Volatile Terpenes and Terpenoids from Workers and Queens of Monomorium chinense (Hymenoptera: Formicidae)

Twenty-one volatile terpenes and terpenoids were found in Monomorium chinense Santschi (Hymenoptera: Formicidae), a native Chinese ant, by using headspace solid-phase microextraction (HS-SPME) coupled with gas-phase chromatography and mass spectrometry (GC-MS), which makes this ant one of the most prolific terpene producers in insect. A sesquiterpene with unknown structure (terpene 1) was the main terpene in workers and neocembrene in queens. Terpenes and terpenoids were detected in poison, Dufour’s and mandibular glands of both workers and queens. Worker ants raised on a terpene-free diet showed the same terpene profile as ants collected in the field, indicating that de novo terpene and terpenoid synthesis occurs in M. chinense.


Introduction
Terpenes and terpenoids are the largest group of natural products, mostly produced by plants, but also identified in other eukaryotes such as fungi, insects, amoebae, marine organisms and even prokaryotes, such as, bacteria [1][2][3]. They have drawn great attention from academia and industry due to not only their economic importance in pharmacy, agriculture, food and perfumery industry, but also their ecological significance in mediating antagonistic and beneficial interactions among organisms [4][5][6].
Approximately 55,000 terpenes have been reported in nature [7]. According to our literature survey, a total of 220 terpenes and terpenoids were reported in 9 orders of insects (Blattodea, Coleoptera, Diptera, Heteroptera, Homoptera, Hymenoptera, Isoptera, Lepidoptera and Phasmatodea, Table S1). Among them, about forty-five terpenes or terpenoids originated from ants (Hymenoptera: Formicidae) (Table S1). Terpene and terpenoids play significant roles as pheromones and defense compounds. In the subfamily Formicinae, a wide variety of monoterpenes are utilized as alarm pheromones, such as citronellal, citronellol, α-pinene, β-pinene, limonene and camphene [8]. In genera of Solenopsis and Monomorium of the subfamily Myrmicinae, farnesenes are often used as trail pheromones [9,10]. In the subfamily Ectatomminae, isogeraniol, a monoterpene might function as a recruitment signal in ant Rhytidoponera metallica [11]. In addition to the pheromonal role, terpenes and terpenoids are used as defensive compounds, such as iridomyrmecin (cyclopentanoid monoterpenes) and iridodials in some ant species in the subfamily Dolichoderinae [12,13]. However, functions of many terpenes
The identities of peaks 10, 13, 17, and 20 could not be finalized because there was no match in RTs with available standards, no match in KIs and AIs with any terpenes and terpenoids in the literature. Therefore the mass spectra of those peaks are presented: peak 10 (terpene 1), [55 (52) The mass spectrum of terpene 1 is similar to that of (5R*,7R*,10S*)-selina-4(14), 11-diene found in Nasutitermes [25]. Peak 20 (terpenoids 1) was considered sesquiterpenoid based on its molecular ion at m/z 232, predicting its molecular formulas C 15 H 20 O 2 . Mass spectra of other unknown terpenes from the ant were provided as well in the supporting material (Figures S10, S13, S17 and S20).

Influence of Diet on Terpene and Terpenoid Profile
TICs of whole-body samples of workers for a field colony (unknown diet), a laboratory colony (controlled diet: mealworm larvae and honey water) and an incipient colonies (terpene-free diet: sucrose water) are shown in Figure 5. Twenty-one terpenes and terpenoids were detected in both the field colonies and the laboratory colonies, in which natural diet were provided, all these compounds were observed also in the incipient colonies, which were fed with the sucrose solution. The squared Mahalanobis distance between field colonies and laboratory colonies, field colonies and incipient colonies, laboratory colonies and incipient colonies was 10.95 (F = 3.65, p = 0.12), 12.86 (F = 4.29, p = 0.09) and 7.88 (F = 2.63, p = 0.18), respectively and all p values were above 0.05, indicating that there was no significant difference of terpene contents among three treatments. Therefore, the terpenes and terpenoids, found in M. chinense workers in different treatments were not sequestered from their dietary sources. Some terpenes and terpenoids found in ant body parts were not detected in gland samples. For example, nine terpenes in the abdomen of workers and queens were not detected in both poison and Dufour's glands, including α-neocallitropsene (peak 6), β-chamigrene (peak 7), γ-curcumene (peak 8), aristolochene (peak 9), (Z)-α-bisabolene (peak 12), terpene 2 (peak 13), β-curcumene (peak 14), βsesquiphellandrene (peak 16) and terpene 3 (peak 17). Reduced abundance of all these compounds due to their evaporation during the dissection process may be the reason why they could not be detected by GC-MS in gland samples.

Influence of Diet on Terpene and Terpenoid Profile
TICs of whole-body samples of workers for a field colony (unknown diet), a laboratory colony (controlled diet: mealworm larvae and honey water) and an incipient colonies (terpene-free diet: sucrose water) are shown in Figure 5. Twenty-one terpenes and terpenoids were detected in both the field colonies and the laboratory colonies, in which natural diet were provided, all these compounds were observed also in the incipient colonies, which were fed with the sucrose solution. The squared Mahalanobis distance between field colonies and laboratory colonies, field colonies and incipient colonies, laboratory colonies and incipient colonies was 10.95 (F = 3.65, p = 0.12), 12.86 (F = 4.29, p = 0.09) and 7.88 (F = 2.63, p = 0.18), respectively and all p values were above 0.05, indicating that there was no significant difference of terpene contents among three treatments. Therefore, the terpenes and terpenoids, found in M. chinense workers in different treatments were not sequestered from their dietary sources.

Discussion
Twenty-one terpenes and terpenoids were detected from workers and twenty from queens of M. chinense. Previous studies showed that Pheidole sinaitica and Solenopsis geminata are the top terpene producers in ants (Hymenoptera: Formicidae) in term of numbers of terpenes and terpenoids detected. For example, the minor workers of P. sinaitica contained a mixture of more than 11 sesquiterpenes (farnesene-type hydrocarbons) [26]. The S. geminata queens produced 11 sesquiterpenes in the venom secretion and among them β-elemene was only tentatively identified [27]. The results indicate that M. chinense is an exceptional terpene producing ant.
In insects, 60 terpenes and terpenoids have been discovered from papilionid larvae (Lepidoptera: Papilionidae) and 53 from termite soldiers (Isoptera: Rhinotermitidae) (Table S1). They seem to be the top terpene and terpenoid producers in insects. In addition to 21 terpenes and terpenoids identified in M. chinense, 10 terpenes and terpenoids have been reported in M. minimum and five in M. pharaonic [28]. These results suggest that Monomorium ants may be one of the most potent terpene producers in insects.
Terpene 1, a sesquiterpene with unknown structure, was the main terpene in workers, followed by β-acoradiene and the remaining terpenes and terpenoids are all minor products. In addition to the major product, nearly half of all characterized monoterpene and sesquiterpene synthases in plants form significant number of minor products [35]. For example, the major sesquiterpene product of valencene synthase was identified as (+)-valencene (49.5% of total product), followed by (−)-7-epi-α-selinene (35.5%) along with five minor products [36]. It is possible that one or few terpene synthases in the workers of M. chinense may be responsible for such a diversity of terpenes and terpenoids.
Usually only one type of gland is involved in terpene and terpenoid production and/or storage in one species of insects, such as osmeteria glands in Papilionid (Lepidoptera: Papilionidae) larvae, frontal glands in termite soldiers (Isoptera: Rhinotermitidae, Serritermitidae, and Termitidae), and metasternal glands in longhorned beetles (Coleoptera: Cerambycidae) [37][38][39]. In contrast, in this study, the terpenes and terpenoids have been detected in three glands, including poison, Dufour's and mandibular glands in M. chinense workers and queens. A list of terpenes or terpenoids in ants with glandular source is summarized in Table S2. Monoterpenes and monoterpenoids have been discovered in rectum, mandibular, Dufour's, poison and pygidial glands in Formicinae, Myrmicinae, Dorylinae and Dolichoderinae. Although sesquiterpenes and diterpenes were mostly found from Dufour's gland, they were also detected in Mandibular, Dufour's or venom glands in Formicinae, Myrmicinae and Nothomyrmeciinae. The multiglandular origin of terpenes and terpenoids may make M. chinense a unique case in family Fomicidae, maybe even in the class Insecta.
Neocembrene was the major terpene produced in the Dufour's gland of M. chinense queens in contrast to its minor abundance in workers. This compound was found in the Dufour's gland in M. pharaonis queens, but not in the workers [24]. Whether neocembrene serves as queen pheromone in M. pharaonis remains questionable because it does not affect sexual brood rearing [40]. Besides two ant species mentioned above, neocembrene was detected in queens of other four species in the genus Monomorium, including M. minimum [41], M. floricola, M. destructor and M. hiten [42], indicating that neocembrene may be a genus-and queen-specific compound in genus Monomorium. Terpenes and terpenoids do not occur often in poison gland. When limonene, a monoterpene, was first found in poison glands of Myrmicaria species, it was considered an unusual case in Formicidae [43]. This study reports that sequiterpenes occur in the poison glands of worker ants. Typical poison gland chemistry of Monomorium species was dominated by alkaloids, which were believed to be the reason for them to successfully compete with the highly aggressive ant species [44,45]. This study reveals that not only alkaloids but also sesquiterpenes occur in the poison glands of M. chinense workers. Along with alkaloids, terpenes from the poison gland may act synergistically to provide higher toxicity or deterrence. However, the specific functions of these terpenes and terpenoids can only be clarified in the future research.
In plants, terpenoids function universally as primary metabolites, such as sterols, carotenoids, quinones, and hormones [46]. However, most of terpenes and terpenoids in plants are restricted to specific lineages and are involved in species-specific ecological interactions as secondary metabolites that may serve roles in plant defense and communication [47]. Terpenes identified in M. chinense, M. pharaonis and M. minimum, do not occur in genera outside Monomorium, indicating these terpenes and terpenoids may also lineage-specific (specialized) terpenoids. Thus, they are most likely also involved in the interaction with other organisms and environment, such as defense against enemies and diseases, or conspecific and heterospecific chemical communications.
All terpenes and terpenoids identified in this study have been found in plants. M. chinense is an omnivorous ant as other species in the genus Monomorium [48], so it was hypothesized that their diet is one potential source of these terpenes and terpenoids. However, ants raised on a terpene-free diet showed the same terpene profile as those of ants fed with natural diets, indicating that de novo terpene synthesis occurs in M. chinense. The terpene biosynthesis of bark beetles and flea beetles is well studied. Both beetles are oligophagous herbivores. Ivarsson et al. provided the first evidence that bark beetle Ips duplicatus can produce their main pheromone component, ipsdienol, a terpene alcohol [49]. Radiolabeling studies provided further evidence of the de novo biosynthesis of terpenes by bark beetles [50]. Geranyl diphosphate synthase of bark beetle Ips pini is the first animal prenyltransferase having terpene synthase activity [21]. No sesquiterpene synthases have been described in insects until the identification of an evolutionarily novel terpene synthase gene family in the striped flea beetle [51]. Terpene and terpenoid biosynthesis in ants have not really been studied by researchers. Considering the significance of terpenes and terpenoids in pharmacy, agriculture, food and perfumery industry, understanding and characterizing terpene synthases in ants may become important, since ant terpene synthase genes may provide us with new opportunities in bioengineering for production of high-valued terpenes and terpenoids. Due to its exceptional ability in terpene production, M. chinense may be a good model insect for study terpene biosynthesis in ants.

Maintenance of Field-Collected Ant Colonies
Nine colonies of M. chinense were collected in Guangzhou, Guangdong, China, and among them 3 colonies were collected from the campus of Guangdong Academy of Agriculture Science (GAAS) in July 2016, 3 from Baiyun district in April 2017 and 3 from Nansha district in April 2017. The colonies were reared in a 45 × 38 × 15 cm plastic container with the inner sides of the wall coated with Fluon F4-1 (Xingshengjie Sci and Tech Co., Ltd., Guangzhou, China) to prevent the escape of ants. Three glass test tubes (2.5 Φ × 19.5 cm) were placed in the container and used as artificial nests. Each tube was filled with 4-5 cm of water and a cotton plug was placed in the tube at the water level to retain the water. Tubes were covered with black paper to shield the light. Colonies were provided with minced mealworm, Tenebrio molitor, a cotton ball saturated with a 20% honey water solution, and a cotton ball with pure water in a Petri dish (7 × 1.5 cm). These colonies were maintained at 26 ± 2 • C and 12:12 (L:D) h photoperiod.

Establishment of Incipient Colonies
Incipient colonies were established by introducing newly dealate queens with 20 workers from the laboratory colonies into a container (45 × 38 × 15 cm). Once young workers emerged in the new colony, the old workers were removed. Colonies were provided with a cotton ball saturated with a 20% sucrose water solution, and a cotton ball with pure water in a Petri dish (7 × 1.5 m). They were maintained at 26 ± 2 • C and 12:12 (L:D) h photoperiod.

Ant Sample Preparation and Extraction by HS-SPME
About 100 live ant workers or 10 queens were put into a 2 mL vial (Agilent Technologies, Santa Clara, CA, USA). In order to facilitate the release of volatiles from the sample into the head space, the vial was placed into a −80 • C refrigerator for 10 min [28]. Headspace solid-phase micro-extraction (HS-SPME) was then conducted on the sample at room temperature (25 ± 1 • C) for 12 h using an 85 µm Polyacrylate SPME fiber (Supelco Inc., Bellefonte, PA, USA). In order to add C 8 to C 20 hydrocarbon standards to the sample, after the sample extraction, the same fiber was used to extract hydrocarbon standards for 1 min in another 2 mL vial. The hydrocarbon standards were prepared by adding 20 µL C 8 to C 20 solution (Sigma-Aldrich, St. Louis, MO, USA, 40 mg/L) into the vial and letting solvent evaporate in a fume hood. In order to facilitate evaporation of the solvent, the capped vial was shaken for 5 s before it was opened in a fume hood. After 1 min of evaporation, the vial was capped and shaken again for 5 s before it was reopened in the fume hood for 9 min. Before each SPME sample extraction, a blank run was performed and the fiber was cleaned in the GC injector for 30 min. There were 5 replicates for each colony.

Determination of Glandular Sources of Terpenes and Terpenoids
Each worker or queen was cut into three major body parts (head, thorax and abdomen) by a razor blade. Each type of body parts was placed into 2 mL vial that was subjected to SPME extraction as described as above. Since terpenes and terpenoids were found in the head and abdomen, the chemistry of the poison gland and Dufour's gland in abdomen and mandibular gland in head were investigated. Because poison gland and Dufour's gland are connected, they were first removed from the body under a stereo microscope (SZ61, Olympus, Tokyo, Japan) by grasping the terminal abdominal segments or the stinger with fine forceps and pulling posteriorly. The poison and Dufour's glands were separated with a dissecting needle. The mandibular gland was removed by grasping the mandible away from head, then separating the gland using a dissecting needle. After separation, each gland was directly placed on the tip of the SPME fiber, which then was inserted into the inject port of the GC-MS system (Agilent Technologies, Santa Clara, CA, USA).

Gas Chromatography and Mass Spectrometry (GC-MS)
The samples were analyzed using GC-MS Agilent 7890A-gas chromatograph coupled with 5975B-mass spectrometer. The analytical conditions were used as follows, splitless injection at 250 • C, DB-5 column (30 m × 0.25 mm i.d., 0.25 µm film thickness), the temperature program was from 60 • C to 246 • C at 3 • C. min −1 . Injector temperature was 220 • C and transfer line temperature 240 • C. The mass spectrometer was operated at 70 eV in the electron impact mode.
Terpenes and terpenoids were identified by comparing retention times (RT), AIs and KIs, and mass spectra of compounds with synthetic standards and compounds in literature [51]  Relative peak area of each terpene or terpenoid was calculated in percentage over the total area of all peaks. To estimate the difference of terpene composition between worker and queens, and the difference among three groups (field colonies, laboratory colonies and incipient colonies), a total of 21 terpene peak relative contents were used as variables in a principal component analysis and the principal components extracted were used as independent variables in the subsequent discriminant analysis and the squared Mahalanobis distances (D2) between the clusters were calculated. There were 3 replicates for each colony. STATISTICA 10.0 (Palo Alto, CA, USA), was used in statistical analyses.

Conclusions
In summary, twenty-one volatile terpenes and terpenoids were found in the Chinese ant, Monomorium chinense using headspace solid-phase micro-extraction (HS-SPME) coupled with gas-phase chromatography and mass spectrometry (GC-MS). The discovery makes M. chinense the most prolific terpene producer in ants. A sesquiterpene with unknown structure terpene 1 and neocembrene are the main terpene in the workers and queens, respectively. Most terpenes and terpenoids were found in the poison, Dufour's and/or mandibular glands. De novo terpenes and terpenoids synthesis are demonstrated in in M. chinense its workers. These findings suggest M. chinense is a novel and promising organism for the study of terpene function and biosynthesis in ants.

Supplementary Materials:
The following are available online. Mass spectra of terpenes and terpenoids showed in figures (Figures S1-S21); Terpenes and terpenoids in insects, terpenes and terpenoids in ants and their glandular source summarized in tables (Tables S1 and S2).