Turkish Journal of Zoology Turk J Zool (2019) 43: 164-170 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1802-25

Sexual dimorphism in antennal sensilla of Parthenium bicolorata

Ishana QADIR, Ayesha QAMAR* Department of Zoology, Aligarh Muslim University, Aligarh, India

Received: 17.02.2018 Accepted/Published Online: 19.02.2019 Final Version: 01.03.2019

Abstract: In the present study, scanning electron microscopy was used to investigate the ultrastructure of sensilla present on the antennae of male and female Zygogramma bicolorata. The antennae in this consist of the scape, pedicel, and nine flagellomeres. Six types of sensilla were identified: sensilla trichodea type 1 and 2 (ST1, ST2), sensilla cavitae (SCa), sensilla chaetica (SCh), and sensilla basiconica type 1 and 2 (SB1, SB2). Sensilla were characterized on the basis of their size and density to find out whether sexual dimorphism exists in this species or not. We observed that the size of the antennomeres was comparatively larger in males than females. Moreover, sensilla chaetica were found on all antennomers and have the qualification of being the longest type, suggesting that they probably have a role in mechanoreception. There was an abundance of sensilla trichodea in both males and females, indicating its function as a chemoreceptor. Sensilla basiconica are believed to sense sex pheromones and hence their abundance was more in males as compared to females.

Key words: Antenna, antennomeres, olfaction, pheromones, sensilla

1. Introduction Zygogramma bicolorata is one of the economically Volatile semiochemicals emitted by plants are the important chrysomelid . It was introduced in principal sensory signals detected by (Crook et India for the control of the highly obnoxious weed al., 2005; Wang et al., 2010; Mamidala et al., 2013). Most . It is highly selective of of the studies concerning detection and perception of its host plant Parthenium, a highly invasive plant volatile semiochemicals have been linked with the sensory believed to be harmful to the welfare of human beings systems of insects, in which antennae play a premier and livestock, as it has been reported to cause severe role (Wigglesworth, 1972). The antenna of the insect is allergies and dermatitis (Patel, 2011). equipped with minute, microscopic, and hair-like sensory There are five larval stages, all of which feed on structures having a defined distribution and morphology, the same plant, followed by a pupal stage that buries called sensilla, and these structures are involved in locating itself in soil for pupation until the adults emerge. the host plant, searching for ovipositional sites, and helping After emerging from the pupae, the adults reach the in intersexual communication between insects (Zacharuk, plant by either walking or flying. For locating a host 1985; Shields, 2008; Zhang et al., 2015). plant, perception of plant odor is very important, as Zacharuk (1985) suggested that sensilla showed a it serves vital functions for the beetle like feeding and well-defined and specific distribution patterns on insect antennae. The sensilla of insects are occupied by an oviposition. The host plant’s volatiles, often referred to arsenal of olfactory neurons, which transmit signals to as plant kairomones, are a source of stimuli for these the odorant binding proteins (Zacharuk, 1980; Buck insects and may facilitate orientation of insects to its and Axel, 1991; Chess et al., 1992; Vosshall et al., 2000; feeding source. Carlson, 2001; Pitts and Zwiebel, 2006), where they are In the present study, scanning electron microscopy recognized by specialized transmembrane G-protein (SEM) was employed to inspect the number, type, coupled receptors, and successional signal transduction and distribution of male and female antennal sensilla can lead to the final quantitative and qualitative detection of Z. bicolorata. This study will be helpful in revealing of volatiles in the insect brain (Galizia and Silke, 2010). possible structure–function relationships, which might Thus, to understand behavioral and ecological adaptations lead to a better comprehension of behavioral and in insects, understanding the structural morphology and adaptive mechanisms of these insects in the process of distribution of antennal sensilla becomes an essential step. host selection. * Correspondence: [email protected] 164

This work is licensed under a Creative Commons Attribution 4.0 International License. QADIR and QAMAR / Turk J Zool

2. Materials and methods the funicle. The funicle is formed by eight nonsoldered Adult Z. bicolorata individuals were taken from the main flagellomeres (F1–F9), followed by the ninth flagellomere colony established in our laboratory at the Department (F9) or the clava (Figure 1). The flagellum is an important of Zoology, Aligarh Muslim University (AMU), India. component of the antenna, where numerous sensilla are The insects for experimental purpose were separated attached. The scanty number of sensilla present on the and housed separately in glass petri dishes. For sample scape, pedicel, and F1 to F4 is in contrast to the abundant preparation, the insects were killed by freezing them at –20 density of sensilla present on the more terminal segments °C for 5 h before sample preparation. Whole heads with (F5 to F9). The average length of the male antennae is antennae were cut off using fine scissors. 2.498 ± 0.079 mm, which is slightly longer than the average 2.1. Microscopy (2.337 ± 0.023 mm) of the female antennae (n = 4) (Table SEM was carried out in the Electronic Microscopy 1). The antennomeres also vary in size (Table 2). The last Laboratory of the University Sophisticated Instrumentation part of the antenna, i.e. the terminal flagellomere (F9), Facility, AMU. Heads with antennae of the frozen beetles appears to be the longest and the widest segment in both were cleaned in 15% ethanolic solution. Dehydration was males (386.7 ± 0.2845 µm) and females (278.3 ± 0.4826 carried out using increasing ethanolic series, i.e. 30%, µm). 50%, 70%, 90%, and 99% v/v ethanol, by placing the heads 3.2. Types, distribution, and abundance of sensilla attached with antennae for 3 min each in the respective Based on sensillary size and shape in males and females solutions. The samples were air-dried overnight at room of Z. bicolorata, one each of sensilla chaetica (SCh) and temperature. Finally, the samples were critically dried sensilla cavitae (SCa), two types of sensilla basiconica (SB1 for 20 min in a critical point dryer and coated with gold and SB2), and two types of sensilla trichodea (ST1 and particles. Three to five males and females were observed ST2) were identified (Figure 2). Their length and width in using a JEOL-JSM8510LV scanning electron microscope. males and females are summarized in Table 3. 2.2. Counting and measurements 3.2.1. Sensilla chaetica (SCh) The antennal segments were measured using a scale built Sensilla chaetica (SCh) are the longest and thickest sensilla into the scanning electron microscope. The density of and are present in both sexes on all antennomeres (Figure sensilla trichodea 1 (ST1) was calculated by counting the 3a; Table 3). These sensilla are straight with a blunt tip and 2 number of sensilla in an area of 60.55 µm on the dorsal side grooved on the lateral surface (Figures 3b and 3c). They of the last two antennomeres. Five to seven measurements are inserted into wide sockets (Figure 3b). The length and were taken for each analysis. diameter of the SCh in females and males are different, with females having a shorter length and smaller diameter 3. Results (Table 3). 3.1. Image and data analysis 3.2.2. Sensilla trichodea type 1 (ST1) Z. bicolorata antennae consisted, from base to tip, of a The sensilla trichodea (ST1) are long (Table 3) and most short and straight scape, followed by a pedicel, which is abundant, found on all antennomeres except the scape longer and wider than the successive antenites that form and pedicel in both males and females. They are tapering and have sharp tips (Figure 4a). Their lateral surfaces have longitudinal grooves and are nearly straight or slightly curved towards the ends. They are inserted into wide sockets (Figure 4a). The lengths of ST1 in females and males are not similar; the female sensilla are longer than those of males (Table 3). Moreover, the diameter is wider in females than in males (Table 3). The density of ST1 is more on the terminal antennomeres (F5–F9) than the basal ones; moreover, their density in female antennae is higher as compared with the males in flagellomeres 8 and 9 (Table 1). 3.2.3. Sensilla trichodea type 2 (ST2) These sensilla are sharp-tipped, with no apparent curvature (Figure 4b). They are tightly inserted into sockets (Figure 4b). These sensilla are present on flagellomeres 5–9. The Figure 1. Whole view of antenna in Zygogramma bicolorata (11 length and diameter show different size distributions in antennomeres). Sc - scape, P - pedicel, F - flagellomere. females and males, as shown in Table 3.

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Table 1. Sensilla density and length of antenna of male and female Z. bicolorata.

Density of sensilla (ST1) (60.55 µm × 60.55 µm) Length of antenna (mean ± SE) Flagellomere 9 (clava) Flagellomere 8 Male Female Male Female Male (mm) Female (mm) 12 ± 1.225 (4) 20.000 ± 1.225 (4) 6.750 ± 0.854 (4) 14.250 ± 1.109 (4) 2.498 ± 0.079 (4) 2.337 ± 0.023 (4)

Table 2. Comparison of length and width of antennomeres between male and female of Z. bicolorata.

Male (µm) (mean ± SE) Female (µm) (mean ± SE)

Antennomeres Length Width Length Width F9 386.7 ± 0.2845 223.6 ± 0.4006 278.3 ± 0.4826 198.3 ± 0.4786 F8 242.9 ± 0.4118 208.2 ± 0.2456 231.7 ± 0.6527 190.2 ± 0.4116 F7 237.2 ± 0.4086 204.3 ± 0.2331 216.3 ± 0.7009 198.8 ± 0.4635 F6 202.4 ± 0.2778 189.5 ± 0.2375 203.1 ± 0.6704 194.7 ± 0.4839 F5 203.3 ± 0.6990 167.4 ± 0.2904 192.1 ± 0.4918 164.2 ± 0.4771 F4 168.1 ± 0.2153 128.9 ± 0.2771 154.3 ± 0.6654 137.4 ± 0.4865 F3 179.5 ± 0.2106 112.7 ± 0.1238 178.3 ± 0.6140 119.3 ± 0.4787 F2 187.6 ± 0.2963 105.1 ± 0.3124 221.8 ± 0.4624 108.3 ± 0.2348 F1 211.8 ± 0.2019 112.4 ± 0.2593 262.4 ± 0.2856 97.42 ± 0.2153 Scape 166.7 ± 0.2121 129.2 ± 0.07642 202.4 ± 0.6520 115.2 ± 0.2319 Pedicel 309.3 ± 0.2285 172.7 ± 0.1890 274.7 ± 0.6476 178.4 ± 0.3178

having longer and broader sensilla than females (Table 3). These sensilla are embedded into wide sockets (Figure 5a). The SB1 are distributed on flagellomeres 5–9 in both sexes. 3.2.5. Sensilla basiconica type 2 (SB2) These sensilla are straight and hair-like, having a grooved surface with blunt contorted tip having the pore at the end (Figures 5b and 5c). In males, the length and diameter of SB2 are greater as compared to females (Table 3). Like SB1, they are also inserted into wide sockets (Figure 5b). They are present mostly on flagellomeres 5–9 in both sexes but their abundance is more in males than in females. 3.2.6. Sensilla cavitae (SCa) The sensilla cavitae (SCa) are characterized by a well- formed pit having diameters 2.324 ± 0.023 and 2.134 ± Figure 2. Sensilla trichodea (ST1, ST2), sensilla chaetica (SCh), 0.043 (Figure 6) in males and females, respectively, without and sensilla basiconica (SB1 and SB2) of Z. bicolorata. a hair-like shaft extending to outside. These sensilla were well distributed on all antennomeres. However, compared to females, these were more abundant in males. 3.2.4. Sensilla basiconica type 1 (SB1) These sensilla have the shortest length among all sensilla 4. Discussion types, are straight and elongated, hair-like, having a smooth In the present study we could classify the antennal sensilla surface with no lateral grooves and ending with a dome- of Z. bicolorata into six types: sensilla trichodea 1 (ST1) like blunt tip (Figure 5a; Table 3). Here also, the length and and 2 (ST2), sensilla chaetica (SCh), sensilla basiconica 1 diameter are different in males and females, with males and 2 (SB1 and SB2), and sensilla cavitae (SCa).

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Table 3. Comparison of types of sensilla with their mean length and diameter of male and female Z. bicolorata.

Length (µm) (mean ± SE) Diameter (µm) (mean ± SE)

Types of Sensilla Male Female Male Female Sensilla chaetica 76.30 ± 4.261 (7) 70.58 ± 1.582 (7) 3.444 ± 0.1825 (7) 3.362 ± 0.1139 (7) Sensilla trichodea-1 40.65 ± 0.7474 (8) 45.30 ± 1.433 (8) 2.960 ± 0.06388 (8) 3.270 ± 0.1296 (8) Sensilla trichodea-2 19.99 ± 0.2871 (8) 10.65 ± 0.2948 (8) 3.035 ± 0.1239 (8) 2.486 ± 0.1393 (8) Sensilla basiconica-1 7.922 ± 0.2067 (9) 6.520 ± 0.2665 (9) 2.762 ± 0.2473 (9) 2.901 ± 0.05109 (9) Sensilla basiconica-2 14.43 ± 0.3987 (9) 10.16 ± 0.4794 (9) 2.810 ± 0.07357 (9) 2.705 ± 0.09696 (9) Sensilla cavitae - - 2.324 ± 0.023 (5) 2.134 ± 0.043 (3)

Figure 3. Whole view of sensilla chaetica (SCh) (a) of female Z. bicolorata; SCh with wide socket and lateral grooves (b) and Figure 4. Sensilla trichodea type 1 (ST1) (a) and sensilla blunt tip (c). trichodea type 2 (ST2) (b) of Z. bicolorata (whole view).

Among all sensilla identified in our study, sensilla various sensilla on the antennal surfaces of other insects trichodea were observed to be the dominant most, abundant in Chrysomelidae, similar observations were reported by a and widespread sensillum type present on the antennal number of workers (Bartlet et al., 1999; Zhang et al., 2013; surface of both male and female Z. bicolorata. These sensilla Zhang et al., 2016). The abundance of ST1 on the terminal were long and straight or slightly curved towards the apex parts recorded in our study may be related to the efficient in both sexes. While analyzing the distribution patterns of signal perception during host location and the other

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Figure 6. Sensilla cavitae (SCa) of male Z. bicolorata (on flagellomere 5).

substrate, and they might also have a prominent role in gustation and mechanoreception. Moreover, the length of SCh in males was more as compared with that of the females (Table 3), which means that they might utilize this property to sense the female-specific surface hydrocarbons and orient themselves to the females. Similar observations were made by several other workers (Saïd et al., 2003; Shields, 2004; Crook et al., 2008). The morphology of ST and SCh were similar to previously described antennal sensilla of several species of curculionid and chrysomelid beetles (Mustaparta, 1973; Hatfield et al., 1976; Alm and Figure 5. Sensilla basiconica type 1 (SB1) (a); sensilla basiconica Hall, 1986; Isidoro and Solinas, 1992). type 2 (SB2) (b); magnified view of the tip of SB-2 (c) in Z. SB1 was typically shorter in comparison to all other bicolorata. sensilla. In the present study, we observed that the density of SB1 was greater in males as compared to females. The abundance of SB on male antenna can be correlated, like important functions. Moreover, the presence of grooves that of ST1, to their function of sex pheromone perception and pores at the base of these sensilla (Figure 4) highlights of mates. SB1 had grooved lateral sides and culminated its involvement in gustatory (Katsuto et al., 2016) and in a contorted apex with a pore on it. Such characteristics olfactory (Hu et al., 2009) reception. can be correlated to its olfactory function (Zacharuk, Sensilla trichodea 2 were relatively scarcer than type 1985; Bartlet et al., 1999). Moreover, these sensilla have 1 but showed almost equal distribution in males and been reported to respond to host plant odors in gas females. The morphology of these two sister sensilla was chromatography coupled to single cell recording (GC- almost similar; however, ST2 differed from ST1 in only SSR) tests (Lopes et al., 2002; Sun et al., 2014; Zhang et al., having a small shaft. Such sensilla are reported to take 2015; Yi et al., 2016). part in olfaction and have been demonstrated to play a In the present study SCa were more abundant in males role of sex pheromone perceptor and respond to host than in the females, as was also observed in Callosobruchus plant odors (Sun et al., 2014; Yi et al., 2016). Transmission rhodesianus by Katsuto et al. (2016). The SCa are designed electron microscopic studies have also confirmed their to function as receptors for a range of stimuli like chemical mechanosensory function on the basis of a single neuron substances, temperature, and humidity (Zacharuk, 1985; innervating the shaft (Merivee et al., 1998; Yi et al., 2016). Katsuto et al., 2016). Sensilla chaetica (SCh) were the most prominent type of Furthermore, the antennae, consistent with previous sensilla. These blunt tipped and aporous sensilla identified observations, were markedly longer in males than females. in the present study might be the first ones to sense the The longer male antennae confer an adaptive significance

168 QADIR and QAMAR / Turk J Zool as they provide more surface area for sense organs to sensillum. We also evaluated the sexual differences in help detect sex pheromones efficiently (Schneider, 1964; the number and distribution of various sensilla. These Dweck 2009; Wang et al., 2016). In the present study, the results will help us not only in the better understanding density of sensilla, especially sensilla trichodea, was found of host selection and courtship behavior of Z. bicolorata to be greater in terminal segments (Figure 2). The greater but also in correlating this aspect with electrophysiological density distribution patterns of individual sensillary types studies. Lastly, this study shall shed some light on the role on terminal flagellomeres provide information about the of sensilla in sensing pheromones, which is expected to importance of these distal segments in behavioral studies lead us to design semiochemical control methods of insect of these insects (de Wilde et al., 1969; de Wilde 1976; pests. Visser and Ave 1978; Sen and Mitchell 1995; Sen and Mitchell, 2001). Acknowledgment In summary, this study was designed to identify and The authors are highly thankful to the University characterize the morphology of antennal sensillum types Sophisticated Instrumentation Facility, Aligarh Muslim on the basis of SEM studies of Z. bicolorata, and we have University (USIF-AMU), for scanning electron provided insights into some probable functions of each microscopic studies and measurements.

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