Morphology and Chemical Analysis of the Metathoracic Scent Glands System in Adelphocoris suturalis (Hemiptera: Miridae)

Abstract The morphological structure of the metathoracic scent glands (MTGs) in Adelphocoris suturalis was observed by utilizing scanning electron microscope (SEM). Also, the secretions of MTGs in male and female were analyzed by using gas chromatography–mass spectrometry (GC-MS). The result showed that the MTGs comprised a reservoir and paired lateral glands, which are connected to a reservoir by duct. The MTGs belong to the diastomian type. A usually depressed channel extends from opening downward the middle of metathorax, a tongue-like structure was covered by bristles and mushroom-shaped cuticular structures, known as evaporative area. In GC-MS investigation, differences were found in quantitative or qualitative compositions of the substances between the two sexes. In our study, hexyl butyrate was the most abundant compound in the MTGs of A. suturalis , comprising ∼85% of total secretions in both female and male, respectively. 4-oxo-(E)-2-hexenal (5.22%) was the second most abundant compound in female MTGs secretions, whereas octacosane (2.42%) followed hexyl butyrate in male MTGs secretions.

The plant bug, Adelphocoris suturalis (Hemiptera: Miridae), has become one of the most important insect pests of cotton, Gossypium hirsutum L., in the Changjiang River Region of China. The damage is gradually increasing with the commercial cultivation of Bt cotton because the use of broad-spectrum insecticide applications were substantial decreased (Lu et al. 2008a(Lu et al. , 2009(Lu et al. , 2010Zhang et al. 2011). The pest causes a substantial economic loss by feeding on cotton organs, causing stunting, and fruit malformation. Because of its high mobility and broad host range, it can rapidly infest a cotton fields from the surrounding vegetation. Conventional chemical control methods often led to the development of pest resistance and environmental pollution. Pheromonebaited traps are an effective tool for monitoring and detecting insect pests because of their efficiency and no harming environment as some other pest management tools (McBrien et al. 1996(McBrien et al. , 1997. However, little is yet known about it in China. There are well-developed metathoracic scent glands (MTGs) and Brindley's glands or secretory setae in some species of the Hemiptera (Aldrich 1988). Brindley's glands were found in some families. Nevertheless, most adult hemipteran insects possess a well-developed MTGs system (Aldrich 1988, Ho andMillar 2001). The external efferent system of the MTGs is considered as an important diagnostic feature in the Miridae (Cassis and Schuh 2012). Previous studies indicated that the MTGs are composed of usually well-developed external efferent system and paired lateral glands (Cassis and Schuh 2012). For most miridae, the position of the peritreme is situated on the metepisternum of the metapleuron and opens in between mesocoxae and metecoxae. A usually depressed channel extends from the peritreme, a tongue-like structure is a smooth surface covered with mushroom-shaped cuticular structures (Cassis and Schuh 2012). However, there are suprageneric differences for the evaporative and the position of the peritreme in the Miridae (Cassis 1995).
Most of adults release a large of strong-smelling and irritating defensive chemicals when they are molested or attacked (Ho and Millar 2001, Durak and Kalender 2007a, Durak 2008. Adult of miridae possess welldeveloped MTGs, which play an important role in defensive and intraspecific communication, such as defense pheromone, aggregation, and sexual behavior (Zhang and Aldrich 2003a). The volatile communication chemicals secreted from MTGs affect insect behavior depending on their concentrations (Farine et al. 1992). Those compounds are mainly composed of short-chain unsaturated aldehydes, straight-chain esters, and acids, such as (E)-2-hexenal, 4-oxo-(E)-2-hexenal, and hexyl butyrate, which have been also found in other families of Hemiptera (Leal et al. 1994;Durak and Millar 2007a,b). Zhang et al. (2011) proved that males of A. suturalis are attracted to the sexually mature virgin females. Moreover, recent studies suggested that some mirid species secrete aggregation or sex pheromones from MTGs (Millar et al. 1997;Staples et al. 2002;Aldrich 2003b, 2008).
In this study, we examined the morphological structure of the MTGs in A. suturalis by using scanning electron microscopy (SEM). In particular, we compared the chemical composition of the MTGs secretions in female and male by using gas chromatography-mass spectrometry (GC-MS). To the best of our knowledge, this was the first report on the morphological structure and secretions of MTGs of A. suturalis, and we also suggested the biological functions of MTGs secretions in A. suturalis by compared other related species. This study established the groundwork for further research on the use of sex pheromone for managing A. suturalis, as well as a basis for further understanding of biosynthesis and storage of communication chemicals in this important pest species.

Materials and Methods
Insect Material. Nymphs of A. suturalis were collected from alfalfa fields in Zhengzhou (Henan Province, China) in June 2010. Insects were maintained at 26 C and 75 6 5% RH, with a photoperiod of 16:8 (L:D) h in plastic cages (10 by 6 by 4 cm; 20 nymphs per cage) fed on green beans, Phaseolis vulgaris L. and a 5% sugar solution (Lu et al. 2008b, Zhang et al. 2011. The emerged adults were separated by gender every day, and same sex were kept in one plastic cage (10 by 6 by 4 cm) with the density of 20 adults per cage before used for further experiments.
Chemical Analysis. The MTGs were removed and immersed in 100 ll analytical grade n-hexane distilled from calcium hydride and stored at À20 C. Two independent series of extracts were made, each sex with 20 glands with 8-10-d-old virgin adults. Extracts were analyzed ($1 ll of the extract) by splitless injection into a GC-MS on an Agilent 7890A GC series equipped with a polar column HP-5 (30 m by 0.32 mm by 0.25 lm) and interfaced to an Agilent 5975C Mass Selective Detector. The GC was programmed at 50 C for 1 min, then rising to 250 C at 10 C/min, and then held for 5 min and postrun 280 C held for 2 min. The carrier gas was helium with a flow rate of 1 ml/min, and the injector and transfer line temperature was 220 and 280 C, respectively. Compounds were tentatively identified by GC-MS and identifications were comparison of the retention times and mass spectra with those of authentic samples. The molecular structure of compounds was determined by a comparison of the recorded mass spectra with the reference spectra of the NIST library and coinjections of the extracts and synthetic compounds on the two columns. The relative proportions of the compounds in the extracts were obtained by integration of the GC-peak areas.

Results
Studies on MTGs by SEM. Micrographs showed that the MTGs of A. suturalis are composed of well-developed reservoir and paired lateral glands (Fig. 1A). The reservoir is long and bag shaped, and the wall of reservoir comprises some sac-like secretory cells and collecting canals (Fig. 1B). The secretions of sac-like secretory are collected by the collecting duct and transferred to the reservoir. The reservoir is connected to the lateral glands by duct. The lateral glands are multitubular (Fig. 1C). The MTGs have paired openings located on the metathoracic basisternum in between mesocoxae and metacoxae (Fig. 1D). A usually depressed channel is long and extends from opening downward the middle of metathorax. The content of MTGs is probably released through the paired openings and spreads over the cuticle that surrounds each of the openings, known as evaporative areas, while the structure is a tongue-like structure. Bristle and mushroom-shaped cuticular structures are found on the surface of the evaporative areas. However, the bristle only locates the lateral area and points laterad (Fig. 1E). The mushroom-shaped cuticular structure is long and narrow, irregular polygonal surrounding around the peritreme of MTGs (Fig. 1F). Generally, mushroom-shaped cuticular structure extends out four to six ridges, and they are linked to other mushroom-shaped cuticular structure. Furthermore, mushroom-shaped cuticular structures are tightly connected to each other by numerous trabeculae.
Analysis of MTGs by GC-MS. The contents of MTGs from A. suturalis are analyzed separately by gender. Differences in quantitative or qualitative compositions of the substances are found between the two sexes. Nineteen different chemical substances are determined in the female, 21 different chemical substances are determined in the male, among which only 15 are common (Table 1). The results indicate that hexyl butyrate (85.44%) is most abundant, and heneicosane (0.07%) is least abundant in the females (Table 1; Fig. 2). Hexyl butyrate (84.12%) is the most abundant, whereas pentadecane (0.06%) is the least abundant in the male (Table 1; Fig. 3).

Discussion
This study examined the morphological structure of A. suturalis MTGs in detail by SEM. Moreover, we analyzed the chemical contents of MTGs in both males and females by utilizing GC-MS. The results indicated that there are not differences in between the two sexes. However, differences in quantitative or qualitative compositions of the MTGs volatiles are found between the two sexes.
As exhibited in Fig. 1, the structure of MTGs in A. suturalis resembled that of Nezara viridula (Gilby and Waterhouse 1967), constituting of paired lateral glands and one reservoir. Moreover, there was no sexual dimorphism in both female and male of A. suturalis. A previous study showed that the shape, size, and development of MTGs in a Reduvid bug varied depending on the nutritional state (Santos-Mallet and De Souza 1990). Here, we observed various differences in the reservoir related to the nutrition state, whereby in nonfed A. suturalis the glands only reached the metathoracic segment. In Hemiptera, there are two types of MTGs on the basis of the number of the opening, the diastomian and the omphalian types (Carayon 1971). Our findings indicated that the MTGs in A. suturalis belong to the diastomian type with paired openings.
In the opening and the evaporation area, there are diverse structures in different families of Hemiptera (Weirauch 2006). Cassis and Schuh (2012) reported that the evaporative areas of MTGs could be extensive in distribution, greatly reduced or absent in miridae. Our study showed that the evaporation area of A. suturalis is extensive in distribution. There are polygonal mushroom-shaped cuticular structures and bristles on the evaporation surface in A. suturalis. Furthermore, the mushroomshaped cuticular structure is present in the Pentatomomorpha, some Cimicomorpha, and Scutelleridae (Weirauch 2006, Durak andKalender 2007b). Remold (1963) reported that the mushroom-shaped cuticular structure could prevent the secretion spreading on the evaporative area because of its high viscosity and structure, which is regarded as obstacles to prevent further spreading. Moreover, Durak and Kalender (2009a) hypothesized that its function of the polygonal mushroom-shaped cuticular structure and ridge might have increased evaporation of the MTGs secretions in Graphosoma lineatum. We found that bristle only located on the outboard of the opening in A. suturalis. In several Reduvilldae, a row of bristles located the anterior lateral surface of each hind coxa. And while walking, the bristles brush over the opening of gland to disseminate the secretions (Schofield andUpton 1978, Weirauch 2006). The polygonal mushroom-shaped cuticular structure might have the functional roles as increasing evaporation of MTGs secretions. However, the function of bristles is unknown.
Our results indicated that the MTGs secretions of both A. suturalis sexes existed some differences in quantity and quality. The results we obtained agreed with the results of other authors. For example, Zhang and Aldrich (2008) reported that both sexes of Phytocoris call have some differences in quantity and quality by analyzing the MTGs content. Furthermore, principal component is hexyl butyrate in male P. call as we observed in A. suturalis. In our study, we found that hexyl isobutyrate, hexyl hexanoate, fumaric acid dec-4-enyl propyl ester, and tetradecane are only found in the female, whereas pentadecane, eicosane, tetracosane, nonadecane, hexadecane, and octacosane are only found in the male. Moveover, chemical analyses of the scent glands have been extensively studied in Hemiptera, e.g. Reduviidae, Scutelleridae, Pentatomidae, and Coreidae (Durak andKalender 2007a, 2009a;Marques et al. 2007;Moraes et al. 2008), which also show that the common compounds between male and female are different in the quantity. However, concerning on the qualitative variations of composition of MTGs secretions of A. suturalis, the biological function for these secretions is questionable and needs further study.
Hexyl butyrate is a major compound of MTGs secretions with diverse function in many mirids (Zhang and Aldrich 2003b). Some studies propose that the esters and aldehydes secreted by the MTGs of Miridae have a dual function depending on their concentration.
In Stenotus rubrovittatus and Lygus rugulipennis, this compound acts as one of sex attractant pheromones (Innocenzi et al. 2001, Yasuda et al. 2008). However, the male secretes to prevent other males from copulating with a female in Phytocoris difficilis, L. lineolaris, and Lygocoris pabulinus (Groot et al. 2001, Zhang et al. 2007, Zhang and Aldrich 2008. Furthermore, Ho and Millar (2002) demonstrated that this compound could attract scavenging chloropid and milichiid flies in Lygus spp. (Zhang and Aldrich 2003c). Hexyl butyrate was identified in both sex of A. suturalis, suggesting that this compound may have a dual function, and we are planning to investigate the functions of hexyl butyrate in A. suturalis both in the lab and the field.
(E)-2-hexenal is identified in female of A. suturalis. It has been attested as a defensive compound in ants, beetles, cockroaches, and true bugs (Staples et al. 2002). However, this compound also acts as an alarm pheromone in Cimex lectularius (Harraca et al. 2010). Moreover, it is found to be an aggregation or repellent pheromone in N. viridula according to the amount (Farine et al. 1992). 4-Oxo-(E)-2-hexenal is identified in both male and female of A. suturalis. It is a defensive compound to protect the nymphs against predation in C. hemipterus and C. lectularius (Harraca et al. 2010, Liedtke et al. 2011. Besides, Eliyahu et al. (2012) reported that the 4-oxo-(E)-2-hexenal is most abundant compound as the defensive secretions of fifth-instar nymphs of Chinavia aseada, Ch. impicticornis, Dichelops melacanthus, and Piezodorus guildinii. However, 4-oxo-(E)-2-hexenal is also identified to be one of sex-attractant pheromones in S. rubrovittatus and L. rugulipennis (Innocenzi et al. 2001, Yasuda et al. 2008. The authors suggest that this compound may have a dual function. Some studies revealed that the 1-hexanol is found in male and female volatiles of Campylomma verbasci and P. calli Knight as our result in the GC-MS investigation (Smith et al. 1991, Zhang andAldrich 2008). Chinta et al. (1994) found that 1-hexanol possesses EAD active for L. lineolaris. Moreover, Wardle et al. (2003) found that it may serve as allelochemics rather than pheromones in the study of inactivity of 1-hexanol against adult L. lineolaris. However, the biological function is still unknown in this insect.
2000; Durak andKalender 2007a, 2009a). In addition, their function is thought to be solvent and released substrates of other volatile compounds in other families of Hemiptera (Gunawardna andHerath 1991, Ho andMillar 2001). And octacosane is found to possess the function in penetrating the cuticle of enemies and delaying evaporation as other alkane (Blum and Brand 1972, Farine et al. 1992, Durak 2008. Krall et al. (1999) indicate that alkane may have some other functions in Cosmopepla bimaculata. Nevertheless, their biological function roles of alkanes in A. suturalis are unknown.
In conclusion, this study is the morphological description and chemical composition analysis of MTGs in A. suturalis. Ester, aldehyde, and alkane are the major compounds in MTGs contents. In mirid, ester, and aldehyde of MTGs, contents are primarily for defense and have role as sex or aggregation pheromones. Moreover, males of A. suturalis could be attractive to females (Zhang et al. 2011). Further studies are necessary to investigate the biological function of ester-aldehyde combination and each compound. In summary, this research has provided useful information on MTGs compounds of A. suturalis, and the results will be essential parts for further studies on functional roles and application in pest control of A. suturalis.