Gynandromorphy, which is characterized by the coexistence of male and female tissues in a single individual, is known in insects (Narita et al. 2010). The gynandromorphic individuals with varying degrees of mosaicism exhibit diverse levels of defects in sex-specific behavior (Yoshizawa et al. 2009). Few studies, however, have focused on the nervous systems responsible for the sexual behavior of gynandromorphs, at least in Hymenoptera (Brockmann and Brückner 1999), despite an abundance of literature on gynandromorphic individuals (Hinojosa-Díaz et al. 2012).

We analyzed an adult gynandromorph of the bumblebee (Bombus ignitus), which emerged on January 6, 2015, from our laboratory colony. The head and thorax exhibited clear dimorphism in the external sexual traits, with male characters observed on the left side and female characters on the right, whereas the gaster exhibited a uniformly male-like appearance (Figure 1a–c; Online Resource 1: Figure S1, Table SI).

Figure 1.
figure 1

“Quarter female mosaic gynandromorph” of B. ignitus. a–c External appearance of the gynandromorph. Dorsal (a), left-side (b), and right-side (c) views. Bar indicates 1 cm. d Dissection of the gaster from the dorsal aspect. The arrows and arrowheads indicate the pair of testes and vas deferens, respectively. Asterisks indicate the pair of accessory glands. The digestive tract has been removed. Bar indicates 2 mm. e Dissection of the genitalia from the gynandromorph. Bar indicates 1 mm. f Bilaterally dimorphic expression of fru in the brain and antennae. Ribosomal protein 49 (rp49) was used as positive control of RT-PCR experiments. L, left; R, right.

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Ten days after the adult eclosion (the timing of sexual maturation), we examined the mating behavior of the gynandromorph and normal male bumblebees by introducing them individually into an observation cage in which ten sexually mature virgin queens were housed (Online Resource 1: Method details). Normal males smoothly exhibited the typical sequence of mating behavior; i.e., approaching a queen, touching her by antennae (inspection), and mounting her with exposed genitalia (copulation attempt) (van Honk et al. 1978), within 20 min of introduction (Table I). In contrast, the gynandromorph only approached and inspected the queens, without apparent mounting behavior during the first 20 min observation period. A clear copulation attempt finally occurred almost 15 min after the start of an additional observation (Table I; Movie, https://doi.org/10.6084/m9.figshare.5758842).

Table I Behavioral response to virgin queens.
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After further 5 days of rearing, we dissected the gynandromorph and verified male genitalia, a pair of testes, vas deferens, and accessory glands in the gaster (Figure 1d, e; Online Resource 1: Figure S2). In contrast, the cephalic labial glands, wherein males produce the scent-marking pheromones to call conspecific new queens (Kubo and Ono 2010), showed clear bilateral dimorphism (Online Resource 1: Figure S3).

The gene fruitless (fru), which encodes a transcription factor located at the bottom of the sexual-differentiation cascade, exhibits the conservation of sex-specific splicing in various insect orders including Hymenoptera (Salvemini et al. 2010). Detailed molecular genetic studies of the fruit fly (Drosophila melanogaster) have revealed that the male-specific Fru isoforms are required to establish the courtship-related neural circuits, including sensory, central, and motor systems (reviewed by Yamamoto et al. 2014). Additionally, the knockdown of fru in a more basal insect, the German cockroach (Blattella germanica), resulted in a great reduction of the male sexual behavior. This strongly suggests the widely conserved role of fru as the master regulator of male sexual behavior in insects (Clynen et al. 2011). Using each brain hemisphere divided along the midline and antennae on both sides of the gynandromorph, we performed RT-PCR with the primers which could amplify fru transcripts of B. ignitus differing in size between the sexes. The gynandromorph showed bilaterally dimorphic expression pattern of fru: a male-type short product (< 500 bp) was selectively amplified from the left-side samples, while from the right side, an approximately 4-kbp amplicon from the brain sample or no clear amplification from the antenna sample was observed to be female-like (Figure 1f; Online Resource 1: Figure S4), implying the left-sided male-type fru-expressing circuit in the gynandromorph.

Visual recognition of a queen is responsible for the approaching behavior by male bumblebees, and an attracted male inspects the cuticle surface of the queen using his antennae. If he detects sex pheromones on the surface of her cuticle, then a copulation attempt could be triggered (van Honk et al. 1978). Therefore, the tendency of the gynandromorph to approach but not attempt copulation could be interpreted as the outcome of the difficulty in the olfactory-dependent transition from early steps of the mating behavior to copulation attempt.

Interestingly, in the fruit fly, even the unilateral activation of the male-type fru-expressing neuron clusters can initiate male-like sexual behavior (Yamamoto et al. 2014). For the bumblebee gynandromorph, one-sided male-type fru-expressing circuit might be sufficient to initiate mating behavior although detailed investigation of the fru-expressing neurons in the brain is needed (Online Resource 1: Figure S5).

We recently found that female (both queen and worker) B. ignitus secretes a chemical compound, which exerts a repulsive effect on conspecific females as a contact chemical (R. Kubo, unpublished data). Thus, we assumed that the gynandromorph received opposing information from its dimorphic antennae during the inspection of a queen. The fru-expressing olfactory receptor neurons housed in the left antenna were activated by the queens’ attractive sex pheromones, whereas the right antenna responded to the repulsive chemical component, resulting in the disturbance of olfactory-dependent transition from approaching to copulation attempt.

Although gynandromorphy occurs at low frequency, detailed analyses would provide us fruitful information regarding the neural mechanisms underlying a variety of sexual behavior on “non-model” insects including hymenopteran species.