Ficus (Urticales: Moraceae) trees are the primary decorative trees used to line avenues in urban landscapes of the southern cities in China and countries in Southeast Asia, and are vital to maintaining ecological balance by actively cycling nutrients and gases (e.g., carbon dioxide and oxygen) and providing a cumulative leaf area for the impingement, absorption, and accumulation of air pollutants (e.g., construction, industrial, and vehicle emissions) that decreases the pollution ratio in the urban atmosphere (Escobedo et al. 2008). However, Ficus microcarpa L. and F. racemosa L. are often extensively damaged by the defoliating pest Phauda flammans (Walker) (Lepidoptera: Zygaenidae) in China (Liu et al. 2014, 2015a, 2015b, 2016) and India (Nageshchandra et al. 1972, Verma and Dogra 1982).

Phauda flammans exhibits two to three generations per year in Nanning City, Guangxi, southern China. Adults primarily are attracted to the host plants during the daytime when they oviposit eggs on leaves. Peaks in the number larvae in the first generation occur from mid-May to late June and in the second generation from early August to mid-October. Larvae can even be found on F. microcarpa leaves during winter months. Pupation most frequently occurs near roots and exposed tussocks on the ground, with only a few individuals pupating in the topsoil. Previous work includes reports of biological characteristics (Liu et al. 2014, 2015a, 2015b; Zheng et al. 2017), occurrence (Nageshchandra et al. 1972, Verma and Dogra 1982), and control (Liu et al. 1985, Zheng et al. 2015). Chemical insecticides remain as the most effective management tactic, but widespread use is no longer feasible due to development of insecticide resistance and negative impacts on biodiversity and environmental health.

Sex pheromone–based monitoring, mass trapping, and mating disruption have been identified as effective alternative control methods (Witzgall et al. 2010). Reproductive behavior in many insects is governed by sex pheromones, which are detected via chemical receptors and expressed in olfactory receptor neurons (Kurtovic et al. 2007). Pheromone identification is frequently part of the study of the electrophysiological response of antennal receptors (Bill et al. 1989), especially the antennal sensilla (Lopes et al. 2002).

In the study presented herein, scanning electron microscopy (SEM) was used to characterize the ultrastructure of the antennal sensilla of adult male and female P. flammans, and to identify the types, distribution, and numbers of antennal sensilla in both sexes of the adults. Our ultimate goal is to provide a basis for subsequent electrophysiological and behavioral studies that will lead to the identification of the P. flammans sex pheromone so that the mechanisms of sexual communication in this species can be determined.

Materials and Methods

Results

Antennae of the female and male moths were filiform, each consisting of a scape, pedicel, and a flagellum of more than 40 flagellomeres (Fig. 1A). The scape was the largest antennal segment for both sexes. In comparing the length of the antennal segments between males and females, the male scape (t = −4.304; df = 8; P = 0.006) and pedicel (t = −3.392; df = 8; P = 0.009) were significantly longer than those of the females, but the flagellum length was not (t = −1.238; df = 58; P = 0.221). In contrast, the width of the scape (t = −1.098; df = 8; P = 0.304) and pedicel (t = −1.000; df = 8; P = 0.347) did not differ between the sexes, but the female flagellum was wider than that of the male (t = −2.167; df = 58; P = 0.034) (Table 1). Each flagellomere could be divided into two main areas (Fig. 1B, C). Scales and sparse sensilla were distributed on the dorsal surface (Fig. 1C, D), while the ventral surface had no scales but was covered with various types of sensilla (Fig. 1B, E).

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Table 1 Mean ± SE length and width of the scape, pedicel, and antennal flagellum of P . flammans.*

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Six types and eight subtypes of sensilla were observed on the flagellar subsegments of the antennae of both sexes, including sensilla trichodea (s. trichodea) (subtypes I and II), sensilla basiconica (s. basiconica) (subtypes I and II), sensilla coeloconica (s. coeloconica), sensilla styloconica (s. styloconica), sensilla chaetica (s. chaetica), and Böhm bristles (B. bristles). Sexual dimorphic characters are presented in Tables 2 and 3, and each of the different sensilla are described in the following paragraphs.

Table 2 Mean ± SE dimensions of the sensilla on the antennae of P. flammans.

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Table 3 Mean ± SE number of sensilla* on the dorsal (D) side and ventral (V) side of P. flammans .

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Table 3 Extended.

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Sensilla trichodea

The s. trichodea was the most abundant type of sensillum on the antennae of both sexes. It was observed on the ventral surface of the entire flagellum and was also found arranged randomly on the dorsal surface. The surfaces of these sensilla were multiporous and showed an annular ridge pattern (Fig. 2B, D) that was divided into two subtypes (Fig. 2A, C). The length (t = 0.209; df = 78; P = 0.514) and diameter (t = 1.159; df = 78; P = 0.250) of subtype I and the length (t = 0.146; df = 78; P = 0.884) and diameter (t = −1.679; df = 78; P = 0.097) of subtype II did not differ between males and females (Table 2). The numbers of subtype I on the ventral surface (t = 1.509; df = 16; P = 0.151) and dorsal surface (t = 0.803; df = 16; P = 0.433) also were similar in both sexes (Table 3). In contrast, the number of subtype II on the ventral surface (t = 2.763; df = 16; P = 0.014) was greater in males than in females, but the number on the dorsal surface (t = −1.307; df = 16; P = 0.210) did not differ (Table 3).

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Sensilla basiconica

The s. basiconica was also divided into two subtypes (I and II) and appeared in large numbers on all flagellomeres in both sexes (Table 3). Subtype I were distributed on the ventral surface of the flagellomeres (Fig. 3A), and only subtype II was observed on the dorsal surface of the antennae (Fig. 4A). Subtype I was ribbed with transverse grooves with some pores (Fig. 3B), and it was significantly longer (t = 2.480; df = 78; P = 0.016) and wider (t = 2.357; df = 78; P = 0.021) in male than in female antennae (Table 2). The number of this subtype on the ventral surface did not differ between the sexes (t = 0.114; df = 16; P = 0.910) (Table 3). Subtype II was longer than subtype I and was also multiporous with transverse grooves (Fig. 4C); the base formed an approximately 70° angle to the antenna and was regularly spaced on the dorsal surface (Fig. 4A, B). The length (t = 0.661; df = 78; P = 0.510) and diameter (t = −0.441; df = 78; P = 0.661) of these sensilla were not significantly different between the sexes (Table 2), and the number of subtype II on the ventral (t = 0.093; df = 16; P = 0.927) and dorsal (t = −0.731; df = 16; P = 0.475) surfaces did not differ significantly between males and females (Table 3).

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Sensilla styloconica

In the upper middle area of each flagellar subsegment, single s. styloconica were often found rising from a cylindrical protrusion to form a peak (Fig. 5C). There were no differences in the length (t = −0.680; df = 78; P = 0.499) and diameter (t = −0.971; df = 78; P = 0.335) of this type of sensillum between males and females (Table 2), but the mean number was much greater in females than in males (t = −3.362; df = 16; P = 0.004) (Table 3).

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Discussion

Although there have been no reports on the antennal sensilla of members of the family Zygaenidae, studies of many other lepidopteran species have shown that the general shape, structure, and types of sensilla of the antenna of adult P. flammans are similar to those described in other species, such as Plodia interpunctella (Ndomo-Moualeu et al. 2014), Catocala remissa Staudinger (Zheng et al. 2014), Holcocerus hippophaecolus Hua (Wang et al. 2015), Monema flavescens Walker (Yang et al. 2016), C. pomonella, C. succedana (Roh et al. 2016), and T. absoluta (Bawin et al. 2017). The functions of all these sensilla have been reported to be primarily sensory, and all of these species are endowed with the sensilla responsible for olfactory detection and the perception of tactile signals (Lena et al. 2005).

Specifically, many studies have shown that s. trichodea are responsible for mechanical and chemical reactions, and these sensilla could be further divided into subtypes by the number of sensory cells or pores on the sensillum wall (Onagbola and Fadamiro 2008), size (Zheng et al. 2014), or other indicators. Two subtypes, which were named “long s. trichodea” by Roh et al. (2016), were identified in our study. Furthermore, s. basiconica are believed to be integrated olfactory stimuli receptors that are sensitive to host volatiles, mates, oviposition attractants, etc. (Rebijith et al. 2016). Our results corroborate the view of Ndomo-Moualeu et al. (2014) that they are composed of two subtypes. Additionally, the multiporous structure suggests that they are key olfactory receptors (Monteforti et al. 2002). Generally, the two s. coeloconica subtypes differ in the presence or absence of a circular fence (Roux et al. 2005), but only subtype II, without the circular fence, was found in our study. These sensilla are apparently sensitive to humidity, temperature, CO₂, olfactory stimuli, etc. (Galvani et al. 2008, Kristoffersen et al. 2006, Kuřavová et al. 2017, Ren et al. 2014). In s. styloconica, the two subtypes are divided based on the presence or absence of apical structures and only the subtype with apical structures, which are associated with the thermo-hygroreceptor function (Steinbrecht and Müller 1991), was found in our study. Several previous studies classified s. chaetica as terminal pore sensilla, suggesting that they function as contact chemoreceptors and mechanoreceptors (Seada 2015), but this pore was not observed in P. flammans. Therefore, s. chaetica may have no receptor function or only a mechanoreceptive function (Wang et al. 2010), such as identifying sounds or air currents (Keil 1999) or protecting other sensilla (Chang et al. 2015). It has been suggested that another specialized sensillum, the B. bristles that only occur on the scape and pedicel in the antennae of P. flammans, might be membrane acceptors on the segment membranes between the scape and pedicel or a mechanosensory unit that monitors the position of the antennae in flight (Krishnan et al. 2012).

Antennal sensilla sexual dimorphism, which is intimately connected to the important tasks of feeding, mate location, dispersal, oviposition, and other functions that are central to the life-history evolution of the species, is an especially ubiquitous form of intraspecific variation in moths (Allen et al. 2011). Faucheux and Kundrata (2017) speculated that sexual differences in the numbers of antennae sensilla may be linked with sex-specific differences in behavior. In P. flammans, sensilla of the male antennae, particularly the s. trichodea, were generally more numerous than on the female antennae (Table 3). Similar results have been reported in Talponia batesi Heinrich (Gómez and Carrasco 2008) and T. absoluta (Bawin et al. 2017). Faucheux et al. (2006) demonstrated that the s. trichodea of the male antennae are the receptors of female sex pheromones, but further study is needed to determine whether there is an inherent relationship between the number or distribution of s. trichodea and sensitivity to chemicals.

Some nocturnal moths have the same filiform antennae as the diurnal P. flammans. Previous studies also reported the s. trichodea, s. basiconica, s. coeloconica, s. styloconica, and s. chaetica sensilla on the antennae of both nocturnal and diurnal moths, that is, T. batesi (Gómez and Carrasco 2008), Synanthedon scitula Harris (Frank et al. 2010), Algedomia coclesalis Walker (Liu et al. 2013), C. remissa (Zheng et al. 2014), and now P. flammans as reported in this study. However, numerous s. auricillica sensilla, which are considered to be olfactory receptors for sex pheromones (Alnter et al. 1977), occur on the antennae of nocturnal moths, but not P. flammans. This difference could be attributed to the environmental adaptability of P. flammans because compound eyes could be used for courtship in some diurnal Zygaenidae, such as Zygaena filipendulae L. and Z. trifolii Esper (Naumann et al. 1999). Therefore, compound eyes may compensate for the lack of olfactory function of the s. auricillica in diurnal Zygaenidae.

In conclusion, this study identifies and characterizes the ultrastructural morphology, abundance, and distribution of the different types of sensilla occurring on the antennae of P. flammans. This information helps in our understanding of the electrophysiological and behavioral elements of chemical communication in P. flammans. In future research, transmission electron microscopy will be used to confirm the functions of the different sensilla identified in this study.