(1R,2S,6R)-Papayanal: A new male-specific volatile compound released by the guava weevil Conotrachelus psidii (Coleoptera: Curculionidae)

Article abstract of DOI:10.1080/09168451.2015.1136877

The guava weevil, Conotrachelus psidii is an aggressive pest of guava (Psidium guajava L.) that causes irreparable damages inside the fruit. The volatile compounds of male and female insects were separately collected by headspace solid-phase microextraction or with dynamic headspace collection on a polymer sorbent, and comparatively analyzed by GC-MS. (1R,2S,6R)-2-Hydroxymethyl-2,6-dimethyl-3-oxabicyclo[4.2.0]octane (papayanol), and (1R,2S,6R)-2,6-dimethyl-3-oxabicyclo[4.2.0]octane-2-carbaldehyde (papayanal) were identified (ratio of 9:1, respectively) as male-specific guava weevil volatiles. Papayanal structure was confirmed by comparison of spectroscopic (EIMS) and chromatographic (retention time) data with those of the synthetic pure compound. The behavioral response of the above-mentioned compounds was studied in a Y-tube olfactometer bioassay, and their role as aggregation pheromone candidate components was suggested in this species.

Full text of DOI:10.1080/09168451.2015.1136877

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
(1R,2S,6R)-Papayanal: a new male-specific
volatile compound released by the guava weevil
Conotrachelus psidii (Coleoptera: Curculionidae)
Alicia Romero-Frías, Yasuhiro Murata, José Maurício Simões Bento & Coralia
Osorio
To cite this article: Alicia Romero-Frías, Yasuhiro Murata, José Maurício Simões Bento & Coralia
Osorio (2016): (1R,2S,6R)-Papayanal: a new male-specific volatile compound released by the
guava weevil Conotrachelus psidii (Coleoptera: Curculionidae), Bioscience, Biotechnology, and
Biochemistry, DOI: 10.1080/09168451.2015.1136877
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Date: 18 February 2016, At: 18:14

Bioscience, Biotechnology, and Biochemistry, 2016
(1R,2S,6R)-Papayanal: a new male-specific volatile compound released by the
guava weevil Conotrachelus psidii (Coleoptera: Curculionidae)
Alicia Romero-Frías1,†, Yasuhiro Murata2, José Maurício Simões Bento3 and
Coralia Osorio1,*,†
2
1Departamento de Química, Universidad Nacional de Colombia, Bogotá, Colombia; Fuji Flavor Co., Ltd., Tokyo,
3
Japan; Departamento de Entomologia, Escola Superior de Agricultura Luiz de Queiroz (ESALQ), Universidade de
São Paulo (USP), Piracicaba, Brazil
Received October 15, 2015; accepted December 14, 2015
http://dx.doi.org/10.1080/09168451.2015.1136877
The guava weevil, Conotrachelus psidii is an
aggressive pest of guava (Psidium guajava L.) that
causes irreparable damages inside the fruit. The
volatile compounds of male and female insects were
separately collected by headspace solid-phase
microextraction or with dynamic headspace collec-
tion on a polymer sorbent, and comparatively
analyzed by GC–MS. (1R,2S,6R)-2-Hydroxymethyl-
2,6-dimethyl-3-oxabicyclo[4.2.0]octane (papayanol),
and (1R,2S,6R)-2,6-dimethyl-3-oxabicyclo[4.2.0]oc-
tane-2-carbaldehyde (papayanal) were identified
(ratio of 9:1, respectively) as male-specific guava
weevil volatiles. Papayanal structure was confirmed
by comparison of spectroscopic (EIMS) and chro-
matographic (retention time) data with those of the
synthetic pure compound. The behavioral response
of the above-mentioned compounds was studied in a
Y-tube olfactometer bioassay, and their role as
aggregation pheromone candidate components was
suggested in this species.
Aggregation pheromones have been studied and
applied in field to monitor or as support control strat-
egy to control several Curculionidae species.3–6) These
kind of pheromones offers a promising method for
direct insect control through mass trapping.7,8) In addi-
tion, previous studies have shown that host plant vola-
tiles are attractive to both sexes of C. psidii.9,10)
Recently Romero-Frías et al.11) identified the terpenes,
β-caryophyllene, and limonene in both insects (C. psidii
males and females) and plants (flower bud and fruit set-
ting guava stages). Therefore, these results suggested a
possible role of these volatiles as host kairomones for
C. psidii in the guava reproductive tissues. Later,
Palacio-Cortés et al.12) reported the release of
(1R,2S,6R)-papayanol by C. psidii males depending on
the photoperiod, which in the presence of plant vola-
tiles is highly attractive to both sexes.
As part of our ongoing research on the search for
semiochemicals that can be successfully used to moni-
tor and/or control pests in tropical fruis (IPM, Inte-
grated Pest Management), the aim of this work was to
study the aggregation pheromone system in C. psidii.
Thus, the isolation, identification, and synthesis of a
new male-specific guava weevil volatile, named
papayanal, is reported here.
Key words: 2,6-Dimethyl-3-oxabicyclo[4.2.0]octane-
2-carbaldehyde; Conotrachelus psidii;
aggregation pheromone; Y-tube olfac-
tometer bioassay
Conotrachelus
psidii
Marshall
(Coleoptera:
Materials and methods
Curculionidae) is one of the most important pests of
guava (P. guajava, Myrtaceae) crops. The mated females
lay eggs in small unripe fruits. During fruit development,
the larvae grow and mature larvae abandon the ripe fruits
and pupate underground. Larval feeding causes an
irreparable damage to the fruit that does not allow its fur-
ther use. The adults of C. psidii are small dark brown
weevils (~6 mm long) with crepuscular habits.1,2) Usu-
ally, guava weevils survive to insecticide applications
because they do not remain exposed, thus making insec-
ticidal control difficult. Hence, complementary strategies
for the management of this pest need to be developed.
Insects.
Male and female C. psidii adults were
collected between January to July 2014 from different
managed crops located in Puente Nacional and Vélez
(Santander, Colombia) using the beating tray method
after shaking branches of the guava plants. Once trans-
ferred to the laboratory, individuals were segregated by
sex according to the procedure reported by Silva-Filho
et al.,13) and maintained separately in plastic boxes at
20 2 °C, at a relative humidity of 80 10% with
pieces of green guava fruits as food and 12-h light
regime.11)
*Corresponding author. Email: cosorior@unal.edu.co
†Facultad de Ciencias, Departamento de Química, Universidad Antonio Nariño
© 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
A. Romero-Frías et al.
Insect volatile collection.
Volatile compound
temperature program. Mass spectra in the negative
mode were acquired with methane as the reactant gas.
Chiral GC analyses were carried out on an Agilent
Technologies 7890A gas chromatograph equipped with
an Astec Chiraldex™ B-PH capillary column (30 m ×
0.25 mm, 0.12 μm film thickness; Sigma–Aldrich,
St.Louis, MO, USA). For (1R,2S,6R)- papayanol and
(1R,2S)-grandisol, the column oven temperature was
programmed from 60 to 100 °C at a rate of 1 °C/min,
and for (1R,2S,6R)-papayanal, it was programmed from
60 to 100 °C at 0.1 °C/min. Optical rotations were
measured with a JASCO Digital polarimeter DIP-370
(Tokyo, Japan). NMR spectra were recorded on a
Bruker Biospin AVANCE III spectrometer (400 MHz,
CDCl3). The 1H (δH 7.24 ppm) and 13C signals of
CHCl3 (δC 77.2) were used as references.
released by C. psidii adults were collected using either
headspace solid-phase microextraction (HS-SPME) or
dynamic headspace (DHS) using polymer-based sor-
bents. For HS-SPME, the weevils (males or females,
separately) were deprived of food, and placed in
100 mL glass flask. The volatile compounds released
by each gender were separately collected on a DVB/
CAR/PDMS fiber (50/30 μm thickness, Supelco, Belle-
fonte, PA, USA) for 12 h during the scotophase. The
analyses were done by triplicate with different insects
each time (10 males or 10 females) upon arrival to the
laboratory. Analysis of background volatiles was per-
formed with the SPME fiber in the flask without
insects. For DHS collection, groups of 30 males and 30
females were separately held on in all-glass aeration
chambers (33 cm high x 4 cm outlet diameter) with
three fresh unripe guava fruits (6–8 g each) at
23–24 °C. The released volatiles were continuously col-
lected during 4 days (minimum time required to detect
volatile compounds), and trapped on glass columns
(10 cm high × 0.5 cm i.d) packed with 0.5 g of Hayesep
D 80/100 (DVB, Supelco, Bellefonte, PA). Charcoal-fil-
tered humidified air was pushed through the aeration
system (1.0 L/min). The adsorbed volatile compounds
were eluted with 500 μL of hexane HPLC grade
(Merck, Darmstadt, Germany). The sample was con-
centrated to about 50 μL under a gentle flow of nitro-
gen, and then analyzed by GC–MS. Blank experiments
were performed under the same conditions, in presence
of fresh unripe guava fruits but without insects.
Reference compounds. Pure reference standards of
β-pinene, limonene, 1,8-cineole, decanal, β-caryophyl-
lene, α-humulene, and (+)-aromadendrene were
purchased from Sigma-Aldrich (Taufkirchen, Germany).
α-Copaene was kindly provided by Dr Ignacio De
Alfonso.14) n-Alkane mix (C8-C26) was acquired from
Merck, Darmstadt, Germany.
Synthesis of (1R,2S,6R)-papayanol and (1R,2S,6R)-
papayanal.
(1R,2S)-Grandisol was obtained by
recrystallization method from (1R*,2S*)-grandisol (cis-
1-methyl-2-isopropenyl-cyclobutane-ethanol, Grandlure
I) (Bedoukian Research Inc, Danbury, CT, USA).
Briefly, (1R*,2S*)-grandisol was acylated with (1S)-
(−)-camphanic acid chloride (Sigma–Aldrich, St.Louis,
MO, USA), the resulting diastereomeric esters were
separable by recrystallization using hexane. After alkali
Analytical procedures.
The volatile compounds
collected by HS-SPME were desorbed into the injection
port of a Shimadzu GC17A coupled to a mass selective
detector QP5050 (Shimadzu, Tokyo, Japan). Desorption
time was set at 5 min. Two capillary columns, RTX-5
and DB-FFAP were used (each 30 m × 0.32 mm i.d.,
0.25 μm film thickness; Restek, Bellefonte, PA, USA,
and J&W Scientific, Chromatographie-Handel Müller,
Fridolfing, Germany, respectively). The column oven
was programmed from 50 °C for 1 min, then raised at
7 °C/min to 250 °C for the DB-FFAP and to 300 °C
for the RTX-5 column and maintained at those temper-
atures for 10 min. The injector temperature was fixed at
250 °C for the DB-FFAP and at 300 °C for the RTX-5
column, using helium as carrier gas at 1.5 mL/min. The
injection port was used in splitless mode. Linear reten-
tion indices were calculated according to the Kovats
method using a mixture of n-alkanes as external refer-
ences. Mass spectral identification was completed via
comparison with either authentic reference standards or
spectra from commercial mass spectral databases
(WILEY and EPA/NIH). MS data in the electron ion-
ization (EI) mode were recorded in a mass range of
30–350 μ, with electron energy of 70 eV and processed
by Class 5000 v 2.2 MS-Workstation software. A Trace
GC Ultra chromatograph connected to a Thermo
Scientific ITQ900 ion trap mass spectrometer (Thermo
Scientific, Austin, TX, USA) was used for the analysis
of volatile samples by chemical ionization (CI-MS)
with a RTX-5 column using the same above-described
25
hydrolysis, the desired (1R,2S)-grandisol (½aꢀD
= +15.8°(C = 1.01, n-hexane)) was obtained.15) The
enantiomeric purity of (1R,2S)-grandisol was estimated
GC analysis on a chiral capillary column to be 94%
e.e. Then, (1R,2S,6R)-papayanol was obtained by reac-
tion of (1R,2S)-grandisol with m-chloroperbenzoic acid
(Tokyo Chemical Industry Co., Tokyo, Japan) as previ-
ously described by Zarbin et al.16) The minor
(1R,2R,6R)-papayanol was removed by chromatography
over silica gel. To oxidize papayanol to its aldehyde
(papayanal),
tetrapropylammonium
perruthenate
(76.0 mg, 0.216 mmol) (Tokyo Chemical Industry Co.,
Tokyo, Japan) was added in three portions to a mixture
of (1R,2S,6R)-papayanol (94% e.e.) (366.5 mg,
2.16 mmol), 4-methylmorpholine N-oxide (380.0 mg,
3.24 mmol) (Wako Pure Chemical Industries, Tokyo,
Japan), and powdered 4Å molecular sieves (760 mg)
(Wako Pure Chemical Industries, Tokyo, Japan) in
dichloromethane (10 ml) at temperature below 30 °C,
and with stirring. After 30 min, the reaction mixture
was filtered through a short pad of silica gel and eluted
with diethyl ether. The solvent was evaporated and the
residue purified by column chromatography over silica
gel. Elution with hexane/diethyl ether (7:3, v/v) gave
197.5 mg (54.4% yield) of (1R,2S,6R)-papayanal (94%
e.e) as a colorless oil. Because (1R,2S)-grandisol (94%
e.e.) contained (1S,2R)-grandisol in a small proportion,

Papayanal as putative male-specific aggregation pheromone
3
1
the synthesized (1R,2S,6R)-papayanal also contained
(1S,2R,6S)-papayanal (Fig. 1).
(1R,2S,6R)-Papayanal. H NMR (400 MHz, CDCl3)
of the synthetic compound: δ [ppm] = 1.12 (s, 3H,
CH3C-11); 1.21 (s, 3H, CH3C-10); 1.37 (ddd, 1H,
2J = 14.0 Hz,3J = 2.3/2.3 Hz, He C-5); 1.47–1.55 (m,
1H, Hendo C-7); 1.58–1.66 (m, 1H, Hexo C-7); 1.59–
1.67 (m, 1H, Hexo C-8); 1.87–1.93 (m, 1H, CH C-1);
1.88–1.96 (m, 1H, Ha C-5); 1.90–2.00 (m, 1H, Hendo
Spectroscopic data.
(1R,2S,6R)-Papayanol. 1H
NMR (400 MHz, CDCl3) of the synthetic compound: δ
[ppm] = 1.12 (s, 3H, CH3 C-11); 1.16 (s, 3H, CH3
C-10); 1.32 (ddd, 1H, 2J = 14.0 Hz, 3J = 2.0/2.0 Hz,
He C-5); 1.43–1.50 (m, 1H, Hendo C-7); 1.55–1.62 (m,
1H, Hexo C-7); 1.57–1.65 (m, 1H, Hexo C-8); 1.73–1.79
(m, 1H, CH C-1); 1.90 (ddd, 1H, 2J = 14.0 Hz,
3J = 13.3/5.1 Hz, Ha C-5); 1.94–2.04 (m, 1H, Hendo C-
2
3
C-8); 3.59 (ddd, 1H, J = 12.2 Hz, J = 12.2/2.2 Hz, Ha
2
3
C-4); 3.77 (ddd, 1H, J = 12.2 Hz, J = 4.9/ 2.3 Hz, He
C-4); 9.44 (s, 1H, CHO C-9). 13C NMR (100 MHz,
CDCl3): δ [ppm] = 17.7 (C-8); 19.2 (C-10); 28.1 (C-
11); 33.6 (C-7); 34.0 (C-5); 36.2 (C-6); 43.3 (C-1);
2
25
8); 3.23 (d, 1H, J = 10.9 Hz, CH2OH); 3.42 (d, 1H,
57.6 (C-4); 78.7 (C-2); 204 (C-9). ½aꢀD
2J = 10.9 Hz, CH2OH); 3.53 (ddd, 1H, 2J = 12.0 Hz,
3J = 13.3/2.2 Hz, Ha C-4); 3.63 (ddd, 1H,
2J = 12.0 Hz, 3J = 5.2/2.1 Hz, He C-4); 13C NMR
(100 MHz, CDCl3): δ [ppm] = 18.0 (C-8); 20.2 (C-10);
28.1 (C-11); 33.6 (C-7); 34.1(C-5); 35.8 (C-6); 45.1
= −201°(C = 1.01, n-hexane). GC/MS (EI, 70 eV) m/z
(%) = 41 (90), 43 (100), 53 (7), 55 (12), 67 (11), 69
(60), 70 (3), 71 (28), 77 (3), 79 (6), 81 (17), 83 (3), 93
(4), 95 (7), 109 (4), 111(17), 121 (5), 139 (60), 140
(10), 153 (0.1).
25
(C-1); 57.5 (C-4); 68.9 (C-9); 72.9 (C-2). ½aꢀD
= −34.4°(C = 1.00, n-hexane). GC/MS (EI, 70 eV) m/z
(%) = 41 (90), 43 (100), 53 (7), 54 (2), 55 (12), 67
(13), 68 (6), 69 (50), 70 (3), 71 (23), 77 (3), 81 (23),
83 (3), 93 (4), 95 (7), 107 (5), 109 (6), 111 (26), 121
(5), 139 (100), 140 (10), 155 (3), 156 (0.3).
Olfactometer assays.
The behavioral response of
male and female C. psidii individuals was studied in a
Y-tube olfactometer bioassay by testing the following
samples: DHS extract of males + host plant (fruit
cis-grandisol ((1R*,2S*)-grandisol, Grandlure I)
Fig. 1. Scheme of the synthesis of the stereochemically defined papayanol and papayanal stereoisomers.

4
A. Romero-Frías et al.
setting), (1R,2S,6R)-papayanol at a concentration of
1 μg/mL, 10 μg/mL, 100 μg/mL, and a mixture of
(1R,2S,6R)-papayanol and (1R,2S,6R)-papayanal in a
ratio of 9:1 (10 μg/mL). The dual choice Y-tube olfac-
tometer was used with a pre-humidified and charcoal-
filtered airflow at a flow of 1 L/min. Odor propagation
simulation tests were performed to visualize the plume
distribution inside the system. The olfactometer con-
sisted of a Y-shaped glass tube of 3.5 cm diameter,
20 cm in length, 10 cm of arms, and a 90° Y angle. All
the treatments were performed during the scotophase
with a red light to avoid any interference of the white
light, at 18 2 °C and a relative humidity of 80
10%. One week before each assay, C. psidii adults
(males and females, with a number varying between 18
to 40 individuals) were collected, placed in plastic
flasks, and deprived of food in a room free from odor-
ants for 24 h before the olfatometric bioassay. In all
cases, a different insect was used for each test. Age
and mating status of the insects were not determined.
The insects (male or female) were introduced one by
one into the base tube of the olfactometer, and its
behavior was observed for 5 min; after that, the next
individual was evaluated until the entire test finished.
A positive choice for the odor source in one of the two
arms was considered when an insect crossed the choice
line, went all over 5 cm into the arm, and remained
there for at least 2 min. Individuals that did not make
any choice during this time were excluded from the
statistical analysis. The relative position of olfactometer
with respect to the light was rotated 180º after each
replicate to avoid positional bias. For each replicate, a
new piece of braided cotton roll (0.5 × 0.5 cm;
Richmond Dental, Charlotte, NC, USA) loaded with
the solutions of tested compounds was used. The evap-
oration time before starting the tests was 10 s. Sample
solutions (2 μL) were tested against hexane HPLC
grade (Merck, Darmstadt, Germany) as solvent control.
After each test, all components of the olfactometer
were cleaned with a detergent solution (Extran alkaline,
Merck, KGaA, Darmstadt, Germany), then rinsed with
water and acetone, and finally dried at 100 °C for
30 min to avoid from cross contamination.
Statistical analyses.
Results of all olfactometer
bioassays were analyzed with a binomial test. The null
hypothesis was considered 50:50 distribution at a sig-
nificance level of p < 0.05. Data analyses were per-
formed using WinSTAT® for Excel 2007 (V.
2012.1.0.84).
Results
Identification of male-specific volatile compounds
released by C. psidii
Two methodologies (HS-SPME and DHS) were eval-
uated for the analysis of volatile compounds released
by adult C. psidii (Table 1). Using DHS technique, a
major number of substances were detected in male
insects, with being papayanol (6) the greater con-
stituent. The terpenes limonene and β-caryophyllene
were detected from male and female guava weevils
both with (DHS) and without (HS-SPME) the presence
of host plant fruits. These compounds were present in
all of the guava reproductive tissues, being quantita-
tively relevant in flower buds and fruit setting, the two
guava stages where C. psidii is found.11) Other terpe-
nes, such as α-copaene, α-humulene and aromaden-
drene, were detected only by DHS and mainly on male
insects (except for α-humulene).
Two male-specific volatile compounds (compounds 4
and 6, IKRTX-5 = 1192 and 1280, IKFFAp = 1713 and
1856, respectively) released by the guava weevil (C.
psidii) were detected in a ratio of 1:9, respectively, dur-
ing GC-MS analysis (Fig. 2). These results were
obtained by collecting Colombian insects in different
periods of year. EI-MS and CI-MS spectra of both
compounds are shown in Fig. 3.
The major compound (6) was identified as
(1R,2S,6R)-papayanol, by GC-MS co-injection with the
synthetic enantiomeric pure sample (Fig. 4(A)), in
agreement with those results reported by for Brazilian
C. psidii.12)
The minor male-specific compound 4, showed a
fragmentation pattern very similar to that of
(1R,2S,6R)-papayanol by mass spectrometry (Fig. 3(B)
and (D)), suggesting in the first instance the presence
Table 1. Volatile compounds released by male and female guava weevils, C. psidii.
Volatile compounds released by C. psidiic (source and method)
Males insects Females insects
HS-SPMEd HS-SPMEd
b
RI
No
Compounda
RTX-5
FFAP
DHSe
DHSe
1
2
3
4
5
6
7
8
9
10
β-Pinene
Limonene
1,8-cineole
Papayanal
Decanal
994
1107
<1200
1190
1713
1480
1856
−
1594
1656
1701
−
+
−
+
+
++++
−
+
−
−
+
+
+
+
+
++++
+
+
+
+
−
+
−
−
−
−
−
++
−
−
−
+
1034
1038
1192
1208
1280
1387
1433
1468
1476
−
−
−
−
−
++
+
Papayanol
α-Copaene
β-Caryophyllene
α-Humulene
(+)-Aromadendrene
−
Notes: aCompound identification was based on retention index and mass spectra comparison with reference compounds.
bRI = retention index.
cVolatile relative peak area (%) as mean of three determinations:+ <5, ++ 5–10, +++ 10–15, ++++ >15%, − = not detected.
dWithout food, 12 h.
eFeeding them by four days.

Papayanal as putative male-specific aggregation pheromone
5
Fig. 2. GC/MS analysis of male-specific volatiles compounds in C. psidii.
Notes: Comparison of total ion chromatograms (RTX-5 column) of volatile compounds obtained from male and female C. psidii insects by
HS-SPME, showing the two male-specific compounds (1R,2S,6R)-papayanal (4), and (1R,2S,6R)-papayanol (6) (numbering corresponds to Table 1).
of a diasteroisomeric compound. However, the ion
fragment at m/z 167 [M-H]- in the CI negative mode
mass spectrum (Fig. 3(A)) clearly evidenced that the
molecular weight of this compound was 168 μ. Thus,
the structure for compound 4 was suggested to be
the 2,6-dimethyl-3-oxa-bicyclo[4.2.0]octane-2-carbalde-
hyde, an analog aldehyde derived from papayanol and
therefore denominated as papayanal. The signals at m/z
139 [M-29]+ and m/z 111 [M-57]+ in the EI mass spec-
trum (Fig. 3(B)) correspond to the loss of formyl and
acrolein (CH2=CH–CHO) radicals from the molecular
ion, respectively. The chemical structure of this
compound was confirmed by GC/MS comparison with
Y-tube olfactometer bioassays
To evaluate the role of papayanol (6) and papayanal
(4) as aggregation pheromones in C. psidii, behavioral
responses of males and females to distinct treatments
were performed in a Y-tube olfactometer (Fig. 5). In
the first treatment, the response of insects to the mix-
ture of male HSD extract and fruit setting guava extract
showed significant differences in attractiveness to both
sexes compared to the control treatment, with attrac-
tiveness being similar for males and females (p < 0.05).
The other treatments showed that (1R,2S,6R)-papayanol
significantly attracts both males and females (p < 0.05)
at a concentration of 100 μg/mL and 10 μg/mL in hex-
ane, but at 1 μg/mL only males were significantly
attracted (p < 0.05). The last bioassay consisted of the
the
synthetic
enantiomerically
pure
standard
(Fig. 4(B)).
Fig. 3. Mass spectra and chemical structures of male-specific volatiles compounds in C. psidii.
Notes: Comparison of (A) CI-MS, and (B) EI-MS of (1R,2S,6R)-papayanal (4) with those of (C) CI-MS, and (D) EI-MS of (1R,2S,6R)-pa-
payanol (6). CI-MS were recorded in negative mode.

6
A. Romero-Frías et al.
evaluation response to the synthetic mixture of
(1R,2S,6R)-papayanol and (1R,2S,6R)-papayanal in the
natural found ratio of 9:1 at a concentration of
10 μg/ml. Significant differences in attractiveness to
both sexes when compared with the control treatment
(p < 0.05) were found.
Discussion
Among two methodologies used to collect the vola-
tile compounds released by C. psidii individuals, DHS
(continuous extraction) allowed getting a major number
of compounds and the extracts needed for olfactometer
bioassays. The fact that male-specific volatiles, papaya-
nol (6) and papayanal (4), were found by different
methodologies confirmed that they are not artifacts
from isolation procedure. The fact that limonene and β-
caryophyllene were found in male as well female C.
psidii addults, suggests the role of these compounds as
kairomones with guava plant. These compounds are
released by the guava fruits and also by C. psidii
insects, thus mediating the communication among two
species. In Coleoptera, sequestration of host com-
pounds for later use as pheromones in their unmodified
form appears to be rare.17) Since pheromone production
is often associated with feeding, it is experimentally
difficult to distinguish between host compounds that
are released by masticated food or feces, and those that
are sequestered by the weevil and released later. How-
ever, kairomones coming from host plant (P. guajava)
could help the C. psidii adults to establish the availabil-
ity of a place to feed and lay their eggs.
As it was mentioned above, the major male-specific
volatile of C. psidii (compound 6) was identified as
(1R,2S,6R)-papayanol and the minor compound (4) was
suggested to be the corresponding aldehyde
(1R,2S,6R)-papayanal. However, the synthesis of
(1R,2S,6R)-papayanal was performed because this com-
pound had not been reported in nature before. For this
Fig. 4. Enantioselective GC analyses of synthetic (1R,2S,6R)-pa-
payanol (A), and (1R,2S,6R)-papayanal (B), and their enantiomeric
mixtures using a Astec Chiraldex™ B-PH column.
Fig. 5. Y-tube olfactometer responses of male and female C. psidii adults to different odour sources.
Notes: The asterisk (*) indicates a statistically significant preference for the binomial test (chi-square test, p < 0.05). Number of replicates is the
sum of number of responsive and non-responsive choices of both, male and female individuals.

Papayanal as putative male-specific aggregation pheromone
7
purpose, the use of ruthenium catalyst tetrapropylam-
monium perruthenate, in conjunction with N-methyl-
morpholine-N′-oxide as a mild oxidant agent18) for the
sensitive alcohol group in papayanol showed to be an
efficient strategy. The 1H and 13C NMR data of
papayanal resembled those of papayanol, except for the
papayanol to attract males (78.3%) and females
(80.8%) of C. psidii without host plant volatiles
(Fig. 5).
This is the first time that (1R,2S,6R)-papayanal is
suggested to have a putative role as aggregation phero-
mone component in guava weevil, however more experi-
mentation is needed to confirm this statement. Field
studies to evaluate the effect of these male-specific
C. psidii volatiles are also needed to better understand
the potential of the male-produced volatile compounds,
here reported, as monitoring strategy of this pest in
guava.
presence
of
carbonyl
group
(δC = 204 ppm;
δH = 9.44 ppm) and the slight deprotection of adjacent
protons H-1 and Hexo C-8). NOESY spectrum showed
the correlation of CH3C-10/H-1 (δH 1.21 and 1.87–
1.93 ppm) and CH3C11/H-1 (δH 1.12 and 1.87–
1.93 ppm) indicating that both methyl groups are posi-
tioned on the same side of papayanal. The correlation
of CH3C-10/Hax-4 (δH 1.21 and 3.59 ppm) and CH3C-
11/Hax-4 (δH 1.12 and 3.59 ppm) also indicated a 1,3-
diaxial relation of both methyl groups with the H-4 that
is in axial position. The correlation of Heq-4 with Heq-5
(δH 3.77 and 1.37 ppm), and Hendo C-7 with Hendo C-8
(δH 1.47–1.55 and 1.90–2.00 ppm) confirmed the rela-
tive position of these protons. To determine the abso-
lute configuration of papayanal, chiral gas
chromatography was used. Comparison of the retention
time of the synthetic sample with the racemic mixture
revealed the natural product to be the enantiomerically
pure (1R,2S,6R)-isomer (Fig. 4(B)).
Acknowledgments
The authors are grateful to Johnny Ruíz, Dr Edgar
Eduardo Daza, and the staff of Centro de Gestión
Agroempresarial del Oriente (SENA, Vélez, Santander,
Colombia) for their valuable cooperation during the
collection of the weevils in the countryside of San-
tander, Colombia. Alicia Romero-Frías thanks financial
support of a scholarship from Departamento Adminis-
trativo de Ciencia, Tecnología e Innovación (COL-
CIENCIAS).
The enantioselective biosynthesis of pheromones
appears to be significant for the behavior and specific
discrimination of insects.19,20) Thus, despite the analy-
sis of DHS extract of male C. psidii was not performed
in the chiral column, the presence of enantiomeric pure
papayanal is reinforced by biogenetic reasons. In two
other Curculionidae species, papaya weevil (Pseudopi-
azurus obesus),21) and banded pine weevil (Pissodes
castaneus),22) the mixture of (1R,2S)-(+)-grandisal and
(1R,2S)-(+)-grandisol, aldehyde and alcohol, were iden-
tified as aggregation pheromones.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was supported by grants from Fundación Para la
Promoción de la Investigación y la Tecnología (Banco de la Repúb-
lica, Convenio 201406), Dirección Nacional de Investigación (DIB) –
Universidad Nacional de Colombia-Sede Bogotá, and INCT
Semioquímicos na Agricultura (Fapesp and CNPq), Brazil.
To confirm the role of (1R,2S,6R)-papayanol and
(1R,2S,6R)-papayanal, Y-tube olfactometer bioassays
were performed (Fig. 5). These results showed that
both sexes of the guava weevil are attracted to male-
produced volatiles, such suggesting the presence of
male-produced aggregation pheromones. After identify-
ing (1R,2S,6R)-papayanol and (1R,2S,6R)-papayanal as
C. psidii male-produced volatiles, different concentra-
tions of synthetic (1R,2S,6R)-papayanol were tested in
the bioassay, as well as the mixture of synthetic
(1R,2S,6R)-papayanol and (1R,2S,6R)-papayanal in the
found natural ratio (9:1). For the case of (1R,2S,6R)-pa-
payanol, the lowest concentration (1 μg/ml) tested
showed a male-specificity in the attraction; in contrast,
at the other concentrations (10 and 100 μg/ml) there
were no preference for any sex, thus the role of this
compound as aggregation pheromone in C. psidii was
clearly confirmed. To this regard, it has been reported
that the response of an insect to a specific volatile com-
pound is significantly influenced by its concentration.23)
The study carried out by Palacio-Cortés et al.12) with
Brazilian population of C. psidii showed that synthetic
papayanol (100 μg/ml) did not attract insects without
host plant volatiles. However, the authors did not use
papayanal in their treatments, contrasting with the
results here presented for Colombian population. It is
important to emphasize the possible synergistic effect
of (1R,2S,6R)-papayanal over bioactivity of (1R,2S,6R)-
ORCID
Coralia Osorio
http://orcid.org/0000-0001-6222-0138
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