The rapid naturalization of Bombus terrestris across the Nemuro Peninsula has led to a decline in two closely related native Japanese species, namely Bombus hypocrita sapporensis and Bombus cryptarum florilegus, both belonging to the common subgenus Bombus. Although it is widely believed that cross-mating of native and non-native species is influenced by the common male sex pheromone in this region, no study has been conducted to substantiate this claim. Thus, we investigated the cross-activities of male sex pheromones between native and non-native bumblebees, as well as the frequencies of cross-mating, using chemical and DNA assays. Our gas chromatography-electroantennographic detector analyses and behavioral tests revealed the presence of sex pheromonal cross-activities between B. terrestris and the two Japanese bumblebees species. Furthermore, DNA analyses revealed the occurrence of cross-mating between native and non-native species in the Nemuro Peninsula. Overall, these results indicate the immediate need for conservation measures to safeguard Japanese bumblebee populations in the Nemuro Peninsula.

Bombus cryptarum florilegus is a rare bumblebee in Japan. We tried to rear this bumblebee under laboratory conditions to investigate its nesting habits and colony development. Five queens out of 15 were successful in establishing colonies, and new queens and males were produced from these colonies. In the laboratory colonies, the average numbers of workers, males, and new queens per colony of B. cryptarum florilegus were 15.2 ± 4.7, 26.0 ± 8.0, and 2.0 ± 2.1, respectively. Although a feral colony producing five hundred cocoons has been reported, B. cryptarum florilegus established small colonies under laboratory conditions. Further study suggests that the improvement of rearing technique is required to produce the large colony of B. cryptarum florilegus.

Previous reports note that males of the Japanese bumblebee Bombus ardens ardens perform nest surveillance to mate with new queens. Here, we report that males of this species also perform patrolling and scent-marking for mating. We observed that many B. ardens ardens males fly together from May to June in circular paths through a wooded area in Tokyo, Japan. The flight activity is bimodal with peaks in the morning and late afternoon. When tethered new queens were presented at a focal point, males approached, touched, or grabbed them but ignored them at a foraging site. Males performed scent-marking on tree leaves only in the early morning (05:30 to 07:30), and compounds from the labial gland of B. ardens ardens males were detected on a scent-marked leaf. Based on these findings and previous reports, we conclude that males of this species have at least two mating tactics or strategies: nest surveillance and patrolling.

Gynandromorphy, which is characterized by the coexistence of male and female tissues in a single individual, is known in insects. Gynandromorphs exhibit diverse levels of defects in sexual behavior. The distribution pattern of both sexes within the nervous systems could be responsible for these differences in behavioral traits; however, most studies have mainly focused on the unique external morphological traits of gynandromorphs, and little attention has been paid to the evaluation of nervous systems, at least in Hymenoptera. In this study, we evaluated a gynandromorphic individual of bumblebee (Bombus ignitus) with a bilaterally dimorphic head and thorax (left side, male; right side, female) and a uniformly masculine gaster. The gynandromorph exhibited abnormal sexual behavior; it normally approached and touched virgin queens but rarely made subsequent copulation attempt. Dissection of the gaster indicated that it possessed a set of male reproductive organs. We analyzed the encephalic and antennal expression patterns of the fruitless (fru) gene, which shows sex-specific alternative splicing conserved in various insect orders and has been thought to act as the master regulator of sexual behavior in male nervous systems. The gynandromorph showed left-side-biased expression of male-type fru transcripts. We discuss a possible mechanism for the observed unusual interruption of sexual behavior of the gynandromorph by reference to the fru expression pattern and our recent findings on the sexually dimorphic response to female-derived pheromones.

AbstractThe rewarding orchid, Cremastra appendiculata (Orchidaceae), has a strong floral scent and is pollinated by long-tongued bumble bee queens (Bombus diversus tersatus Smith; Hymenoptera: Apidae). The response of queens of B. diversus tersatus to the scent of C. appendiculata was investigated using a gas chromatography–electroantennographic detector system and Y-tube olfactometer. Gas chromatography–electroantennographic detector analysis of flower extracts showed that seven compounds (nerol, methyl decanoate, methyl dodecanoate, methyl tetradecanoate, methyl hexadecanoate, ethyl dodecanoate, and ethyl tetradecanoate) elicited antennal responses from B. diversus tersatus queens. Further, Y-tube olfactometer test results showed that queens significantly preferred both the flower extracts and synthetic mixture of seven EAD active compounds compared with pentane (a solvent control). These results suggest that floral scents play an important role in attracting queens and promote successful pollination.

To explore the role of the volatiles emitted from male labial gland (LG) of the bumblebee Bombus ardens ardens, we investigated the responses of virgin queens and males to volatiles using a gas chromatography–electroantennographic detector (GC–EAD) system and Y-tube olfactometer. GC–EAD analysis revealed that citronellol, the main compound detected in the male LG, caused clear electrophysiological responses in the antennae of B. a. ardens virgin queens and males although two minor compounds elicited antennal responses when applied in a high concentration. Behavioral tests using a Y-tube olfactometer showed that queens and males were significantly attracted to both LG extracts and citronellol more than to the solvent alone. This is the first study to demonstrate that citronellol as a major compound of male scent-marking pheromone in B. a. ardens functions as a sex attractant for queens. The results also suggest that this compound has another function as a trail marker used by males.

We identified mermithid nematodes infecting a post-hibernating Japanese bumblebee ( Bombus pseudobaicalensis Vogt) queen in Nemuro, Hokkaido, Japan. The infected queen did not lay eggs or feed on pollen during laboratory rearing. In addition, the queen usually crawled under the straw set in the rearing box, which differed from the typical behavior of a normal queen. Intra-abdominal observation revealed that, compared with the ovaries of a normal queen, those of the infected queen were undeveloped. Infection by mermithid nematodes may result in the sterilization of the bumblebee queen. The molecular phylogenetic analysis revealed that the mermithid species infecting the Japanese bumblebee queen differed from the Pheromermis species reported as a parasite of Hymenopteran insects, and that they were closely related to mermithid nematodes found in spiders and Ovomermis sinensis Chen, a parasite of the tobacco budworm larva Helicoverpa armigera Hübner. These findings constitute the first report of mermithid nematodes infecting Japanese bumblebee.

Gynandromorphy that has both male and female features is known in many insect orders, including Hymenoptera. In most cases, however, only external morphology and behavioral aspects have been studied. We found a gynandromorph of bumblebee, Bombus ignitus , that showed almost bilateral distribution of external sexual traits, with male characters observed on the left side and female characters on the right side. This individual never exhibited sexual behavior toward new queens. The dissection of the head part showed that it had bilaterally dimorphic labial glands, only the left of which was well developed and synthesized male-specific pheromone components. In contrast, the gynandromorph possessed an ovipositor and a pair of ovaries in the abdominal part, suggesting that it had a uniformly female reproductive system. Furthermore, we characterized several internal organs of the gynandromorph by a molecular biological approach. The expression analyses of a sex determination gene, doublesex , in the brain, the fat bodies, the hindgut, and the ovaries of the gynandromorph revealed a male-type expression pattern exclusively in the left brain hemisphere and consistent female-type expression in other tissues. These findings clearly indicate the sexual discordance between external traits and internal organs in the gynandromorph. The results of genetic analyses using microsatellite markers suggested that this individual consisted of both genetically male- and female-type tissues.

The epiparasitic plant Monotropastrum humile (D. Don) Hara is pollinated by workers of the long-tongued bumblebee, Bombus diversus diversus (Smith). The interaction between the floral scents of this plant and bumblebee was investigated using electrophysiological and behavioral techniques. Three components (linalool, -terpineol and geraniol) elicited strong responses in antennae from B.d. diversus workers and are innate attractants, suggesting floral scents play an important role in attracting these pollinators and successful pollination.

The volatile components from the labial glands of males of six Japanese bumblebee species were analyzed and compared. Clear species-specificity was found. Ethyl dodecanoate was identified as the major component from the glands of Bombus (Bombus) hypocrita hypocrita and Bombus (Bombus) hypocrita sapporoensis while dihydrofarnesal and dihydrofarnesol were the major components from Bombus (Bombus) ignitus. Citronellol and trans,trans-farnesol were found from Bombus (Pyrobombus) ardens ardens and Bombus (Diversobombus) diversus diversus, respectively. trans,trans-Farnesol was also found from Bombus (Diversobombus) diversus tersatus in Hokkaido. Such differences strongly suggest that these chemicals play an important role in reproductive isolation between sympatric species of Japanese bumblebees.

A method to promote flower visiting by honeybees Apis mellifera L. (Hymenoptera; Apidae) on eggplants was examined by conditioning honeybees to associate eggplant floral scents with sugar-water rewards. The fragrance components of eggplant floral scents were analyzed by coupling solid-phase micro-extraction techniques and gas chromatography-mass spectrometry. The main fragrance component of the whole flowers and pollen of eggplants was identified as geranylacetone. The effect of scents on foraging preferences was investigated by feeding honeybee colonies with sugar water including geranylacetone during the evening before the test day. Foraging honeybees from geranylacetone-conditioned colonies tended to visit feeding sites with geranylacetone scent more than feeding sites with linalool scent and only sugar water. Twelve geranylacetone-conditioned honeybee colonies were placed in eggplant greenhouses. Foraging honeybees from all 12 colonies started visiting eggplant flowers the following day. Unconditioned colonies fed only sugar water were tested as a control. In 5 of 12 unconditioned colonies, foraging honeybees started visiting eggplant flowers the following day. In the other 7 unconditioned colonies, foraging honeybees waited more than 2 days before starting to visit eggplant flowers. We conclude that conditioning honeybees to associate geranylacetone—the main fragrance component of eggplant floral scents—with sugar-water rewards promotes flower visiting.