Sex-linked transcription factor involved in a shift of sex-pheromone preference in the silkmoth Bombyx mori

Abstract

In the sex-pheromone communication systems of moths, odorant receptor (Or) specificity as well as higher olfactory information processing in males should be finely tuned to the pheromone of conspecific females. Accordingly, male sex-pheromone preference should have diversified along with the diversification of female sex pheromones; however, the genetic mechanisms that facilitated the diversification of male preference are not well understood. Here, we explored the mechanisms involved in a drastic shift in sex-pheromone preference in the silkmoth Bombyx mori using spli mutants in which the genomic structure of the gene Bmacj6, which encodes a class IV POU domain transcription factor, is disrupted or its expression is repressed. B. mori females secrete an ∼11:1 mixture of bombykol and bombykal. Bombykol alone elicits full male courtship behavior, whereas bombykal alone shows no apparent activity. In the spli mutants, the behavioral responsiveness of males to bombykol was markedly reduced, whereas bombykal alone evoked full courtship behavior. The reduced response of spli males to bombykol was explained by the paucity of bombykol receptors on the male antennae. It was also found that, in the spli males, neurons projecting into the toroid, a compartment in the brain where bombykol receptor neurons normally project, responded strongly to bombykal. The present study highlights a POU domain transcription factor, Bmacj6, which may have caused a shift of sex-pheromone preference in B. mori through Or gene choice and/or axon targeting.

Moths possess highly diverse and complex sex-pheromone communication systems. A combination of compounds and fine blending confer high species specificity, crucial to the reproductive isolation of moths (1, 2). To date, great efforts have been made to clarify the genes responsible for generating pheromone diversity by using pheromone strains within a species or closely related species. Recently, it was shown that allelic variation in the fatty-acyl reductase gene or selective transcription of desaturase genes is responsible for female sex-pheromone variation in the moth genus Ostrinia (3–5). In contrast to the progress made in understanding the genes responsible for diversification of pheromone production, the molecular mechanisms that shift sex-pheromone preferences in male moths are not well understood.

An interesting genetic feature of sex-pheromone preference in moths is its sex linkage. In most moths, the sex chromosome’s constitution is Z/W in females and Z/Z in males (6). Some of the genes encoding sex-pheromone receptors, which are important factors determining the preference of the males, are reported to be Z-linked in Bombyx (7–9), Heliothis (10), and Ostrinia (9, 11). In Ostrinia nubilalis, a shift in male behavioral responses in the two pheromone strains is controlled by a locus called Resp that maps to the Z chromosome (12, 13).

The silkmoth Bombyx mori has been used as a model for studying sex-pheromone communication systems in moths. B. mori females secrete an ∼11:1 mixture of bombykol [(E,Z)-10,12-hexadecadien-1-ol] and bombykal [(E,Z)-10,12-hexadecadien-1-al] from the pheromone gland (14). Bombykol alone elicits full courtship behavior in males, whereas bombykal alone shows no apparent activity (14). Odorant receptors (Ors) for bombykol (BmOr1) and bombykal (BmOr3) are pheromone receptors identified in Lepidoptera (7, 8). The genes encoding these Ors reside on the Z chromosome (7–9). The Ors are expressed in two specialized chemosensory neurons in the long sensilla trichodea on the male antenna (8). Olfactory receptor neurons (ORNs) responding to bombykol and bombykal project to the macroglomerular complex (MGC) in the brain of male moths, where the information on pheromone reception is integrated (15). The MGC of B. mori consists of three subdivisions: the toroid, cumulus, and horseshoe. ORNs responding to bombykol and bombykal send their axons to the toroid and cumulus, respectively (16).

A large variety of mutants are available in B. mori, representing all major stages of development (the egg, larva, pupa, and adult) (17). The availability of complete genome data for B. mori (18–20) combined with these mutants provides unparalleled opportunities to isolate and analyze the genes governing biologically important traits in Lepidoptera. To date, genes responsible for over 20 mutants have been identified by positional cloning or candidate gene approaches (21).

The Z-chromosome–linked mutant spli is characterized by a soft and pliable larval body. We previously reported that (i) a 66- to 96-kb sequence is deleted from the Z chromosome in the spli mutant and that (ii) only Bmacj6, a Drosophila acj6 homolog encoding a class IV POU domain transcription factor, is computationally predicted in the deleted sequence (22). We concluded that disruption of Bmacj6 is associated with the spli phenotype (22). Subsequently, analysis with full-length cloning of Bmacj6 has clarified that, among the four exons of the gene, exons 2–4, which encode a POU domain, are missing in the spli mutant (Fig. S1). In Drosophila, acj6 mutants exhibit abnormal olfactory behavior and reduced mobility (23–25), and genetic and electrophysiological analyses suggested that acj6 determines Or gene choice and axon targeting of ORNs (24, 26).

Here, we report that (i) the Bt mutant, originally characterized by an abnormal feeding behavior, bears an allele of spli (spli^Bt); (ii) behavioral responsiveness of the male spli mutants (spli and spli^Bt) to bombykol is markedly reduced in association with the paucity of bombykol receptors on the antennae; (iii) full courtship behavior of spli males is evoked by bombykal alone; and (iv) neurons projecting into the toroid, a compartment in the brain where bombykol receptor neurons normally project, respond to bombykal.

Results

Identification of a spli Allele.

The Z-chromosome–linked mutant Bt is known for abnormal feeding on plants other than mulberry (27). We noted that the larval body of this mutant was soft and pliable, which are characteristics of the spli mutant. Crossing of Bt males (Bt/Bt) with normal females (+/W) and spli females (spli/W) suggested that the Bt mutant bears an allele of spli, which we designated spli^Bt (Table S1). RT-PCR analysis of Bmacj6, disruption of which is associated with the spli phenotype (22) (Fig. S1), showed that, in contrast to the expression in the nervous system and some other tissues in the normal strain, no expression was observed in any tissues tested in the spli^Bt mutant (Fig. 1). Despite the nonexpression, however, no mutation that may account for it was identified in the 1- to 2-kb sequences in and around the four exons in the spli^Bt allele (accession no. AB635375–635378). We considered that spli^Bt is an allele of Bmacj6, the mRNA expression of which was repressed by an unknown mechanism.

Fig. 1.

RT-PCR analysis of Bmacj6 in p50T (+/+, +/W) and the mutant strain (spli^Bt/spli^Bt, spli^Bt/W). An, antenna of male moth; Br, brain; Cn, central nerve; Fa, fat body; In, integument; Si, silk gland; Ma, Malpighian tubules; Mg, midgut; Mp, larval mouth part; Ov, ovary; Te, testis; Wd, wing disk. Mp was derived from both males and females. Tissues except for Te and An were obtained from females. M, molecular size marker (100-bp ladder). a, b, and c indicate 1.5 kb, 1 kb, and 500 bp, respectively. Forward and reverse primers were designed in exons 1 and 4, respectively. Ribosomal protein L3 (RpL3) was used as a positive control. Primers for RpL3 are listed in ref. 37.

Behavioral Response of spli Males to Bombykol and Bombykal.

Similar to normal male moths, spli males became excited and performed mating dances when exposed to female moths. To examine whether the pheromone recognition system of the spli mutants is normal, we observed the behavioral responses of the spli male moths to bombykol and bombykal (Fig. 2 A and B). A small fraction of the p50T (+/+) and spli/+ males responded to 1 ng bombykol, and all responded to 1,000 ng bombykol (Fig. 2A). In contrast, spli and spli^Bt males did not respond to ≤100 ng of bombykol and only about 50% responded to 1,000 ng (Fig. 2A). This indicated that spli and spli^Bt males were considerably less sensitive to bombykol than p50T (+/+) and spli/+ males. The p50T (+/+) and spli/+ males did not respond to bombykal at all (Fig. 2B). In contrast, spli and spli^Bt males started responding to bombykal at 10 ng, and 80–100% responded to 1,000 ng bombykal (Fig. 2B). Fig. 2C shows the differential responses. When the male spli moths (spli/spli) and the male gray moths with a normal spli locus (+^spli/+^spli, mln/mln) were placed around two vials releasing either bombykal or bombykol, the normal moths were specifically attracted to bombykol, whereas the spli males were specifically attracted to bombykal (Movie S1). These findings indicate that bombykal, rather than bombykol, elicited courtship behavior in the spli mutants.

Fig. 2.

Behavioral responses of B. mori mutants to bombykol and bombykal. (A and B) The percentage of moths that responded within 30 s from the onset of stimulation is shown. Six moths were used in each experiment, and three experiments were performed at each concentration. Red diamond, p50T (+/+); yellow triangle, spli/+; blue circle, spli/spli; black square, spli^Bt/spli^Bt. Error bars represent ± SD (n = 3). (C) White wing moths are spli males (spli/spli, +^mln/+^mln; mln, melanism gene), and gray wing moths are males with a normal spli locus (+^spli/+^spli, mln/mln). Left and right vials contain bombykal (100 ng/μL) and bombykol (50 ng/μL), respectively.

Electrophysiological and Molecular Analysis of the spli Male Antennae.

To elucidate the cause of the diminished behavioral response of spli males to bombykol, we measured electroantennogram (EAG) responses of male spli moths to bombykol and bombykal. Whereas the response of spli and spli^Bt males to bombykal was similar to the response of p50T (+/+) and spli/+ males (Fig. 3A), the response to bombykol was significantly reduced compared with the response of p50T (+/+) and spli/+ males (Fig. 3A). This result suggested that the expression of the odorant receptor for bombykol (BmOr1) was specifically decreased in the spli mutants. To test this possibility, we determined the expression levels of BmOr1 and BmOr3 in the male antenna by quantitative RT-PCR (qRT-PCR). Whereas expression levels of BmOr3 in the spli and spli^Bt males were almost the same as those in p50T (+/+) and spli/+ males (Fig. 3B), 354- and 1,085-fold reductions in BmOr1 expression were observed in the spli and spli^Bt males, respectively (Fig. 3B). These results suggest that the attenuated EAG response of the spli male to bombykol is attributable to the paucity of BmOr1 in its antenna.

Fig. 3.

Electrophysiological and molecular analyses of the spli male antenna. (A) Typical electroantennographic responses of the normal and spli moths to bombykol and bombykal excerpted from the results of GC-EAD analyses. (B) Quantitative RT-PCR analysis of BmOr1 and BmOr3 expression in the antennae of spli mutants.

Odor Response Properties of Projection Neurons Innervating the Toroid.

Mistargeting of ORNs is found in the null mutant of acj6, suggesting that acj6 plays an important role in the axon targeting of ORNs in Drosophila (26). This led us to suspect that the spli, a deletion mutant of the acj6 homolog (Bmacj6), may have a defect in the targeting of ORNs. Therefore, we analyzed the odor response properties of the projection neurons (PNs) in the antennal lobe of spli male moths (Fig. 4). In normal males, PNs innervating the toroid, a compartment in the MGC, showed excitation in response to bombykol but not to bombykal (16) (Fig. 4 B and F). Interestingly, PNs innervating the toroid in spli males showed little or no response to bombykol but a strong response to bombykal at the dose tested (Fig. 4 B and F). These findings suggest that PNs innervating the toroid received input from ORNs expressing BmOr3 in spli males. In the spli moths, no structural changes as exemplified by the toroid:cumulus volume ratio were identified in the antennal lobe (Fig. 4 D and E).

Fig. 4.

Glomerular organization in the antennal lobe and responses of projection neurons innervating the toroid. (A) Confocal image of a projection neuron innervating the toroid in the spli brain. The neuron had smooth branches mainly in the toroid and sent the axonal projection to the inferior lateral protocerebrum (ILPC), the same termination site as in the WT. D, dorsal; L, lateral; OG, ordinary glomeruli. Dotted line shows the shape of the toroid in this optical section. (B) Intracellular recording from a toroid-projecting neuron in response to bombykol (bol) and bombykal (bal). An air puff without odors was used as a blank stimulus. (C) Confocal images of antennal lobe structures in spli. Background staining with Lucifer yellow was performed to distinguish the borders between glomeruli (38). C, cumulus; D, dorsal; L, lateral; OG, ordinary glomeruli; T, toroid. The number at the upper right (25.2 μm) indicates the depth from the surface of the brain. (D) 3D reconstruction of the toroid (yellow) and cumulus (green) in the spli antennal lobe. (E) Comparison of the toroid:cumulus ratio between the spli and normal moths (n = 5 for each group; P = 0.4, Mann–Whitney U test). (F) Neuronal response of projection neurons innervating the toroid in the p50 and spli moths. The number of spikes within 1 s from the onset of stimulus was counted. *P < 0.01, **P < 0.005, Mann–Whitney U test. n = 6 for spli and n = 5 for p50.

Discussion

A major unresolved issue in the evolution of sexual communication systems in moths is how the pheromone recognition systems in the male moths diversified in association with the diversification of female sex pheromones. Two pheromone strains of the European corn borer, O. nubilalis, have served as models to study this problem. O. nubilalis Z-strain females produce a 3:97 blend of E11- and Z-11-tetradecenyl acetate (E/Z11-14:OAc) to which males respond, whereas, in the E strain, females produce a 99:1 blend of E/Z11-14:OAc to which males respond (28, 29). Genetic analysis of these strains indicated that the difference in behavioral response in the male is determined by a single sex-linked major gene (12). Furthermore, neuroanatomical analysis of the brains of these strains demonstrated a sex-linked change in the topology of axon targeting of ORNs responding to E11- and Z11-14:OAc (30) and a change in the volume ratio of medial and lateral MGCs (31). Despite this impressive progress, the molecular mechanisms controlling these neural changes remain to be clarified.

The expression levels of BmOr1 in the antennae of the spli and spli^Bt males were markedly reduced, whereas those of BmOr3 were not affected (Fig. 3B). Bmacj6 was the only gene computationally predicted to reside in the 66- to 96-kb sequence deleted from the Z chromosome of the spli mutant (22) (Fig. S1), and Bmacj6 was not expressed in the spli^Bt mutant (Fig. 1). Given that Drosophila Acj6 directly regulates the expression of an Or gene subset by binding to upstream sequences of relevant genes (32), Bmacj6 is also likely to be involved in Or gene choice. Although our results do not exclude the possibility that alteration in the promoter region of BmOr1 caused the reduction in expression in the spli and spli^Bt males, we think that this is unlikely for two reasons. (i) BmOr1 and the 66- to 96-kb deletion, including Bmacj6, are >3.6 Mb apart (20) and the cis element controlling the expression of BmOr1 resides 3.7 kb upstream (33), and (ii) it is hard to consider that the same mutation occurred independently in the two mutant strains, which had been established on the basis of unrelated phenotypes, a soft and pliable larval body (spli) and abnormal feeding behavior (spli^Bt).

Each long sensillum trichodeum on the male antenna comprises two ORNs, one expressing the bombykol receptor (BmOr1) sends its axon to the toroid, and the other expressing the bombykal receptor (BmOr3) sends its axon to the cumulus (8, 33) (Fig. S2A). However, PNs innervating the toroid in the spli brain responded to bombykal, not to bombykol (Fig. 4 B and F). At least two different explanations are possible for this phenomenon. First, axons of BmOr3 neurons might have anomalously projected to the toroid (Fig. S2B). Second, BmOr3 might be ectopically expressed in place of BmOr1 in the ORNs innervating the toroid (Fig. S2C). Both hypotheses well explain the phenomena, including the behavioral responses of spli males to high concentrations of bombykol, but an increase in the expression of BmOr3, which would have been expected if the ectopic expression hypothesis were applied, was not observed in the spli males (Fig. 3B). Further study is needed to obtain an overall picture of the anomaly in the spli mutants.

Since the earliest studies on the sex pheromone of B. mori were done, why a small amount of bombykal is produced by females has remained a mystery (14). Given that a null mutation in Bmacj6 causes males to prefer bombykal, Bmacj6 is not required for a hypothetical bombykal-mediated mate recognition system. This raises the possibility that the production of a trace amount of bombykal is a remnant of the ancestral bombykal-mediated system. In this scenario, the ancestral Bmacj6 may have been involved in functions other than pheromone recognition, and mutations in Bmacj6 itself or its target gene involve Bmacj6 in the pheromone recognition. Although the few bombycid moths examined to date do not produce bombykal, several species belonging to the Sphingidae and Saturniidae, families closely related to Bombycidae, use bombykal as a sex-pheromone component [http://www.tuat.ac.jp/~antetsu/List_of_Sex_Pheromones_in_English(2011.2.2).pdf]. More extensive research on bombycid sex pheromones is awaited.

The present study reports the involvement of a transcription factor in a shift of sex-pheromone preference in moths. It is intriguing that such a shift resulted from a sex-linked mutation in the gene for a transcription factor, Bmacj6, rather than from direct mutations in the genes encoding odorant receptors or odorant-binding proteins. The molecular functions of acj6, Or gene choice, and/or axon targeting appear to be conserved across the insect orders, at least between Diptera and Lepidoptera. In addition to olfactory defects, null mutants of acj6 or Bmacj6 show additional defects. Drosophila acj6 mutants are less active than the wild type (24, 25), and the larval bodies of Bmacj6 mutants are soft and pliable. In the antenna of Drosophila, 10 splicing variants of acj6 were reported (34); similarly, 6 splicing variants were isolated from B. mori antenna (Table S2). In Drosophila, different splice forms have different functions in different ORNs, and as few as two amino acid differences between splice forms resulted in different functions (34). Therefore, we speculate that unique point mutations that specifically alter the function of acj6 homologs in the olfactory system could lead to a change in olfactory behavior in wild moth species without affecting other functions and decreasing their fitness. The roles of acj6 homologs and other POU-domain transcription factors in the divergence of olfactory behavior, including sexual signaling and host–plant selection, should be investigated using moth species representing diverse taxa.

Materials and Methods

Silkworm Strain.

The B. mori strains used as standards were p50 and p50T. The Institute of Genetic Resources, Kyushu University supplied strain n41 with the spli mutation. The Bt mutant was obtained from the Institute of Sericulture. The larvae were reared on fresh mulberry leaves.

Behavioral Experiments.

Behavioral responses of male moths to bombykol and bombykal were observed using a transparent plastic box (diameter: 15 cm) shown in Fig. S3. Six moths placed individually in small plastic cups were deployed concentrically in the box. Purified air was introduced into the box through a glass pipette, which contained a piece of filter paper impregnated with a defined amount of bombykol or bombykal. The outflow from the box was recovered in a plastic bag to prevent contamination of ambient air. Moths that initiated wing fluttering within 30 s from the onset of stimulation were regarded as responsive.

Chemicals and Analytical Instruments.

The authentic standard for bombykol [(E,Z)-10,12-hexadecadien-1-ol] was a gift from Dr. S. Matsuyama (University of Tsukuba, Tsukuba, Japan). Bombykal [(E,Z)-10,12-hexadecadien-1-al] was prepared from bombykol by pyridinium chlorochromate oxidation. Gas chromatography–electroantennographic detection (GC-EAD) was performed as described previously (35). A 5:1 mixture of bombykol (1.15 nmol) and bombykal (0.23 nmol) was injected into the GC system so that the EAD responses of normal (p50T) male moths to these chemicals were nearly equal.

Quantitative RT-PCR.

Total RNA was isolated from the antennae (n = 10 for each sample) using an RNeasy mini kit (Qiagen) and then treated with DNase I (Qiagen). First-strand cDNA was synthesized using a PrimeScript first-strand cDNA Synthesis kit (TaKaRa) with an oligo(dT) primer. Our qRT-PCR experiments were performed with 2× Power SYBR Green PCR Master Mix (Applied Biosystems) using an ABI PRISM 7000 sequence detection system (Applied Biosystems). Primers designed by Wanner et al. (36) were used for amplifying BmOr1, BmOr3, and BmRPS3, the latter used as a standard.

Intracellular Recording and Staining.

A moth was held in a plastic gadget with the head immobilized by a notched plastic yoke set between the head and thorax. The brain was exposed by opening the head capsule and removing the large tracheae, and the intracranial muscles were removed to eliminate brain movement. The antennal lobe was surgically desheathed to facilitate the insertion of a glass microelectrode. Electrodes were filled with 5% Lucifer yellow CH (Sigma) for staining neurons after the recordings. Electrode resistance was ∼70–150 Mohm. A silver ground electrode was placed in the body, and the brain was superfused with saline solution [140 mM NaCl, 5 mM KCl, 7 mM CaCl₂, 1 mM MgCl₂, 4 mM NaHCO₃, 5 mM trehalose, 5 mM N-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, and 100 mM sucrose (pH 7.3)]. Electrical responses of projection neurons were monitored with an oscilloscope and recorded on a DAT recorder (RD-125T; TEAC) at a sampling rate of 24 kHz. The recorded signals were transferred to a computer through an A/D converter (Quick Vu 2; TEAC). The odorant was applied to a piece of filter paper (1 × 2 cm) in a glass stimulant cartridge (tip diameter: 5.5 mm), and the tip of the cartridge was positioned 1.5 cm from the antenna. Air was introduced into the cartridge through a charcoal filter, and each stimulus was applied at a velocity of 500 mL/min (∼35 cm/s).

3D Reconstruction of Single Neurons.

Neurons used for recording were stained by the iontophoretic injection of Lucifer yellow with a constant hyperpolarizing current (−1 to −3 nA) for 1–5 min. The brain was fixed for 4–10 h at 4 °C in 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4) with 10% sucrose, dehydrated with an ethanol series, and cleared in methyl salicylate at room temperature. Each stained neuron was examined using a confocal imaging system (LSM510; Carl Zeiss) with excitation at 458 nm. Serial optical sections were acquired at 0.7-μm intervals through the entire depth of the neuron, and 3D reconstructions of the labeled neurons were generated using AVIZO 6.0 (Visage Imaging).

Cloning and Sequencing of Bmacj6.

Genomic DNA was extracted from the fifth instar larvae using a DNeasy Blood and Tissue kit (Qiagen). Total RNA was prepared using TRIzol (Invitrogen). The sequence of the Bmacj6 transcript was determined by nested RT-PCR. PCR was performed using Ex Taq (Takara) under the following conditions: 94 °C for 1 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min followed by 72 °C for 10 min. PCR products were cloned into a pGEM-T easy vector (Promega) and sequenced using an ABI3130xl genetic analyzer (Applied Biosystems). The 5′-end and 3′-end sequences of Bmacj6 were determined with a CapFishing full-length cDNA premix kit (Seegene) using poly(A)+ RNA prepared from a larval head and purified with an Oligotex-dT-30 Super mRNA purification kit (Takara). Primer sequences used in this study are shown in Table S3.