Lepidoptera: Noctuidae

Journal of Insect Science

RESEARCH

Insight Into the Ultrastructure of Antennal Sensilla of Mythimna separata (Lepidoptera: Noctuidae) Xiang-Qian Chang,1,2 Shu Zhang,2 Liang Lv,2 and Man-Qun Wang1,3 1

College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China Hubei Province Key Laboratory for Crop Diseases, Insect Pests and Weeds Control, Institute of Plant Protection & Soil Science, Hubei Academy of Agricultural Sciences, Wuhan 430068, People’s Republic of China 3 Corresponding author, e-mail: [email protected] 2

Subject Editor: Roland Muehlethaler J. Insect Sci. (2015) 15(1): 124; DOI: 10.1093/jisesa/iev103

ABSTRACT. The oriental armyworm, Mythimna separata (Walk), is one of the most serious pests of cereals in Asia and Australasia. The structure and distribution of the antennal sensilla of M. separata were studied by scanning electron microscopy and transmission electron microscopy. The results showed that antennae of both female and male M. separata are filiform in shape. Three groups and seven morphological sensillum types were recorded in both sexes, including uniporous sensilla (sensilla chaetica), multiporous sensilla (sensilla trichodea, basiconica, coeloconica, and styloconica), and aporous sensilla (sensilla squamiformia and Bo¨hm bristles). S. trichodea, which were the most abundant sensilla, was made of three subtypes (ST I, ST II, and ST III) according to external features and two subtypes of s. basiconica (SB I and SB II) and s. coeloconica (SCo I and SCo II) were identified, respectively. Sexual dimorphisms in sensilla of M. separata were mainly perceived as the variations in the numbers of several sensilla subtypes. Also, the possible functions of the antennal sensilla were discussed. These results contribute to our understanding of the function of antennae in the behavior of M. separata. Key Words: oriental armyworm, antenna sensilla, SEM, TEM

The oriental armyworm, Mythimna separata (Walk), is one of the most serious pests of cereals in Asia and Australasia (Burgess 1987; Chen et al. 1989, 1995) and attacks more than 33 plant species in eight families, resulting in heavy crop losses (Sharma and Davies 1983). Although the sex pheromone of M. separata (Z11-16: Ald) has been identified (Zhu et al. 1987, Kou et al. 1992) and monitoring methods have been developed using it as a lure (Jung et al. 2013), there is no information on the physiology of the olfaction sensory apparatus on the antennae of this species. Insect antennae play an important role in the behavior of insects, such as in finding habitats and mates (Chapman 1998). Also, lots of sensory organs, or sensilla, occur on the antennae of insects, in the form of hairs, pegs, pits or cones which function as chemoreceptors (gustatory and olfactory), mechanoreceptors, thermoreceptors, hygroreceptors, and CO2 receptors (Keil 1999, Stange and Stowe 1999). Morphology and structure observations of insect sensillum are the basement of the function study, and by now, antennal sensilla have previously been described for many insect species using scanning electron microscopy (SEM) or/ and transmission electron microscopy (TEM) (Onagbola et al. 2008, Rebora et al. 2008, Drilling and Klass 2010, Sun et al. 2011, Galvania et al. 2012, Zhou et al. 2015). According to the morphology, the sensilla were termed as trichoid, chaetica, coeloconica, basiconca, Bo¨hm bristles, etc. (Keil 1999). Sensilla trichoid, basiconic, and coeloconica were thought sensitive to many kinds of chemical stimulus (Kaissling 1986, Isidoro et al. 1998, Onagbola and Fadamiro 2008). Styloconica and one type of coeloconica have been suggested involving in the perception of humidity, temperature, heat, and CO2 (Altner et al. 1983) and might play a role in preventing desiccation (Kristoffersen et al. 2006). Bo¨hm’s bristles have been thought sensing the position and movements of the antennae (Merivee et al. 2002). To improve our understanding of their function and of the ways that semiochemicals may mediate mate and host location in M. separata, the antennal sensory structures of M. separata were studied by SEM and TEM.

Materials and Methods M. separata larvae were obtained from fields in Yicheng (111 570 E; 31 260 N), People’s Republic of China, in May 2013 and reared on wheat (Huamai 2152) shoots at 24 6 1 C under a photoperiod of 12:12 (L:D) h. After emergence, female and male adults were provided with a 10% sucrose solution, and 2–3-d-old adults were used for the observations. Before SEM and TEM examination, the antennae were cut from the head and cleaned twice in an ultrasonic bath (250 W) for 5 s. SEM and TEM observations were made following the methods of Sun et al. (2011). Briefly, for SEM, the antennae were dehydrated through a graded ethanol series of 30%, 45%, 60%, 75%, 90%, and 95% (for 15 min each) and then fully dehydrated twice in 100% ethanol solution (for 15 min each time). Following drying to the critical point, the antennae preparations were mounted on a holder using double-sided adhesive tape and sputter coated with gold/palladium (40:60). Fifteen antennae from separate males and females were examined in total, using a Hitachi jsm-6390l (Hitachi, Tokyo, Japan) SEM at 20 kV. For TEM, the antennae were fixed in 2% glutaraldehyde and 1% sucrose in 0.1 M cacodylate buffer for 3 h. The samples were washed in 0.1 M cacodylate buffer and then post-fixed in 1% osmium tetraoxide in 0.1 M cacodylate buffer for 2 h, all at pH 7.0 and 25 C. The antennae were embedded in epon after dehydration in a graded series of ethanol, 50%, 70%, 80%, and 95% (for 30 min each), and twice at 100% (for 30 min each time). Epon was polymerized at 60 C for 48 h. Serial sections of 80 nm were cut on a Leica Super Nova ultramicrotome with a diamond knife and collected on formvar-coated nickel grids. Sections were contrasted with uranylacetate and stained with 1% toluidine blue. The sample grids were observed using a Hitachi H-7500 TEM (Hitachi, Tokyo, Japan). Terminology and Data Processing. The morphological terms used here follow those defined in the literature (Schneider 1964, Altner 1977, Zacharuk 1985). The antennal length, diameter, and sensillum counts for each segment, and the length and diameter of the sensilla were directly

C The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America. V

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measured from the printed SEM and TEM images. Images were enhanced using Adobe Photoshop 7.0. The number and size differences between females and males were analyzed using Student’s t-tests.

Results General Description of Antennae of M. separata. Antennae of female and male moths are both thread-like and consist of a scape, a pedicel, and a flagellum. The dorsal surface of each segment is covered by two rows of squamae. The flagellum length of females (10.08 6 0.68 mm) and males (9.96 6 0.76 mm) were similar (t ¼ 0.59, P ¼ 0.56), while the number of antennal segments of female flagella (76.8 6 3.0) were greater than that of the male (73.8 6 3.8) (t ¼ 3.17, P < 0.05). Antennal Sensillum Types and Distribution. On the ridge-shaped surface of the antennae (Fig. 2A1), seven different types of sensilla were observed on both male and female antennae: sensilla chaetica, sensilla trichodea (s. trichodea), sensilla basiconica (s. basiconica), sensilla coeloconica (s. coeloconica), sensilla styloconica (s. styloconica), sensilla squamiformia (s. squamiformia), and Bo¨hm bristles. Based on the presence of pores on the sensillum wall, they could be divided into three groups: uniporous sensilla (s. chaetica), multiporous sensilla (s. trichodea, s. basiconica, s. coeloconica, and s. styloconica), and aporous sensilla (s. squamiformia and Bo¨hm bristles). S. chaetica/SC. S. chaetica arose from a doughnut-shaped base, grooved longitudinally when observed at high magnification (Fig. 1A1) and were significantly longer and wider than the other sensilla (Table 2). The TEM micrographs of s. chaetica showed a thick wall and no pores (Fig. 1B), except with a terminal pore (Fig. 1C). Both of female and male antennae had six s. chaetica surrounding each segment except that 12 s. Chaetica were found on the last segment of the antennae (Table 1), and the length of s. chaetica distributed on the

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latero-ventral was longer than that of those on the medio-dorsal or on the medio-ventral (Fig. 1A2; Table 2). S. trichodea/ST. S. trichodea-like hairs filled the surface socket opening and obvious ring-like striations occurred from the middle part to the apex, becoming smooth at the base (Fig. 2A1). The TEM micrographs of their cross-sections showed they have thick cuticle walls and a few pores (Fig. 2B1 and B2). Three different types were identified based on their external features. S. trichodea I occurred on the lateral regions of the proximal and median segments and were “S” curved, usually stood perpendicular to the antennae, and had grooves on the surface (Table 1). In male antennae, s. trichodea I clustered in three or four rows (Fig. 2A2 and A3) and decreased gradually in length from the lateral edge toward the center of the segment within each row. The number of s. trichodea I decreased after about 40 segments. However, in female antennae, s. trichodea were dispersed (Fig. 2A4) and only occurred on the first 40 segments of the antennae (Table 1). S. trichodea II, which were shorter than s. trichodea I (Table 2), curved from the base in a “C-shape” with grooves on the surface, and were not arranged in rows (Fig. 2A2; Table 1). S. trichodea III had grooves on the surface, were either slightly curved or not, and lay almost parallel to the antennal flagellum, usually appearing in the ventral region of each segment (Figs. 2A5, 3A2, and 4A2; Table 1). The total number of s. trichodea I of males was significantly higher than that of females (t ¼ 72.85, P < 0.05), while the numbers of s. trichodea II and III of females were significantly higher than those of males (t ¼ 21.85, P < 0.05; t ¼ 35.30, P < 0.05) (Table 1). The length of s. trichodea I was similar between females and males, while the width of females was larger than that of males. The length of s. trichodea II of males was longer than that of females, while those in females were wider than in males. S. trichodea III were longer in

Fig. 1. SEM micrographs of three types of s. chaetica (A1, A2). A1 shows the groove on s. chaetica. A2 shows two combined segments of the distal antennae. TEM micrographs of s. chaetica (B), showing its thick and nonporous wall. CW, cuticular wall; SC, s. chaetica.

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CHANG ET AL.: ANTENNAL SENSILLA OF MYTHIMNA SEPARATA

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Fig. 2. SEM micrographs of s. trichodea in male (A1, A2), showing its distribution characters and shape of three types (A1, A2, A3, A4, and A5). (B1) The SEM micrographs of the groove on s. trichodea. TEM micrographs of s. trichodea (B2, B3), showing its thick wall and less pores and dendrites in it. D, dendrites; CW, cuticle wall; P, pores. ST I, s. trichodea type I; ST II, s. trichodea type II; ST III, s. trichodea type III. (C) TEM micrographs of the ridge shape of surface. females than in males, and they were wider in males than in females (Table 2). S. basiconica/SB. The bases of s. basiconica were flat, arose from round pits, and did not totally fill the insertion socket, which had faint herringbone striations (Fig. 3A1 and A2). TEM photomicrographs of these sensilla showed thin cuticle walls and conspicuous pores and dentrites (Fig. 3B). S. basiconica were randomly located on the lateral and

ventral surfaces of the segment (Table 1). Based on the morphology, there are two types of s. basiconica. S. basiconica I arose from the socket and were slightly curved or not (Fig. 3A1). The number of s. basiconica I of males was significantly higher than that of females (t ¼ 28.61, P < 0.05) (Table 1). S. basiconica II had bases vertical to the flagellum and were sharply curved (Fig. 3A2). The number of s. basiconica II of females

244.7 6 9.9 233.0 6 6.1 10.3 6 1.5 11.7 6 1.5 — — — — — — — — — — — — — — — — — — — — — — 12.0 6 0.0 12.0 6 0.0 12.0 6 0.0 12.0 6 0.0 12.0 6 0.0 12.0 6 0.0 12.0 6 0.0 12.0 6 0.0 12.0 6 0.0 12.0 6 0.0 10.0 6 0.0 10.0 6 0.0 6.0 6 0.0 6.0 6 0.0 2.0 6 0.0 2.0 6 0.0 — — — — — — 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 1.0 6 0.0 0.0 6 0.0 0.0 6 0.0 — — — — — — 0.0 6 0.0 0.0 6 0.0 0.0 6 0.0 0.0 6 0.0 1.0 6 0.0 1.3 6 0.8 0.0 6 0.0 1.6 6 0.9 0.0 6 0.0 0.0 6 0.0 2.0 6 0.0 0.0 6 0.0 0.0 6 0.0 2.0 6 0.0 0.0 6 0.0 0.0 6 0.0 46.1 6 1.8 66.4 6 3.4 — — — — 10.0 6 4.7 12.0 6 4.5 8.0 6 2.4 16.8 6 7.8 11.7 6 6.0 15.2 6 2.3 15.3 6 7.0 15.5 6 4.5 14.3 6 0.6 16.4 6 5.4 11.0 6 5.0 14.5 6 2.5 12.5 6 3.3 10.3 6 2.5 2.3 6 0.6 1.0 6 0.0 769.0 6 29.6 1,023.7 6 52.8 — — — — 1.8 6 0.8 0.8 6 0.4 2.4 6 0.9 1.3 6 0.8 2.2 6 1.0 3.6 6 1.8 1.0 6 0.0 3.0 6 1.3 1.7 6 0.6 1.6 6 0.9 6.3 6 3.2 2.8 6 1.7 3.3 6 1.5 2.3 6 0.6 0.0 6 0.0 1.0 6 0.0 183.0 6 7.0 154.7 6 8.0 Flagellum

Pedicel

s. trichodea II s. trichodea I

— — — — — — — — — — — — — — — — — — — — 6.0 6 0.0 17.6 6 1.5 79.8 6 11.3 16.0 6 2.4 3.0 6 0.7 6.0 6 0.0 64.4 6 19.7 52.2 6 15.7 10.0 6 5.8 5.0 6 2.2 6.0 6 0.0 21.0 6 1.4 84.8 6 6.3 14.2 6 2.0 4.2 6 1.3 6.0 6 0.0 71.3 6 13.5 51.2 6 23.7 12.5 6 4.1 11.2 6 4.2 6.0 6 0.0 17.3 6 9.1 59.2 6 14.9 20.8 6 11.0 4.3 6 1.6 6.0 6 0.0 61.2 6 19.0 34.4 6 21.6 11.6 6 2.4 9.2 6 3.3 6.0 6 0.0 6.0 6 2.0 57.3 6 16.8 17.3 6 12.1 8.0 6 5.3 6.0 6 0.0 44.2 6 10.9 46.7 6 18.6 8.0 6 3.3 5.3 6 2.5 6.0 6 0.0 0.0 6 0.0 33.3 6 3.1 12.0 6 3.6 7.3 6 5.0 6.0 6 0.0 31.8 6 11.6 43.6 6 9.2 14.4 6 8.8 7.2 6 0.8 6.0 6 0.0 0.0 6 0.0 39.0 6 18.4 12.7 6 4.2 3.0 6 1.7 6.0 6 0.0 16.8 6 3.9 43.8 6 12.4 9.5 6 6.0 9.3 6 4.7 6.0 6 0.0 0.0 6 0.0 34.5 6 17.5 12.5 6 1.9 7.5 6 2.5 6.0 6 0.0 14.0 6 6.1 20.0 6 2.0 7.3 6 4.6 8.7 6 2.3 12.0 6 0.0 0.0 6 0.0 10.7 6 2.5 4.0 6 2.0 1.7 6 0.6 12.0 6 0.0 9.7 6 3.2 11.0 6 2.6 1.3 6 0.6 1.3 6 0.6 — 796.5 6 30.7 3,788.6 6 145.9 1,104.8 6 42.6 372.3 6 14.3 — 3,190.0 6 164.7 2,893.6 6 149.4 714.8 6 36.9 551.2 6 28.5 $ # $ # 1–10 $ # 11–20 $ # 21–30 $ # 31–40 $ # 41–50 $ # 51–60 $ # 61–70 $ # Apical $ # Estimated total per $ antennal flagellum #

Sex s. chaetica Parts of antennal segment

Scape

s. trichodea III s. basiconica I s. basiconica II s. coeloconica I s. coeloconica II s. styloconica s. squamiformia Bo¨hm bristles


Table 1. Average number of sensillar on different parts of M. separata antennal segments

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was significantly higher than that of males (t ¼ 13.56, P < 0.05) (Table 1). S. coeloconica/SCo. S. coeloconica had a double wall and a centrally placed peg surrounded by 15–17 inward-facing tapering spines (Fig. 4A1). S. coeloconica were usually present on the ventral surface of both female and male antennae (Table 1). Based on the appearance of the spines, s. coeloconica can be divided into two types. S. coeloconica I had spines, and there was a peg in the center surrounded by 15–17 inward facing spines (Figs. 4B and 5A1). The number of s. coeloconica I of males was significantly higher than that of females (t ¼ 21.43, P < 0.05) (Table 1). The average length and width of s. coeloconica I were similar between females and males (Table 2). S. coeloconica II were without spines, with the random occurrence of a peg in the center of the pit, and occurred on the ventral surface of some segments (Figs. 1A2 and 4A2). The average length and width were 5.85 6 0.74 and 1.23 6 0.26 lm for females, respectively, and 5.92 6 0.38 and 1.19 6 0.34 lm for males, respectively. The total number of s. coeloconica II of females was significantly fewer than that of males (t ¼ 26.86, P < 0.05). S. styloconica/SSt. The bases of s. styloconica were sculptured, smooth on top, with two to three cavity-peg complexes at their tips (Fig. 5A). Each segment had one s. styloconica located in the middle of the leading edge of the ventral region of each segment (Table 1). The number of s. styloconica of females and males was similar, but they were significantly longer and thinner in females (Table 2). S. squamiformia/SSq. S. squamiformia had nonporous walls like squamae but were narrower than squamae (Fig. 6B). Each segment of the female and male antennae had about 2–12 s. squamiformia (Table 1). The length and width of s. squamiformia on females and males did not differ significantly (Table 2). Because all s. squamiformia were covered by squamae, their number could not be clearly counted. Bo¨hm bristles/BB. Bo¨hm bristles only existed on the scape and the pedicel of the antennae in clusters (Fig. 7A1; Table 1). Bo¨hm bristles were spine-like structures with smooth cuticles standing almost perpendicular to the antennal surface (Fig. 7A2). Cross section of the sensilla showed no pore on the wall and the little spine surround the sensillum were seta (Fig. 7B1 and B2). Both females and males had similar numbers of the Bo¨hm bristles (Table 1).

Discussion Seven sensillum types identified in M. separata were similar to those reported in most Lepidoptera, including multiporous s. trichodea, s. basiconica, s. coeloconica, and s. styloconica; uniporous s. chaetica; and aporous s. squamiformia and Bo¨hm bristles (Jefferson et al. 1970, Flower and Helson 1974, Liu and Liu 1984, Lavoie-Dornik and McNeil 1987, Castrejoń-Go´mez et al. 1999, Zohry 2008, Diongue et al. 2013). S. chaetica were found distributed around each antennal segment of M. separata except the last segment which had a higher number of s. chaetica, and the similar distribution of this sensilla have been reported in Pseudaletia unipuncta (Flower and Helson 1974), Helicoverpa armigera (Diongue et al. 2013), and Spodoptera littoralis (Seada 2015). S. chaetica were in general recognized as mechnoreceptors (Keil and Steinbrecht 1984), while as uniporous (terminal pore) sensilla, it also have been suggested to have contact/chemoreceptor functions (Seada 2015). And the characteristic that s. chaetica were significantly higher and wider than other sensilla suggests that this sensilla might have a protection role for other sensilla except as mechnoreceptors or contact/ chemoreceptors. Pores, or even continuous pores, were found on the walls of s. trichodea and basiconica, on the central peg of s. coeloconica, and on the apices of s. styloconica. Pores on the walls of sensilla indicate that they play a role in sensing chemical stimuli and in olfactory functions (Keil and Steinbrecht 1984). S. trichodea are the most abundant sensilla on the antennae of M. separata, and studies of many other lepidopteran

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Table 2. Mean 6 SD sizes of the main antennal sensilla of M. separata antennal segments Length (lm) $ Medio-dorsal s. chaetica 83.41 6 8.68 Latero-ventral s. chaetica 98.46 6 12.23 Medio-ventral s. chaetica 53.73 6 15.18 s. trichodea I 44.69 6 9.77 s. trichodea II 30.97 6 4.63 s. trichodea III 31.69 6 6.05 s. basiconica I 17.42 6 2.57 s. basiconica II 7.38 6 2.38 s. coeloconica Ia 8.28 6 0.80 s. coeloconica II 5.85 6 0.74 s. styloconica 20.69 6 3.94 s. squamiformia 34.03 6 2.17 Bo¨hm bristles I 14.81 6 2.56 Bo¨hm bristles II 3.90 6 0.72 a The mean length of pit diameter.

Width (lm) #

86.54 6 8.47 106.47 6 16.07 57.08 6 16.91 44.11 6 7.49 34.21 6 7.34 28.54 6 4.79 16.29 6 3.50 8.95 6 2.39 8.55 6 0.66 5.32 6 0.69 17.31 6 2.59 34.24 6 1.64 14.63 6 3.11 4.20 6 0.85

$ t ¼ 1.04, P ¼ 0.3083 t ¼ 1.86, P ¼ 0.0700 t ¼ 0.79, P ¼ 0.4303 t ¼ 2.56, P ¼ 0.7988 t ¼ 2.04, P ¼ 0.0459 t ¼ 2.03, P ¼ 0.0465 t ¼ 1.08, P ¼ 0.2899 t ¼ 1.61, P ¼ 0.1216 t ¼ 1.43, P ¼ 0.1591 t ¼ 1.54, P ¼ 0.1423 t ¼ 3.20, P ¼ 0.0027 t ¼ 0.23, P ¼ 0.8192 t ¼ 0.16, P ¼ 0.8751 t ¼ 0.78, P ¼ 0.4460

#

3.78 6 0.42 5.77 6 0.65 5.05 6 0.77 1.78 6 0.45 1.95 6 0.32 1.57 6 0.27 1.30 6 0.23 1.84 6 0.42

3.35 6 0.41 6.88 6 0.67 5.64 6 0.53 2.05 6 0.38 1.65 6 0.32 1.59 6 0.27 1.35 6 0.38 1.82 6 0.29

t ¼ 2.67, P ¼ 0.0122 t ¼ 5.56, P ¼ 0.0000 t ¼ 3.39, P ¼ 0.0013 t ¼ 2.53, P ¼ 0.0141 t ¼ 3.70, P ¼ 0.0005 t ¼ 0.31, P ¼ 0.7563 t ¼ 0.47, P ¼ 0.6460 t ¼ 0.12, P ¼ 0.9061

1.23 6 0.26 5.36 6 6 0.76 2.81 6 0.32 2.21 6 0.20 0.64 6 0.18

1.00 6 0.30 6.39 6 1.15 3.02 6 0.30 2.15 6 0.20 0.54 6 0.08

t ¼ 1.80, P ¼ 0.0901 t ¼ 3.34, P ¼ 0.0019 t ¼ 1.46, P ¼ 0.1650 t ¼ 0.66, P ¼ 0.5165 t ¼ 1.55, P ¼ 0.1432

Fig. 3. SEM micrographs two types of s. basiconica (A1, A2). TEM micrographs of s. basiconica (B), showing its thin wall and continuous pores and dendrites. D, dendrites; CW, cuticle wall; P, pores; SB I, s. basiconica type I; SB II, s. basiconica type II.

Fig. 4. SEM micrographs of s. coeloconica (A1, A2,). (B) The entire transverse section of s. coeloconica central peg. D, dendrites; CW, cuticle wall; P, pores; SCo I, s. coeloconica type I; SCo II, s. coeloconica type II.

Fig. 5. SEM (A) and TEM (B) micrographs of s. styloconica. CW, cuticle wall; P, pores; SSt, s. styloconica.

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Fig. 6. SEM micrographs of s. squamiformia and squamae on the dorsal regions of the antennae surface (A). TEM micrographs of squamae (B). SSq, s. squamiformia.

Fig. 7. SEM micrographs of Bo¨hm bristle (A1, A2), B1 and B2 showing the cross section of Bo¨hm bristle and seta, respectively, on the scape and pedicel of the antennae.

species have shown that these sensilla could be divided into more subtypes according to the number of sensory cells (Hallberg et al. 1994) or the presence (or absence) of pores on the sensillum wall (Onagbola et al. 2008), or depended on the distribution and length (Ren et al. 2014). In our study, it is difficult to pair SEM photos of three subtypes of s. trichodea with TEM photos, respectively, and s. trichodea were divided into three subtypes according the shape of the sensilla. In our study, sexual dimorphism of s. trichodea was found, the number of s. trichodea I were higher than that of females. Many studies on noctuid

moths have reported that only males have s. trichodea I (“long s. trichodea”) (Flower and Helson 1974), but s. trichodea I are actually seen in female M. separata (Fig. 2A4). Similar with other insect species, s. trichodea I, which called long s. trichodea in other papers, might associate with olfactory reception of sex pheromones (Kaissling 1979, Zacharuk 1985, Steinbrecht et al. 1992). S. basiconica are considered to be olfactory receptors to many kinds of chemical stimulus (Kaissling 1986, Isidoro et al. 1998, Onagbola and Fadamiro 2008), though s. basiconica were neglected in some

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Noctuidae moths (Flower and Helson 1974, Calatayud et al. 2006, Zheng et al. 2014). In M. separata antennae, two subtypes of this sensillum were identified. Continuous pores in the wall of this sensillum suggested they were able to detect various chemical compounds. S. coeloconica were typical double-wall multiporous sensilla with special topology, which commonly exist on the antennae of Lepidoptera with little variance (Zhang et al. 2001, Sun et al. 2011, Ndomo et al. 2014, Zheng et al. 2014, Seada 2015). Two subtypes of this sensillum were identified in M. separata antennae, and like the s. coeloconica in other insect species, they might function as chemo- or thermoreceptors (Roux et al. 2005, Gao et al. 2007). And the sexual differences in the numbers of s. basiconica and coeloconica might associate with sex-specific differences in behavior of females and males. S. styloconica identified in M. separata antennae were similar to those described in P. unipuncta (Lavoie-Dornik and McNeil 1987), H. armigera (Diongue et al. 2013), and S. littoralis (Seada 2015). Pores were observed on the tip indicated their chemoreceptor function, and in fact, Flower and Helson (1974) and Zohry (2008) called this sensillum as “taste rod.” Two aporous sensilla, s. squamiformia and Bo¨hm bristles, were identified in M. separata antennae, these sensilla devoid of wall pores suggested they should be as non-olfactory role. S. squamiformia are commonly present in lepidopteran insects (Sun et al. 2010, Ndomo et al. 2014, Zheng et al. 2014, Seada 2015), and they were thought might as mechanoreceptors or as wind velocity receptors (Dyer and Seabrook 1978). Like many other insects, Bo¨hm bristles only occur on the scape and pedicel in M. separata antennae, and they have been suggested that Bo¨hm bristles might be as membrane acceptors on the segment membranes between the scape and pedicel, or be as mechanoreceptors (Cuperus 1983), sensing the position and movements of the antennae (Merivee et al. 2002). In conclusion, this study identified and characterized the distribution of the different sensillum types of M. separata. This information will enable a better understanding of the behavior of this moth and future studies using electrophysiological recordings coupled with behavioral studies could enable the clarification of the processes involved in chemical communication between individual M. separata.

Acknowledgments This study was supported by the Public Welfare Project from Ministry of Agriculture (201403031) and Fundamental Research Funds for the Central Universities (2013PY046).

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