A SEM study of antennal sensilla in Maladera orientalis Motschulsky (Coleoptera: Scarabaeidae: Melolonthinae)

Micron 119 (2019) 17–23

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A SEM study of antennal sensilla in Maladera orientalis Motschulsky (Coleoptera: Scarabaeidae: Melolonthinae) ⁎

Kai-Min Shaoa,b, Yan Suna,b,c, Wen-Kai Wanga, , Li Chenb,

T

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a

School of Agriculture, Yangtze University, Jingzhou, Hubei 434025, China State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China c Zhengzhou Botanical Garden, Zhengzhou 450042, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Antenna Morphology Sensilla Scanning electron microscopy Scarab Beetle

The external morphology of antenna and fine structure of its sensilla of Maladera orientalis were studied using scanning electron microscopy. The antenna consists of scape, pedicel, funicle and a club composed of three lamellae. Funicle has five, sometimes, four segments. Böhm bristles, three subtypes of sensilla chaetica, one subtype of sensilla trichodea, one subtype of sensilla coeloconica, four subtypes of sensilla basiconica, and one subtype of sensilla placodea were described. No sexual differences were found in number, type and distribution of sensilla on all antennomeres. The most notable feature of sensilla on antennae of M. orientalis is the presence of long and narrow strip-like sensilla placodea on both sides of all club lamellae. These porous sensilla placodea are slightly depressed below the lamella surface and tend to lie in parallel with the lamella axe. They are similar to sensilla placodea found in hymenopteran species but very different from those round plate-like sensilla placodea occurring on the lamellae of scarab beetles.

1. Introduction Many scarab beetle species (Coleoptera: Scarabaeidae), especially plant-feeding chafers in the subfamilies Cetoniinae, Melolonthinae, Dynastinae, and Rutelinae, are economically important pests in agriculture, horticulture, and forestry. These scarab beetles have easily recognizable antennae, which are usually 10-segmented, with the last 3–7 segments forming a lamellate club (Ahrens and Vogler, 2008). The lamellae can open like a fan during mate, host plant, and oviposition site location in female and/or male beetles, or fold together to protect sensilla on their inner surfaces. Due to their olfactory function, the ultrastructure of sensilla on lamellae of scarab species in the tribes Cetoniini, Dynastini, Hopliini, Melolonthini, Rutelini, Coprini, and Geotrupini have received much attention (Zauli et al., 2016). Sensilla of scarab beetles in Melolonthinae, for instance, Antitrogus parvulus Britton (Allsopp, 1990), Dasylepida ishigakiensis Niijima et Kinoshita (Tanaka et al., 2006), Lepidiota mansueta Burmeister (Handique et al., 2017), Lepidiota negatoria Blackburn (Allsopp, 1990), Macrodactylus mexicanus (Burmeister), Macrodactylus nigripes (Bates) (Romero-López et al., 2017), Phyllophaga anxia (LeConte) (Ochieng et al., 2002), Phyllophaga obsoleta Blanchardand (Romero-López et al., 2004), Phyllophaga opaca Moser (Romero-López and Morón, 2013), Phyllophaga ravida Blanchard (Romero-López et al., 2010), and many species in the tribe Hopliini

⁎

(Romero-López et al., 2013), have been extensively studied and characterized. The genus Maladera includes almost 600 inconspicuous species with a trilamellate antennal club (Ahrens and Vogler, 2008). Maladera orientalis Motschulsky is a common and widespread polyphagous scarab beetle throughout most parts of China in the tribe Sericini of the subfamily Melolonthinae (Ahrens, 2005; Liu et al., 2015). World distribution of M. orientalis also includes Eastern Siberia and Japan (Ahrens, 2006). Overwintering adults emerge at dusk in April and May, attack a wide variety of crops and trees, and sometimes cause severe defoliation on tree leaves in early season. After mating, the females oviposit in the soil and the hatching larvae feed on plant litter and roots until they pupate in late season. Because of large population in peanut field, M. orientalis cause serious losses in peanut production in China (Gao et al., 2017). The aims of this work were to illustrate general morphology of the antennae, and to describe ultrastructure and distribution of the sensilla on the whole antenna by using scanning electron microscopy (SEM). The presence of a possible sexual dimorphism in sensilla responsible for pheromone and plant volatile reception was also analyzed to propose their putative function in chemical communication, as has been suggested in other species of Melolonthinae and related Rutelinae. The results of this study would be essential for future electrophysiological

Corresponding authors. E-mail addresses: [email protected] (W.-K. Wang), [email protected] (L. Chen).

https://doi.org/10.1016/j.micron.2019.01.004 Received 5 December 2018; Received in revised form 8 January 2019; Accepted 8 January 2019 0968-4328/ © 2019 Published by Elsevier Ltd.

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support and mobility to the terminal club. The last three segments (L1, L2 and L3) were modified into three plates (= lamellae) which could be expanded like a fan or folded together into a club (Fig. 2). Measurements of different antennal segments revealed no sexual difference in length of each segment (Table 1). Males (357.25 ± 16.28 μm) had slightly, but not significantly (t = 0.48, P = 0.64), longer funicle than females (348.00 ± 10.17 μm). Also, there was no sexual difference in antenna length as that of females measured 1098.62 ± 16.17 μm and that of males 1088.39 ± 21.56 μm (t = 0.57, P = 0.58). We also observed some individuals that had both antennae with 4 segments of funicle (Figs. 1C-D). The length of the 5-segment funicle relative to that of the 4-segment funicle was slightly longer, but the difference was not significant (data not shown).

and behavioral studies of the antennal sensory system involved in mate, host plant, and oviposition site location of M. orientalis. 2. Materials and methods Female and male adults of M. orientalis were collected manually from Beijing“s Olympic Forest Park while they were feeding in the evening in April. They were killed by freezing (−20 °C), and the antennae of 12 females and 12 males were cut off with a microknife and preserved in 70% ethanol separately. Specimens were cleaned in an ultrasonic cleaner for 2 min and rinsed in water, and then dehydrated once in a graded ethanol series of 75, 85, 95%, and thrice in absolute ethanol, in each case for 10 min. After this dehydration process, the proximal, middle, and distal lamellae forming each antennal club of half of specimens were separated under a stereomicroscope and grouped according to sex and lamellar side. All specimens were then mounted to double-sticky tapes on aluminum stubs, sputter-coated with gold (in an E-1045 Sputter Coater), and examined with a SU8010 SEM (Hitachi, Japan) at accelerating voltage 5 kV. Micrographs were taken of the antennae and sensilla, and the dimensions of the sensilla were measured. We followed terminology of Romero-Lopez et al. (2010) and Zauli et al. (2016) to classify the sensilla types. Abundance and the distribution of the antennal sensilla types were compared between females and males. Data on antennal segments and sensilla dimension for M. orientalis adult females and males were analyzed with Student’s t-tests (SPSS 21.0 statistic package), and all tests were considered significant at α = 0.05. All values reported were mean ± standard error.

3.2. Sensilla on the antenna Six types of morphologically distinct sensilla were identified on the antennae of female and male M. orientalis, including Böhm bristles (BB), three subtypes of sensilla chaetica (SCh), sensilla trichodea (STr), sensilla coeloconica (SCo), four subtypes of sensilla basiconica (SBa), and sensilla placodea (SP). 3.2.1. Böhm bristles Böhm bristles are thorn-like (Fig. 3A), smooth-walled, straight, and tapered towards the apical part with the base inserted in a wide socket (length 14.37 ± 1.85 μm, width at base 3.19 ± 0.15 μm). They project at about 45° from the antennal surface. They are present around the base of scape (Fig. 3A, Table 2).

3. Results

3.2.2. Sensilla chaetica Sensilla chaetica subtype 1 (SCh1) are spine-like (Figs. 3B, 3D), straight and tapered towards the apical part with the base inserted in a wide socket (length 107.42 ± 2.79 μm, width at base 6.44 ± 0.14 μm). Strip-like pipes are piled regularly on the whole surface of these sensilla (Fig. 3D). They project from the antennal surface vertically, which are arranged in groups on the lateral side of the scape in both sexes (Fig. 3B, Table 2). Sensilla chaetica subtype 2 (SCh2) are hair-like, straight and tapered towards the apex (Fig. 3B, 3C and 3E). The base is inserted in a narrow socket, and projects at about 60–90° from the antennal surface. The

3.1. General morphology of the antenna The antenna of both sexes of M. orientalis exhibited a typical lamellate structure comprising of 10 segments, arranged in four distinctive parts from base to apex, scape, pedicel, funicle and a club (Fig. 1). The basal segment, scape, which is attached to the head, was expanded at the apex. The pedicel was short and nearly cylindrical, loosely inserted inside the scape. The funicle comprised 5 segments as F1, F2 and F3 were cylindrical, and F4 and F5 became wide to give

Fig. 1. SEM micrographs of female and male antennae of M. orientalis. S: scape; P: pedicel; F: funicle; L1–L3: lamellae of the antennal club. (A) Female with 10segment antenna; (B) Male with 10-segment antenna; (C) Female with 9-segment antenna; (D) Male with 9-segment antenna. 18

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Fig. 2. SEM micrographs of the surface of the three lamellae in the antennae. (A–D) L1; (E–H) L2; (I–L) L3. ♀: Female; ♂: Male; a: proximal side; b: distal side.

4.70 ± 0.14 μm, width at base 2.12 ± 0.06 μm) and cone-shaped, showing a short peg sunken in a cuticular socket (Fig. 4C). The surface is smooth with sparse pores. Sensilla basiconica subtype 2 (SBa2) are long-rod shaped (length 19.45 ± 0.32 μm, width at base 3.37 ± 0.08 μm, Fig. 4D). Numerous pores are distributed over the surface in SBa2. Sensilla basiconica subtype 3 (SBa3) are short-cone shaped (length 9.36 ± 0.19 μm, width at base 2.56 ± 0.06 μm, Fig. 4E). The surface of SBa3 is characterized by an irregularly worn, rough microsculpture. Many pores are found on the proximal portion of the hair shaft. The distal portion of the hair splits into finger-like structures. The tip of the sensilla exhibits terminal opening. Sensilla basiconica subtype 4 (SBa4) are characterized by cuticular finger-like projections and tapered towards the apex (length 10.57 ± 0.15 μm, width at base 2.41 ± 0.04 μm, Fig. 4F). The surface of basal portion is smooth without pores. They are distributed principally on the distal side of the proximal and middle lamellae and both sides of the distal lamella in relatively low numbers (Table 2).

Table 1 Length of antennal segments of adult female and male M. orientalis (μm, n = 6). Antennal segment

Female

Male

P

Scape Pedicel Funicle 1 Funicle 2 Funicle 3 Funicle 4 Funicle 5 Lamella 1 Lamella 2 Lamella 3

488.61 ± 4.98 133.29 ± 2.75 100.14 ± 2.71 66.20 ± 4.09 65.43 ± 2.55 69.86 ± 2.81 44.07 ± 1.83 612.36 ± 8.94 597.27 ± 7.67 536.80 ± 5.64

469.65 ± 10.08 133.88 ± 4.61 98.45 ± 4.08 64.80 ± 1.42 69.96 ± 3.57 70.36 ± 7.68 53.65 ± 4.63 603.36 ± 21.34 598.96 ± 18.60 547.96 ± 17.36

0.13 0.77 0.74 0.76 0.33 0.95 0.09 0.70 0.94 0.55

length of SCh2 varies largely, ranging from 20.43 to 393.79 μm. The surface is worn-looking with irregular projections. These hairs project from the antennal surface at 60-90°. They are distributed on the apical portion of the scape, pedicel and funicle F3 and F4 (Figs. 3B-C, Table 2). Sensilla chaetica subtype 3 (SCh3) are spine-like (Fig. 3E), straight and tapered towards the apical part with the base inserted in a narrow socket (length 24.59 ± 4.36 μm, width at base 2.06 ± 0.23 μm). There are multiple branches on the surface of shaft. These bristles are present on the funicle F4-F5 (Figs. 3C, 3 F).

3.2.6. Sensilla placodea Sensilla placodea are densely distributed on both sides of all club lamellae and cover almost entire area of lamellae. These sensilla are slightly depressed below the lamellar surface and tend to be aligned in parallel with the longitudinal axis of the lamella. There are about 2500 SP on the lamellae (Table 2). They are long and narrow strip with high pore density (Fig. 5). The diameter of pores is about 20 nm. The density of pores on SP ranges 40.4–66.0 pores/μm2, and there is no difference in pore density between sexes (Table 3). SP vary in length from 47.73 to 324.70 nm.

3.2.3. Sensilla trichodea Sensilla trichodea have a long hair-like structure that occurs along the peripheral edges of all cub lamellae and sometimes on the outer surface the third lamella of both sexes (Figs. 2, 3C and 3 G, length 81.76 ± 3.35 μm, width at base 4.25 ± 0.26 μm). They appear broader at the basal region and taper apically. Their surface is smooth. Under high magnification, however, STr can be seen as having a grooved cuticular surface. These sensilla project from the antennal surface at 45-60°.

4. Discussion The antennae of M. orientalis show the typical lamellicorn shape of scarab beetles (Leal, 1998; Meinecke, 1975), composed of nine or ten antennomeres, including a basal scape, a pedicel, a funicle composed of four or five antennomeres and a club composed of three lamellae. Both sexes of M. orientalis have antennae of similar size, shape and structure. This is different from several melolonthid species of scarab beetles with distinct sexual dimorphism in the antennae (Handique et al., 2017; Mutis et al., 2014; Romero-López et al., 2004; Tanaka et al., 2006). The incidence of gynandromorphy, i.e., one antenna is apparently different from the other one from the same beetle, has been reported for D. ishigakiensis in very low frequency. In the present study, some individuals of M. orientalis appeared with 9-segment antennae. However, we have not observed any beetles having asymmetrical antennae that represent a gynandromorphy as previously mentioned in M. orientalis (Liu et al., 1997). The physiological significance of 9-segment antennae

3.2.4. Sensilla coeloconica Sensilla coeloconica are short (length 2.18 ± 0.69 μm, width at base 1.90 ± 0.03 μm) onion-like pegs (Figs. 4A-B), sunken in a wide circular socket. They are observed only on pedicel in low numbers in both sexes (Table 2). 3.2.5. Sensilla basiconica The lamellae bear 4 subtypes of sensilla basiconica, which are often intermingled together in the central areas of the inner surface of the proximal and middle lamellae and both surfaces of the distal lamella (Table 2). They are completely absent on peripheral edges of all lamellae. Sensilla basiconica subtype 1 (SBa1) are short (length 19

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Fig. 3. SEM micrographs of the sensilla types present in the antennae of M. orientalis. (A) Böhm bristles (BB) on scape; (B) Sensilla chaetica on scape and pedicel; (C) Sensilla chaetica on pedicel and funicle, and sensilla trichodea on lamellae; (D) Sensilla chaetica subtype 1 (SCh1); (E) Sensilla chaetica subtype 2 (SCh2); (F) Sensilla chaetica subtype 3 (SCh3); (G) Sensilla tricodea (STr).

Amorphochelus retusus Klug (Romero-López et al., 2013), D. ishigakiensis (Tanaka et al., 2006), H. elegans (Mutis et al., 2014), L. mansueta (Handique et al., 2017), Mac. mexicanus (Burmeister), Mac. nigripes (Bates) (Romero-López et al., 2017), Phylloph. anxia (Ochieng et al., 2002), and Phyllope. horticola (Ågren, 1985). Böhm bristles, sensilla chaetica and trichodea are considered to be related with mechanoreceptor functions (Schneider, 1964). They may have a role in the proprioception of the antenna position, movement and in regulation of body position by perception of mechanical stimuli, such as touch, air currents, sound, and gravity (Mutis et al., 2014). Sensilla coeloconica occur exclusively on pedicel in both sexes of M. orientalis. These peg-like sensilla are very similar to those described on antenna lamellae in other scarab species: Cotinis nitida Burmeister (Baker and Monroe, 2005), D. ishigakiensis (Tanaka et al., 2006), Or. rhinoceros (Renou et al., 1998), Os. eremita (Zauli et al., 2016). However, SCo have not been described on pedicel of scarab beetle previously. Similar to other scarab beetles, M. orientalis adults emerge from the ground in warm evenings in the late spring season. SCo on pedicel

awaits further investigation. Böhm bristles found only at the base of the scape in M. orientalis are probably homologous in all insects (Schneider, 1964). They are present at the base of the scape and the pedicel in a cetonid beetle, Osmoderma eremita (Scopoli) (Zauli et al., 2016). Three subtypes of sensilla chaetica are observed on scape, pedicel and funicle. These bristles are widespread sensilla occurring in scarab beetles, such as An. parvulus (Allsopp, 1990), Hylamorpha elegans (Burmeister) (Mutis et al., 2014), L. mansueta (Handique et al., 2017), L. negatoria (Allsopp, 1990), Os. eremita (Zauli et al., 2016), Oryctes rhinoceros (L.) (Renou et al., 1998), Phyllopertha horticola L. (Ågren, 1985), and Popillia japonica Newman (Kim and Leal, 2000). Sensilla trichodea occur on the edges of all lamellae in both sexes of M. orientalis. They have a hair-like structure that occur along the peripheral edges and on the outer surface of lamellae, and are much longer than sensilla chaetica as evidenced in P. obsoleta (Romero-López et al., 2004), P. opaca (Romero-López and Morón, 2013), and P. ravida (Romero-López et al., 2010). Sensilla trichodea have been observed on the lamellae of many other scarab beetles, 20

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chemical communication and perception of plant volatiles (Bengtsson et al., 2009, 2011; Renou et al., 1998; Stensmyr et al., 2001). Putative olfactory sensilla basiconica are present in low numbers on the inner surfaces of the proximal and middle lamellae and on both sides of the distal lamella in M. orientalis. Their contribution to olfactory sensitivity and specificity in antennae could be very limited. Sensilla placodea in scarab beetles have been reported to be involved in the chemical perception of pheromones (Kim and Leal, 2000; Larsson et al., 1999; Leal and Mochizuki, 1993; Nikonov and Leal, 2002; Nikonov et al., 2002; Wojtasek et al., 1998) and green leaf volatiles (Hansson et al., 1999; Larsson et al., 2001; Nikonov et al., 2001). In previous studies regarding scarab beetles, porous sensilla placodea, usually cup-like, are common on all lamellae (reviewed by Zauli et al., 2016). However, sensilla placodea in very different look are found in large number on the both sides of all lamellae of M. orientalis. They are long and narrow strips instead of round cuticular plates in previously reported scarab beetles. Considering the high number of these sensilla, olfactory processing must take place in sensilla placodea with numerous pores. The terminal ends of dendritic branches of receptor neurons presumably reach right below this pore system to transduce sensory signal. These sensilla placodea found on the lamellae of M. orientalis are similar to the elongated sensilla placodea described in hymenopteran species. On the antennae of parasitic wasps, sensilla placodea are slightly elevated above the antennal surface, and sometimes are surrounded by grooved gap and cuticular ridges (Bleeker et al., 2004; Das et al., 2011; Onagbola and Fadamiro, 2008). Sexual dimorphism of antennal structure has been reported in several species of Melolonthini: An. parvulus (Allsopp, 1990), D. ishigakiensis (Tanaka et al., 2006), Holotrichia diomphalia Bates (Sun et al., 2007), L. mansueta Burmeister (Handique et al., 2017), Mac. mexicanus (Burmeister), Mac. nigripes (Bates) (Romero-López et al., 2017), Phylloph. obsoleta, Phylloph. opaca, and Phylloph. ravida (Romero-López and Morón, 2013). In these scarab beetles, much greater number of pheromone-sensitive sensilla placodea in males than in females could be related to long-range pheromone reception (Kim and Leal, 2000). In M. orientalis, however, we did not observe any apparent sexual difference in the presence and distribution of olfactory sensilla. The lack of sexual dimorphism could be associated with short-range pheromone reception, suggesting that there are no any specialized pheromone-sensitive sensilla in M. orientalis (Allsopp, 1990; McQuillan and Semmens, 1990; Zauli et al., 2016).

Table 2 Number and distribution pattern of antennal sensilla on antennomeres of M. orientalis (n = 6). Sensilla type Böhm bristle Chaetica

Subtype

Female

Male

Segments

SCh1 SCh2

13.50 ± 0.52 16.80 ± 0.37 17.83 ± 0.47

12.60 ± 0.43 17.40 ± 1.03 16.67 ± 0.61

SBa1

3.30 ± 0.34 37.25 ± 0.85 5.63 ± 0.47 22.67 ± 3.17

2.80 ± 0.13 38.50 ± 1.32 5.00 ± 0.47 26.00 ± 2.64

SBa2 SBa3

18.00 ± 1.53 23.33 ± 1.45

17.33 ± 2.73 19.67 ± 1.20

SBa4

37.00 ± 2.65

45.33 ± 4.06

416.83 ± 50.52*

392.29 ± 27.89*

Scape Scape Scape, Pedicel, F3, F4 F4, F5 Lamellae Pedicel L1b, L2b, L3a, L3b L1b, L2b L1b, L2b, L3a, L3b L1b, L2b, L3a, L3b Lamellae

SCh3 Trichodea Coeloconica Basiconica

Placodea

F: Funicle; L: Lamella; a: proximal side; b: distal side; *: data for the proximal side of L2.

may respond to changes in humidity and temperature functioning as hygro- and thermo-receptors. An olfactory function was suggested for sensilla coeloconica on lamellae in the reception of plant volatiles (Kim and Leal, 2000; Ochieng et al., 2002). However, no sensilla coeloconica were observed on all lamellae of M. orientalis. Four subtypes of sensilla basiconica occur on both sides of all club lamellae except the outer surface of the proximal lamella in both sexes of M. orientalis. They are common sensilla in scarab beetles, An. parvulus (Allsopp, 1990), L. negatoria (Allsopp, 1990), C. nitida (Baker and Monroe, 2005), Mac. mexicanus, Mac. nigripes (Romero-López et al., 2017), Phylloph. obsoleta, Phylloph. opaca, and Phylloph. ravida (RomeroLópez and Morón, 2013), Po. japonica (Kim and Leal, 2000), and many species in Hopliinae (Romero-López et al., 2013). Porous feature of sensilla basiconica suggests an olfactory function. The grooved shaft of SBa4 is quite similar to sensilla coeloconica. It is very likely that SBa4 play a role in detecting acids as grooved sensilla basiconica in firebrat Thermobia domestica (Missbach et al., 2014) and grooved pegs in mosquitoes (Bowen, 1995). The lamellar club of scarabs, on which the olfactory sensilla are concentrated, is the most important sensorial zone involved in sexual

Fig. 4. SEM micrographs of sensilla coeloconica and basiconica. (A–B) Sensilla coeloconica (SCo) on pedicel; (C) Sensilla basiconica subtype 1 (SBa1); (D) Sensilla basiconica subtype 2 (SBa2); (E) Sensilla basiconica subtype 3 (SBa3); (F) Sensilla basiconica subtype 4 (SBa4). 21

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Fig. 5. SEM micrographs of sensilla placodea (SP). (A–B): SP; (C) High-resolution SEM of SP showing pore distribution (white arrowhead). Table 3 Density of cuticular pores on lamellae of both sexes of M. orientalis (pores/μm2, n = 6). Lamella/side L1 L2 L3

Female Outer Inner Proximal Distal Inner Outer

48.5 50.5 40.4 66.0 54.5 57.9

± ± ± ± ± ±

Male 6.6 2.6 1.3 5.9 4.1 7.5

47.7 49.5 43.6 59.3 48.5 64.7

1063–1076. Bengtsson, J.M., Khbaish, H., Reinecke, A., Wolde-Hawariat, Y., Negash, M., Seyoum, E., Hansson, B.S., Hillbur, Y., Larsson, M.C., 2011. Conserved, highly specialized olfactory receptor neurons for food compounds in 2 congeneric scarab beetles, Pachnoda interrupta and Pachnoda marginata. Chem. Senses 36, 499–513. Bleeker, M.A.K., Smid, H.M., Van Aelst, A.C., Van Loon, J.J.A., Vet, L.E.M., 2004. Antennal sensilla of two parasitoid wasps: a comparative scanning electron microscopy study. Microsc. Res. Tech. 63, 266–273. Bowen, M.F., 1995. Sensilla basiconica (grooved pegs) on the antennae of female mosquitos: electrophysiology and morphology. Entomol. Exp. Appl. 77, 233–238. Das, P., Chen, L., Sharma, K.R., Fadamiro, H.Y., 2011. Abundance of antennal chemosensilla in two parasitoid wasps with different degree of host specificity may explain sexual and species differences in their response to host-related volatiles. Microsc. Res. Tech. 74, 900–909. Gao, Y., Li, G., Li, K., Lei, C., Huang, Q., 2017. Comparison of the trapping effect and antioxidant enzymatic activities using three different light sources in cockchafers. Environ. Sci. Pollut. Res. - Int. 24, 27855–27861. Handique, G., Bhattacharyya, B., Baruah, A.A.L.H., Boruah, R., 2017. Antenna morphology and sensilla microstructure of the male and female scarab beetle, Lepidiota mansueta Burmeister (Coleoptera: Scarabaeidae). Invertebr. Reprod. Dev. 61, 200–205. Hansson, B.S., Larsson, M.C., Leal, W.S., 1999. Green leaf volatile-detecting olfactory receptor neurones display very high sensitivity and specificity in a scarab beetle. Physiol. Entomol. 24, 121–126. Kim, J.Y., Leal, W.S., 2000. Ultrastructure of pheromone-detecting sensillum placodeum of the Japanese beetle, Popillia japonica Newmann (Coleoptera: Scarabaeidae). Arthropod Struct. Dev. 29, 121–128. Larsson, M.C., Leal, W.S., Hansson, B.S., 1999. Olfactory receptor neurons specific to chiral sex pheromone components in male and female Anomala cupreabeetles (Coleoptera: Scarabaeidae). J. Compar. Physiol. A-Neuroethol. Sensory Neural Behav. Physiol. 184, 353–359. Larsson, M.C., Leal, W.S., Hansson, B.S., 2001. Olfactory receptor neurons detecting plant odours and male volatiles inAnomala cuprea beetles (Coleoptera: Scarabaeidae). J. Insect Physiol. 47, 1065–1076. Leal, W.S., 1998. Chemical ecology of phytophagous scarab beetles. Annu. Rev. Entomol. 43, 39–61. Leal, W.S., Mochizuki, F., 1993. Sex pheromone reception in the scarab beetle Anomala cuprea: enantiomeric discrimination by sensilla placodea. Naturwissenschaften 80, 278–281. Liu, G.-R., Zhang, Y.-W., Wang, R., 1997. The Colour Illustrated of Common Lamellicornia beetles of Northern China. China Forestry Press, Beijing. Liu, W.-G., Eberle, J., Bai, M., Yang, X.-K., Ahrens, D., 2015. A phylogeny of Sericini with particular reference to Chinese species using mitochondrial and ribosomal DNA (Coleoptera: Scarabaeidae). Org. Divers. Evol. 15, 343–350. McQuillan, P.B., Semmens, T.D., 1990. Morphology of antenna and mouthparts of adult Adoryphorus couloni(Burmeister) (Coleoptera: Scarabaeidae: Dynastinae). J. Aust. Entomol. Soc. 29, 75–79. Meinecke, C.-C., 1975. Riechsensillen und systematik der Lamellicornia (Insecta, Coleoptera). Zoomorphologie 82, 1–42. Missbach, C., Dweck, H.K., Vogel, H., Vilcinskas, A., Stensmyr, M.C., Hansson, B.S., Grosse-Wilde, E., 2014. Evolution of insect olfactory receptors. eLife 3, e02115. Mutis, A., Palma, R., Parra, L., Alvear, M., Isaacs, R., Morón, M., Quiroz, A., 2014. Morphology and distribution of sensilla on the antennae ofHylamorpha elegans Burmeister (Coleoptera: Scarabaeidae). Neotrop. Entomol. 43, 260–265. Nikonov, A.A., Leal, W.S., 2002. Peripheral coding of sex pheromone and a behavioral antagonist in the Japanese beetle, Popillia japonica. J. Chem. Ecol. 28, 1075–1089. Nikonov, A.A., Valiyaveettil, J.T., Leal, W.S., 2001. A photoaffinity-labeled green leaf volatile compound ‘tricks’ highly selective and sensitive insect olfactory receptor neurons. Chem. Senses 26, 49–54. Nikonov, A.A., Peng, G., Tsurupa, G., Leal, W.S., 2002. Unisex pheromone detectors and pheromone-binding proteins in scarab beetles. Chem. Senses 27, 495–504. Ochieng, S.A., Robbins, P.S., Roelofs, W.L., Baker, T.C., 2002. Sex pheromone reception in the scarab beetle Phyllophaga anxia(Coleoptera: Scarabaeidae). Ann. Entomol. Soc. Am. 95, 97–102. Onagbola, E.O., Fadamiro, H.Y., 2008. Scanning electron microscopy studies of antennal sensilla ofPteromalus cerealellae (Hymenoptera: Pteromalidae). Micron 39, 526–535. Renou, M., Tauban, D., Morin, J.P., 1998. Structure and function of antennal pore plate sensilla ofOryctes rhinoceros (L.) (Coleoptera : Dynastidae). Int. J. Insect Morphol.

P ± ± ± ± ± ±

5.5 5.1 4.7 12.9 2.3 12.4

0.93 0.87 0.54 0.66 0.24 0.67

In conclusion, our results demonstrated that M. orientalis has typical club-shaped scarab antennae, with olfactory sensilla mainly concentrated on the three lamellar segments comprising the antennal club. Mechanosensitive sensilla (BB, SCh and STr) and chemosensitive sensilla (SBa and SP), as well as sensilla for the detection of humidity and temperature (SCo) are present on the antennae of M. orientalis. The elongate multiporous sensilla placodea, long and narrow strip-like, are very different from those round-plate sensilla found on both sides of all club lamellae in previous studies. They are likely associated with detection of sex pheromone and host plant volatiles. Future observations using transmission electron microscopy of the cross sections of the lamella and electrophysiological investigations are therefore necessary to ascertain the ultrastructure and the specific role of sensilla placodea in M. orientalis. Acknowledgements The authors wish to express sincere thanks to the reviewers for valuable comments on the manuscript. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31171847 and 31460474) and the Key Research Program of the Chinese Academy of Sciences (Grant No. KSZD-EW-Z-021-3-4). There is no conflict of interest for any of the authors. References Ågren, L., 1985. Architecture of a lamellicorn flagellum (Phyllopertha horticola, Scarabaeidae, Coleoptera, Insecta). J. Morphol. 186, 85–94. Ahrens, D., 2005. The phylogeny of Sericini and their position within the Scarabaeidae based on morphological characters (Coleoptera: Scarabaeidae). Syst. Entomol. 31, 113–144. Ahrens, D., 2006. Cladistic analysis ofMaladera (Omaladera): implications on taxonomy, evolution and biogeography of the Himalayan species (Coleoptera: Scarabaeidae: Sericini). Org. Divers. Evol. 6, 1–16. Ahrens, D., Vogler, A.P., 2008. Towards the phylogeny of chafers (Sericini): analysis of alignment-variable sequences and the evolution of segment numbers in the antennal club. Mol. Phylogenet. Evol. 47, 783–798. Allsopp, P.G., 1990. Sexual dimorphism in the adult antennae of Antitrogus parvulus Britton and Lepidiota negatoriaBlackburn (Coleoptera: Scarabaeidae: Melolonthinae). Aust. J. Entomol. 29, 261–266. Baker, G.T., Monroe, W.A., 2005. Sensilla on the adult and larval antennae of Cotinis nitida (Coleoptera: Scarabaeidae). Microsc. Microanal. 11, 170–171. Bengtsson, J., Wolde-Hawariat, Y., Khbaish, H., Negash, M., Jembere, B., Seyoum, E., Hansson, B., Larsson, M., Hillbur, Y., 2009. Field attractants for Pachnoda interrupta selected by means of GC-EAD and single sensillum screening. J. Chem. Ecol. 35,

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Stensmyr, M.C., Larsson, M.C., Bice, S., Hansson, B.S., 2001. Detection of fruit- and flower-emitted volatiles by olfactory receptor neurons in the polyphagous fruit chafer Pachnoda marginata(Coleoptera: Cetoniinae). J. Comp. Physiol. A Sens. Neural Behav. Physiol. 187, 509–519. Sun, F., Hu, J.-H., Wang, G.-L., Peng, L., 2007. Ultrastructure of olfactory sensilla ofHolotrichia diomphalia Bates (Coleoptera: Melolonthidae). Acta Entomol. Sin. 50, 675–681. Tanaka, S., Yukuhiro, F., Wakamura, S., 2006. Sexual dimorphism in body dimensions and antennal sensilla in the white grub beetle,Dasylepida ishigakiensis (Coleoptera: Scarabaeidae). Appl. Entomol. Zool. (Jpn.) 41, 455–461. Wojtasek, H., Hansson, B.S., Leal, W.S., 1998. Attracted or repelled?—a matter of two neurons, one pheromone binding protein, and a chiral center. Biochem. Biophys. Res. Commun. 250, 217–222. Zauli, A., Maurizi, E., Carpaneto, G.M., Chiari, S., Svensson, G.P., Di Giulio, A., 2016. Antennal fine morphology of the threatened beetle Osmoderma eremita(Coleoptera: Scarabaeidae), revealed by scanning electron microscopy. Microsc. Res. Tech. 79, 178–191.

Embryol. 27, 227–233. Romero-López, A.A., Morón, M.A., 2013. Sexual dimorphism in antennae of Mexican species of Phyllophaga (Coleoptera: Scarabaeoidea: Melolonthidae). In: Moriyama, H. (Ed.), Sexual Dimorphism. IN TECH Publisher, Croatia, pp. 17–34. Romero-López, A.A., Arzuffi, R., Valdez, J., Morón, M.A., Castrejón-Gómez, V., Villalobos, F.J., 2004. Sensory organs in the antennae of Phyllophaga obsoleta(Coleoptera: Melolonthidae). Ann. Entomol. Soc. Am. 97, 1306–1312. Romero-López, A., Morón, M., Valdez, J., 2010. Sexual dimorphism in antennal receptors of Phyllophaga ravida Blanchard (Coleoptera: Scarabaeoidea: Melolonthidae). Neotrop. Entomol. 39, 957–966. Romero-López, A.A., Carrillo-Ruiz, H., Moron, M.A., 2013. Morphological diversity of antennal sensilla in Hopliinae (Coleoptera: Scarabaeoidea: Melolonthidae). Academic Journal of Entomology 6, 20–26. Romero-López, A.A., Benítez-Herrera, L.N., Martínez-Bonilla, O.K., Yanes-Gómez, G., Aragón-Sánchez, M., 2017. Comparative study of distribution of antennal chemoreceptors of Macrodactylus of Mexico. Southwest. Entomol. 42, 111–119. Schneider, D., 1964. Insect antennae. Annu. Rev. Entomol. 9, 103–122.

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