Male-Specific Substances Possibly Contributing to Nuptial Flight of the Japanese Carpenter Ant Camponotus Japonicus (Hymenoptera: Formicidae)

Male-Specific Substances Possibly Contributing to Nuptial Flight of the Japanese Carpenter Ant Camponotus Japonicus (Hymenoptera: Formicidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Male-Specific Substances Possibly Contributing to Nuptial Flight of the Japanese Carpenter Ant Camponotus Japonicus (Hymenoptera: Formicidae) Shunya Habe, Shigeru Matsuyama, Natsumi Kanzaki, Keiko Hamaguchi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4002899/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Sep, 2024 Read the published version in Journal of Chemical Ecology → Version 1 posted 9 You are reading this latest preprint version Abstract The nuptial flight of ants usually occurs during certain periods of the year. Alate females and males fly out of their nests to mate simultaneously. In the genus Camponotus , sex-specific chemicals are deposited in the male head capsule; however, their roles in nuptial flight have not yet been clarified. This study aimed to elucidate the functions of male-specific chemicals in the Japanese carpenter Camponotus japonicus . First, we identified three chemicals attributed to the male as authentic chemicals: methyl salicylate (MS), methyl 6-methyl salicylate (MMS), and methyl anthranilate (MA). Electroantennogram (EAG) responses to these three chemicals were confirmed to occur in male and female alates. Subsequently, the amounts of MMS and MA in the alate male head capsule were compared before and after flight under laboratory conditions. The amounts of both substances were significantly reduced after flight, suggesting that males secreted these substances during flight. Thereafter, a field trap experiment was conducted in three fields of the Kyoto Prefecture during the nuptial flight season in 2021 using MMS and MA as bites. The number of captured alate males was significantly higher than that of the females, suggesting that these compounds primarily attracted males rather than females. Considering the field conditions, if the local concentration of these chemicals is increased by male aggregation, females may be attracted because EAG responses were also observed in females. Our findings represent a first step toward understanding chemically mediated male lek formation during the process of male aggregation syndrome in this species. Camponotus japonicus nuptial flight methyl salicylate (MS) methyl 6-methyl salicylate (MMS) methyl anthranilate (MA). Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Mating flight, also known as nuptial flight, is an important reproductive event in most ant species (Cole 1983 ; Hölldobler and Wilson 1990 ; Negroni 2016; Eyer and Vargo 2021 ). To increase the chance of interbreeding, flight usually occurs in many conspecific colonies simultaneously during certain concentrated periods of the year when local weather conditions are appropriate (Boomsma and Leusink 1981 ) to provide an opportunity to meet and interbreed male and female alates from different colonies. The typical mating patterns fall into either female-calling syndrome (Hölldobler and Bartz 1985 ; Heinze and Tsuji 1995 ) or male-aggregation syndrome (Hölldobler and Wilson 1990 ). In the female-calling syndrome, females are often wingless, therefore, do not travel far from the nest and stand on the ground to release sex pheromones to call male alates (Hölldobler 1976 ; Villet et al. 1989 ; Mori et al. 2001 ; Ruano and Tinaut 2005 ; Peeters and Aron 2017 ). For the male-aggregation syndrome, male alates from many colonies congregate at specific mating sites that have prominent landscape features, such as sun-flecked clearings, hilltops, and tree crowns, whereas female alates fly into male swarms from long distances to mate (Cherix et al. 1993 ; O’Neill 1994 ; Wuellner 2000 , Peeters and Ito 2001 ; Peeters et al. 2013 ). Male alates release aggregates and sex pheromones to call conspecific male and female alates, respectively. Most species have one syndrome, either female calling or male aggregation; however, some species show both types of syndrome. (Franks et al. 1991 ). Female-produced sex pheromones in ants were first reported in the redwood ant Formica lugubris , in which n -undecene was identified as the major component, although the workers also possessed the same compound that acted as an alarm pheromone among the workers (Walter et al. 1993 ). According to Cherix et al. ( 1991 , 1993 ), mating occurs on the ground, and females fly off or cut off their wings after mating. Similar behavior has been confirmed in slave-making Polyergus ants, and methyl 6-methyl salicylate, 3-ethyl-4-methylpentanol, and methyl 3-ethyl-4-methylpentanoate have been identified as sex pheromones ( P. breviceps : Greenberg et al. 2004 ; P. rufescens : Castracani et al. 2005 , 2008 ; P. samurai : Iwamoto et al. 2020 ). In contrast, the red-imported fire ant, Solenopsis invicata , performs nuptial flights and matings 150–300 m above the ground (Markin et al. 1971 ; Milio et al. 1988 ). Both male and female alates possess 2-ethyl-3,6-dimethylpyrazine in their mandibular glands, which notably decreases after a flight (Choi and Vander Meer 2015). The same compound acts as an alarm pheromone among workers but may have a different ethological function as a sex pheromone for alates. In addition to these cases, phenomena that could be interpreted as male aggregation syndrome have been observed in several ant species; however, the male-derived sex pheromone component remains unknown. In Camponotus ants, males and females engage in nuptial flights by soaring aloft. During these flights, chemical communication mediated by sexual pheromones is postulated to occur between the male and female counterparts. The putative substances implicated in this process are compounds present in male mandibular glands (Torres et al. 2001 ; Brand et al. 1973 ). Although these compounds are hypothesized to promote female flight during nuptial flight (Hölldobler and Maschwitz 1965 ), studies on the behavioral effects of alate male compounds on alate females found in the air or near nest entrances are limited. The role of male ants within the nest is not well understood (Hölldobler and Wilson 1990 ), but as the primary role of male ants is reproduction, male mandibular gland secretion is likely to be important for their nuptial flight. This study aimed to elucidate both the structure and ethological functions of the chemical characteristics of alate males in the Japanese carpenter ant Camponotus japonicus and to investigate the possibility of chemical communication during their nuptial flight. Similar to other carpenter ants, males and females of this species enter the air during nuptial flight. Generally, such males are believed to form “lek” during nuptial flight to attract females; however, whether this is also applicable to C. japonicus remains unclear. Male-specific chemicals may provide lek-forming information. To investigate this possibility, we compared volatile chemicals among workers, soldiers, alate males, and females, focusing on their head capsules, where male-specific chemicals were detected in a preliminary analysis, and the changes in the amount of male-specific chemicals were quantified before and after the nuptial flight under controlled conditions. Furthermore, we used field trap experiments to evaluate the attraction effects of these volatile components on males and females during nuptial flight. MATERIALS AND METHODS Ants. Camponotus japonicus was collected from two different fields: the Kobe University campus (KU), Kobe, Japan, and the Kansai Research Center, Forestry and Forest Products Research Institute (FFPRI), Kyoto, Japan. Materials were collected from KU during the mating season from May to June 2008. The nest entrances were covered with unglazed flower pots, and approximately 30–50 female and male alates, with workers aggregated on the internal wall of the pot, were collected before flight. All these ants were transferred to a plastic bag, and then most of them were immediately stored in a freezer at -25°C. At FFPRI, four nests under a rock, concrete block, or metal plate were collected for three years of the flight season, May 2021–2023. The surface cover (rock, block, or plate) was removed, and colony members were collected either manually or using a vacuum cleaner. The members consisted of fertile queens, workers, and broods, and female and male alates were fed an appropriate amount of mealworms Tenebrio molitor , skim milk, and 50% maple syrup solution twice a week. The collected colonies were maintained separately in plastic containers of different sizes, depending on the number of members. Small plastic cases with wet plaster bottoms that served as artificial nests were placed in each container. A red plastic plate was placed on top of the artificial nest, and the inner walls of the container were coated with fluon (AGC Japan) to prevent the ants from escaping. They were kept in the dark at 15°C to avoid providing stimuli that could induce nuptial flights. Extraction and Collection of Volatile Chemicals. The ant bodies killed by freezing were dissected into three parts: head, thorax, and abdomen. Each part was ground individually, and the chemical substances were extracted with 1 mL of n -hexane for 3 min. Each extract was validated by electroantennography (EAG) and gas chromatography (GC) analyses. Apart from solvent extraction, each part was placed in a 2 mL glass vial and exposed to a 100 µm PDMS-SPME fiber (SPELCO, Sigma-Aldrich) for 1 min to collect volatile odors. The SPME fibers were conditioned at 250 ℃ for 1 min according to the prescribed method. The odor-adsorbed SPME fiber was immediately subjected to gas chromatography-mass spectrometry (GC-MS) and gas chromatography-elecytroantennogram detection (GC-EAD) analyses. EAG and GC Analyses . The EAG responses were recorded using an IDAC-2 (Syntech, Kirchzarten, Germany) and processed using software (AutoSpike ver. 3.9, Syntech). The antenna of each caste was removed from the head and cut into pedicels. The flagellum was placed on a silver electrode using an electrically conductive gel (ECG cream; Kenz). For stimulation, a puff of air containing the sample vapor was delivered using a glass syringe (15 mm i.d., 10 mL). One microliter of a hexane solution containing 0.01 head equivalent of the test sample was applied to the plunger head. After the solvent evaporated, the plunger was inserted into the barrel, leaving 1 mL of air in the syringe, which was kept for 5 min at room temperature before puffing. One milliliter of air containing the sample vapor was puffed into an airflow (300 mL/min, 18 cm/s) and delivered to the antenna. This was repeated 10–15 times using antennas from multiple individuals. GC analyses were conducted with Shimadzu GC 2014 equipped with an apolar capillary column DB-1HT (15 m length, 0.25 mm i.d., 0.1 µm film thickness) and a flame ionization detector. Helium was used as a carrier gas at a flow rate of 1 mL/min. The injection was made at splitless mode for 1 min at 300 ℃. The column oven temperature was set at 50 ℃ for 5 min, programmed to 300 ℃ at 10 ℃/min, then held at the final temperature for 5 min. The data were analyzed using a Shimadzu CR-8A chromatograph. GC-MS analysis was performed on a high-resolution mass spectrometer JMS-600H coupled with an Agilent GC 6890N equipped with an apolar capillary column HP-1MS (25 m length, 0.25 mm i.d., 0.25 µm film thickness). Helium was the carrier gas at a flow rate of 1 mL/min, and the injection was made in splitless mode for 1 min at 250°C. The column oven temperature was set at 40 ℃ for 1 min, programmed to 250 ℃ at 10 ℃/min, then held at the final temperature for 8 min. Both the interface temperature and the ion source temperature were set at 250 ℃. The EI mass spectra were obtained at 70 eV, and the scan range was m/z 40–500. GC-EAD analyses were conducted with Agilent GC6890N, which was tuned to bifurcate into FID and EAD by OP 275 Pro II sniffing port (GL Sciences Inc.). The FID signal was recorded and analyzed with Chemstation software (Ver. A10.01, Agilent Technologies). EAD signal was simultaneously acquired from an EAD probe (Syntech) through IDAC-2 data acquisition controller (Syntech) and recorded with AutoSpike (ver 3.9, Syntech). A nonpolar capillary column DB-5MS (30 m length, 0.25 mm i.d., 0.25 µm film thickness) was used for analyses. Both the injection port and flame ionization detector (FID) port were set at 280°C. The column oven temperature was initially set at 100 ℃, programmed to 320 ℃ at 10 ℃/min, then held at the final temperature for 3 min. Lab and field trials. To investigate the changes in the amounts of substances peculiar to alate males before and after flight, 45 male alates were equally separated into three groups. The males in the first group were immediately frozen and killed to pick up the head parts to quantify male-specific substances by GC analysis prior to flight. The males of the second and third groups were each placed in a small petri dish (Φ9 × 2 cm) as artificial nest, then in a large case (10.5 × 20 × 6 cm), and the large cases were left in the laboratory (2.5 × 2.5 × 5 m) in turn and allowed to fly freely. For the third group, 15 female alates were placed in a small case of the same size and then together in the same large case. After 1 h, all male alates were recaptured from the laboratory to quantify male-specific substances after the flight. All of these were freeze-killed, and chemicals were extracted from each head in 100 µL of n-hexane for 1 h, and then 50 ng of n-pentadecane (Nacalai Tesque) was added to each as an internal standard for quantification. Field trials were conducted to capture flying alate ants during the nuptial flight season in May 2021 at the Kamigamo Experimental Station (KES) and Uji campus (UC) of Kyoto University, Kyoto, and the FFPRI. As the chemical bait, 50 µL of hexane solution of the mixture of 250 µg of methyl 6-methyl salicylate (MMS) and 100 µg of methyl-2-aminobenzoate (MA) was applied on the rubber septa placed in the center on the adhesive sheets (24 × 30 cm) in the Delta traps (Sankei Chemical Co. Ltd.). A 50 µL of hexane alone was applied as the negative control. Pairs of traps with bait and controls were placed at a height of 1.5 to 2 m above the ground and at least 2 m apart. A total of 22 pairs of traps (4 pairs at KES, 8 pairs at UC, and 10 pairs at FFPRI) were set from May 2 to 28 in 2021. Both test and control sample solutions were replenished daily between 12:00 and 14:00. Damaged or dirty traps were replaced if necessary, and insects captured on each adhesive sheet were recorded daily for counting. Preparation of Candidate Chemical Compounds. Methyl salicylate (MS) and MA were purchased from Tokyo Chemical Industry Co., Ltd. MMS was prepared by the saponification of commercially available ethyl 6-methyl salicylate (4.0 g, 22.2 mmol, Tokyo Chemical Industry, Co., Ltd.) followed by methyl esterification with diazomethane. Ethyl 6-methyl salicylate (4.0 g, 22.2 mmol) was dissolved in ethanol (100 mL) in a round-bottomed one-neck flask. KOH pellets (12.3 g, 220 mmol, 10 equiv) were then added to the solution, which was further refluxed for 6 h using a Dimroth condenser. When the saponification was complete (as monitored by TLC), the solvent was evaporated using a rotary evaporator until it became crystalline. The mixture was then transferred to a separatory funnel with the aid of ice water and extracted with hexane (30 mL) to separate the unreacted ethyl ester. The aqueous layer was then acidified with 2N-HCl, extracted three times with diethyl ether, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated to yield crude 6-methylsalicylic acid, which reacted with diazomethane without purification. Crude 6-methylsalicylic acid was dissolved in diethyl ether and treated with an ether solution of diazomethane prepared analogously using the method described in the Organic Synthesis. After the diazomethane treatment, the solvent was carefully evaporated to give methyl 6-methyl salicylate (3.45 g, 20.8 mmol, 94% yield), which was further purified by SiO 2 column chromatography to yield pure material (3.14 g, 18.9 mmol, 85.1% yield). GC-MS [ m/z (%)]: 51 (6), 53 (5), 77 (12), 78 (15), 79 (5), 105 (14), 106 (25), 134 (100), 135 (23), 166 (39), 167 (4). GC: 11.050 min (HP-1MS), 13.863 min (FFAP). Statistical Analyses. Regarding laboratory trials, one-way analysis of variance (ANOVA) with post-hoc Tukey HSD (Honestly Significant Difference) was used to compare pre-and post-flight chemical possession of alate males with and without alate females in the same container. Regarding field trials, the effects of compound presentation on trap capture were summarized in a 2 × 2 table at each or all three fields KES, FFPRI, and UC, and Fisher’s exact tests were performed in one-sided confidence intervals. All these statistical analyses were conducted with a freeware, js-STAR XR+ (release 1.7.2.j)(Tanaka and Nappa 2022 ) RESULTS Identification of Male-Specific Chemicals. Comparative GC analysis of hexane extracts of body parts from alates and workers confirmed the volatile chemical characteristics of the male head (Fig. 1A-D). Peak a, which was common in workers, soldiers, and alate females, was identified as n -undecane. The characteristic male volatiles, including at least three compounds, B, C, and D, were also captured by the PDMS-SPME fiber (Fig. 1E), which exhibited molecular ions with m/z 152, 166, and 151, respectively. Based on the fragmentation patterns, they were identified as salicylic acid methyl ester (methyl salicylate), methyl salicylate with a methyl-substituted benzene ring at an unknown position (methyl x-methylsalicylate), and 2-aminobenzoic acid methyl ester (methyl 2-aminobenzate). Compound B was confirmed as methyl salicylate by GC co-injection with an authentic compound. For compounds B and C, all candidate isomers were synthesized, and their GC retention times and mass spectra were recorded (Table 1). The GC retention time of compound C was closest to that of methyl 6-methyl salicylate and similar to that of methyl 2-aminobenzoate (methyl anthranilate). EAG Responses Against the Quantitative Major Compounds . The typical and reproducible EAG responses of alate and worker ants are shown in Fig. 2. In all those castes, alate male head extract (MHE) at 0.01–0.02 ant equivalent, as well as synthesized MMS and MA at 0.01 ant equivalent, evoked EAG responses. Although the workers’ EAG responses to MA were also confirmed, the data are not shown. Both alate female head extract (FHE) and worker’s head extract (WHE), as well as n- undecane (UD), evoked EAG responses at 0.02 ant equivalent in the workers and the alate females but not in the alate males (data not shown). The alate female antennae tended to be more responsive to MHE than to FHE or WHE. The EAG responses of alate and worker ants were confirmed to be components of MMS and MA, both in MHE and the mixture of synthetic MMS and MA (Fig. 3). Lab Trials to Compare Pre- and Post-Flight Male Chemicals. Fig. 4 compares the MMS and MA contents of alate males before and after the flight. Quantitative GC analysis estimated pre-flight MMS and MA to be 38.55 ± 23.59 and 13.68 ± 7.56 μg per male (n=10), whereas post-flight MMS and MA were 14.98 ± 23.59 and 6.59 ± 2.59 μg (n=9) in the absence of the females, but 16.93 ± 9.70 and 8.93 ± 5.48 μg (n=10) in the presence of the females, respectively. MMS was significantly decreased after flight regardless of the presence of the females ( P <0.05, ANOVA with post hoc Tukey test). MA showed a similar trend but was significantly reduced when flying in the absence of females. Field Trials to Capture Female and Male Alates by M6M and MA . Within the three fields for bait trap experiments, a total of 12 sets of traps were used for KES (four sets) and UC (10 sets), but only one trap treated with MMS and MA from FFPRI captured one male alate (Table 2). Alate ants were not captured regardless of the presence or absence of MMS and MA treatment. However, at the UC, multiple alate males were captured in four of ten trap sets only when treated with MMS and MA. By focusing on the number of captures at the UC, the number of traps treated with MMS and MA was significantly higher than that of the control traps (Two-way ANOVA, P <0.05). Significant differences were also observed in the number of captures by the target caste (two-way ANOVA, P <0.05), and alate males tended to be captured in large numbers (multiple comparisons using Bonferroni, P <0.05). DISCUSSION We identified three volatile compounds in male C. japonicus collected using SPME: MS, MMS, and MA (Fig. 1 and Table 1 ). These compounds were also detected in worker ants; however, their relative amounts were small, and n -undecane was the main component. Therefore, these three volatiles were considered to have male characteristics (Fig. 1 ). EAG responses were confirmed in all alate males, females, and workers to these three male-derived components (Figs. 2 and 3 ); however, EAG responses were not confirmed in males to head extracts from females or workers. This suggests that male-specific behavior during nuptial flight, that is, male lek formation, is caused by male-characteristic compounds. Because alate females also respond to male-characteristic compounds, these would function as communicative signals related to mating. Quantitative analysis revealed that the average value of MMS, the most abundant compound, decreased from 38.54 ± 23.59 µg per male to 14.98 ± 7.13 µg per male before and after flight (Fig. 3 ). This is probably because alate males secrete the compound during nuptial flights, which promotes alate male swarming or attracts alate females. This hypothesis was tested in a field trap experiment that compared the number of ants captured in sticky board traps with and without the addition of male compounds. Although the number of captures was not overwhelming, significantly more alate males were captured in traps with male compounds (Fig. 3 ). Although further verification is required, these results indicate that these male compounds may function as pheromones to attract alate males during nuptial flight in this species. MMS, identified as the characteristic compound in males of C. japonicus , has previously been reported to be present in males of more than 20 species of American Camponotus (Brand et al. 1973 a, b; Llyod et al. 1984; Torres et al. 2001 ). Some of these species produce detectable quantities of volatile compounds only in males, suggesting that male-specific compounds may provide a way of comprehending their caste-specific flights during mating. Workers in some Camponotus species also possess this compound, which is believed to function in alarm communication (Brand et al. 1973 a. 1999). These results will likely also apply to C. japonicus . MMS has also been reported in the mandibular gland content of workers of the ponerine ant (Duffield and Blum, 1975a, b; Longhurst et al. 1980 ) and those of both workers and queens of Polyergus species (Greenberg et al. 2004 ). The Polyergus queen serves as a sex pheromone that attracts alate males (Greenberg et al. 2004 ). More recently, MMS has been identified as a major component of the trail pheromone of Tetramorium impurum (Morgan et al. 1990 )d tsushimae (Nakamura et al. 2019 ), and they secrete this compound from the poison and Dufour’s gland, respectively. In carpenter ants, although the relationship between MMS and the mandibular gland has not been confirmed, the localization of MMS (that is, the compound was isolated from the head capsule) suggests that it is deposited in and secreted from the mandibular gland. Laboratory bioassays of C. japonicus confirmed a significant decrease in the amount of MMS retained by alate males after flight (Fig. 3 ), suggesting that alate males release this compound during their flight. A similar phenomenon has been confirmed in Solenopsis invicta , in which alate males and females possess 2-ethyl-3,6-dimethylpyrazine in their mandibular glands (Choi and Vander Meer 2015). The pyrazine compound was significantly reduced in both males and females after nuptial flights, suggesting its potential role as a sex pheromone in nuptial flight behaviors, such as male-lek formation. Although this type of comparative analysis is rare for ant species, notably, similar trends were observed in S. invicta as well as C. japonicus . Furthermore, in this laboratory experiment, male flight altitude was evidently lower than that during conjugal flight, suggesting that flight behavior may be an important stimulus for the release of male pheromones. Such a case has been reported in Dasylepida beetle, where flight behavior stimulates female sex pheromone secretion (Fukuya et al. 2009 ). Significantly more males were captured in traps treated with an artificial blend of male alate compounds. This suggests that the blend has an attractive effect on flying alate males. However, field assays require review and validation because the number of catches per trap is generally low. Possibly, this is a result of difficulties in developing the necessary bioassays for high-flying ants, as noted by Choi and Vander Meer (2015) for fire ants that mate at approximately 150 m in the air. Abe ( 1973 ) reported the nuptial flight behavior of C. japonicus ; however, its mating height is not well understood. Although Woyciechowski ( 1990 ) stated that the male-lek-forming type of nuptial flight is risky, it is advantageous for avoiding inbreeding. To verify this, understanding the site where the male lek is formed is necessary, but this has not been possible in many species other than Myrmica ants (Kannnowski 1963; Elmes 1982 ; Woyciechowski 1990 ). To elucidate the pheromonal communication involved in nuptial flight, constructing a bioassay method based on the location of lek formation is fundamental and apparently necessary to perform the bioassays at the right time and right place on the day of nuptial flights. MMS, MS, and MA are characteristic of alate males but are not specific. As all castes showed EAG responses to these compounds, they may be context-dependent multifunctional pheromones. Choi and Vander Meer (2015) reported that all the adult castes of S. invicta have 2-ethyl-3,6-dimethylpyrazine in their mandibles, which is used as a sex pheromone between the alate males and females but functions as an alarm pheromone among workers. This context-dependent multifunctionality is considered to allow social insects to develop complex chemical communication systems and use a small number of chemicals to maintain social behavior (Mitaka and Akino 2021 ). Regarding C. japonicus , chemical information is input into the brain through three compounds. Therefore, behaviorally and physiologically, verifying changes in function between castes is essential. The results obtained from this experiment do not directly observe pheromone communication during the mating flight of C. japonicus . They are merely inferred through chemical analysis. Therefore, continuous detailed field observations will be necessary for future studies. Declarations FUNDING This work was supported by JSPS KAKENHI Grant Numbers JP20K06073. Competing Interests The authors have no relevant financial or non-financial interests to disclose. AUTHOR CONTRIBUTIONS Shunya Habe designed the study, the main conceptual ideas, the proof outline, and collected the data. Shigeru Matsuyama synthesized the candidate chemicals applied in the experiments. Natsumi Kanzaki, Keiko Hamaguchi, Mamiko Ozaki, and Toshiharu Akino aided in interpreting the results and worked on the manuscript. Mamiko Ozaki and Toshiharu Akino supervised the project. Shunya Habe wrote the manuscript with support from Toshiharu Akino. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Abe T (1973) On the behavior of the ant Camponotus japonicus at the nuptial flight. Kontyu 41:333–341 Boomsma JJ, Leusink A (1981) Weather conditions during nuptial flights of four European ant species. 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(2004) Sex pheromone of queens of the slave making ant, Polyergus breviceps . J Chem Ecol 30: 1297–1303. https://doi.org/10.1023/B:JOEC.0000030300.11787.01 Greenberg L, Johnson CA, Trager JC, et al. (2018) Sex attractant pheromones of virgin queens of sympatric slave-making ant species in the genus Polyergus , and their possible roles in reproductive isolation. J Chem Ecol 44: 547–555. https://doi.org/10.1007/s10886-018-0966-9 Heinze J, Tsuji K (1995) Ant reproductive strategies. Res Popul Ecol 37: 135–149. https://doi.org/10.1007/BF02515814 Hölldobler B (1976) The behavioral ecology of mating in harvester ants (Hymenoptera: Formicidae: Pogonomyrmex). Behav Ecol Sociobiol 1: 405–423. https://doi.org/10.1007/BF00299401 Hölldobler B, Maschwitz U (1965) Der hochzeitsschwarm der rossameise Camponotus herculeanus L. Zeitsehrift fiir vergleiehende Physiologie 50: 551–68. https://doi.org/10.1007/BF00355658 Hölldobler B, Bartz SH (1985) Sociobiology of reproduction in ants. Exp Behav Ecol Sociobiol 31: 237–257 Hölldobler B, Wilson EO (1990) The ants. Harvard University Press, Cambridge, Massachusetts. Iwamoto N, Nakamuta K, Millar JG (2020) Sex attractant pheromone of the Japanese slave-making ant, Polyergus samurai . J Chem Ecol 46: 520–523. https://doi.org/10.1007/s10886-020-01179-1 Kannowski PB (1963) The flight activities of formicine ants. Symp Genet Biol 12: 74–102 Lloyd HA, Blum MS, Duffield RM (1975) Chemistry of the male mandibular gland secretion of the ant, Camponotus clarithorax . Insect Biochem 5: 489–494. https://doi.org/10.1016/0020-1790(75)90032-3 Lloyd HA, Schmuff NR, Hefetz A (1984) Chemistry of the male mandibular gland secretion of the carpenter ant, Camponotus thoracicus fellah emery. Comp Biochem Physiol B: Comp Biochem 78: 687–689. https://doi.org/10.1016/0305-0491(84)90119-6 Longhurst C, Baker R, Howse PE, (1980) A comparative analysis of mandibular gland secretions in the ant tribe Tetramoriini. Insect Biochem 10: 107–112. https://doi.org/10.1016/0020-1790(80)90046-3 Markin GP, Dillier JH, Hill SO, et al. (1971) Nuptial flight and flight ranges of the imported fire ant, Solenopsis saevissima richteri (Hymenoptera: Formicidae). J Georgia Entomol Soc 6: 145–156 Milio J, Lofgren CS, Williams DF (1988) Nuptial flight studies of field-collected of Solenopsis invicta Buren. In Trager JC (ed) Advances in Myrmecology, Brill, Leiden, pp 419–431. https://doi.org/10.1163/9789004630765_030 Mitaka Y, Akino T (2021) A Review of termite pheromones: Multifaceted, context-dependent, and rational chemical communications. Front Ecol Evol 8: 595614. https://doi.org/10.3389/fevo.2020.595614 Morgan ED, Jackson BD, Ollett DG, Sales GW (1990) Trail pheromone of the ant Tetramorium impurum and model compounds: Structure-activity comparisons. J Chem Ecol 16: 3493–3510. https://doi.org/10.1007/BF00982113 Mori A, Grasso DA, Visicchio R, et al. (2001) Comparison of reproductive strategies and raiding behaviour in facultative and obligatory slave-making ants: the case of Formica sanguinea and Polyergus rufescens. Insectes Soc. 48: 302–314. https://doi.org/10.1007/PL00001782 Nakamura T, Harada K, Akino T (2019) Identification of methyl 6‑methylsalicylate as the trail pheromone of the Japanese pavement ant Tetramorium tsushimae (Hymenoptera: Formicidae). Appl Entomol Zool 54: 297–305. https://doi.org/10.1007/s13355-019-00626-0 Negroni MA, Jongepier E, Feldmeyer B, et al. (2016) Life history evolution in social insects: a female perspective. Curr Opin Insect Sci 16: 51–57. https://doi.org/10.1016/j.cois.2016.05.008 O’Neill KM (1994) The male mating strategy of the ant Formica subpolita Mayr (Hymenoptera: For): Swarming, mating, and predation risk. J Entomol 101: 93–108. https://doi.org/10.1155/1994/38217 Payne TL, Blum MS, Duffield RM (1975) Chemoreceptor responses of all castes of a carpenter ant to male-derived pheromones. Ann Entomol Soc Am 68: 385–386. https://doi.org/10.1093/aesa/68.2.385 Peeters C, Aron S (2017) Evolutionary reduction of female dispersal in Cataglyphis desert ants. Biol J Linn Soc 122: 58–70. https://doi.org/10.1093/biolinnean/blx052 Peeters C, Ito F (2001) Colony dispersal and the evolution of queen morphology in social Hymenoptera. Annu Rev Entomol 46: 601–630. https://doi.org/10.1146/annurev.ento.46.1.601 Peeters C, Lin CC, Quinet Y, et al. (2013) Evolution of a soldier caste specialized to lay unfertilized eggs in the ant genus Crematogaster (subgenus Orthocrema). Arthropod Struct Dev 42: 257–264. https://doi.org/10.1016/j.asd.2013.02.003 Ruano F, Tinaut A (2005) Mating behaviour in a slave-making ant, Rossomyrmex minuchae (Hymenoptera, Formicidae). Naturwissenschaften 92: 328–331. https://doi.org/10.1007/s00114-005-0633-1 Tanaka S, Nappa (2022) js-STAR XR+. https://www.kisnet.or.jp/nappa/software/star/info/new.htm, lasy accessed on Sep11. 2023 Torres JA, Snelling RR, Canals M (2001) Seasonal and nocturnal periodicities in ant nuptial flights in the tropics (Hymenoptera: Formicidae). Sociobiology 37: 601–626 Villet M, Crewe R, Robertson H (1989) Mating behavior and dispersal in Paltothyreus tarsatus Fabr. (Hymenoptera, Formicidae). J. Insect Behav 2: 413-417. https://doi.org/10.1007/BF01068065 Walter F, Fletcher DJC, Chautems D, et.al. (1993) Identification of the sex pheromone of an ant, Formica lugubris (Hymenoptera, Formicidae). Naturwissenschaften 80: 30–34. https://doi.org/10.1007/BF01139755 Woyciechowski M (1990) Mating behaviour in the ant Myrmica rubra (Hymenoptera, Formicidae). Acta Zool Cracov 33: 565–574 Wuellner CT (2000) Male aggregation by Solenopsis richteri Forel (Hymenoptera: Formicidae) and associated mating behavior in Argentina. J Insect Behav 13: 751–756. https://doi.org/10.1023/A:1007852229186 Tables Tables 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files TABLES.docx Cite Share Download PDF Status: Published Journal Publication published 21 Sep, 2024 Read the published version in Journal of Chemical Ecology → Version 1 posted Editorial decision: Revision requested 13 May, 2024 Reviews received at journal 08 May, 2024 Reviewers agreed at journal 16 Apr, 2024 Reviews received at journal 14 Apr, 2024 Reviewers agreed at journal 27 Mar, 2024 Reviewers invited by journal 27 Mar, 2024 Editor assigned by journal 27 Mar, 2024 Submission checks completed at journal 25 Mar, 2024 First submitted to journal 01 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4002899","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":284704267,"identity":"1c095e0c-bf9a-41a1-ad70-7421905c1224","order_by":0,"name":"Shunya Habe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBACAwkGNiBlw8zGzHwAyJCQIVZLGjsfO1sCSAsPsVoO88vx8xiABAhrMZduf/a4sI1Zmo2Z5/OrGzUWPAzsh49uwKfFcs4Zc+OZbWzGbMy826xzjgEdxpOWdgOvw27ksEnztvEkg7QY57ABtUjwmBHQkv4MqEWivo2Z55lxzj+itCSYAbUYAAOZh/lxbhsxWu6cMZPmOZcA1MJmxpzbJ8HDRtAvt9ufSfOU/WeW7z/8+HPOtzo5fvbDx/BqAQNGNjDFJgEmCSoHgz9gkvkDcapHwSgYBaNgpAEARoE9Bp4qu4MAAAAASUVORK5CYII=","orcid":"","institution":"Kyoto Institute of Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shunya","middleName":"","lastName":"Habe","suffix":""},{"id":284704269,"identity":"25731238-512e-4790-b4ed-c8275b3663d4","order_by":1,"name":"Shigeru Matsuyama","email":"","orcid":"","institution":"University of Tsukuba","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shigeru","middleName":"","lastName":"Matsuyama","suffix":""},{"id":284704271,"identity":"551bbbcd-f5b0-4bef-b6e9-2b86ac053ca2","order_by":2,"name":"Natsumi Kanzaki","email":"","orcid":"","institution":"Forestry and Forest Products Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Natsumi","middleName":"","lastName":"Kanzaki","suffix":""},{"id":284704273,"identity":"0c82731c-f394-4be1-8886-22b454cadb34","order_by":3,"name":"Keiko Hamaguchi","email":"","orcid":"","institution":"Forestry and Forest Products Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Keiko","middleName":"","lastName":"Hamaguchi","suffix":""},{"id":284704274,"identity":"e5f81869-e0c0-4e1b-8696-994cddf1e1a5","order_by":4,"name":"Mamiko Ozaki","email":"","orcid":"","institution":"Nara Women's University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mamiko","middleName":"","lastName":"Ozaki","suffix":""},{"id":284704276,"identity":"1d58ffb5-7bb2-49ef-a80f-e1738c701347","order_by":5,"name":"Toshiharu Akino","email":"","orcid":"","institution":"Kyoto Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Toshiharu","middleName":"","lastName":"Akino","suffix":""}],"badges":[],"createdAt":"2024-03-01 10:33:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4002899/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4002899/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10886-024-01548-0","type":"published","date":"2024-09-21T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53668205,"identity":"8c7e88fb-110e-4386-936c-30a22cc4526b","added_by":"auto","created_at":"2024-03-28 17:17:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47380,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis results of volatile compounds from the head of\u003cem\u003e C. japonicus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA~C are mass chromatogram of volatiles collected from a crushed head of \u003cem\u003eC. japonicus\u003c/em\u003e: A=Alate male, B=Alate female, C=Worker. D is mass chromatogram of volatiles collected by SPME from crushed head of the \u003cem\u003eC.japonicus\u003c/em\u003e. E is Mass spectra of peak b, c, d. Peak identifications: a=undecane, b=methylsalicilate, c=methyl 6-methylsalicilate, d=methyl anthranilate\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4002899/v1/b75358610aee899e4a4f192b.jpg"},{"id":53668208,"identity":"28a45fcb-29b1-4bbd-988b-891c0daae8f2","added_by":"auto","created_at":"2024-03-28 17:17:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80045,"visible":true,"origin":"","legend":"\u003cp\u003eEAG responses of antennae from each caste of \u003cem\u003eC. japonicus \u003c/em\u003eto various volatile compounds\u003c/p\u003e\n\u003cp\u003eMA: methyl anthranilate, MMS: methyl 6 methyl salicilate, MS: methyl salicylate, MHE: alate male head extract, FHE: alate female head extract, WHE: worker head extract\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4002899/v1/04a3702e9307f2b3b746affc.jpg"},{"id":53668206,"identity":"f0672d6c-5483-47a8-b2f2-375fb7532e9e","added_by":"auto","created_at":"2024-03-28 17:17:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32413,"visible":true,"origin":"","legend":"\u003cp\u003eGC-EAD responses of \u003cem\u003eC. japonicus\u003c/em\u003e antennae to volatile compounds from male heads\u003c/p\u003e\n\u003cp\u003ePeak c is methyl 6-methylsalicilate, peak d is methyl anthranilate.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4002899/v1/cff393c5b93f0990947cc33f.jpg"},{"id":53668209,"identity":"c13015b2-fdb7-419c-99d0-63f5c8857a15","added_by":"auto","created_at":"2024-03-28 17:17:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24269,"visible":true,"origin":"","legend":"\u003cp\u003eAmounts of methyl-6-methylsalicylic acid and methyl-anthranilic acid per male of \u003cem\u003eC. japonicus \u003c/em\u003ebefore and after flight in laboratory\u003c/p\u003e\n\u003cp\u003eTen alate males were confirmed in the same container as 10 workers, or 10 workers and 10 females (Alate female – and +, respectively). Different letters mean statistically significant differences (p \u0026lt; 0.05, ANOVA with post-hoc Tukey’s test)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4002899/v1/6e4b3e0482a0ffc3d07c730d.jpg"},{"id":65104008,"identity":"16501905-7c08-4e5d-9bc7-18bc248928a8","added_by":"auto","created_at":"2024-09-23 16:10:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":532163,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4002899/v1/126c1d87-f0a9-4bb3-87a5-fcfc8801409f.pdf"},{"id":53668207,"identity":"aeb982be-4e07-4404-a473-551ca5647f91","added_by":"auto","created_at":"2024-03-28 17:17:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":261943,"visible":true,"origin":"","legend":"","description":"","filename":"TABLES.docx","url":"https://assets-eu.researchsquare.com/files/rs-4002899/v1/a55bd7a3f7463975ad1981cf.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eMale-Specific Substances Possibly Contributing to Nuptial Flight of the Japanese Carpenter Ant Camponotus Japonicus (Hymenoptera: Formicidae)\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMating flight, also known as nuptial flight, is an important reproductive event in most ant species (Cole \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; H\u0026ouml;lldobler and Wilson \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Negroni 2016; Eyer and Vargo \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To increase the chance of interbreeding, flight usually occurs in many conspecific colonies simultaneously during certain concentrated periods of the year when local weather conditions are appropriate (Boomsma and Leusink \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) to provide an opportunity to meet and interbreed male and female alates from different colonies. The typical mating patterns fall into either female-calling syndrome (H\u0026ouml;lldobler and Bartz \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Heinze and Tsuji \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) or male-aggregation syndrome (H\u0026ouml;lldobler and Wilson \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). In the female-calling syndrome, females are often wingless, therefore, do not travel far from the nest and stand on the ground to release sex pheromones to call male alates (H\u0026ouml;lldobler \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Villet et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Mori et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Ruano and Tinaut \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Peeters and Aron \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For the male-aggregation syndrome, male alates from many colonies congregate at specific mating sites that have prominent landscape features, such as sun-flecked clearings, hilltops, and tree crowns, whereas female alates fly into male swarms from long distances to mate (Cherix et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; O\u0026rsquo;Neill \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Wuellner \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Peeters and Ito \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Peeters et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Male alates release aggregates and sex pheromones to call conspecific male and female alates, respectively. Most species have one syndrome, either female calling or male aggregation; however, some species show both types of syndrome. (Franks et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFemale-produced sex pheromones in ants were first reported in the redwood ant \u003cem\u003eFormica lugubris\u003c/em\u003e, in which \u003cem\u003en\u003c/em\u003e-undecene was identified as the major component, although the workers also possessed the same compound that acted as an alarm pheromone among the workers (Walter et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). According to Cherix et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1991\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), mating occurs on the ground, and females fly off or cut off their wings after mating. Similar behavior has been confirmed in slave-making \u003cem\u003ePolyergus\u003c/em\u003e ants, and methyl 6-methyl salicylate, 3-ethyl-4-methylpentanol, and methyl 3-ethyl-4-methylpentanoate have been identified as sex pheromones (\u003cem\u003eP. breviceps\u003c/em\u003e: Greenberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; P. \u003cem\u003erufescens\u003c/em\u003e: Castracani et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; P. \u003cem\u003esamurai\u003c/em\u003e: Iwamoto et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In contrast, the red-imported fire ant, \u003cem\u003eSolenopsis invicata\u003c/em\u003e, performs nuptial flights and matings 150\u0026ndash;300 m above the ground (Markin et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Milio et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). Both male and female alates possess 2-ethyl-3,6-dimethylpyrazine in their mandibular glands, which notably decreases after a flight (Choi and Vander Meer 2015). The same compound acts as an alarm pheromone among workers but may have a different ethological function as a sex pheromone for alates. In addition to these cases, phenomena that could be interpreted as male aggregation syndrome have been observed in several ant species; however, the male-derived sex pheromone component remains unknown.\u003c/p\u003e \u003cp\u003eIn Camponotus ants, males and females engage in nuptial flights by soaring aloft. During these flights, chemical communication mediated by sexual pheromones is postulated to occur between the male and female counterparts. The putative substances implicated in this process are compounds present in male mandibular glands (Torres et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Brand et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). Although these compounds are hypothesized to promote female flight during nuptial flight (H\u0026ouml;lldobler and Maschwitz \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1965\u003c/span\u003e), studies on the behavioral effects of alate male compounds on alate females found in the air or near nest entrances are limited. The role of male ants within the nest is not well understood (H\u0026ouml;lldobler and Wilson \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), but as the primary role of male ants is reproduction, male mandibular gland secretion is likely to be important for their nuptial flight.\u003c/p\u003e \u003cp\u003eThis study aimed to elucidate both the structure and ethological functions of the chemical characteristics of alate males in the Japanese carpenter ant \u003cem\u003eCamponotus japonicus\u003c/em\u003e and to investigate the possibility of chemical communication during their nuptial flight. Similar to other carpenter ants, males and females of this species enter the air during nuptial flight. Generally, such males are believed to form \u0026ldquo;lek\u0026rdquo; during nuptial flight to attract females; however, whether this is also applicable to \u003cem\u003eC. japonicus\u003c/em\u003e remains unclear. Male-specific chemicals may provide lek-forming information. To investigate this possibility, we compared volatile chemicals among workers, soldiers, alate males, and females, focusing on their head capsules, where male-specific chemicals were detected in a preliminary analysis, and the changes in the amount of male-specific chemicals were quantified before and after the nuptial flight under controlled conditions. Furthermore, we used field trap experiments to evaluate the attraction effects of these volatile components on males and females during nuptial flight.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cem\u003eAnts. Camponotus japonicus\u003c/em\u003e was collected from two different fields: the Kobe University campus (KU), Kobe, Japan, and the Kansai Research Center, Forestry and Forest Products Research Institute (FFPRI), Kyoto, Japan. Materials were collected from KU during the mating season from May to June 2008. The nest entrances were covered with unglazed flower pots, and approximately 30\u0026ndash;50 female and male alates, with workers aggregated on the internal wall of the pot, were collected before flight. All these ants were transferred to a plastic bag, and then most of them were immediately stored in a freezer at -25\u0026deg;C. At FFPRI, four nests under a rock, concrete block, or metal plate were collected for three years of the flight season, May 2021\u0026ndash;2023. The surface cover (rock, block, or plate) was removed, and colony members were collected either manually or using a vacuum cleaner. The members consisted of fertile queens, workers, and broods, and female and male alates were fed an appropriate amount of mealworms \u003cem\u003eTenebrio molitor\u003c/em\u003e, skim milk, and 50% maple syrup solution twice a week. The collected colonies were maintained separately in plastic containers of different sizes, depending on the number of members. Small plastic cases with wet plaster bottoms that served as artificial nests were placed in each container. A red plastic plate was placed on top of the artificial nest, and the inner walls of the container were coated with fluon (AGC Japan) to prevent the ants from escaping. They were kept in the dark at 15\u0026deg;C to avoid providing stimuli that could induce nuptial flights.\u003c/p\u003e \u003cp\u003e \u003cem\u003eExtraction and Collection of Volatile Chemicals.\u003c/em\u003e The ant bodies killed by freezing were dissected into three parts: head, thorax, and abdomen. Each part was ground individually, and the chemical substances were extracted with 1 mL of \u003cem\u003en\u003c/em\u003e-hexane for 3 min. Each extract was validated by electroantennography (EAG) and gas chromatography (GC) analyses. Apart from solvent extraction, each part was placed in a 2 mL glass vial and exposed to a 100 \u0026micro;m PDMS-SPME fiber (SPELCO, Sigma-Aldrich) for 1 min to collect volatile odors.\u003c/p\u003e \u003cp\u003eThe SPME fibers were conditioned at 250 ℃ for 1 min according to the prescribed method. The odor-adsorbed SPME fiber was immediately subjected to gas chromatography-mass spectrometry (GC-MS) and gas chromatography-elecytroantennogram detection (GC-EAD) analyses.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEAG and GC Analyses\u003c/em\u003e. The EAG responses were recorded using an IDAC-2 (Syntech, Kirchzarten, Germany) and processed using software (AutoSpike ver. 3.9, Syntech). The antenna of each caste was removed from the head and cut into pedicels. The flagellum was placed on a silver electrode using an electrically conductive gel (ECG cream; Kenz). For stimulation, a puff of air containing the sample vapor was delivered using a glass syringe (15 mm i.d., 10 mL). One microliter of a hexane solution containing 0.01 head equivalent of the test sample was applied to the plunger head. After the solvent evaporated, the plunger was inserted into the barrel, leaving 1 mL of air in the syringe, which was kept for 5 min at room temperature before puffing. One milliliter of air containing the sample vapor was puffed into an airflow (300 mL/min, 18 cm/s) and delivered to the antenna. This was repeated 10\u0026ndash;15 times using antennas from multiple individuals.\u003c/p\u003e \u003cp\u003eGC analyses were conducted with Shimadzu GC 2014 equipped with an apolar capillary column DB-1HT (15 m length, 0.25 mm i.d., 0.1 \u0026micro;m film thickness) and a flame ionization detector. Helium was used as a carrier gas at a flow rate of 1 mL/min. The injection was made at splitless mode for 1 min at 300 ℃. The column oven temperature was set at 50 ℃ for 5 min, programmed to 300 ℃ at 10 ℃/min, then held at the final temperature for 5 min. The data were analyzed using a Shimadzu CR-8A chromatograph.\u003c/p\u003e \u003cp\u003eGC-MS analysis was performed on a high-resolution mass spectrometer JMS-600H coupled with an Agilent GC 6890N equipped with an apolar capillary column HP-1MS (25 m length, 0.25 mm i.d., 0.25 \u0026micro;m film thickness). Helium was the carrier gas at a flow rate of 1 mL/min, and the injection was made in splitless mode for 1 min at 250\u0026deg;C. The column oven temperature was set at 40 ℃ for 1 min, programmed to 250 ℃ at 10 ℃/min, then held at the final temperature for 8 min. Both the interface temperature and the ion source temperature were set at 250 ℃. The EI mass spectra were obtained at 70 eV, and the scan range was \u003cem\u003em/z\u003c/em\u003e 40\u0026ndash;500.\u003c/p\u003e \u003cp\u003eGC-EAD analyses were conducted with Agilent GC6890N, which was tuned to bifurcate into FID and EAD by OP 275 Pro II sniffing port (GL Sciences Inc.). The FID signal was recorded and analyzed with Chemstation software (Ver. A10.01, Agilent Technologies). EAD signal was simultaneously acquired from an EAD probe (Syntech) through IDAC-2 data acquisition controller (Syntech) and recorded with AutoSpike (ver 3.9, Syntech). A nonpolar capillary column DB-5MS (30 m length, 0.25 mm i.d., 0.25 \u0026micro;m film thickness) was used for analyses. Both the injection port and flame ionization detector (FID) port were set at 280\u0026deg;C. The column oven temperature was initially set at 100 ℃, programmed to 320 ℃ at 10 ℃/min, then held at the final temperature for 3 min.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLab and field trials.\u003c/em\u003e To investigate the changes in the amounts of substances peculiar to alate males before and after flight, 45 male alates were equally separated into three groups. The males in the first group were immediately frozen and killed to pick up the head parts to quantify male-specific substances by GC analysis prior to flight. The males of the second and third groups were each placed in a small petri dish (Φ9 \u0026times; 2 cm) as artificial nest, then in a large case (10.5 \u0026times; 20 \u0026times; 6 cm), and the large cases were left in the laboratory (2.5 \u0026times; 2.5 \u0026times; 5 m) in turn and allowed to fly freely. For the third group, 15 female alates were placed in a small case of the same size and then together in the same large case. After 1 h, all male alates were recaptured from the laboratory to quantify male-specific substances after the flight. All of these were freeze-killed, and chemicals were extracted from each head in 100 \u0026micro;L of n-hexane for 1 h, and then 50 ng of n-pentadecane (Nacalai Tesque) was added to each as an internal standard for quantification.\u003c/p\u003e \u003cp\u003eField trials were conducted to capture flying alate ants during the nuptial flight season in May 2021 at the Kamigamo Experimental Station (KES) and Uji campus (UC) of Kyoto University, Kyoto, and the FFPRI. As the chemical bait, 50 \u0026micro;L of hexane solution of the mixture of 250 \u0026micro;g of methyl 6-methyl salicylate (MMS) and 100 \u0026micro;g of methyl-2-aminobenzoate (MA) was applied on the rubber septa placed in the center on the adhesive sheets (24 \u0026times; 30 cm) in the Delta traps (Sankei Chemical Co. Ltd.). A 50 \u0026micro;L of hexane alone was applied as the negative control. Pairs of traps with bait and controls were placed at a height of 1.5 to 2 m above the ground and at least 2 m apart. A total of 22 pairs of traps (4 pairs at KES, 8 pairs at UC, and 10 pairs at FFPRI) were set from May 2 to 28 in 2021. Both test and control sample solutions were replenished daily between 12:00 and 14:00. Damaged or dirty traps were replaced if necessary, and insects captured on each adhesive sheet were recorded daily for counting.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePreparation of Candidate Chemical Compounds.\u003c/em\u003e Methyl salicylate (MS) and MA were purchased from Tokyo Chemical Industry Co., Ltd. MMS was prepared by the saponification of commercially available ethyl 6-methyl salicylate (4.0 g, 22.2 mmol, Tokyo Chemical Industry, Co., Ltd.) followed by methyl esterification with diazomethane. Ethyl 6-methyl salicylate (4.0 g, 22.2 mmol) was dissolved in ethanol (100 mL) in a round-bottomed one-neck flask. KOH pellets (12.3 g, 220 mmol, 10 equiv) were then added to the solution, which was further refluxed for 6 h using a Dimroth condenser. When the saponification was complete (as monitored by TLC), the solvent was evaporated using a rotary evaporator until it became crystalline. The mixture was then transferred to a separatory funnel with the aid of ice water and extracted with hexane (30 mL) to separate the unreacted ethyl ester. The aqueous layer was then acidified with 2N-HCl, extracted three times with diethyl ether, washed with brine, dried over Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated to yield crude 6-methylsalicylic acid, which reacted with diazomethane without purification. Crude 6-methylsalicylic acid was dissolved in diethyl ether and treated with an ether solution of diazomethane prepared analogously using the method described in the Organic Synthesis. After the diazomethane treatment, the solvent was carefully evaporated to give methyl 6-methyl salicylate (3.45 g, 20.8 mmol, 94% yield), which was further purified by SiO\u003csub\u003e2\u003c/sub\u003e column chromatography to yield pure material (3.14 g, 18.9 mmol, 85.1% yield). GC-MS [\u003cem\u003em/z\u003c/em\u003e (%)]: 51 (6), 53 (5), 77 (12), 78 (15), 79 (5), 105 (14), 106 (25), 134 (100), 135 (23), 166 (39), 167 (4). GC: 11.050 min (HP-1MS), 13.863 min (FFAP).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical Analyses.\u003c/em\u003e Regarding laboratory trials, one-way analysis of variance (ANOVA) with post-hoc Tukey HSD (Honestly Significant Difference) was used to compare pre-and post-flight chemical possession of alate males with and without alate females in the same container. Regarding field trials, the effects of compound presentation on trap capture were summarized in a 2 \u0026times; 2 table at each or all three fields KES, FFPRI, and UC, and Fisher\u0026rsquo;s exact tests were performed in one-sided confidence intervals. All these statistical analyses were conducted with a freeware, js-STAR XR+ (release 1.7.2.j)(Tanaka and Nappa \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cem\u003eIdentification of Male-Specific Chemicals.\u0026nbsp;\u003c/em\u003eComparative GC analysis of hexane extracts of body parts from alates and workers confirmed the volatile chemical characteristics of the male head (Fig. 1A-D). Peak a, which was common in workers, soldiers, and alate females, was identified as \u003cem\u003en\u003c/em\u003e-undecane. The characteristic male volatiles, including at least three compounds, B, C, and D, were also captured by the PDMS-SPME fiber (Fig. 1E), which exhibited molecular ions with m/z 152, 166, and 151, respectively. Based on the fragmentation patterns, they were identified as salicylic acid methyl ester (methyl salicylate), methyl salicylate with a methyl-substituted benzene ring at an unknown position (methyl x-methylsalicylate), and 2-aminobenzoic acid methyl ester (methyl 2-aminobenzate). Compound B was confirmed as methyl salicylate by GC co-injection with an authentic compound. \u0026nbsp; For compounds B and C, all candidate isomers were synthesized, and their GC retention times and mass spectra were recorded (Table 1). The GC retention time of compound C was closest to that of methyl 6-methyl salicylate and similar to that of methyl 2-aminobenzoate (methyl anthranilate).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEAG Responses Against the Quantitative Major Compounds\u003c/em\u003e. The typical and reproducible EAG responses of alate and worker ants are shown in Fig. 2. In all those castes, alate male head extract (MHE) at 0.01\u0026ndash;0.02 ant equivalent, as well as synthesized MMS and MA at 0.01 ant equivalent, evoked EAG responses. Although the workers\u0026rsquo; EAG responses to MA were also confirmed, the data are not shown. Both alate female head extract (FHE) and worker\u0026rsquo;s head extract (WHE), as well as \u003cem\u003en-\u003c/em\u003eundecane (UD), evoked EAG responses at 0.02 ant equivalent in the workers and the alate females but not in the alate males (data not shown). The alate female antennae tended to be more responsive to MHE than to FHE or WHE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe EAG responses of alate and worker ants were confirmed to be components of MMS and MA, both in MHE and the mixture of synthetic MMS and MA (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLab Trials to Compare Pre- and Post-Flight Male Chemicals.\u0026nbsp;\u003c/em\u003eFig. 4 compares the MMS and MA\u0026nbsp;contents of\u0026nbsp;alate males before and after\u0026nbsp;the flight.\u0026nbsp;Quantitative GC analysis estimated pre-flight MMS and MA to be 38.55 \u0026plusmn; 23.59 and 13.68 \u0026plusmn; 7.56 \u0026mu;g per male (n=10), whereas post-flight MMS and MA were 14.98 \u0026plusmn; 23.59 and 6.59 \u0026plusmn; 2.59 \u0026mu;g (n=9) in the absence of the females, but 16.93 \u0026plusmn; 9.70 and 8.93 \u0026plusmn; 5.48 \u0026mu;g (n=10) in the presence of the females, respectively. MMS was significantly decreased after flight regardless of the presence of the females (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ANOVA with post hoc Tukey test). MA showed a similar trend but was significantly reduced when flying in the absence of females.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eField Trials to Capture Female and Male Alates by M6M and MA\u003c/em\u003e. Within the three fields for bait trap experiments, a total of 12 sets of traps were used for KES (four sets) and UC (10 sets), but only one trap treated with MMS and MA from FFPRI captured one male alate (Table 2). Alate ants were not captured regardless of the presence or absence of MMS and MA treatment. However, at the UC, multiple alate males were captured in four of ten trap sets only when treated with MMS and MA. By focusing on the number of captures at the UC, the number of traps treated with MMS and MA was significantly higher than that of the control traps (Two-way ANOVA, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eSignificant differences were also observed in the number of captures by the target caste (two-way ANOVA, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), and alate males tended to be captured in large numbers (multiple comparisons using Bonferroni, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe identified three volatile compounds in male \u003cem\u003eC. japonicus\u003c/em\u003e collected using SPME: MS, MMS, and MA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These compounds were also detected in worker ants; however, their relative amounts were small, and \u003cem\u003en\u003c/em\u003e-undecane was the main component. Therefore, these three volatiles were considered to have male characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). EAG responses were confirmed in all alate males, females, and workers to these three male-derived components (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e); however, EAG responses were not confirmed in males to head extracts from females or workers. This suggests that male-specific behavior during nuptial flight, that is, male lek formation, is caused by male-characteristic compounds. Because alate females also respond to male-characteristic compounds, these would function as communicative signals related to mating. Quantitative analysis revealed that the average value of MMS, the most abundant compound, decreased from 38.54\u0026thinsp;\u0026plusmn;\u0026thinsp;23.59 \u0026micro;g per male to 14.98\u0026thinsp;\u0026plusmn;\u0026thinsp;7.13 \u0026micro;g per male before and after flight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This is probably because alate males secrete the compound during nuptial flights, which promotes alate male swarming or attracts alate females. This hypothesis was tested in a field trap experiment that compared the number of ants captured in sticky board traps with and without the addition of male compounds. Although the number of captures was not overwhelming, significantly more alate males were captured in traps with male compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although further verification is required, these results indicate that these male compounds may function as pheromones to attract alate males during nuptial flight in this species.\u003c/p\u003e \u003cp\u003eMMS, identified as the characteristic compound in males of \u003cem\u003eC. japonicus\u003c/em\u003e, has previously been reported to be present in males of more than 20 species of American \u003cem\u003eCamponotus\u003c/em\u003e (Brand et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1973\u003c/span\u003ea, b; Llyod et al. 1984; Torres et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Some of these species produce detectable quantities of volatile compounds only in males, suggesting that male-specific compounds may provide a way of comprehending their caste-specific flights during mating. Workers in some \u003cem\u003eCamponotus\u003c/em\u003e species also possess this compound, which is believed to function in alarm communication (Brand et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1973\u003c/span\u003ea. 1999). These results will likely also apply to \u003cem\u003eC. japonicus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eMMS has also been reported in the mandibular gland content of workers of the ponerine ant (Duffield and Blum, 1975a, b; Longhurst et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1980\u003c/span\u003e) and those of both workers and queens of \u003cem\u003ePolyergus\u003c/em\u003e species (Greenberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The \u003cem\u003ePolyergus\u003c/em\u003e queen serves as a sex pheromone that attracts alate males (Greenberg et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). More recently, MMS has been identified as a major component of the trail pheromone of \u003cem\u003eTetramorium impurum\u003c/em\u003e (Morgan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1990\u003c/span\u003e)d \u003cem\u003etsushimae\u003c/em\u003e (Nakamura et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and they secrete this compound from the poison and Dufour\u0026rsquo;s gland, respectively. In carpenter ants, although the relationship between MMS and the mandibular gland has not been confirmed, the localization of MMS (that is, the compound was isolated from the head capsule) suggests that it is deposited in and secreted from the mandibular gland.\u003c/p\u003e \u003cp\u003eLaboratory bioassays of \u003cem\u003eC. japonicus\u003c/em\u003e confirmed a significant decrease in the amount of MMS retained by alate males after flight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting that alate males release this compound during their flight. A similar phenomenon has been confirmed in \u003cem\u003eSolenopsis invicta\u003c/em\u003e, in which alate males and females possess 2-ethyl-3,6-dimethylpyrazine in their mandibular glands (Choi and Vander Meer 2015). The pyrazine compound was significantly reduced in both males and females after nuptial flights, suggesting its potential role as a sex pheromone in nuptial flight behaviors, such as male-lek formation. Although this type of comparative analysis is rare for ant species, notably, similar trends were observed in \u003cem\u003eS. invicta\u003c/em\u003e as well as \u003cem\u003eC. japonicus\u003c/em\u003e. Furthermore, in this laboratory experiment, male flight altitude was evidently lower than that during conjugal flight, suggesting that flight behavior may be an important stimulus for the release of male pheromones. Such a case has been reported in \u003cem\u003eDasylepida\u003c/em\u003e beetle, where flight behavior stimulates female sex pheromone secretion (Fukuya et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSignificantly more males were captured in traps treated with an artificial blend of male alate compounds. This suggests that the blend has an attractive effect on flying alate males. However, field assays require review and validation because the number of catches per trap is generally low. Possibly, this is a result of difficulties in developing the necessary bioassays for high-flying ants, as noted by Choi and Vander Meer (2015) for fire ants that mate at approximately 150 m in the air. Abe (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1973\u003c/span\u003e) reported the nuptial flight behavior of \u003cem\u003eC. japonicus\u003c/em\u003e; however, its mating height is not well understood. Although Woyciechowski (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) stated that the male-lek-forming type of nuptial flight is risky, it is advantageous for avoiding inbreeding. To verify this, understanding the site where the male lek is formed is necessary, but this has not been possible in many species other than \u003cem\u003eMyrmica\u003c/em\u003e ants (Kannnowski 1963; Elmes \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Woyciechowski \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). To elucidate the pheromonal communication involved in nuptial flight, constructing a bioassay method based on the location of lek formation is fundamental and apparently necessary to perform the bioassays at the right time and right place on the day of nuptial flights.\u003c/p\u003e \u003cp\u003eMMS, MS, and MA are characteristic of alate males but are not specific. As all castes showed EAG responses to these compounds, they may be context-dependent multifunctional pheromones. Choi and Vander Meer (2015) reported that all the adult castes of \u003cem\u003eS. invicta\u003c/em\u003e have 2-ethyl-3,6-dimethylpyrazine in their mandibles, which is used as a sex pheromone between the alate males and females but functions as an alarm pheromone among workers. This context-dependent multifunctionality is considered to allow social insects to develop complex chemical communication systems and use a small number of chemicals to maintain social behavior (Mitaka and Akino \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Regarding \u003cem\u003eC. japonicus\u003c/em\u003e, chemical information is input into the brain through three compounds. Therefore, behaviorally and physiologically, verifying changes in function between castes is essential. The results obtained from this experiment do not directly observe pheromone communication during the mating flight of \u003cem\u003eC. japonicus\u003c/em\u003e. They are merely inferred through chemical analysis. Therefore, continuous detailed field observations will be necessary for future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFUNDING\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI Grant Numbers JP20K06073. Competing Interests The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eAUTHOR CONTRIBUTIONS\u003c/p\u003e\n\u003cp\u003eShunya Habe designed the study, the main conceptual ideas, the proof outline, and collected the data. Shigeru Matsuyama synthesized the candidate chemicals applied in the experiments. Natsumi Kanzaki, Keiko Hamaguchi, Mamiko Ozaki, and Toshiharu Akino aided in interpreting the results and worked on the manuscript. Mamiko Ozaki and Toshiharu Akino supervised the project. Shunya Habe wrote the manuscript with support from Toshiharu Akino. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbe T (1973) On the behavior of the ant \u003cem\u003eCamponotus japonicus\u003c/em\u003e at the nuptial flight. Kontyu 41:333\u0026ndash;341 \u003c/li\u003e\n\u003cli\u003eBoomsma JJ, Leusink A (1981) Weather conditions during nuptial flights of four European ant species. Oecologia 50: 236\u0026ndash;241. https://doi.org/10.1007/BF00348045\u003c/li\u003e\n\u003cli\u003eBrand JM, Duffield RM, MacConnell JG, et al. (1973) Caste-specific compounds in male Carpenter ants. Science 179: 388\u0026ndash;389. https://doi.org/10.1126/science.179.4071.388\u003c/li\u003e\n\u003cli\u003eBrand JM, Fales HM, Sokoloski FA, et al. (1973) Identification of mellein in the mandibular gland secretions of Carpenter ants. 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Harvard University Press, Cambridge, Massachusetts.\u003c/li\u003e\n\u003cli\u003eIwamoto N, Nakamuta K, Millar JG (2020) Sex attractant pheromone of the Japanese slave-making ant, \u003cem\u003ePolyergus samurai\u003c/em\u003e. J Chem Ecol 46: 520\u0026ndash;523. https://doi.org/10.1007/s10886-020-01179-1\u003c/li\u003e\n\u003cli\u003eKannowski PB (1963) The flight activities of formicine ants. Symp Genet Biol 12: 74\u0026ndash;102\u003c/li\u003e\n\u003cli\u003eLloyd HA, Blum MS, Duffield RM (1975) Chemistry of the male mandibular gland secretion of the ant, \u003cem\u003eCamponotus clarithorax\u003c/em\u003e. Insect Biochem 5: 489\u0026ndash;494. https://doi.org/10.1016/0020-1790(75)90032-3\u003c/li\u003e\n\u003cli\u003eLloyd HA, Schmuff NR, Hefetz A (1984) Chemistry of the male mandibular gland secretion of the carpenter ant, \u003cem\u003eCamponotus thoracicus\u003c/em\u003e fellah emery. Comp Biochem Physiol B: Comp Biochem 78: 687\u0026ndash;689. https://doi.org/10.1016/0305-0491(84)90119-6\u003c/li\u003e\n\u003cli\u003eLonghurst C, Baker R, Howse PE, (1980) A comparative analysis of mandibular gland secretions in the ant tribe Tetramoriini. Insect Biochem 10: 107\u0026ndash;112. https://doi.org/10.1016/0020-1790(80)90046-3\u003c/li\u003e\n\u003cli\u003eMarkin GP, Dillier JH, Hill SO, et al. (1971) Nuptial flight and flight ranges of the imported fire ant, \u003cem\u003eSolenopsis saevissima richteri\u003c/em\u003e (Hymenoptera: Formicidae). J Georgia Entomol Soc 6: 145\u0026ndash;156 \u003c/li\u003e\n\u003cli\u003eMilio J, Lofgren CS, Williams DF (1988) Nuptial flight studies of field-collected of \u003cem\u003eSolenopsis invicta \u003c/em\u003eBuren. In Trager JC (ed) Advances in Myrmecology, Brill, Leiden, pp 419\u0026ndash;431. https://doi.org/10.1163/9789004630765_030\u003c/li\u003e\n\u003cli\u003eMitaka Y, Akino T (2021) A Review of termite pheromones: Multifaceted, context-dependent, and rational chemical communications. Front Ecol Evol 8: 595614. https://doi.org/10.3389/fevo.2020.595614\u003c/li\u003e\n\u003cli\u003eMorgan ED, Jackson BD, Ollett DG, Sales GW (1990) Trail pheromone of the ant \u003cem\u003eTetramorium impurum \u003c/em\u003eand model compounds: Structure-activity comparisons. J Chem Ecol 16: 3493\u0026ndash;3510. https://doi.org/10.1007/BF00982113\u003c/li\u003e\n\u003cli\u003eMori A, Grasso DA, Visicchio R, et al. (2001) Comparison of reproductive strategies and raiding behaviour in facultative and obligatory slave-making ants: the case of \u003cem\u003eFormica sanguinea\u003c/em\u003e and Polyergus rufescens. 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Insect Behav 2: 413-417. https://doi.org/10.1007/BF01068065\u003c/li\u003e\n\u003cli\u003eWalter F, Fletcher DJC, Chautems D, et.al. (1993) Identification of the sex pheromone of an ant, \u003cem\u003eFormica lugubris\u003c/em\u003e (Hymenoptera, Formicidae). Naturwissenschaften 80: 30\u0026ndash;34. https://doi.org/10.1007/BF01139755\u003c/li\u003e\n\u003cli\u003eWoyciechowski M (1990) Mating behaviour in the ant \u003cem\u003eMyrmica rubra\u003c/em\u003e (Hymenoptera, Formicidae). Acta Zool Cracov 33: 565\u0026ndash;574\u003c/li\u003e\n\u003cli\u003eWuellner CT (2000) Male aggregation by \u003cem\u003eSolenopsis richteri\u003c/em\u003e Forel (Hymenoptera: Formicidae) and associated mating behavior in Argentina. J Insect Behav 13: 751\u0026ndash;756. https://doi.org/10.1023/A:1007852229186\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-chemical-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joce","sideBox":"Learn more about [Journal of Chemical Ecology](https://www.springer.com/journal/10886)","snPcode":"10886","submissionUrl":"https://submission.nature.com/new-submission/10886/3","title":"Journal of Chemical Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Camponotus japonicus, nuptial flight, methyl salicylate (MS), methyl 6-methyl salicylate (MMS), methyl anthranilate (MA).","lastPublishedDoi":"10.21203/rs.3.rs-4002899/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4002899/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe nuptial flight of ants usually occurs during certain periods of the year. Alate females and males fly out of their nests to mate simultaneously. In the genus \u003cem\u003eCamponotus\u003c/em\u003e, sex-specific chemicals are deposited in the male head capsule; however, their roles in nuptial flight have not yet been clarified. This study aimed to elucidate the functions of male-specific chemicals in the Japanese carpenter \u003cem\u003eCamponotus japonicus\u003c/em\u003e. First, we identified three chemicals attributed to the male as authentic chemicals: methyl salicylate (MS), methyl 6-methyl salicylate (MMS), and methyl anthranilate (MA). Electroantennogram (EAG) responses to these three chemicals were confirmed to occur in male and female alates. Subsequently, the amounts of MMS and MA in the alate male head capsule were compared before and after flight under laboratory conditions. The amounts of both substances were significantly reduced after flight, suggesting that males secreted these substances during flight. Thereafter, a field trap experiment was conducted in three fields of the Kyoto Prefecture during the nuptial flight season in 2021 using MMS and MA as bites. The number of captured alate males was significantly higher than that of the females, suggesting that these compounds primarily attracted males rather than females. Considering the field conditions, if the local concentration of these chemicals is increased by male aggregation, females may be attracted because EAG responses were also observed in females. Our findings represent a first step toward understanding chemically mediated male lek formation during the process of male aggregation syndrome in this species.\u003c/p\u003e","manuscriptTitle":"Male-Specific Substances Possibly Contributing to Nuptial Flight of the Japanese Carpenter Ant Camponotus Japonicus (Hymenoptera: Formicidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-28 17:17:43","doi":"10.21203/rs.3.rs-4002899/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-13T21:12:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-08T18:05:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b308d2c4-e850-4246-a2a5-2f8d678c2e6b","date":"2024-04-16T16:55:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-14T05:36:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122d9d64-6668-41a4-87ea-5a0001d75063","date":"2024-03-28T02:43:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-27T20:54:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-27T20:18:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-25T23:06:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Chemical Ecology","date":"2024-03-01T10:23:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-chemical-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joce","sideBox":"Learn more about [Journal of Chemical Ecology](https://www.springer.com/journal/10886)","snPcode":"10886","submissionUrl":"https://submission.nature.com/new-submission/10886/3","title":"Journal of Chemical Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3088a016-7cca-4af0-a0cd-e5b0cb41248e","owner":[],"postedDate":"March 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:01:36+00:00","versionOfRecord":{"articleIdentity":"rs-4002899","link":"https://doi.org/10.1007/s10886-024-01548-0","journal":{"identity":"journal-of-chemical-ecology","isVorOnly":false,"title":"Journal of Chemical Ecology"},"publishedOn":"2024-09-21 15:57:23","publishedOnDateReadable":"September 21st, 2024"},"versionCreatedAt":"2024-03-28 17:17:43","video":"","vorDoi":"10.1007/s10886-024-01548-0","vorDoiUrl":"https://doi.org/10.1007/s10886-024-01548-0","workflowStages":[]},"version":"v1","identity":"rs-4002899","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4002899","identity":"rs-4002899","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}