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.
- SourceAvailable from: Peggy L Bunger[Show abstract] [Hide abstract]
ABSTRACT: Sex pheromone communication, acting as a prezygotic barrier to mating, is believed to have contributed to the speciation of moths and butterflies in the order Lepidoptera. Five decades after the discovery of the first moth sex pheromone, little is known about the molecular mechanisms that underlie the evolution of pheromone communication between closely related species. Although Asian and European corn borers (ACB and ECB) can be interbred in the laboratory, they are behaviorally isolated from mating naturally by their responses to subtly different sex pheromone isomers, (E)-12- and (Z)-12-tetradecenyl acetate and (E)-11- and (Z)-11-tetradecenyl acetate (ACB: E12, Z12; ECB; E11, Z11). Male moth olfactory systems respond specifically to the pheromone blend produced by their conspecific females. In vitro, ECB(Z) odorant receptor 3 (OR3), a sex pheromone receptor expressed in male antennae, responds strongly to E11 but also generally to the Z11, E12, and Z12 pheromones. In contrast, we show that ACB OR3, a gene that has been subjected to positive selection (ω = 2.9), responds preferentially to the ACB E12 and Z12 pheromones. In Ostrinia species the amino acid residue corresponding to position 148 in transmembrane domain 3 of OR3 is alanine (A), except for ACB OR3 that has a threonine (T) in this position. Mutation of this residue from A to T alters the pheromone recognition pattern by selectively reducing the E11 response ∼14-fold. These results suggest that discrete mutations that narrow the specificity of more broadly responsive sex pheromone receptors may provide a mechanism that contributes to speciation.Proceedings of the National Academy of Sciences 08/2012; 109(35):14081-6. · 9.81 Impact Factor
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ABSTRACT: Male moths can accurately perceive the sex pheromone emitted from conspecific females by their highly accurate and specific olfactory sensory system. Pheromone receptors are of special importance in moth pheromone reception because of their central role in chemosensory signal transduction processes that occur in olfactory receptor neurons in the male antennae. There are a number of pheromone receptor genes have been cloned, however, only a few have been functionally characterized. Here we cloned six full-length pheromone receptor genes from Helicoverpa armigera male antennae. Real-time PCR showing all genes exhibited male-biased expression in adult antennae. Functional analyses of the six pheromone receptor genes were then conducted in the heterologous expression system of Xenopus oocytes. HarmOR13 was found to be a specific receptor for the major sex pheromone component Z11-16:Ald. HarmOR6 was equally tuned to both of Z9-16: Ald and Z9-14: Ald. HarmOR16 was sensitively tuned to Z11-16: OH. HarmOR11, HarmOR14 and HarmOR15 failed to respond to the tested candidate pheromone compounds. Our experiments elucidated the functions of some pheromone receptor genes of H. armigera. These advances may provide remarkable evidence for intraspecific mating choice and speciation extension in moths at molecular level.PLoS ONE 01/2013; 8(4):e62094. · 3.53 Impact Factor
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ABSTRACT: Male moths locate their mates using species-specific sex pheromones emitted by conspecific females. One striking feature of sex pheromone recognition in males is the high degree of specificity and sensitivity at all levels, from the primary sensory processes to behavior. The silkmoth Bombyx mori is an excellent model insect in which to decipher the underlying mechanisms of sex pheromone recognition due to its simple sex pheromone communication system, where a single pheromone component, bombykol, elicits the full sexual behavior of male moths. Various technical advancements that cover all levels of analysis from molecular to behavioral also allow the systematic analysis of pheromone recognition mechanisms. Sex pheromone signals are detected by pheromone receptors expressed in olfactory receptor neurons in the pheromone-sensitive sensilla trichodea on male antennae. The signals are transmitted to the first olfactory processing center, the antennal lobe (AL), and then are processed further in the higher centers (mushroom body and lateral protocerebrum) to elicit orientation behavior toward females. In recent years, significant progress has been made elucidating the molecular mechanisms underlying the detection of sex pheromones. In addition, extensive studies of the AL and higher centers have provided insights into the neural basis of pheromone processing in the silkmoth brain. This review describes these latest advances, and discusses what these advances have revealed about the mechanisms underlying the specific and sensitive recognition of sex pheromones in the silkmoth.Frontiers in Physiology 01/2014; 5:125.
Sex-linked transcription factor involved in a shift
of sex-pheromone preference in the silkmoth
Tsuguru Fujiia, Takeshi Fujiib, Shigehiro Namikic, Hiroaki Abed, Takeshi Sakuraic, Akio Ohnumae, Ryohei Kanzakic,
Susumu Katsumaa, Yukio Ishikawab, and Toru Shimadaa,1
aLaboratory of Insect Genetics and Bioscience, Department of Agricultural and Environmental Biology, University of Tokyo, Tokyo 113-8657, Japan;
bLaboratory of Applied Entomology, Department of Agricultural and Environmental Biology, University of Tokyo, Tokyo 113-8657, Japan;cResearch
Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan;dLaboratory of Insect Functional Biochemistry, Department
of Biological Production, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan; andeInstitute of Sericulture, Ami, Ibaraki
Edited by John G. Hildebrand, University of Arizona, Tucson, AZ, and approved September 26, 2011 (received for review June 9, 2011)
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 facili-
tated the diversification of male preference are not well un-
derstood. 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 re-
duced 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.
Z chromosome | olfactory receptor | atavism | speciation | food preference
fine blending confer high species specificity, crucial to the re-
productive isolation of moths (1, 2). To date, great efforts have
been made to clarify the genes responsible for generating pher-
omone diversity by using pheromone strains within a species or
closely related species. Recently, it was shown that allelic vari-
ation in the fatty-acyl reductase gene or selective transcription of
desaturase genes is responsible for female sex-pheromone varia-
tion 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).
oths possess highly diverse and complex sex-pheromone
communication systems. A combination of compounds and
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 tri-
chodea 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, re-
A large variety of mutants are available in B. mori, repre-
senting 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 bi-
ologically important traits in Lepidoptera. To date, genes re-
sponsible 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 com-
putationally predicted in the deleted sequence (22). We con-
cluded 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
Author contributions: Tsuguru Fujii, Takeshi Fujii, S.N., H.A., A.O., R.K., S.K., Y.I., and
T. Shimada designed research; Tsuguru Fujii, Takeshi Fujii, S.N., and T. Sakurai performed
research; Tsuguru Fujii, Takeshi Fujii, S.N., and Y.I. analyzed data; and Tsuguru Fujii,
Takeshi Fujii, S.N., and Y.I. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the
GenBank database (accession nos. AB623137–AB623142 and AB635375–AB635378).
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 1, 2011
| vol. 108
| no. 44www.pnas.org/cgi/doi/10.1073/pnas.1107282108
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 (spliBt);
(ii) behavioral responsiveness of the male spli mutants (spli and
spliBt) to bombykol is markedly reduced in association with the
paucity of bombykol receptors on the antennae; (iii) full court-
ship 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
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) sug-
gested that the Bt mutant bears an allele of spli, which we desig-
nated spliBt(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 spliBtmutant (Fig. 1). Despite the non-
expression, 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 spliBtallele (accession no. AB635375–635378). We consid-
ered that spliBtis an allele of Bmacj6, the mRNA expression of
which was repressed by an unknown mechanism.
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 spliBtmales did not respond to ≤100 ng
of bombykol and only about 50% responded to 1,000 ng (Fig.
2A). This indicated that spli and spliBtmales 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 spliBtmales 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.
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 electroan-
tennogram (EAG) responses of male spli moths to bombykol and
bombykal. Whereas the response of spli and spliBtmales to
bombykal was similar to the response of p50T (+/+) and spli/+
males (Fig. 3A), the response to bombykol was significantly re-
duced 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 de-
termined the expression levels of BmOr1 and BmOr3 in the male
antenna by quantitative RT-PCR (qRT-PCR). Whereas expres-
sion levels of BmOr3 in the spli and spliBtmales 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 spliBtmales, 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.
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).
A major unresolved issue in the evolution of sexual communi-
cation systems in moths is how the pheromone recognition sys-
tems in the male moths diversified in association with the di-
versification 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, neuroana-
tomical 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
spliBtmales were markedly reduced, whereas those of BmOr3
were not affected (Fig. 3B). Bmacj6 was the only gene compu-
p50T(+/+ or +/W)
(spliBt/spliBt, spliBt/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.
RT-PCR analysis of Bmacj6 in p50T (+/+, +/W) and the mutant strain
Fujii et al. PNAS
| November 1, 2011
| vol. 108
| no. 44
tationally predicted to reside in the 66- to 96-kb sequence de-
leted from the Z chromosome of the spli mutant (22) (Fig. S1),
and Bmacj6 was not expressed in the spliBtmutant (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. Al-
though our results do not exclude the possibility that alteration in
the promoter region of BmOr1 caused the reduction in expres-
sion in the spli and spliBtmales, we think that this is unlikely for
two reasons. (i) BmOr1 and the 66- to 96-kb deletion, including
Expression levels relative to BmRPS3
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
Electrophysiological and molecular analyses of the spli male antenna. (A) Typical electroantennographic responses of the normal and spli moths to
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, spliBt/spliBt. 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.
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
| www.pnas.org/cgi/doi/10.1073/pnas.1107282108Fujii et al.
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 in-
dependently in the two mutant strains, which had been estab-
lished on the basis of unrelated phenotypes, a soft and pliable
larval body (spli) and abnormal feeding behavior (spliBt).
Each long sensillum trichodeum on the male antenna com-
prises 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 ex-
pression of BmOr3, which would have been expected if the ec-
topic 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 re-
quired 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 spe-
cies belonging to the Sphingidae and Saturniidae, families closely
related to Bombycidae, use bombykal as a sex-pheromone com-
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 in-
triguing 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 be-
tween splice forms resulted in different functions (34). There-
fore, 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
e t a r g
n i r i f n
k i p
20 mV, 1 s
10 mV, 1 s
u l u
c - d i o r o
o i t a r e
u l o
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 pro-
tocerebrum (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 in-
nervating 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.
Glomerular organization in the antennal lobe and responses of projection neurons innervating the toroid. (A) Confocal image of a projection neuron
Fujii et al. PNAS
| November 1, 2011
| vol. 108
| no. 44
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 malemothsto 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 stimula-
tion 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 experi-
ments 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 re-
sistance 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 com-
puter through an A/D converter (Quick Vu 2; TEAC). The odorant was ap-
plied 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 hyper-
polarizing 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 sa-
licylate 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.
ACKNOWLEDGMENTS. We thank M. R. Goldsmith for critical review. The
silkworm strains and DNA clones were provided by the National Bioresource
Project, Ministry of Education, Culture, Sports, Science and Technology,
Japan. We are grateful to M. Kawamoto for his technical assistance. This
work was supported by Grants-in-Aid for Scientific Research (Grants
21248006 and 22128004), the Agri-genome Research Program (Ministry of
Agriculture, Forestry and Fisheries, Japan), and the Professional Program for
Agricultural Bioinformatics (Ministry of Education, Culture, Sports, Science
and Technology, Japan). Tsuguru Fujii is a recipient of the Japan Society for
the Promotion of Science Fellowship for Young Scientists.
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Fujii et al.PNAS
| November 1, 2011
| vol. 108
| no. 44
Fujii et al. 10.1073/pnas.1107282108
are underlined. Six splicing variants were isolated from the moth antennae (Table S2). Two types of splice donor sites (a and b) in exon 3 and five types of splice
acceptor sites (c–g) in exon 4 were identified. (B) Amino acid sequence alignment of Bmacj6, Acj6, and GA002017. On the basis of amino acid sequence
similarity, GA002017 was predicted to be the Bombyx homolog of Acj6 (1). (C) Exons 2–4 are deleted in the spli mutant. The Z chromosome of the spli mutant is
split into two parts, Z3and Z4. A fragment of chromosome 5 is incidentally connected to Z4. A 66- to 96-kb sequence is deleted by a breakage event in the Z
chromosome (1). Introns 1 (39 kb), 2 (33 kb), and 3 (23 kb) of Bmacj6 occupy most of the deleted region.
Molecular analysis of Bmacj6. (A) Exon/intron structure of Bmacj6. Regions encoding the POU IV box, POU-specific domain, and POU homeodomain
1. Fujii T, et al. (2010) Identification and molecular characterization of a sex chromosome rearrangement causing a soft and pliable (spli) larval body phenotype in the silkworm, Bombyx
mori. Genome 53:45e54.
(BmOr1) neurons and bombykal receptor (BmOr3) neurons project to the toroid and cumulus, respectively. Bombykol alone elicits full male courtship behavior,
whereas bombykal alone shows no apparent activity. (B) Mistargeting hypothesis. Axons of BmOr3 neurons anomalously project to the toroid. The reduced
response of spli moths to bombykol is explained by the paucity of BmOr1. (C) Ectopic expression hypothesis. Decreased expression of BmOr1 is compensated by
the ectopic expression of BmOr3 in the same neurons. The smaller Or1 indicates reduced expression.
Hypotheses explaining the induction of full male courtship behavior by bombykal in the spli mutants. (A) In normal males, bombykol receptor
Fujii et al. www.pnas.org/cgi/content/short/11072821081 of 3
Fig. S3. Devices used to analyze the behavioral response to sex pheromones. Materials and Methods has detailed descriptions.
phenotype identified in the Bt strain
Crossing experiment used to map the soft and pliable
Mating scheme NormalSoft and pliable NormalSoft and pliable
+/W × Bt/Bt
spli/W × Bt/Bt
Table S2. Bmacj6 splice variants
1, 2, 3a, 4c
1, 2, 3a, 4d
1, 2, 3b, 4e
1, 3a, 4d
1, 2, 4f
1, 2, 4g
Six Bmacj6 moth antenna splice variants were identified; a and b indicate
the different splice donor sites, and c–g indicate the different splice acceptor
Table S3.Primers used in this study
Sequence (5′–3′ end) Object
First RT-PCR for Bmacj6
Nested RT-PCR for Bmacj6
5′ RACE for Bmacj6
3′ RACE for Bmacj6
Genomic PCR for exon 1 of Bmacj6
Genomic PCR for exon 2 of Bmacj6
Genomic PCR for exon 3 of Bmacj6
Genomic PCR for exon 4 of Bmacj6
Fujii et al. www.pnas.org/cgi/content/short/1107282108 2 of 3
(100 ng/μL) and bombykol (50 ng/μL), respectively.
Mating behavior of spli (white because of +mln) and normal (+spli; gray because of the melanism) moths. Left and right vials contain bombykal
Fujii et al. www.pnas.org/cgi/content/short/11072821083 of 3