Odorant-binding proteins and olfactory coding in the solitary bee Osmia cornuta

Article abstract of DOI:10.1007/s00018-013-1308-2

Solitary bees are major pollinators but their chemical communication system has been poorly studied. We investigated olfactory coding in Osmia cornuta from two perspectives, chemical and biochemical. We identified (E)-geranyl acetone and 2-hexyl-1,3-dioxolane, specifically secreted by females and males, respectively. A transcriptome analysis of antennae revealed 48 ORs (olfactory receptors), six OBPs (odorant-binding proteins), five CSPs (chemosensory proteins), and a single SNMP (sensory neuron membrane protein). The numbers of ORs and OBPs are much lower than in the honeybee, in particular, C-minus OBPs are lacking in the antennae of O. cornuta. We have expressed all six OBPs of O. cornuta and studied their binding specificities. The best ligands are common terpene plant odorants and both volatiles produced by the bee and identified in this work.

Full text of DOI:10.1007/s00018-013-1308-2

Cell. Mol. Life Sci. (2013) 70:3029–3039
DOI 10.1007/s00018-013-1308-2
Cellular and Molecular Life Sciences
RESEARCH ARTICLE
Odorant-binding proteins and olfactory coding in the solitary bee
Osmia cornuta
Xue-Wei Yin · Immacolata Iovinella · Roberto Marangoni ·
Federica Cattonaro · Guido Flamini · Simona Sagona ·
Long Zhang · Paolo Pelosi · Antonio Felicioli
Received: 19 November 2012 / Revised: 13 February 2013 / Accepted: 18 February 2013 / Published online: 20 March 2013
©
Springer Basel 2013
Abstract Solitary bees are major pollinators but their
chemical communication system has been poorly studied.
We investigated olfactory coding in Osmia cornuta from
two perspectives, chemical and biochemical. We identified
expressed all six OBPs of O. cornuta and studied their bind-
ing specificities. The best ligands are common terpene plant
odorants and both volatiles produced by the bee and identi-
fied in this work.
(E)-geranyl acetone and 2-hexyl-1,3-dioxolane, specifi-
cally secreted by females and males, respectively. A tran-
scriptome analysis of antennae revealed 48 ORs (olfactory
receptors), six OBPs (odorant-binding proteins), five CSPs
Keywords Osmia cornuta · Solitary bees · Chemical
communication · Transcriptome · Odorant-binding
proteins · Olfactory receptors
(chemosensory proteins), and a single SNMP (sensory neu-
ron membrane protein). The numbers of ORs and OBPs are
much lower than in the honeybee, in particular, C-minus
OBPs are lacking in the antennae of O. cornuta. We have
Abbreviations
OR
Olfactory receptor
OBP
Odorant-binding protein
CSP
Chemosensory protein
SNMP
1-NPN
IPTG
Sensory neuron membrane protein
N-Phenyl-1-naphthylamine
Isopropyl-1-thio-β-d-galacto-pyranoside
X.-W. Yin and I. Iovinella have contributed equally to this work.
Electronic supplementary material The online version of this
article (doi:10.1007/s00018-013-1308-2) contains supplementary
material, which is available to authorized users.
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel
electrophoresis
GC/MS
Amel
Bter
Mrot
Ocor
Gas-chromatography/mass spectrometry
Apis mellifera
Bombus terrestris
Megachile rotundata
Osmia cornuta
X.-W. Yin · I. Iovinella · P. Pelosi
Department of Agriculture, Food and Environment,
University of Pisa, Pisa, Italy
R. Marangoni
Department of Biology, University of Pisa, Pisa, Italy
F. Cattonaro
IGA Technology Services Srl, Udine, Italy
Introduction
G. Flamini
The study of olfaction in honeybees has received much
attention due to the complex chemical language these social
insects use for exchanging information between members of
the colony [1–12].
Solitary bees that build individual nests reasonably need
a simpler olfactory system to perform a limited number
of tasks, such as finding a mate and monitoring the envi-
ronment. Very little information is available on chemical
Department of Pharmacy, University of Pisa, Pisa, Italy
S. Sagona · A. Felicioli (*)
Department of Veterinary Sciences, University of Pisa, Pisa, Italy
e-mail: a.felicioli@vet.unipi.it
X.-W. Yin · L. Zhang
Key Lab for Biological Control of the Ministry of Agriculture,
China Agricultural University, Beijing, China
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X.-W. Yin et al.
communication in solitary bees concerning both semio-
chemicals and proteins mediating their perception [13–16].
However, solitary bees represent an extremely important
subject of investigation due to their key role in pollinating
a great variety of plants of economic interest [17]. Moreo-
ver, there is great concern about the recent decline of their
populations [18, 19]. In particular, solitary bees of the genus
Osmia are reared and used as alternative fruit pollinators
gene. Again, this work clearly demonstrated that the lack of
a single gene encoding OBP is enough to upset the olfactory
performance of the fly [37]. Research performed with two spe-
cies of aphids has provided evidence that two OBPs, out of the
dozen that represent the total repertoire of these proteins in
aphids, are involved in the recognition of the alarm message
conveyed by the specific pheromone β-farnesene [38, 39].
Shifting the focus from olfactory receptors to OBPs pre-
sents the great advantage that OBPs, being small and solu-
ble, are proteins very easy to study both at the structural
level and regarding their capacity of binding pheromones
and odorants. In fact, a very large number of OBPs have
been described in different insect species and their ligand
specificities to pheromones and general odorants has been
mapped [34]. Moreover, the three-dimensional structures of
18 OBPs, four of them from A. mellifera, have been solved,
both using X-ray crystallography and NMR [40–43], thus
paving the way to designing mutants and understanding at
the microscopic level specific interactions between these
proteins and their ligands. Finally, another class of small
soluble proteins, CSPs (chemosensory proteins) is found in
the lymph of chemosensilla [34, 44–51]. Although different
in amino acid sequence and structure from OBPs [52–54],
several aspects suggest that the two classes of proteins
might be similar in function. However, evidence for a pos-
sible requirement of CSPs in olfactory transduction has not
been provided.
[20–23]. Finally, a comparison between the repertoires
of proteins involved in chemical communication between
social and solitary bees could be highly informative and
indicate which proteins are specifically involved in exchang-
ing chemical information between individuals of the same
nest in the former species.
Recent research has shown that three classes of proteins are
required for a correct functioning of the olfactory system in
insects, membrane-bound olfactory receptors (ORs), so-called
sensory neuron membrane protein (SNMP) whose specific
mode of action is still unclear, and soluble odorant-binding
proteins (OBPs). Experiments have demonstrated that silenc-
ing genes encoding proteins of each of these three classes
abolishes or modifies electrophysiological and/or behavioral
response of insects to olfactory stimuli [24–28]. Olfactory
receptors, belonging to the family of seven-helices transmem-
brane proteins, have been regarded as the biochemical ele-
ments responsible for detecting and discriminating olfactory
stimuli [29–31]. According to the current view, direct binding
of odorant molecules to ORs initiates a series of events lead-
ing to depolarization of the olfactory neurons. SNMP are also
membrane proteins belonging to the family of two-transmem-
brane helical domains. There are generally only one or two
genes encoding such proteins in each insect species and their
function still remains unclear. Most interesting is the specific
role of OBPs that has emerged on the basis of recent research.
OBPs are small soluble proteins of the chemosensillar lymph
In this paper, we report on the identification of the differ-
ent classes of proteins involved in olfaction in the solitary
bee Osmia cornuta, based on a transcriptome analysis of
antennal RNA, and the characterization of some representa-
tive OBPs. We have also identified sex-specific semiochem-
icals of O. cornuta that represent good ligands for the OBPs
of the same species. Finally, we have expressed five of the
six OBPs of O. cornuta and mapped their ligand-binding
specificities to a series of compounds, including the newly
identified semiochemicals.
[32–34]. Only recently an OBP of Drosophila melanogaster,
LUSH, has been demonstrated to be required for detection of
the specific pheromone cis-vaccenyl acetate [25]. Moreover, a
direct interaction of LUSH with its related receptor has been
hypothesized on the basis of structural and electrophysiologi-
cal data [35]. Other studies have investigated the contribu-
tion of OBPs in the recognition of specific odor messages in
behavior assays [36–39]. The unusual attraction of Drosophila
sechellia to the distinctive smell of a tropical plant Morinda
citrifolia, otherwise repulsive for other Drosophila spe-
cies, has been related to two specific OBPs. Exchanging the
genes encoding these proteins between D. sechellia and other
Drosophila species, the behavior of these species towards
the smelly plant resulted to be strongly modified, sometimes
reaching opposite results with respect to the wild types [36].
Another work addressed the same issue in a more general way,
studying the behavior to the same set of odors of a number of
mutants of D. melanogaster, each defective in a specific OBP
Materials and methods
Reagents
All enzymes were from New England Biolabs. Oligonucle-
otides were custom synthesized at Eurofins MWG GmbH,
Ebersberg, Germany. All other chemicals were purchased
from Sigma-Aldrich and were of reagent grade.
Insects
O. cornuta bees were reared in the experimental field of the
Apidology and Biochemistry Laboratory at the Department
of Veterinary Sciences, University of Pisa. Cocoons were
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Olfaction in solitary bees
3031
collected from artificial nests during the month of Novem-
ber 2011, after the bees had reached the imago stage and
were ready to enter diapause. Cocoons were then kept at
Analysis of sequences
Sequence similarity search for assembled contigs was per-
formed using BLAST v. 2.2.25 (utility tBLASTn, standard
penalties for DNA substitutions) and the sequences of hon-
eybee Apis mellifera and the solitary bee, Megachile rotun-
data as templates.
4
°C until February 2012, then brought outdoors until
emergence.
RNA extraction and cDNA synthesis
Alignments were performed with ClustalW using a Blo-
sum62 matrix and the following values: gap open 5, gap
extension 0.05, and NJ (neighbor-joining) clustering, and
similarity trees were constructed based on such alignments.
Total RNA was extracted with TRI Reagent (Sigma), follow-
ing the manufacturer’s protocol. cDNA was prepared from
total RNA by reverse transcription, using 200 units of Super-
Script III Reverse Transcriptase (Invitrogen) and 0.5 μg of
an oligo-dT primer in 50 μl of total volume. The mixture
also contained 0.5 mM of each dNTP (GE-Healthcare),
Polymerase chain reaction
7
5 mM KCl, 3 mM MgCl , 10 mM DTT, and 0.1 mg/ml
Aliquots of 1 μl of crude cDNA were amplified in a Bio-
Rad Gene Cycler thermocycler, using 2.5 U of Thermus
aquaticus DNA polymerase (GE Healthcare), 1 mM of
each dNTP (GE Healthcare), 1 μM of each PCR primer,
2
BSA in 50 mM Tris–HCl, pH 8.3. The reaction mixture was
incubated at 50 °C for 60 min and the product was directly
used for PCR amplification or stored at −20 °C.
5
0 mM KCl, 2.5 mM MgCl and 0.1 mg/ml BSA in 10 mM
2
Transcriptome sequencing and assembly
Tris–HCl, pH 8.3, containing 0.1 % v/v Triton X-100. At the
′ end, we used specific primers corresponding to the
5
For transcriptome analysis, total RNA was extracted from
a pooled sample of antennae collected in the morning
soon after emergence from 100 males and 100 females of
O. cornuta.
sequence encoding the first six amino acids of the mature
protein. The primers also contained an NdeI restriction site
for ligation into the expression vector and providing at the
same time the ATG codon for an additional methionine in
position 1. For OBP1, we used a BamHI site instead of NdeI
with the addition of an extra base (G) to obtain the sequence
in frame with the starting codon. At the 3′ end, specific prim-
ers were used, encoding the last six amino acids, followed
by a stop codon and an EcoRI restriction site (an XhoI site
in the case of OBP2 and OBP4) for ligation into the expres-
sion vector. Therefore, we used the following primers for
the each protein (enzyme restriction sites are underlined):
RNA samples were processed using TruSeq RNA-seq
Sample Prep kit from Illumina (Illumina, Inc., CA, USA).
Briefly, the poly-A-containing mRNA molecules were puri-
fied from a total of 2 μg of total RNA using poly-T oligo-
attached magnetic beads, fragmented into small pieces
using divalent cations under elevated temperature, cDNA
was synthesized by reverse transcription and standard blunt-
ending plus add ‘A’ was performed. Then Illumina TruSeq
adapters with indexes were ligated to the ends of the cDNA
fragments. After ligation reaction and separation of not
ligated adapters, samples were amplified by PCR to selec-
tively enrich those cDNA fragments in the library having
adapter molecules at both ends.
fwOcorOBP1: 5′-GGATCCGCTCGATCAAGATGCCG
T-3′
rvOcorOBP1:5′-GAATTCCTAGTTGAAAAGCTTCTG
-3′
The sample was loaded on Illumina flowcell and clus-
ters created by Illumina cBot. Clusters were sequenced at
ultra-high throughput on Illumina HiSeq2000 (Illumina
Inc.) obtaining 228.8 millions of pair-end reads, 100 bp
long. The Illumina reads were trimmed by quality scores
using a modified version of the Mott algorithm (limit 0.05),
as implemented on CLC Genomics Workbench (CLC-Bio,
Denmark). The reads were next trimmed at the ends on
the basis of the presence of ambiguous nucleotides (typi-
cally N) by using the same software (maximum value set at
fwOcorOBP2: 5′-CATATGGATGAGGATATAATAAAA
GT-3′
rvOcorOBP2:5′-CTCGAGTCAAGGGAAGAAGAACG
TCTC-3′
fwOcorOBP3:5′-CATATGCTCACTGAGGAACAAA
A-3′
rvOcorOBP3:5′-GAATTCTTAGTTGAGTACGTTGA
A-3′
fwOcorOBP4: 5′-CATATGGATGAGGATATAATAAAA
GT-3′
2
). Then, high-quality reads were assembled into contigs
rvOcorOBP4: 5′-CTCGAGTCAGGCAGGGGTTTTCA
GGTC-3′
fwOcorOBP5: 5′-CATATGGAGCCTGACGATGTTT-3′
rvOcorOBP5: 5′-GAATTCTTAGTTTTCCATGAAACA
TTT-3′
using CLC Genomics Workbench v. 4.9 (CLC Bio, Den-
mark) at default parameters. RNA sequencing was per-
formed at IGA Technology Services Srl Service Provider
(
Udine, Italy).
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X.-W. Yin et al.
fwOcorOBP6: 5′-CATATGGTCTCGGGTGATCTCGA
3′
100 ml with Tris buffer and dialyzed three times against
Tris buffer.
-
rvOcorOBP6:5′-GAATTCTTAAAGAATGTGCGTTCG
CG-3′
Purification of the protein was accomplished by com-
binations of chromatographic steps anion-exchange resins
DE-52 (Whatman) and QFF (GE Healthcare), along with
standard protocols previously adopted for other OBPs
[55, 56].
After a first denaturation step at 95 °C for 5 min, we
performed 35 amplification cycles (1 min at 95 °C, 30 s at
5
7
0 °C, 1 min at 72 °C) followed by a final step of 7 min at
2 °C. Amplification products of about 400 bp, in agree-
Fluorescence measurements
ment with the expected sizes were obtained in all cases.
Emission fluorescence spectra were recorded on a Jasco FP-
750 instrument at 25 °C in a right-angle configuration, with
a 1-cm light path quartz cuvette and 5-nm slits for both exci-
tation and emission. The protein was dissolved in 50 mM
Tris–HCl buffer, pH 7.4, while ligands were added as 1 mM
methanol solutions.
Cloning and sequencing
The crude PCR product was ligated into a pGEM (Pro-
mega) vector without further purification, using a 1:5
(plasmid:insert) molar ratio and incubating the mixture
overnight, at room temperature. After transformation of
E. coli XL-1 Blue competent cells with the ligation product,
positive colonies were selected by PCR using the plasmid’s
primers SP6 and T7 and grown in LB/ampicillin medium.
DNA was extracted using the Plasmid MiniPrep Kit (Euro-
clone) and custom sequenced at Eurofins MWG (Ebersberg,
Germany).
Ligand-binding experiments
The affinity of the fluorescent ligand 1-NPN to each protein
was measured by titrating a 2 μM solution of the protein with
aliquots of 1 mM ligand in methanol to final concentrations
of 2–16 μM. The probe was excited at 337 nm and emission
spectra were recorded between 380 and 450 nm, following
the established protocol [57]. The affinity of other ligands
was measured in competitive binding assays, where a solu-
tion of the protein and 1-NPN, both at the concentration of
2 μM (except for OBP3 that was used at the concentration
of 5 μM) was titrated with 1 mM methanol solutions of each
competitor to final concentrations of 2–16 μM. Dissociation
constant for 1-NPN and the stoichiometry of binding was
obtained processing the data with Prism software. Disso-
ciation constants of the competitors were calculated from
the corresponding IC50 values (concentrations of ligands
halving the initial fluorescence value of 1-NPN), using the
equation: KD = [IC50]/1 + [1−NPN]/K1−NPN, [1−NPN]
being the free concentration of 1-NPN and K1−NPN being
the dissociation constant of the complex protein/1-NPN.
Cloning in expression vectors
pGEM plasmid containing the appropriate sequence was
digested with NdeI and EcoRI restriction enzymes for 2 h
at 37 °C and the digestion product was separated on an aga-
rose gel. The obtained fragments were purified from gel
using QIAEX II Extraction kit (Qiagen) and ligated into the
expression vector pET5b or pET30b (Novagen, Darmstadt,
Germany), previously linearized with the same enzymes.
The resulting plasmid was sequenced and shown to encode
the mature protein.
For OBP4 that could not be expressed following the
standard procedure, we used a synthetic gene, custom pre-
pared at MWG-Eurofins (Ebersberg, Germany).
Preparation of the recombinant protein
Analysis of volatiles
For expression of recombinant protein, pET vectors con-
taining the sequence encoding the appropriate OBP was
used to transform BL21(DE3)pLysS E. coli cells. Protein
expression was induced by the addition of IPTG to a final
concentration of 0.4 mM when the culture had reached a
value of OD600 = 0.8. Cells were grown for further 2 h
at 37 °C, then harvested by centrifugation and sonicated.
After centrifugation, the expressed proteins were obtained
in soluble form, except for OBP2 and OBP6 that were
present as inclusion bodies. To solubilize them, the pellet
from 1 l of culture was dissolved in 10 ml of 8 M urea,
Male antennae, excised soon after emergence, or whole
female live individuals were housed in 3-ml vials closed by
a Teflon-coated rubber. The headspace was analyzed using
Supelco SPME devices coated with polydimethylsiloxane
(PDMS, 100 μm) that were inserted through the rubber sep-
tum and exposed to the headspace for 30 min at room tem-
perature. The fiber was then withdrawn into the needle and
transferred to the injection port of the GC–MS system consti-
tuted by a Varian CP-3800 gas-chromatograph equipped with
a DB-5 capillary column (30 m × 0.25 mm; coating thickness
0.25 μm) and a Varian Saturn 2000 ion trap mass detector.
Analytical conditions were: injector and transfer line at 250
1
mM DTT in 50 mM Tris buffer, pH 7.4, then diluted to
1
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Olfaction in solitary bees
3033
and 240 °C, respectively; oven temperature was programmed
from 60 to 240 °C at 3 °C/min; carrier gas helium at 1 ml/min;
splitless injection. Identification of the constituents was based
on comparison of the retention times with those of authentic
samples, comparing their linear retention indices (LRI) rela-
tive to the series of n-hydrocarbons, and by computer match-
ing against commercial (NIST 98 andADAMS 95) and home-
made library mass spectra built up from pure substances and
components of known essential oils and MS literature data.
Identity of the two key compounds, 2-hexyl-1,3-dioxolane
and (E)-geranylacetone, was confirmed by comparison of GC
retention times and mass spectra of authentic samples ana-
lyzed in the same conditions.
expressed in the antennae of O. cornuta to identify OBPs,
ORs, and other proteins involved in chemical communi-
cation, and to compare its repertoire with that known for
the social bee A. mellifera. This Transcriptome Shotgun
Assembly project has been deposited at DDBJ/EMBL/Gen-
Bank under the accession GAGH00000000. The version
described in this paper is the first version, GAGH01000000.
Therefore, we have extracted RNA from a pooled samples
of antennae from 100 males and 100 females. Illumina high-
quality pair-end reads were assembled into 85,143 contigs
(average contig length = 914.5, N50 = 8,265, L50 = 1,932).
We then performed a BLAST search on the assembled
contigs to identify OBPs, ORs, SNMPs, and CSPs, using
BLAST v. 2.2.25 (utility tBLASTn, standard penalties for
DNA substitutions) and the sequences of the honeybee
Apis mellifera and the solitary bee, Megachile rotundata as
templates.
This search yielded six complete sequences encod-
ing OBPs (acc. numbers: KC464549-54 for OBP1-OBP6)
aligned in Fig. 1 and five for CSPs, reported in Figure S1,
together with their orthologues in A. mellifera and M. rotun-
data. For both species of solitary bees we assigned to CSPs
the same number of their A. mellifera orthologues. Only
for CSP5 of A. mellifera we did not find an orthologue in
O. cornuta. As this CSP was only found in the eggs and ova-
ries of the honeybee [58], we were not surprised to find that
a similar protein was missing in our antennal transcriptome
analysis.
Synthesis of 2-hexyl-1,3-dioxolane
Heptanal (5.7 g, 50 mmol) was refluxed with 10 ml of ethylene
glycol and a trace of p-toluenesulfonic acid in 50 ml of ben-
zene for 8 h in a Dean-Stark apparatus. Extraction with diethyl
ether, evaporation of the solvent and distillation of the residue
under reduced pressure afforded pure 2-hexyl-1,3-dioxolane.
Results
Identification of olfactory genes in the antennae
of O. cornuta
As genome information is not available for solitary bees,
we decided to perform a transcriptome analysis of the genes
We also identified a single SNMP, highly similar to those
of A. mellifera (acc. n. P86905) and M. rotundata (acc. n.
Fig. 1 Alignment of the six OBPs identified in the antennal tran-
scriptome of O. cornuta. The sequences are highly divergent and
only share the six cysteines of insects OBPs signature. In the similar-
ity tree, their amino acid sequences (names in red font) are compared
with those of the 21 OBPs of A. mellifera (blue) 17 of B. terrestris
XP_003397885, XP_003397906, XP_003397921, XP_003398555,
XP_003398556, XP_003399482, XP_003401135, XP_003401479,
XP_003401480, XP_003402792, XP_003403020; M. rotundata:
XP_003700064–XP_003700067, XP_003700069, XP_003700073,
XP_003707077, XP_003707078, XP_003701453, XP_003704755,
XP_003708550. For the last two species, the nomenclature adopted is
OBP followed by the last three digit of the accession number
(green), and 11 of M. rotundata (magenta). Accession numbers are:
O. cornuta OBP1-6: KC464549-54; B. terrestris: XP_003393962,
XP_003397012, XP_003397647, XP_003397865, XP_003397877,
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3
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X.-W. Yin et al.
XP_003699727). The alignment is shown in Figure S2.
As reported in the“Introduction”, these proteins have been
described as transmembrane polypeptides associated with
chemosensory neurons of insects and have been shown to
be required for a correct functioning of the olfactory system
in Drosophila [26–28].
Concerning ORs, we found sequence segments with sig-
nificant similarity to the 170 ORs of A. mellifera [4] and/or
to the 123 of M. rotundata in 48 contigs. We could identify
designed on both ends of this reconstructed sequence, we
were not able to obtain any amplification product in PCR
experiments. Instead, we successfully amplified a segment
of cDNA when we used primers designed on the originally
identified OBP. Sequencing of this product, after cloning in
pGEM, confirmed the unusual sequence identified by bioin-
formatic analysis.
Volatile compounds produced by O. cornuta
2
6 complete or partial sequences of at least 200 amino acids,
that are aligned in Figure S3. In particular, one of the com-
plete sequences (OcorOR945) presents high similarity with
so-called Orco, the common olfactory receptors, that forms
heterodimers with other specific ORs [59, 60]. In Figure S4,
OcorOR945 is aligned with its orthologues in A. mellifera
At the same time, we addressed our attention to poten-
tial semiochemicals produced by O. cornuta and possibly
involved in short-range courtship signal function. Based
on earlier studies of the courtship in this species [61], we
suspected that volatiles could be secreted by the antennae
of males during pre-copulation courtship. This idea was
supported by the presence of pores on the antennal cuticle
observed only in males and connected to secretory cells
(91 % identity) and M. rotundata (89 % identity).
Odorant-binding proteins
[61]. Moreover, it was reported in the same paper that pre-
As for OBPs, we only found six genes encoding proteins of
this family in the antennae of O. cornuta, compared with
copulatory courtship behavior during mating involves com-
plex stereotyped sequence of periodical motions of the male
antennae without any contact with the female. We therefore
analyzed the headspace of male antennae and whole live
females, collected soon after emergence. The gas-chroma-
tographic analyses (Figure S5) show in both cases simple
profiles with a limited number of peaks. In particular, one
of these peaks, only present in the male antennal sample,
where it represents the most abundant component, has been
identified as the cyclic acetal of heptanal with ethylene
glycol. The structure has been confirmed with an authen-
tic sample, synthesized from heptanal and ethylene glycol.
The female gas-chromatographic profile, on the other hand,
contains a prominent specific component, identified as
(E)-geranylacetone, also by comparison with an authentic
commercial sample. The identification of these two bee-
associated odors is still preliminary to conclude that such
volatiles actually mediate chemical communication between
sexes. However, these results provided two potential ligands,
synthesized by the bees in a sex-specific function, to utilize
in binding experiments.
2
1 in the genome of A. mellifera [6], of which 15 have been
detected in the antennae of foragers [6] (12 of them identi-
fied at the protein level [11]). It is also worth observing that
the OBPs found in O. cornuta are all classic OBPs (although
OBP6 presents a different arrangement of the six cysteines),
while the antennae of foragers honeybee express all eight
sequences of the C-minus family, besides seven classic
OBPs. The sequences of the six OBPs of O. cornuta are
aligned in Fig. 1, that also reports a similarity tree including
those of social bees A. mellifera and Bombus terrestris as
well as the 11 OBPs of another solitary bee, M. rotundata.
Accession numbers for the OBPs of these two species are
reported in the legend of Fig. 1.
Four OBPs could be considered as orthologues between
O. cornuta and A. mellifera, namely OcorOBP1 and
AmelOBP2 (65 % identity), OcorOBP2 and AmelOBP5
(52 %), OcorOBP3 and AmelOBP13 (42 %), OcorOBP6
and AmelOBP11 (45 %), while the other two, OcorOBP4
and OcorOBP5 are less closely related to AmelOBP2 (29
and 22 %, respectively).
A particular note deserves OcorOBP6, that we could
easily identify in the transcriptome. Most of its sequence
is very similar to OBP11 of the honeybee, up to the fourth
cysteine of the conserved motif. The C-terminal segment,
instead, markedly differs from those of other OBPs, but still
contains the two remaining cysteines, although in non-con-
served positions (Fig. 1). We suspected this could be due to a
misreading of the sequences and looked for a more familiar
C-terminus in the same contig. In fact, this search produced
a sequence that could reasonably be the missing C-ter-
minus of our protein. This looked like a most reasonable
explanation, except for the fact that, using specific primers
Bacterial expression of OBPs
We then decided to express the OBPs of O. cornuta and
analyze their binding properties with relationship to poten-
tial semiochemicals for this species. Based on the recently
recognized key role of OBPs in decoding olfactory mes-
sages [25, 35–39], we decided to focus our study on these
soluble proteins rather than on ORs, also given the lim-
ited number of OBPs in the antennae of O. cornuta and
the relative ease in handling such proteins. All six OBPs
have been successfully expressed in bacteria in high yields
and were obtained in their soluble forms, except for OBP2
1
3

Olfaction in solitary bees
3035
and OBP6, present as inclusion bodies. Solubilization in
these cases was accomplished by denaturation with urea
and DTT, followed by slow renaturation through exten-
sive dialysis. This protocol affords the proteins in active
form, as previously demonstrated in several cases [55, 56].
Purification was then easily accomplished for OBPs 1–5
through a first chromatographic fractionation on anion-
exchange resin DE-52 (Whatman), followed by a second
separation on the same resin or on a QFF (GE-Healthcare).
This protocol afforded samples of the proteins electropho-
retically pure and free from DNA. Electrophoretic analyses
relative to expression and purification of these OBPs are
reported in Figure S6. OBP6, instead, proved difficult to
purify. In fact, every attempt to fractionate the solubilized
protein through a DE-52 column resulted in complete loss
of the protein. We reasoned that, because of their unusual
position, two of the last three cysteines could be present
in their reduced forms and therefore available to form
intermolecular bridges, eventually resulting in insoluble
oligomers. Given these difficulties and the unconventional
sequence of OBP6, we decided to limit our study to the
other five OBPs.
Fig. 2 Ligand-binding properties of the five expressed OBPs of
O. cornuta. a Binding of the fluorescent probe 1-NPN to purified
OBPs. Protein solutions (2 μM in Tris buffer, pH 7.4) were titrated
with 1 mM solution of 1-NPN in methanol to final concentrations
of 2–16 μM. Values are averages of three replicates. Calculated dis-
sociation constants were: OBP1: 1.4 μM (SD 0.35), OBP2:1.6 μM
the same order of the values reached at the concentration of 12 μM.
c Graphical summary of the dissociation constants measured with the
indicated ligands and the five OBPs of O. cornuta. For more imme-
diate visualization, the affinity constants are reported instead of the
dissociation constants. For ligands producing displacement too weak
to allow evaluation of the dissociation constants, we have reported a
minimum nominal value corresponding to a KD of 50 μM. In addi-
tion to the reported ligands, 1-octen-3-ol, 2-heptanone and menthol
did not show measurable affinity to any of the five OBPs. d Struc-
tures of the ligands used in binding experiments. Geranyl acetone
and 2-hexyl-1,3-dioxolane are the volatiles secreted by female whole
body and male antennae, respectively
(SD 0.48), OBP3: 0.9 μM (SD 0.41), OBP4: 3.7 μM (SD 0.36),
OBP5: 1.2 μM (SD 0.25). b Displacement of 1-NPN from its com-
plex with OBP1 by some representative ligands. A mixture of protein
and 1-NPN, both at 2 μM in Tris buffer, pH 7.4, was titrated with
1
2
mM solution of each ligand in methanol to final concentrations of
-16 μM. The ligands are listed from the weakest to the stronger in
1
3

3
036
X.-W. Yin et al.
Ligand-binding experiments
values of dissociation constants. All the chemical structures
of the ligands utilized are reported in Fig. 2d.
The purified OBPs were used in ligand-binding experiments
with both compounds produced by male and female bees
and identified in our GC/MS analysis, as well as a num-
ber of natural and synthetic chemicals, that helped defining
the structural requirements for strong binding to each OBP.
The fluorescent probe, usually adopted in such studies with
insect OBPs (1-NPN: N-phenyl-1-naphthylamine), binds all
five recombinant OBPs with dissociation constants in the
micromolar range (Fig. 2a).
Using 1-NPN as the fluorescent reporter, we then meas-
ured the affinity of a number of pure volatile compounds
in competitive binding assays. Besides the two volatiles
secreted by O. cornuta males and females, we included in
our study several terpenoids and other compounds com-
monly found in essential oils of leaves (3-hexen-1-ol and
nonanal), and flowers (linalool, citral, citronellal, geranyl
acetate), plant-derived aromatic compounds (eugenol, ethyl
cinnamate, butyl cinnamate), components of honeybee
pheromones (coniferyl alcohol, homovanillic acid, 2-hep-
tanone) and other derivatives with structural similarities to
the above compounds. We generally measured moderate to
good affinities of OBPs 1, 3, and 5 to terpenoids and other
natural compounds of medium size, including the two vola-
tiles secreted by our solitary bees (2-hexyl-1,3-dioxolane
and (E)-geranylacetone). Figure 2b shows as an example
the displacement curves obtained with OBP1 and few rep-
resentative ligands, while all the measured affinity constants
are shown in graphical form in Fig. 2c. Table 1 reports the
Discussion
The antenna of the solitary bee O. cornuta contains genes
encoding at least 48 ORs, six OBPs, one SNMP, and five
CSPs.
The number of ORs seems much smaller than the 170
of A. mellifera and the 123 reported in another solitary
bee, M. rotundata. However, the presence of several partial
sequences among the identified ORs of O. cornuta suggests
that their number could be higher.
For OBPs, instead, we can confidently assume that all the
members of this family present in the antennae have been
identified, based on the fact that OBPs are expressed at lev-
els that are orders of magnitude higher than ORs. It is also
an interesting coincidence that O. cornuta and A. mellifera
express similar numbers of genes encoding classic OBPs in
their antennae (6 and 7, respectively), while all the eight
C-minus OBPs of the honeybee (a sub-class characterized
by a motif of only four cysteines), all present in the antennae
also at the protein level [11], are missing in O. cornuta. In
the other solitary bee M. rotundata, all the 11 OBPs so far
reported belong to the classic sub-group. We could specu-
late that this set of C-minus OBPs might be used by the hon-
eybee in exchanging information between castes and indi-
viduals of the colony, a task not required in solitary bees.
This hypothesis is also suggested by the observation that
Table 1 Dissociation constants
Ligand
OBP1
OBP2
6.3
OBP3
OBP4
OBP5
(micromolar) of the complexes
between ligands and five
recombinant OBPs
of O. cornuta
Linalool
Citronellal
Citral
1.8
2.5
2.5
2.0
3.0
4.0
3.5
2.3
2.5
3.7
5.0
9.2
10
7.7
7.9
8.2
2.5
3
,7-DMO acetate
7.5
Eugenol
Coniferyl alcohol
Geranyl acetate
Retinal
9.2
6.3
6.2
9.2
3.3
9.2
7.5
5.8
7.1
8.6
13
9.1
Geranyl acetone
6.14
2
-Hexyl-1,3-dioxolane
Decanoic acid
Homovanillic acid
Ethyl cinnamate
Butyl cinnamate
Cinnamaldehyde
Nonanal
10
8.0
12
3.7
13.6
5.5
14.7
All the listed compounds
3.0
8.0
12
have been tested with all five
proteins. Dissociation constants
are reported only where [IC]50
values could be measured
(Z)-3-Hexen-1-ol
2
-Ethyl fenchol
10
1
3

Olfaction in solitary bees
3037
C-minus OBPs are largely and abundantly expressed in the
mandibular glands of honey bees, that are known to secrete
intraspecific pheromones, across all castes and ages [12].
In solitary bees, instead, chemical communication
between conspecifics is limited to sex recognition and court-
ship. Very little is known about sex pheromones in solitary
bees, and no information is available for the Osmia genus.
We had previously reported on the presence of secretory
glands and pores on the male antenna of O. cornuta and the
role played by the antennae during pre-copulatory courtship,
suggesting that these organs could be the source of semio-
chemicals [61]. The major compounds we have detected in
the headspace analysis of male antennae and female whole
body exhibit good affinity to three of the OBPs of O. cor-
nuta, suggesting some activity in the bees, although the
recognition of a pheromonal function for these compounds
would require more extensive and detailed investigation.
Ligand-binding experiments indicate that OBP2 and
OBP4 seem rather specific towards few selected odorants,
while the other three exhibit a broad spectrum of binding.
Such a poor degree of specificity between odorants and
OBPs is not surprising when we consider that only those
ligands inducing a specific conformational change in the
protein may lead to a physiological signal generated by a
cooperative action with a specific olfactory receptor [35].
Therefore, a compound able to start this series of events,
even if its affinity with the OBP is not very high, could rep-
resent a more efficient semiochemical than other stronger
ligands. At the physiological level, we are well aware that
each semiochemical can activate several glomeruli in the
antennal lobe with different intensities. It is this complex
olfactory map that is recognized by the brain, rather than the
activation of a single glomerulus. Therefore, if OBPs are the
first proteins to interact with semiochemicals, the complex-
ity observed at the level of the antennal lobe is likely gener-
ated at the peripheral level by multiple interactions of the
same odorant with a number of OBPs.
It has been well established that olfaction, both in verte-
brates and in insects, utilizes a combinatorial code, where
each chemical is able to activate several sensors, produc-
ing responses of different intensities, while each sensor can
respond to several stimuli. The existence of a combinatorial
code at the level of OBPs has been evidenced in recent stud-
ies, showing that a correct olfactory image is built on the
contributions of several OBPs [37, 38].
Generally speaking, a combinatorial code, based on
broadly tuned sensors is the best strategy for a perception
system using a limited number of elements. The best exam-
ple is offered by the human color vision. Even if it is based
on just three rhodopsins, each broadly responding to a large
part of the visible spectrum, the system as a whole is able to
discriminate thousands of color shades. Finally, we cannot
ignore the fact that OBPs in some cases may interact [62],
producing heterodimers with novel binding behavior, thus
enlarging the number of actual detectors of the chemosen-
sory system.
Acknowledgments Y. X.-W. thanks the University of Pisa for a
1
-year scholarship supporting his visit at the laboratory of A. Felicioli.
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