicate that three distinct groups (subtypes) of trichoid cific morphological sets of antennal sensilla (Ploomi et al., 2003; sensilla are present in the antennae of both E. postvittana and P. octo. Sukontason et al., 2004; Xiangqun et al., 2014). For example, squami- The presence of different morphological types of antennal trichoid formia sensilla are characteristic in butterflies (Xiangqun et al., 2014), sensilla has been reported in several other tortricid moths, such as G. short basiconic sensilla in flies (Sukontason et al., 2004, 2007), placoid molesta (George and Nagy, 1984), C. pomonella, and C. succedana (Roh sensilla in wasps (Bleeker et al., 2004), and campaniformia sensilla in et al., 2016), as well as moths in other genera including Ostrinia nubilalis beetles (Ploomi et al., 2003). Although a specific biological structure is (Crambidae) (Cornford et al., 1973; Hallberg et al., 1994), Helicoverpa frequently related to a specific function, it is not well understood how assulta (Noctuidae) (Koh et al., 1995), and H. armigera (Noctuidae) each type of olfactory sensilla is correlated to a specific olfactory sen- (Diongue et al., 2013). By contrast, only one morphological type of sory function. A well-known relationship between the shape of sensilla trichoid sensilla was present in both sexes of Z. dixolophella (Gómez and their function is the pheromone detection function of male-specific et al., 2003). It has been suggested that the olfactory sensory function is trichoid sensilla in moths (Hansson et al., 1995; Cossé et al., 1998; different among distinct morphological types of trichoid sensilla, as Baker et al., 2004). Even in this case, however, it is unclear if the long demonstrated in some moths by electrophysiological studies of the re- hair-shape structure is an outcome of long-term evolutionary morpho- sponse pro files of individual ORNs (Hansson et al., 1995; Cossé et al., logical optimization, although it has been suggested that the densely 1998; Baker et al., 2004; Pophof et al., 2005; Lee et al., 2006) and aligned long hairs maximize the efficiency of capturing airborne pher- molecular studies of the distribution of odorant receptors in different omone molecules (Gómez et al., 2003; Diongue et al., 2013; Roh et al., sensilla (Maida et al., 2005). 2016). Therefore, the olfactory function of each morphological subtype of The presence of numerous nanoscale pores or longitudinal grooves trichoid sensilla is likely to be different from those of other subtypes in on the sensillum surface is a good morphological indicator of olfactory E. postvittana and P. octo. However, the exact olfactory sensory

17 G.H. Roh et al. Micron 106 (2018) 7–20 functions of different morphological types of trichoid sensilla are lar- (Roh et al., 2016). However, in other moth species, such as T. bisselliella gely unknown in these species. Likewise, the functional implication of (Tineidae), Agathiphaga vitiensis (Agathiphagidae), and H. nebulella the differences in the profiles of morphological types of trichoid sensilla (Pyralidae), basiconic sensilla are more abundant in females than in between species is yet to be elucidated. males (Faucheux, 1985, 1990a, 1991). Our results indicate that there is sexual dimorphism in the profile of morphological types of trichoid sensilla in E. postvittana and P. octo, 5.5. Auricillica demonstrating that a distinct group (subtype T-I) of trichoid sensilla is male-specific. However, another group (subtype T-II) of trichoid sen- Auricillic sensilla appear to share common morphological features silla was more abundant in female antennae in both species. In two across different moths. The narrow or wide rabbit ear-shaped auricillic other tortricid species, i.e., C. pomonella and C. succedana, subtype II sensilla identified in E. postvittana and P. octo in our study have been and III trichoid sensilla are more abundant in females than in males reported in other moth species, such as Cydia nigricana (Tortricidae) (Roh et al., 2016). Additionally, subtype III is more abundant in females (Wall, 1978), O. nubilalis (Hallberg et al., 1994), Scoliopteryx libatrix than in males in H. assulta (Koh et al., 1995). Male-specific morpholo- (Noctuidae) (Anderson et al., 2000), Z. dixolophella (Gómez et al., gical types of trichoid sensilla are common in moths, such as H. assulta 2003), C. cactorum (Pophof et al., 2005), C. pomonella, and C. succedana (Koh et al., 1995), Z. dixolophella (Gómez et al., 2003), H. armigera (Roh et al., 2016). (Diongue et al., 2013), C. pomonella, and C. succedana (Roh et al., The presence of auricillic sensilla in both males and females of E. 2016), and electrophysiological (Hansson et al., 1995; Cossé et al., postvittana and P. octo may indicate their sensory role in the detection of 1998; Baker et al., 2004), molecular (Steinbrecht et al., 1992; Rogers food and host plant-related volatiles, as suggested previously in other et al., 2001; Krieger et al., 2004; Forstner et al., 2009), and behavioral moths (Ebbinghaus et al., 1997; Anderson et al., 2000; Larsson et al., (McDonough et al., 1993; El-Sayed et al., 2011) studies have indicated 2002; Ansebo et al., 2005; Pophof et al., 2005). However, some aur- that their main function is the detection of conspecific female sex icillic sensilla in the male codling moth C. pomonella contain ORNs pheromone. specialized for the detection of conspecific female sex pheromone Therefore, these results suggested that the main function of the (Ansebo et al., 2005), although the detection of female sex pheromone male-specific trichoid sensilla detected in E. postvittana and P. octo in appears to be limited to male-specific long trichoid sensilla in other our study is the detection of female sex pheromones. moths, such as C. pomonella (Ebbinghaus et al., 1997), Helicoverpa zea The most abundant and longest of the male-specific trichoid sensilla (Noctuidae) (Cossé et al., 1998), Heliothis subflexa (Noctuidae), and may exhibit highly sensitive detection of active compounds in the ol- Heliothis virescens (Noctuidae) (Baker et al., 2004). The presence of factory system of each species. Although the male-specific trichoid different morphological groups of auricillic sensilla has been reported sensilla exhibit different physiological types among species using multi- in other moth species, such as Eriocrania semipurpurella (Eriocraniidae) component female sex pheromone, as demonstrated in other moths (Larsson et al., 2002). (Hansson et al., 1995; Cossé et al., 1998), these sensilla could not be The number of auricillic sensilla did not differ between the sexes in classified further based on their external morphology. Although the E. postvittana or P. octo. This result was similar to those obtained for sensory function of trichoid sensilla present in both male and female other tortricid moths, like T. batesi (Gómez and Carrasco, 2008). moths is largely unknown, they are likely to play a role in detecting host However, the number is dependent on sex in other species. For ex- and non-host plant volatiles, as demonstrated in a moth, Cactoblastis ample, auricillic sensilla are more abundant in females of C. pomonella cactorum (Lepidoptera: Pyralidae), for which female-specific short tri- and C. succedana (Roh et al., 2016), and in males of H. nebulella choid sensilla contain ORNs specialized for detecting plant volatiles (Faucheux, 1991) and Tineola bisselliella (Tineidae) (Faucheux, 1985). (Pophof et al., 2005). 5.6. Coeloconica 5.4. Basiconica The presence of coeloconic sensilla in antennae is also common The shape and surface morphology of basiconic sensilla identified in across moths. Peg-shaped coeloconic sensilla surrounded by a series of the antennae of E. postvittana and P. octo are similar to those of sensilla circular inwardly pointed microtrichia are typical in tortricid moth in other moths (Koh et al., 1995; Razowski and Wojtusiak, 2004; species, such as T. batesi (Gómez and Carrasco, 2008), C. pomonella, and Faucheux et al., 2006; Sun et al., 2011; Roh et al., 2016). The presence C. succedana (Roh et al., 2016), and are also found in Bombyx mori of numerous nanoscale pores on the cuticular surface of basiconic (Lepidoptera: Bombycidae) (Hunger and Steinbrecht, 1998), Z. dix- sensilla observed in E. postvittana and P. octo in our study and in other olophella (Gómez et al., 2003), Synempora andesae (Lepidoptera: moths (Sun et al., 2011; Roh et al., 2016) suggests their olfactory Neopseustidae) (Faucheux et al., 2006), and H. armigera (Diongue et al., function. However, the implication of the differences in the shape and 2013). distribution of pores for their sensory function is unclear. Although nanoscale pores, which are typical in all other morpho- Some electrophysiological studies have indicated that basiconic logical types of olfactory sensilla, are not present in coeloconic sensilla, sensilla of moths contain ORNs for the detection of plant volatile the olfactory function of coeloconic sensilla is relatively well under- compounds (Hallberg et al., 1994; Pophof et al., 2005). Similar to our stood based on electrophysiological studies (Hallberg et al., 1994; findings, the presence of different morphological types of basiconic Pophof, 1997; Pophof et al., 2005). sensilla on antennae has been reported in other tortricid moths, such as Ultrastructural observations of coeloconic sensilla have indicated G. molesta (George and Nagy, 1984), C. pomonella, and C. succedana that odor molecules enter the sensillum lymph cavity through the (Roh et al., 2016), as well as Z. dixolophella (Pyralidae) (Sun et al., longitudinal grooves along the sensory peg (Steinbrecht, 1997; Hunger 2011) and Tineola bisselliella (Tineidae) (Faucheux, 1985). The presence and Steinbrecht, 1998). The likely olfactory function and presence in of the same subtypes of basiconic sensilla in both male and female both sexes suggest that the coeloconic sensilla present in the antennae antennae also implies their sensory role in volatile detection for both of E. postvittana and P. octo perceive odorants from food and host plants. sexes of E. postvittana and P. octo, as suggested in other moth species for Subtype I coeloconic sensilla were more abundant in females than in which they have roles in detecting odors from host plants (Anderson males in E. postvittana, but were more abundant in males than in fe- et al., 1995; Pophof et al., 2005; Sun et al., 2011). males in P. coto. In the tortricid moths T. batesi (Gómez and Carrasco, Our results indicate that the distribution of basiconic sensilla is si- 2008) and H. nebulella (Faucheux 1991), there are no differences in the milar in males and females of both species. Basiconic sensilla are more number of coeloconic sensilla between the sexes. However, there are abundant in males than in females in both C. pomonella and C. succedana more coeloconic sensilla in females than in males in other tortricid

18 G.H. Roh et al. Micron 106 (2018) 7–20 species, such as C. pomonella and C. succedana (Roh et al., 2016), and in Bleeker, M.A.K., Smid, H.M., Aelst, A.C., van Loon, J.J.A., Vet, L.E.M., 2004. Antennal Z. dixolophella (Gómez et al., 2003). The coeloconic sensilla were more sensilla of two parasitoid wasps: a comparative scanning electron microscopy study. – fi Micros. Res. Tech. 63, 266 273. abundant in P. octo than in E. postvittana. A similar interspeci c dif- Brockerhoff, E.G., Suckling, D.M., Ecroyd, C.E., Wagstaff, S.J., Raabe, M.C., Dowell, R.V., ference in the number of coeloconic sensilla within the same family has Wearing, C.H., 2011. Worldwide host plants of the highly polyphagous, invasive also been found in Agathiphagidae, i.e., Agathiphaga vitiensis bears more Epiphyas postvittana (Lepidoptera: Tortricidae). J. Econ. Entomol. 104, 1514–1524. Broeckling, C.D., Salom, S.M., 2003. Antennal morphology of two specialist predators of coeloconic sensilla than A. queenslandensis (Faucheux 1990a). hemlock woolly adelgid, Adelges tsugae Annand (Homoptera: Adelgidae). Ann. The sensory role of different types of coeloconic sensilla and the Entomol. Soc. Am. 96, 153–160. implications of the differences in their distribution between two species Corcoran, J.A., Jordan, M.D., Thrimawithana, A.H., Crowhurst, R.N., Newcomb, R.D., is unclear. 2015. The peripheral olfactory repertoire of the lightbrown apple moth, Epiphyas postvittana. PLoS One 10, e0128596. Cornford, M.E., Rowley, W.A., Klun, J.A., 1973. Scanning electron microscopy of antennal 5.7. Chaetica and styloconica sensilla of the european corn borer, Ostrinia nubilalis. Ann. Entomol. Soc. Am. 66, 1079–1088. fi Cossé, A.A., Todd, J.L., Baker, T.C., 1998. Neurons discovered in male Helicoverpa zea Chaetic and styloconic sensilla identi ed in E. postvittana and P. octo antennae that correlate with pheromone-mediated attraction and interspecific an- in our study are common in moths (Roh et al., 2016; Wee et al., 2016). tagonism. J. Comp. Physiol. A 182, 585–594. Similar to other moths, nanoscale pores are not present on the cuticular Cuperus, P.L., 1985. Inventory of pores in antennal sensilla of Yponomeuta spp. (Lepidoptera: Yponomeutidae) and Adoxophyes orana F. V. R. (Lepidoptera: Please surface of chaetic and styloconic sensilla in these species; therefore, change such as "Yponomeutidae) and Tortricidae). Int. J. Insect Morphol. Embryol. they do not appear to have olfactory function, as suggested in other 14, 347–359. moths, such as Noctua pronuba (Noctuidae), H. nebulella (Pyralidae), Danthanarayana, W., 1975. The bionomics, distribution and host rang of the light brown apple moth, Epiphyas postvittana (Walk.) (Tortricidae). Aus. J. Zool. 23, 419–437. and Dyseriocrania subpurpurella (Eriocraniidae) (Faucheux, 1990b, Diongue, A., Yang, J.T., Lai, P.Y., 2013. Biomorphometric characteristics of different 1991, 2010). types of sensilla detected on the antenna of Helicoverpa armigera by scanning electron Some chaetic sensilla present in antennae or other body parts dis- microscopy. J. Asia Pac. Entomol. 16, 23–28. Ebbinghaus, D., Lösel, P.M., Lindemann, M., Scherkenbeck, J., Zebitz, C.P.W., 1997. play one or a few pores at the apex (Zacharuk, 1985), suggesting their Detection of major and minor sex pheromone components by the male codling moth involvement in contact chemoreception (Zacharuk, 1985; Jørgensen Cydia pomonella (Lepidoptera: Tortricidae). J. Insect Physiol. 44, 49–58. et al., 2007) as well as a mechanosensory function (Zhang et al., 2001). El-Sayed, A.M., Mitchell, V.J., Manning, L.M., Suckling, D.M., 2011. New sex pheromone Some aporous styloconic sensilla have been identified as thermos-hy- blend for the light brown apple moth, Epiphyas postvittana. J. Chem. Ecol. 37, 640–646. groreceptors (Altner and Loftus, 1985). Faucheux, M.J., Kristensen, N.P., Yen, S.H., 2006. The antennae of neopseustid moths: morphology and phylogenetic implications, with special reference to the sensilla – 6. Conclusion (Insecta, Lepidoptera, Neopseustidae). Zool. Anz. 245, 131 142. Faucheux, M.J., 1985. Morphology and distribution of antennal sensilla in the female and male clothes moth, Tineola bissellella Humm. (Lepidoptera, Tineidae). Can. J. Zool. In conclusion, our results demonstrated the presence of four mor- 63, 355–362. phological types of olfactory sensilla (s. trichodea, s. basiconica, s. Faucheux, M.J., 1990a. Antennal sensilla in adult Agathiphaga vitiensis Dumbl. and A. queenslandensis Dumbl. (Lepidoptera: Agathiphagidae). Int. J. Insect Morphol. auricillica, and s. coeloconica) and two other types (s. chaetica and s. Embryol. 19, 257–268. styloconica) in the antennae of E. postvittana and P. octo. The former Faucheux, M.J., 1990b. External structure of sensilla on the male and female flagellum of four types could be further classified into subtypes based on their de- Noctua pronuba L. (Lepidoptera: Noctuidae). Ann. Soc.delet "An Soc" Ent Fr (ns) 26, 173–184. tailed shapes and surface morphology. Faucheux, M.J., 1991. Morphology and distribution of sensilla on the cephalic appen- These results clearly indicate that the morphological types of ol- dages, tarsi and ovipositor of the Eurasian sunflower moth, Homoeosoma nebulella factory sensilla and their distribution in antennae are species-specific Den. & Schiff. (Lepidoptera: Pyralidae). Int. J. Insect Morphol. Embryol. 20, 291–307. fi Faucheux, M.J., 2010. Antennal sensilla in the female of Dyseriocrania subpurpurella and sex-speci c. Our analyses of the morphology and distribution of (Haworth, 1828) (Lepidoptera Eriocraniidae). Replacement of aporous sensilla antennal sensilla will provide useful information for studies of the chaetica by uniporous sensilla chaetica. Bul v het Kon Belg Inst v Nat Ent 80, 51–58. physiological and behavioral function of each type of sensilla, which Forstner, M., Breer, H., Krieger, J., 2009. A receptor and binding protein interplay in the will improve our understanding of the chemical communication system detection of a distinct pheromone component in the silkmoth Antheraea polyphemus. Int. J. Biol. Sci. 5, 745–757. in these species. Frasnelli, E., Anfora, G., Trona, F., Tessarolo, F., Vallortigara, G., 2010. Morphofunctional asymmetry of the olfactory receptors of the honeybee (Apis mellifera). Behav. Brain – Acknowledgements Res. 209, 221 225. Gómez, R.V.C., Carrasco, J.V., 2008. Morphological characteristics of antennal sensilla in Talponia batesi (Lepidoptera: Tortricidae). Ann. Entomol. Soc. Am. 101, 181–188. We would like to thank Anne Barrington, a research associate at Gómez, V.R.C., Nieto, G., Valdes, J., Castrejón, F., Rojas, J.C., 2003. The antennal sensilla Plant and Food Research for providing leaf-roller moth’s pupae and of Zamagiria dixolophella Dyar (Lepidoptera: Pyralidae). Ann. Entomol. Soc. Am. 96, 672–678. Mrs. Ji-Ae Kim at Korea Research Institute of Bioscience and Gao, Y., Luo, L.Z., Hammond, A., 2007. Antennal morphology, structure and sensilla Biotechnology for her kind help in taking SEM images. This study was distribution in Microplitis pallidipes (Hymenoptera: Braconidae). Micron 38, 684–693. partially supported by Rural Development Administration, Republic of George, J.A., Nagy, E.A., 1984. Morphology, distribution, and ultrastructureal differences of sensilla trichodea and basiconica on the antennae of the oriental fruit moth, Korea (grant no. PJ01175602). Grapholitha molesta (Busck) (Lepidoptera: Totricidae). Int. J. Insect Morphol. 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