Revisiting the Male-Produced Aggregation Pheromone of the Lesser Mealworm, Alphitobius diaperinus (Coleoptera, Tenebrionidae): Identification of a Six-Component Pheromone from a Brazilian Population

Article abstract of DOI:10.1021/acs.jafc.6b02235

The lesser mealworm, Alphitobius diaperinus Panzer 1797 (Coleoptera: Tenebrionidae), is a cosmopolitan insect pest affecting poultry production. Due to its cryptic behavior, insecticide control is usually not efficient. Thus, sustainable and effective methods would have an enormous and positive impact in poultry production. The aim of this study was to confirm the identity of the male-produced aggregation pheromone for a Brazilian population of A. diaperinus and to evaluate its biological activity in behavioral assays. Six male-specific compounds were identified: (R)-limonene (1), (E)-ocimene (2), 2-nonanone (3), (S)-linalool (4), (R)-daucene (5), all described before in an American population, and a sixth component, (E,E)-α-farnesene (6), which is apparently exclusive to a Brazilian population. Y-Tube bioassays confirmed the presence of a male-produced aggregation pheromone and showed that all components need to be present in a similar ratio and concentration as emitted by male A. diaperinus to produce a positive chemotactic response.

Full text of DOI:10.1021/acs.jafc.6b02235

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Revisiting the Male-Produced Aggregation Pheromone of the Lesser
Mealworm, Alphitobius diaperinus (Coleoptera, Tenebrionidae):
Identification of a Six-Component Pheromone from a Brazilian population
Marla Juliane Hassemer, Josué SantAna, Miguel Borges, David M. Withall, John A. Pickett, Márcio
Wandré Morais de Oliveira, Raúl Alberto Laumann, Michael A. Birkett, and Maria Carolina Blassioli-Moraes
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02235 • Publication Date (Web): 05 Aug 2016
Downloaded from http://pubs.acs.org on August 5, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 31
Journal of Agricultural and Food Chemistry
TITLE
Revisiting the MaleꢀProduced Aggregation Pheromone of the Lesser Mealworm,
Alphitobius diaperinus (Coleoptera, Tenebrionidae): Identification of a Sixꢀ
Component Pheromone from a Brazilian Population
AUTHORS
Marla J. Hassemer1,2,3, Josué Sant’Ana2; Miguel Borges3; David Withall4, John A.
Pickett4, Márcio W. M. de Oliveira3; Raul A. Laumann3, Michael A. Birkett4, Maria C.
BlassioliꢀMoraes3*
AFFILIATIONS
1UnB – Universidade de Brasília, Zoologia Campus Universitário Darcy Ribeiro
Brasilia, DF, 70910ꢀ900, Brasil
2Universidade Federal do Rio Grande do Sul, Agronomia Avenida Bento Gonçalves,
7712 Porto Alegre, RS, 90040ꢀ060, Brasil
3EMBRAPA Recursos Genéticos e Biotecnologia, Parque Estação Biológica – W5
Norte, 70770ꢀ917, Brasília, Brazil.
4Biological Chemistry and Crop Protection Department, Rothamsted Research,
Harpenden, Herts., AL5 2JQ, UK.
AUTHOR INFORMATION
*Corresponding Author. Tel.: +55 61 3448ꢀ4932; Fax: +55 61 3448ꢀ4762; Eꢀmail:
carolina.blassioli@embrapa.br
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 31
1
ABSTRACT
2
3
The lesser mealworm, Alphitobius diaperinus Panzer 1797 (Coleoptera: Tenebrionidae),
is a cosmopolitan insect pest affecting poultry production. Due to its cryptic behavior,
insecticide control is usually not efficient. Thus, sustainable and effective methods
would have an enormous and positive impact in poultry production. The aim of this
study was to confirm the identity of the maleꢀproduced aggregation pheromone for a
Brazilian population of A. diaperinus, and to evaluate its biological activity in
behavioral assays. Six maleꢀspecific compounds were identified: (R)ꢀlimonene (1), (E)ꢀ
ocimene (2), 2ꢀnonanone (3), (S)ꢀlinalool (4), (R)ꢀdaucene (5), all described before in an
American population, and a sixth component, (E,E)ꢀαꢀfarnesene, which is apparently
exclusive to a Brazilian population. YꢀTube bioassays confirmed the presence of a
maleꢀproduced aggregation pheromone and showed that all components need to be
present in a similar ratio and concentration as emitted by male A. diaperinus to produce
a positive chemotactic response.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
KEYWORDS: Alphitobius diaperinus, Aggregation pheromone, Lesser mealworm
2
ACS Paragon Plus Environment

Page 3 of 31
Journal of Agricultural and Food Chemistry
19
INTRODUCTION
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
The lesser mealworm, Alphitobius diaperinus Panzer 1797 (Coleoptera: Tenebrionidae),
is a cosmopolitan insect pest affecting poultry production.1ꢀ3 Modern broiler facilities
offer suitable environmental conditions for insect proliferation, including high
temperatures, dark and sheltered sites, moisture and food availability, and consequently,
high A. diaperinus larvae and adult densities are found, aggregating predominately
under feeders and along house edges.4ꢀ6 Bacteria, virus and fungi can infect A.
diaperinus at all stages of the life cycle, and so these insects are potential disease
vectors affecting avine health.7ꢀ11 Control of A. diaperinus in poultry houses is currently
undertaken using insecticide application, causing potential contamination of poultry and
affecting meat quality. Additionally, due to the cryptic behavior of this pest, insecticide
control is usually not efficient. Several studies have been conducted with the aim of
developing alternative methods for lesser mealworm control, thereby minimizing
reliance on the use of insecticides,12ꢀ14 including semiochemicals that modify A.
diaperinus behavior.15ꢀ18 A five component, maleꢀproduced aggregation pheromone for
a North American population of A. diaperinus was reported.15 This pheromonal blend
was tested in poultry houses, capturing more adults and larvae than control traps,15,16
indicating effectiveness of these compounds in lesser mealworm management. In view
of reported incidences of semiochemical diversity in geographically distinct insect
populations,19ꢀ24 the aim of this study was to confirm the identity of the maleꢀproduced
aggregation pheromone for a Brazilian population of A. diaperinus, and to evaluate its
biological activity in behavioral assays.
41
42
MATERIALS AND METHODS
43
44
45
46
47
48
49
50
51
Chemicals. Hexane for HPLC (≥ 97%) and diethyl ether were purchased from Sigma
Aldrich and reꢀdistilled before use. (R)ꢀLinalool (95%) was purchased from Sigmaꢀ
Aldrich (Steinheim, Germany), (R)ꢀlimonene and (S)ꢀlimonene (95%) were purchased
from TCIꢀAmerica (Portland, USA). 2ꢀNonanone (99%) was provided by Jeffrey R.
Aldrich Consulting LLC (Santa Cruz, USA). (E,E)-αꢀFarnesene was synthesized in 3
steps from isoprene and sulphur dioxide by modifying the procedure reported by Spicer25
.
Thus, sulphur dioxide (30 ml) was condensed into a pressure flask cooled to ꢀ78˚C
containing isoprene (10 g, 146.80 mmol) and hydroquinone (0.5 g, 4.68 mmol) before
being sealed and stirred for 7 days at room temperature. The reaction flask was cooled
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 31
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
to ꢀ78˚C, opened and the sulphur dioxide allowed to evaporate overnight in a fume
hood. The residue was dissolved in methanol, filtered and concentrated under vacuum.
The crude material was recrystallized from hot methanol to provide 3ꢀmethylsulpholene
1
(13.7 g, 71% yield) as a white crystalline solid. HꢀNMR (CDCl3, 500 MHz): 5.70 (m,
1H, SO2CH2CH=C), 3.81 (m, 2H, SO2CH2=C), 3.69 (m, 2H, SO2CH2C(CH3)), 1.90 (s,
3H, CH3). To a solution of 3ꢀmethylsulpholene (1.32 g, 10.00 mmol) and geranyl
bromide (4.34 g, 20.00 mmol) in THF (20 mL), cooled to ꢀ98˚C under N2, was added
lithium bis (trimethylsilyl) amide (1.67 g, 10.00 mmol) in THF (7 mL). The resulting
solution was stirred for 10 min before being allowed to warm to room temperature over
a further 30 min. The reaction was quenched with saturated NH4Cl and the THF
removed under vacuum. The residue was dissolved in EtOAc before being washed with
water, dried (MgSO4) and concentrated under vacuum. The crude product was purified
on silica gel (20% EtOAc in petroleum ether) to give the product (514 mg, 21% yield)
1
as a colorless oil. HꢀNMR (CDCl3, 500 MHz): 5.70 (m, 1H, SO2CH2CH=C), 5.24 (m,
1H, (CH3)2C=CHCH2CH2C(CH3)=CH), 5.09 (m, 1H, (CH3)2C=CH), 3.69 (m, 2H,
SO2CH2), 3.53 (m, 1H, SO2CH), 2.59 (m, 2H, (CH3)2C=CHCH2CH2C(CH3)=CHCH2),
2.12 – 2.03 (m, 4H, (CH3)2C=CHCH2CH2), 1.88 (s, 3H, SO2CH2CH=C(CH3)), 1.69 (s,
3H, (CH3)2C=CH), 1.68 (s, 3H, (CH3)2C=CHCH2CH2C(CH3)=CH), 1.62 (s, 3H,
(CH3)2C=CH). A solution of 2ꢀ(Eꢀ3,7ꢀdimethylꢀ2,6ꢀoctadiene)ꢀ3ꢀmethylsulpholene
(514 mg, 2.02 mmol) in dry pyridine (10 mL) was heated to 150˚C under N2 for 3
hours. The reaction mixture was cooled, poured into 1M HCl and extracted with
petroleum ether. The combined organics were dried (MgSO4) and concentrated under
vacuum. The crude product was purified on florisil (100% petroleum ether) to give
(E,E)-αꢀfarnesene (330 mg, 80%) as a colorless oil (> 95% pure by GC). 1HꢀNMR
(CDCl3, 500 MHz): 6.27 (dd, 1H, J = 17.5, 10.7 Hz, CH2=CH) 5.49 (t, 1H, J = 7.4 Hz,
CH2=CHC(CH3)=CH)
),
5.17
–
5.01
(m,
3H,
(CH3)2C=CHCH2CH2C(CH3)=CHCH2CH=C(CH3)CH=CH2), 4.96 (d, 1H, J = 10.6 Hz,
CH2=CH), 2.85 (t, 2H, J = 7.2 Hz, (CH3)2C=CHCH2CH2C(CH3)=CHCH2), 2.09 (m,
2H, (CH3)2C=CHCH2), 2.02 (m, 2H, (CH3)2C=CHCH2CH2), 1.79 (s, 3H,
CH2=CHC(CH3)),
1.71
(s,
3H,
(CH3)2C=CH),
1.66
(s,
3H,
(CH3)2C=CHCH2CH2C(CH3)), 1.63 (s, 3H, (CH3)2C=CH). (R)ꢀDaucene was
synthesized from (R)ꢀcarotol obtained from carrot seeds.26,27,15 Carrot seeds (15.6 g) of
Brasília variety were extracted for 8 hours in nꢀhexane at ambient temperature. The
hexane extract was filtered and evaporated under vacuum to provide yellow pale oil
4
ACS Paragon Plus Environment

Page 5 of 31
Journal of Agricultural and Food Chemistry
86
87
(571.5 mg), which was subjected to liquid chromatography over silica gel (15 g, Sigma
Aldrich, 80/100 mesh). The oil (250 mg) was eluted sequentially with hexane (3×10
mL) and 98:2 diethyl ether:hexane (10×10 mL). The carotol structure was confirmed by
NMR and GCꢀMS analysis and was obtained in 98% purity (by GC analysis). 1HꢀNMR
(CDCl3, 500 MHz): 5.31 (m, 1H, (CH3)C=CH), 2.25 (br d, 1H, J = 16 Hz,
(CH3)C=CHCH2), 2.07 (m, 2H, CH=C(CH3)CH2), 1.94 (m, 1H, CH=C(CH3)CH2CH2),
1.79 (m, 2H, (CH3)2CHCH), 1.72 – 1.47 (m, 8H), 1.29 (m, 1H, (CH3)2CHCHCH2CH2),
1.14 (bs, 1H, OH), 0.99 (d, 3H, J = 6.7 Hz, (CH3)2CH), 0.94 (m, 6H, (CH3)2CH and
C(OH)C(CH3)). [α]20 = + 27.5 (c. 0.76, CHCl3; literature [α]20 = + 29.6).27 Carotol
88
89
90
91
92
93
94
D
D
95
(102.96 mg) was then treated with thionyl chloride in pyridine,27 with (R)ꢀdaucene
96
(82.32 mg) obtained with 87% purity (by GCꢀanalysis) following the same procedure of
97
Levissales and Rudler (1967).
1HꢀNMR (CDCl3, 500 MHz): 5.42 (m, 1H,
98
(CH3)C=CH), 2.66 (sept, 1H, J = 6.7 Hz, (CH3)2CH), 2.39 (m, 1H, (CH3)2CHCCH2),
2.16 (m, 2H, CH=C(CH3)CH2), 2.07 – 1.92 (m, 3H, (CH3)C=CHCH2 and
(CH3)2CHCCH2), 1.82 (m, 1H, CH=C(CH3)CH2CH2), 1.74 (s, 3H, (CH3)C=CH2),
1.61 – 1.42 (m, 3H, CH=C(CH3)CH2CH2 and (CH3)2CHCCH2CH2), 0.97 (d, 3H, J = 6.7
Hz, (CH3)2CH), 0.93 (d, 3H, J = 6.7 Hz, (CH3)2CH), 0.91 (s, 3H, CH3C).
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
[α]20D = + 22.0 (c. 0.53, CHCl3; literature (S)ꢀdaucene [α]20 = – 24.9, CHCl3).28
D
(S)ꢀLinalool was obtained from coriander seed essential oil. Thus, seeds of coriander,
Coriandrum sativum L. (2.6 kg) were extracted by hydrodistillation providing paleꢀ
colored oil (2.2 g). GCꢀFID analysis showed that the major component (85%) was
linalool. The oil (0.6 g) was subjected to liquid chromatography on silica gel (30 g,
SigmaꢀAldrich, 80/100 mesh) with sequential elution using petroleum ether (5 x 20 mL)
and 95:5 petroleum ether: ethyl acetate (20 x 20 mL) to obtain linalool of 98% purity.
The enantiomeric purity of the linalool obtained from the coriander seed oil was
analyzed using a chiral column βꢀDEX 325, as described above. The (S) to (R) ratio was
determined to be 86:14 by GC. (E)ꢀOcimene was obtained in high purity from a
commercially available sample of ocimene (Sigma Aldrich) containing a 30:65 mix of
(Z) and (E) isomers. A sample of the ocimene (100 mg) was chromatographed on 0.5 g
(
SiO2 Sigma—Aldrich, 80/100 mesh) impregnated with 25% AgNO3 (SigmaꢀAldrich,
St. Louis, USA), with elution using 97.5: 2.5 hexane: diethyl ether (15 x 1 mL). The
ratio between both isomers was determined by GCꢀFID analysis using a DBꢀ5MS
column (30 m length, 0.25 mm ID, 0.25 ꢁm film; Supelco, Bellefonte, PA, USA) and a
splitless injector, with helium as the carrier gas. The oven temperature program began at
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 31
120
121
122
50 °C for 2 min, increased at 15 °C/min to 250 °C and then this temperature was
maintained for 20 min. (E)ꢀOcimene was obtained in 98% purity (by GC analysis) and
(Z)ꢀocimene in 95 % purity.
123
124
125
126
127
128
129
130
131
132
133
Insects. Adults of Alphitobius diaperinus were obtained from a commercial poultry
farm in PAD/DF, Brasília, DF, Brazil (15°59'40.5"S 47°37'22.8"W). The insects were
reared at Embrapa Genetics Resources and Biotechnology, Brasília, DF, Brazil
(15°43'48.0"S 47°53'59.5"W) in plastic boxes (40 x 20 cm) containing a fine layer of
wood shavings and corrugated cardboard on top29. Adults and larvae were fed with a
commercial chicken feed three times a week (composition: ground corn, soybeans,
wheat, rice meal, meat meal, limestone, salt, vitamin premix, probiotics and minerals)
and were provided with water daily. Boxes were kept in environmentally controlled
o
room [(27 ± 2 C, 60ꢀ80% RH and 16:8 h (D/L) photoperiod]. For bioassays and
volatile collections, pupae were separated by sex and placed in new boxes. The genders
were distinguished by differences in pupal abdominal appendages.30
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
Semiochemical collection. Two hundred 10ꢀdayꢀold virgin male and female beetles
were placed separately in glass chambers (500 mL), containing a fine layer of
vermiculite (approximately 1 cm) and a steel mesh (4×10 cm), that were used as shelter.
Humidified and purified air (charcoal filter 20ꢀ40 mesh) was drawn through the flasks at
300 mL/min using a vacuum pump. Volatiles from the insects were trapped every 24 h
using porous polymer adsorbent (100 mg of PorapakꢀQ 60ꢀ80 mesh, Sigma Aldrich®).
To avoid contaminants, volatile collections were conducted without food. To guarantee
insects were well fed during collections, and minimize release of defense
compounds15,18 each group of 200 insects was replaced every day. Individuals that died
were replaced. Volatile collections were performed consecutively for 60 days and the
trapped volatiles were eluted from adsorbent using 500 ꢁL of nꢀhexane and preꢀ
concentrated to 100 ꢁL under a gentle flow of N2. The samples were stored at ꢀ20oC
until use. The volatiles from the fine layer of vermiculite (approximately 1 cm) and a
steel mesh (4×10 cm) (control) were also collected during consecutive 60 days to check
for possible contaminants.
149
150
151
152
Olfactometry. Behavioral bioassays were conducted using a Yꢀtube olfactometer
manufactured in a square acrylic block (26.0 x 23.0 cm) with a Yꢀshaped cavity (1.5 cm
thickness) sandwiched between two glass plates. The apparatus trunk measured 12.0 cm
and each arm measured 10.5 cm of length.31 Bioassays were carried out in a controlled
6
ACS Paragon Plus Environment

Page 7 of 31
Journal of Agricultural and Food Chemistry
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
environment room at 25 + 1 ºC and 60 + 10% RH, on a white bench under red artificial
lighting (514 Lux). Charcoalꢀfiltered and humidified air was pushed into the system at
0.6 L/min and pulled out at 0.3 L/min. This ‘pushꢀpull’ system prevents entry of
contaminating volatiles from the exterior. A single virgin A. diaperinus (15 days old)
was introduced individually at the olfactometer trunk base, and observed for 300 sec.
Both sexes were assayed at the same time in two olfactometer sets, until a total of 40
males and 40 females had responded. If no choice was made in 4 minutes, the bioassay
was aborted and the insect recorded as nonꢀresponding.32 The following observations
were recorded: (1) first choice, considered when beetles entered 2.0 cm into the arm; (2)
residence time, measured as the mean of total bioassay time spent in each arm of the
olfactometer. Each individual was sampled only once and the filter papers were
replaced after every five repetitions. At this time the Yꢀtube olfactometer was
exchanged, cleaned with detergent, distilled water and acetone. Silicone tubing, filter
papers and glass syringes were baked in an oven for at least 12 h at 45 ºC prior to use.
In the first experiment, the chemotaxis behavior of A. diaperinus towards the odor of
live conspecifics was assessed. Each olfactometer arm was connected to a 20 mL glass
syringe containing either 20 virgin sexually mature females, 20 virgin sexually mature
males, 10 of each sex, or air (control). The insects in the syringe were allowed to
acclimatize for 30 minutes before experiments started, and were replaced after every 10
repetitions. In the second experiment, the chemotaxis behavior of A. diaperinus
towards collected male volatiles, synthetic solutions or hexane (control) was recorded.
The following treatments were evaluated as odor stimuli: male extract at a
concentration of 1 insect equivalent/µL (IE/µL) and synthetic solution (SS) containing
all compounds produced by males at 0.1, 1 and 10 IE/µL [(R)ꢀlimonene, (E)ꢀocimene,
2ꢀnonanone, (S)ꢀlinalool, (R)ꢀdaucene and (E,E)ꢀαꢀfarnesene)]. Each olfactometer arm
was connected to a 10 mL glass syringe containing a filter paper (0.5 cm width, 1.0 cm
length, 205 µm thicknesses) treated with 10 µL of each treatment. In order to determine
whether all compounds were necessary to modify A. diaperinus behavior, a third
experiment was conducted using synthetic solutions missing one of the identified
components. A fourth experiment was undertaken to evaluate the influence of nonꢀ
naturally occurring isomers, and for this, four different blends containing the six
components (mix), with one of the components present as a different isomer, were
evaluated. The isomers evaluated were: (S)ꢀlimonene, (Z)ꢀocimene, (R)ꢀlinalool, and a
mixture of farnesene isomers. (S)ꢀDaucene was not tested because it was unavailable.
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 31
187
188
189
190
191
192
193
194
195
196
Chemical Analyses. GCꢀFID analyses of collected volatile extracts were performed
using a gas chromatograph (Shimadzu 17A) equipped with a DBꢀ5MS column (30 m
length, 0.25 mm i.d., 0.25 ꢁm film thickness; Supelco, Bellefonte, PA, USA). The
carrier gas was helium. The oven temperature program was programmed to start at 50
°C for 2 min, increase at 5 °C/min to 180 °C, and then increase at 10 ºC/min to 250 °C,
with the final hold time of 20 min. One microliter of each selected sample was injected
in splitless mode; the injector temperature was 250 °C, and the detector temperature was
270 °C. Compounds were quantified by comparing GC peak areas with the peak area of
the internal standard (IS) (nꢀtetracosane, IS was prepared at a final concentration of
1 ꢁg/mL).
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
For qualitative analysis, selected volatile extracts were analyzed by coupled GCꢀMS
using an Agilent MSD 5975C quadrupole mass spectrometer coupled to a gas
chromatograph (GCꢀMS Agilent, 7890A) equipped with a DBꢀ5 column (30 m length,
0.25 mm ID, 0.25 ꢁm film; Supelco, Bellefonte, PA, USA) and a splitless injector, with
helium as the carrier gas, using the same temperature program described for GCꢀFID
analysis. Ionization was performed by electron impact (70 eV; source temperature
200°C). Data were collected using ChemStation software (Agilent Technologies).
Tentative identifications were made by comparison of the target spectra with library
databases (NIST and Wiley 2008), with published spectra and the retention indices
(RI).33,34 Confirmation of the identifications was done by GC peak enhancement with
authentic standards. The absolute configuration of limonene and linalool produced by
males was determined by enantioselective gas chromatography using a chiral GC
column (30 mm×0.25 mm i.d., 0.25 ꢁm, βꢀDEX 325 matrix nonꢀbonded with 25% 2,3ꢀ
di-O-acetyl-6-OꢀTBDMSꢀβꢀcyclodextrin in SPBꢀ20 poly (20% phenyl/ 80%
dimethylsiloxane phase) (Supelco, USA). The oven temperature was programmed as
follows: 50 °C for 2 min, increase at 2 °C/min until 210 °C and hold for 10 min.
Injections were made in splitless mode with helium as the carrier gas (1.5 mL/min),
injector temperature at 250 °C, and detector temperature at 270 °C. To confirm the
identity of (E,E)ꢀαꢀfarnesene, male volatile extracts and authentic standards were also
analyzed and coꢀinjected using a DBꢀWAX column (30 m length, 0.25 mm i.d., 1.0 ꢁm
film thickness; Supelco, Bellefonte, PA, USA). The carrier gas was helium. The oven
temperature program began at 50 °C for 2 min, increased at 5 °C/min to 180 °C, and
then increased at 10 ºC/min to 250 °C; this temperature was maintained for 20 min. One
8
ACS Paragon Plus Environment

Page 9 of 31
Journal of Agricultural and Food Chemistry
220
221
microliter of each selected sample was injected in splitless mode; the injector
temperature was 250 °C, and the detector temperature was 270 °C.
222
223
224
Statistical analysis. The choices made by the insects in the bioassays were analyzed by
chiꢀsquare test and the residence time was analyzed by Wilcoxon’s matchedꢀpairs test,
by using the statistical program R 2.14.0,35 with 95% of reliability.
225
226
RESULTS
227
228
229
230
231
232
233
234
235
Chemical analyses. Chemical analysis of the headspace volatiles collected from both
genders of A. diaperinus indicated that the males produced six volatile organic
compounds that were not present in the volatile collections from females or from
vermiculite and steel mesh (control) (Figure 1). GCꢀMS analysis of the male volatile
extracts, comparison of the spectra with NIST, GC peak enhancement and
enantioselective GC with authentic standards confirmed the identity of the compounds
to be (R)ꢀlimonene (1), (E)ꢀocimene (2), 2ꢀnonanone (3), (S)ꢀlinalool (4), (R)ꢀdaucene
(5) and (E,E)ꢀαꢀfarnesene (6) (Table 1). The mean production of all six compounds was
consistent across all samples analyzed (N = 60) (Table 1).
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
Olfactometry bioassays. Behavior bioassays using live A. diaperinus as the odor
source showed that both genders were attracted to the odor from live adult males
compared to air (control) (Female, χ2 = 12.80, P < 0.001; Male, χ2 = 9.80, P = 0.03)
(Figure 2A and C), and spent more time in the arm containing odor from live male
adults (Female, W = 705, P < 0.001; Male, W = 681, P < 0.001) (Figure 2B and D). The
same behavior was observed when the odors of both live genders were compared to air
[first choice (Female, χ2 = 9.81, P = 0.002; Male, χ2 = 9.72, P = 0.002) (Figure 2A and
C); residence time (Female, W = 70, P < 0.001; Male, W = 681, P < 0.001)] (Figure 2B
and D). When the odor from live females was evaluated, females were not attracted (χ2
= 0.81, P = 0.37, n = 40) (Figure 2A), and males were attracted to air (χ2 = 5.03, P =
0.02, n = 40) (Figure 2C), while for the residence time, both females and males showed
no preference between odor from live females and air (Female, W = 421, P = 0.85, n =
40; Male W = 431, P = 0.74, n = 40) (Figure 2B and D).
Both genders preferred odor from male volatile collections when compared to the
control n-hexane (Female, χ2 = 9.05, P = 0.003; Male, χ2 = 4.68, P = 0.03) (Figure 3A
and C) and spent more time in the arm containing the odor from male volatile
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 31
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
collections (Female, W = 703.5, P < 0.001; Male, W = 674, P < 0.001) (Figure 3B and
D). Males and females were attracted to odor emitted from a synthetic solution
containing the all six maleꢀspecific components [(R)ꢀlimonene, (E)ꢀocimene, 2ꢀ
nonanone, (S)ꢀlinalool, (R)ꢀdaucene, and (E,E)ꢀαꢀfarnesene] in the same ratio produced
by males and at concentration of 1 IE/ꢁL compared to odor from hexane (Table 2,
Figure 3A and C), and spent more time in the arm containing the odor from the
synthetic solution (Table 2, Figure 3B and D). However, insects showed no preference
for the synthetic solution when tested at 0.1 IE/µL or 10 IE/µL (Table 2, Figure 3A and
C) over the solvent control (P > 0.05), and did not spend more time in the arm
containing the odor from the synthetic solution (P > 0.05) (Table 2, Figure 3B and D).
Usually when each maleꢀspecific compound was tested individually at 1 IE/µL males
and females showed no significant behavior activity, except for (S)ꢀlinalool and (R)ꢀ
daucene (Table 2, Figure 4). Males and females were not significantly attracted to
synthetic pheromone blends with one component missing (Table 2, Figure 5), or when
one of the sixꢀcomponent blend was incorporated as an incorrect isomer (Table 2,
Figure 6).
269
DISCUSSION
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
Males of the Brazilian population of the lesser mealworm, A. diaperinus, have been
shown to produce and emit six maleꢀspecific volatile compounds. Five of the six
compounds identified were also described as components of the aggregation pheromone
of this species in North America,15 i.e., (R)ꢀlimonene, (E)ꢀocimene, 2ꢀnonanone, (S)ꢀ
linalool and (R)ꢀdaucene. The sixth component identified in this study, (E,E)ꢀαꢀ
farnesene, is apparently exclusive to a Brazilian population. YꢀTube bioassays
confirmed the presence of a maleꢀproduced aggregation pheromone, with all six
identified compounds required for pheromone activity. Furthermore, components need
to be present in a similar ratio and concentration as emitted by male A. diaperinus to
produce a positive chemotactic response in laboratory conditions. The bioassays
conducted with odor from live females corroborated the chemical analysis, showing that
live females do not produce the attractant that males produce to attract males and
females, and that their odor does not interfere with the maleꢀproduced aggregation
pheromone. Males and females were not attracted to synthetic blends with concentration
10 times more or less compared to the amount produced naturally by males. A.
10
ACS Paragon Plus Environment

Page 11 of 31
Journal of Agricultural and Food Chemistry
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
diaperinus, as well as for other Coleoptera species Phyllotreta cruciferae Goeze
(Chrysomelidae)36 and the boll weevil Anthonomus grandis Boheman (Curculionidae)37
did not show attraction to enantiomers of aggregation pheromone components. For
pheromoneꢀmediated behavior in most species, and particularly by aggregation
pheromones, a naturally occurring pheromone enantiomer is more attractive than the
nonꢀnaturally occurring enantiomer.38 The effect of racemic blends differs between
species.38 In some instances, the unnatural enantiomer does not interfere with the
response to the active enantiomer, but in other species the presence of the enantiomer
can reduce or eliminate the response.38 The stereochemistry of optically active
pheromone components was elucidated in this work and matched those reported.15
(E,E)ꢀαꢀFarnesene has been reported as an important semiochemical that acts as an
attractant and kairomone for Coleoptera39,40 and Hymenoptera.41 This compound is also
present in pheromone blends of Diptera, Hemiptera, Hymenoptera, Isoptera and
Lepidoptera,42ꢀ48 and here, we report the first instance of its appearance as an
aggregation pheromone component for a member of the Coleoptera.
300
301
302
303
304
305
306
307
In addition to the composition difference, the ratio between the components produced
by males from the Brazilian population was different from the North American
population, i.e., for the Brazilian population the major component was (S)ꢀlinalool,
whilst for the North American population, the major component is (E)ꢀocimene. Wind
tunnel bioassays showed that only three components of the A. diaperinus aggregation
pheromone are necessary to attract both genders of North American populations,17
whereas our results showed that Brazilian populations need all six maleꢀspecific
compounds for pheromoneꢀmediated behavior.
308
309
310
311
312
313
314
315
316
317
Differences in pheromonal blend might be due to geographical isolation. For Coleoptera
(beetles) this phenomenon was reported for pine bark beetles, Ips pini Say (Scolytidae),
where eleven years after the first pheromone identification, a new component was
isolated from another population.19,20,49,50 For Hawaiian and Australian populations of
cane weevil borers, Rhabdoscelus obscurus Boisduval22,51 and also for bark beetles, Ips
subelongatus Motschulsky, where population divergence in aggregation pheromone
responses was reported.24 This phenomenon is not exclusive to Coleoptera. Differences
between populations also occur, for example, in the sex pheromone of the European
Corn Borer, Ostri nia nubilalis Hübner (Lepidoptera: Pyralidae)21 and a trail pheromone
present in stingless bees, Trigona corvina Cockerell (Hymenoptera: Apidae).23
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 31
318
319
320
321
322
323
324
325
326
327
328
329
330
In the United States, the pheromonal blend of North American A. diaperinus showed
promising results in trapping experiments in poultry houses, with pheromone traps
attracting three times more adults and larvae than control traps.15,16 Our results suggest
that if the North American pheromone blend were used in Brazil, the mixture would
probably not be effective for A. diaperinus management. Problems with deployment of
sex pheromones for control of insect pests across different geographical regions have
been reported elsewhere. For fall armyworm, Spodoptera frugiperda Smith
(Lepidoptera: Noctuidae), interpopulational differences in sex pheromone components
between sympatric regions presents difficulties in the application of this technology.
52,53
Thus, for control of Brazilian populations, careful consideration for use of the six
components blend must be applied, whereas for other populations outside of North
America and Brazil, pheromone composition must be verified prior to deployment in
trapping systems.
331
332
333
334
335
336
337
338
339
One of the major problems in poultry production in Brazil is to keep poultry litter free
of A. diaperinus. Since extensive chemical control is unaffordable and often involves
replacement of poultry litter, sustainable and less expensive methods to control and
manage A. diaperinus populations in poultry houses would have an enormous and
positive impact in Brazil. Further studies are required to investigate the pheromone
composition in different populations of A. diaperinus around the world in order to
provide control of this cosmopolitan pest. For Brazilian populations, the next step is to
test the feasibility of using pheromone based traps in poultry houses and quantify the
economic and social impact of reduced chemical control upon poultry production.
340
341
342
ACKNOWLEDGMENTS: The authors express their special thanks to Anildo Cunha
Jr., and Gerson Neudi Scheuermann, for providing information about A. diaperinus.
343
344
12
ACS Paragon Plus Environment

Page 13 of 31
Journal of Agricultural and Food Chemistry
345
REFERENCES
346
347
(1) Axtell, R.C.; Arends J.J. Ecology and Management of Arthropod Pests of Poultry.
Ann. Rev. Entomol. 1990, 35, 101ꢀ126.
348
349
350
(2) Dunford, J.C.; Kaufman P.E. 2006. Lesser Mealworm, Litter Beetle, Alphitobius
diaperinus (Panzer) (Insecta: Coleoptera: Tenebrionidae). University of Florida IFAS
Acessed on January 8, 2016 from http://edis.ifas.ufl.edu/pdffiles/IN/IN66200.pdf.
351
352
(3) Japp, A.K.; Bicho, C.L.; Silva A.V.F. Importância e Medidas de Controle para
Alphitobius diaperinus em Aviários. Cienc. Rural. 2010, 40, 1668ꢀ1673.
353
354
355
(4) Salin, C.; Delettre, Y.R.; Cannavacciuolo M.; Vernon P. Spatial Distribution of
Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) in the Soil of a Poultry
House along a Breeding Cycle. Eur. J. Soil Biol. 2000, 36, 107ꢀ115.
356
357
358
359
(5) Lambkin, T.A.; Kopittke, R.A.; Rice, S.J.; Bartlett J.S.; Zalucki M.P. Distributions
of Lesser Mealworm (Coleoptera: Tenebrionidae) in Litter of a Compacted EarthꢀFloor
Broiler House in Subtropical Queensland, Australia. J. Econ. Entomol. 2007, 100, 1136ꢀ
1146.
360
361
362
(6) Lambkin, T.A.; Kopittke, R.A.; Rice, S.J.; Bartlett, J.S.; Zalucki, M.P. Factors
Affecting Localized Abundance and Distribution of Lesser Mealworm in EarthꢀFloor
Broiler Houses in Subtropical Australia. J. Econ. Entomol. 2008, 101, 61ꢀ67.
363
364
365
(7) McAllister, J.C.; Steelman, C.D.; Newberry, L.A.; Skeeles, J.K. Isolation of
Infectious Bursal Disease Virus from the Lesser Mealworm, Alphitobius diaperinus
(Panzer). Poult. Sci. 1995, 74, 45ꢀ49.
366
367
368
(8) Goodwin, M.A.; Waltman, W.D. Transmission of Eimeria, Viruses and Bacteria to
Chicks: Darkling Beetles (Alphitobius diaperinus) as Vectors of Pathogens. J. Appl.
Poultry Res. 1996, 5, 51–55.
369
370
371
372
(9) Hazeleger, W.C.; Bolder, M.N.; Beumer, R.R.; JacobsꢀReitsma, W.F. Darkling
Beetles (Alphitobius diaperinus) and their Larvae as Potential Vectors for the Transfer
of Campylobacter jejuni and Salmonella enterica serovar paratyphi B Variant Java
Between Successive Broiler Flocks. Appl. Environ. Microbiol. 2008, 74, 6887–6891.
373
374
(10) Roche, A.J.; Cox, N.A.; Richardson, L.J.; Buhr, R.J.; Cason, J.A.; Fairchild, B.D.;
Hinkle, N.C. Transmission of Salmonella to Broilers by Contaminated Larval and Adult
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 31
375
376
Lesser Mealworms, Alphitobius diaperinus (Coleoptera: Tenebrionidae). Poult. Sci.
2009, 88, 44ꢀ48.
377
378
379
380
(11) Leffer, A.M.; Kuttel, J.; Martins, L.M.; Pedroso, A.C.; AstolfiꢀFerreira, C.S.;
Ferreira, F.; Ferreira, A.J.P. Vectorial Competence of Larvae and Adults of Alphitobius
diaperinus in the Transmission of Salmonella enteritidis in Poultry. Vector-Borne and
Zoonotic Dis. 2010, 10, 481ꢀ487.
381
382
383
384
(12) ChernakiꢀLeffer, A.M.; SosaꢀGòmez, D.R.; Almeida, L.M. Selection for
Entomopathogenic Fungi and LD50 of Metarhizium anisopliae (Metsch.) Sorok. for the
Lesser Mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae). Braz.
J. Poultry Sci. 2007, 9, 187ꢀ191.
385
386
387
388
(13) Pezowicz, E.; Mazurkiewicz, A.; Tumialis, D. Sensitivity of Imago and Larvae of
the Lesser Mealworm Alphitobius diaperinus (Panzer 1797) in a Sawdust Litter to
Selected Species and Strains of Steinernematidae and Heterorhabditidae under
Laboratory Conditions. Anim. Sci. 2013, 52, 161ꢀ166.
389
390
391
(14) Prado, G.P.; Stefani, L.M.; Silva, A.S.; Smaniotto, L.F.; Garcia, F.R.M.; Moura
N.F. Alphitobius diaperinus (Coleoptera: Tenebrionidae) Susceptibility to Cunila
angustifolia Essential Oil. J. Med. Entomol. 2013, 50, 1040ꢀ1045.
392
393
394
(15) Bartelt, R.J.; Zilkowski, B.W.; Cossé, A.A.; Steelman, C.D.; Singh, N. Maleꢀ
Produced Aggregation Pheromone of the Lesser Mealworm Beetle, Alphitobius
diaperinus. J. Chem. Ecol. 2009, 35, 422ꢀ434.
395
396
397
(16) Singh, N.; Johnson, D.T. Attractiveness of an Aggregation Pheromone Lure and
Chicken Droppings to Adults and Larvae of Alphitobius diaperinus (Coleoptera:
Tenebrionidae). J. Econ. Entomol. 2012, 105, 2196ꢀ206.
398
399
400
401
(17) Cossé, A.A.; Zilcowski, B.W. Behavioural Responses of Lesser Mealworm
Beetles, Alphitobius diaperinus, (Coleoptera: Tenebrionidae) to Pheromone
Components Using a Wind Tunnel Dual Choice Walking Bioassay. J. Insect behav.
2015, 28, 202ꢀ210.
402
403
404
405
(18) Hassemer, M.J.; Sant'Ana, J.; de Oliveira, M.W.; Borges, M.; Laumann, R.A.;
Caumo, M.; BlassioliꢀMoraes, M.C. Chemical Composition of Alphitobius diaperinus
(Coleoptera: Tenebrionidae) Abdominal Glands and the Influence of 1,4ꢀbenzoquinones
on its Behavior. J. Econ. Entomol. 2015, 108, 2107ꢀ2116.
14
ACS Paragon Plus Environment

Page 15 of 31
Journal of Agricultural and Food Chemistry
406
407
408
(19) Lanier, G.N.; Claesson, A.; Stewart, T.; Piston, J.; Silverstein, R.M. Ips pini: The
Basis for Interpopulational Differences in Pheromone Biology. J. Chem. Ecol. 1980, 6,
677ꢀ687.
409
410
411
(20) Teale, S. A.; Webster, F.X.; Zhang, A.; Lancer, G.N. Lanierone: A New
Pheromone Component from lps pini (Coleoptera: Scolytidae) in New York. J. Chem.
Ecol. 1991, 17, 1159ꢀ1176.
412
413
414
(21) Dopman, E. B.; Bogdanowicz, S. M.; Harrison, R.G. Genetic Mapping of Sexual
Isolation Between E and Z Pheromone Strains of the European Corn Borer (Ostrinia
nubilalis). Genetics. 2004, 167, 301–309.
415
416
417
418
(22) Muniappan, R.; Bamba, J.; Cruz, J.; Reddy, G.V.P. Field Response of Guam
Populations of the New Guinea Sugarcane Weevil, Rhabdoscelus obscurus (boisduval)
(Coleoptera: Curculionidae), to Aggregation Pheromones and Food Volatiles.
Micronesica. 2004, 37, 57ꢀ68.
419
420
421
(23) Jarau, S.; Dambacher, J.; Twele, R.; Aguilar, I.; Francke, W.; Ayasse, M. The Trail
Pheromone of a Stingless Bee, Trigona corvina (Hymenoptera, Apidae, Meliponini),
Varies between Populations. Chem. Senses. 2010, 35, 593ꢀ601.
422
423
424
(24) Chen, D.; Li1, L.; Zhang, Q.; Zhang, S.; Wang, H.; Zhang, Z.; Zhao, L.; Kong1, X.
Population Divergence of Aggregation Pheromone Responses in Ips subelongatus in
Northeastern China. Insect Sci. 2015, 00, 1744ꢀ7917.
425
426
(25) Spicer, J.A.; Brimble, M.A.; Rowan, D.D. Oxidation of αꢀFarnesene. Aust. J.
Chem. 1993, 46, 1929–1940.
427
428
429
(26) Sýkora, V.; Novotný, L.; Holub, M.; Herout, V.; Šorm, F. On terpenes. CXVII.
The Proof of Structure of Carotol and Daucol. Collect. Czech. Chem. Commun. 1961,
26, 788ꢀ802.
430
431
(27) Levisalles, J.; Rudler, H. Terpénoides. II Carotol. I: Stéreochimedu Carotol. Bull.
Soc. Chim. Fr. 1967, 365, 2059ꢀ2066.
432
433
434
435
(28) Bennett, N.B.; Stoltz, B.M. A Unified Approach to the Daucane and Sphenolobane
Bicyclo[5.3.0]decane Core: Enantioselective Total Syntheses of Daucene, Daucenal,
Epoxydaucenal B, and 14ꢀparaꢀAnisoyloxydaucꢀ4,8ꢀdiene. CHEM-EUR J, 2013, 19,
17745– 17750.
436
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 31
437
438
(29) Rice, S.J.; Lambkin, T.A. A New Culture Method for the Lesser Mealworm,
Alphitobius diaperinus. J. Appl. Entomol. 2009, 133, 67ꢀ72.
439
440
441
(30) Esquivel, J.F.; Crippen, T.L.; Ward, L.A. Improved Visualization of Alphitobius
diaperinus (Panzer) (Coleoptera: Tenebrionidae)ꢀPart I: Morphological Features for Sex
Determination of Multiple Stadia. Psyche: J. Entomol. 2012, 2012, 1ꢀ7.
442
443
444
(31) Moraes, M.C.; Millar, J.G.; Laumann, R.A.; Sujii, E.R.; Pires, C.S.; Borges, M.
Sex Attractant Pheromone from the Neotropical RedꢀShouldered Stink Bug, Thyanta
perditor (F.). J. Chem. Ecol. 2005, 31, 1415ꢀ1427.
445
446
447
(32) Borges, M.; Millar J.G.; Laumann, R.A.; Moraes, M.C.B. A Maleꢀproduced sex
Pheromone from the Neotropical Redbanded Stink Bug, Piezodorus guildinii (W.). J
Chem. Ecol. 2007, 33, 1235ꢀ1248.
448
(33) NIST. 2011. Chemistry WebBook. http://webbook.nist.gov/chemistry/.
449
450
(34) Pherobase. Database of pheromone and other semiochemicals. Accessed on
January 19, 2016 from http://www.pherobase.com/database/kovats/kovatsꢀindex.php.
451
452
(35) R Development Core Team. 2011. R: A Language and Environment for Statistical
Computing.R Foundation for Statistical Computing, Vienna, Austria.
453
454
455
456
(36) Tóth, M.; Csonka, E.; Bartelt, R. J.; Cossé, A.A.; Zilkowski, B.W.; Muto, S.; Mori,
K. Pheromonal Activity of Compounds Identified from male Phyllotreta cruciferae:
Field Tests of Racemic Mixtures, Pure Enantiomers, and Combinations with Allyl
Isothiocyanate. J. Chem. Ecol. 2010, 31, 2705ꢀ2720.
457
458
459
(37) Dickens, J.C.; Mori, K. Receptor Chirality and Behavioural Specificity of the Boll
Weevil, Anthonomus grandis Boh. (Coleoptera: Curculionidae), for its Pheromone, (+)ꢀ
Grandisol. J. Chem. Ecol. 1989, 15, 517ꢀ528.
460
461
(38) Mori K. Significance of Chirality in Pheromone Science. Bioorg. Medic. Chem.
2007, 15, 7505–7523.
462
463
464
(39) Sun, X.L.; Wang, G.C.; Cai, X.M.; Jin, S.; Gao, Y.; Chen, Z.M. The Tea Weevil,
Myllocerinus aurolineatus, is Attracted to Volatiles Induced by Conspecifics. J. Chem.
Ecol. 2010, 36, 388ꢀ395.
465
466
(40) Yasui, H.; Akino, T.; Fukaya, M.; Wakamura, S.; Ono, H. Sesquiterpene
Hydrocarbons: Kairomones with a Releaser Effect in the Sexual Communication of the
16
ACS Paragon Plus Environment

Page 17 of 31
Journal of Agricultural and Food Chemistry
467
468
Whiteꢀspotted Longicorn Beetle, Anoplophora malasiaca (Thomson) (Coleoptera:
Cerambycidae). Chemoecol. 2008, 18, 233ꢀ242.
469
470
471
472
(41) Michereff, M.F.; Laumann, R.A.; Borges, M.; MichereffꢀFilho, M.; Diniz, I.R.;
Neto, A.L.; Moraes, M.C. Volatiles Mediating a PlantꢀHerbivoreꢀNatural Enemy
Interaction in Resistant and Susceptible Soybean Cultivars. J. Chem. Ecol. 2011, 37,
273ꢀ85.
473
474
475
476
(42) Heath, R.R.; Landolt, P.J.; Tumlinson, J.H.; Chambers, D.L.; Murphy, R.E.;
Doolittle, R.E.; Dueben, B.D.; Sivinski, J.; Calkins, C.O. Analysis, Synthesis,
Formulation, and Field Testing of Three Major Components of Male Mediterranean
Fruit Fly Pheromone. J. Chem. Ecol. 1991, 17, 1925ꢀ1940.
477
478
(43) Nault, L.R.; Bowers, W.S. Multiple Alarm Pheromones in Aphids. Entomol. Exp.
Appl. 1974, 17, 455ꢀ457.
479
480
481
(44) Aldrich, J.R.; Waite, G.K.; Moore, C.; Payne, J.A.; Lusby, W.R.; Kochansky, J.P.
Maleꢀspecific volatiles from Nearctic and Australasian true bugs (Heteroptera:
Coreidae and Alydidae). J. Chem. Ecol. 1993, 19, 2767ꢀ2781.
482
483
484
485
(45) Khrimian, A.; Fay, H.A.C.; Guzman, F.; Chauhan, K.; Moore, C.; Aldrich, J.R.
Pheromone of the BananaꢀSpotting Bug, Amblypelta lutescens lutescens Distant
(Heteroptera: Coreidae): Identification, Synthesis, and Field Bioassay. Psyche. 2012,
2012, 1ꢀ8.
486
487
488
(46) Cane, J.H. Chemical Evolution and Chemosystematics of the Dufonr's Gland
Secretions of the LactoneꢀProducing Bees (Hymenoptera: Colletidae, Halictidae, and
Oxaeidae). Evolution. 1983, 37, 657ꢀ674.
489
490
491
(47) Schulz, S.; Boppre, M.; VaneꢀWright R.I. Specific Mixtures of Secretions from
Male Scent Organs of African Milkweed Butterflies (Danainae). Phil. Trans. R. Soc.
Lond. B. 1993, 342, 161ꢀ181.
492
493
494
(48) Šobotník, J.; Hanus, R.; Kalinová, B.; Piskorski, R.; Cvacka, J.; Bourguignon, T.;
Roisin, Y. (E,E)ꢀαꢀfarnesene, an Alarm Pheromone of the Termite Prorhinotermes
canalifrons. J. Chem. Ecol. 2008, 34, 478ꢀ486.
495
496
(49) Symonds, M.R.E.; Elgar, M.A. The Mode of Pheromone Evolution: Evidence from
Bark Beetles. Proc. R. Soc. London Ser. B, Biol. Sci. 2004, 271, 839ꢀ846.
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 31
497
498
499
(50) Miller, D. R.; Borden, J.H.; Slessor, K.N. Interꢀ and Intrapopulation Variation of
the Pheromone, Ipsdienol Produced by Male Pine Engravers, Ips pini (Say) (Coleoptera:
Scolytidae). J. Chem. Ecol. 1989, 15, 233ꢀ247.
500
501
502
503
(51) GiblinꢀDavis, R.M.; Gries, R.; Crespi, B.; Robertson, L.N.; Hara, A.H.; Gries, G.;
O'Brien, C.W.; Pierce Jr, H.D. Aggregation Pheromones of two Geographical Isolates
of the New Guinea Sugarcane Weevil, Rhabdoscelus obscurus. J. Chem. Ecol. 2000, 26,
2763ꢀ2780.
504
505
506
(52) Andrade, R.; Rodriguez, C.; Oehlschlagera, A. C. Optimization of a Pheromone
Lure for Spodoptera frugiperda (Smith) in Central America. J. Braz. Chem. Soc. 2000,
11, 609ꢀ613.
507
508
509
510
(53) BatistaꢀPereira, L.G.; Stein, K.; de Paula, A.F.; Moreira, J.A.; Cruz, I.; Figueiredo,
M.L.L.; Perri, J.; Corrêa, A. Isolation, Identification, Synthesis, and Field Evaluation of
the Sex Pheromone of the Brazilian Population of Spodoptera frugiperda. J. Chem.
Ecol. 2006, 32, 1085–1099.
511
512
FUNDING SOURCES
513
514
515
516
517
518
519
520
We thank the Fitotecnia PostꢀGraduate Program of the Federal University of Rio
Grande do Sul for the use of their facility. This work received financial support from the
Coordination of Superior Level Staff Improving (CAPES) through a grant to Marla J.
Hassemer and from the National Council for Scientific and Technological Development
(CNPq), the Brazilian Corporation of Agricultural Research (EMBRAPA) and Research
Support Foundation of the Federal District (FAPꢀDF). Rothamsted Research receives
grantꢀaided support from the Biotechnology and Biological Sciences Research Council
(BBSRC) of the United Kingdom.
521
18
ACS Paragon Plus Environment

Page 19 of 31
Journal of Agricultural and Food Chemistry
522
FIGURE CAPTIONS
523
524
Table 1. Mean quantity ± standard error (SE) (ng/insect/day) and retention index of each
male specific compound using DBꢀ5MS and DBꢀWAX GC columns.
525
526
527
528
529
530
531
Table 2. Statistical analysis of the first choice and residence time data for female and
male Alphitobius diaperinus in Yꢀtube olfactometer bioassays with different synthetic
solutions, at 0.1, 1 or 10 Insect Equivalent (IE)/µL, containing components of male
aggregation pheromone against nꢀhexane (control). Analyses were carried out using chiꢀ
square test for first choice and Wilcoxon’s matchedꢀpairs test for residence time.
*P<0.05, **P<0.01 and ***P<0.001.
532
533
534
535
536
537
538
539
540
541
542
543
544
Figure 1. Gas chromatograms of volatile collections of glass chambers containing
vermiculite (control), Alphitobius diaperinus females plus vermiculite, and A.
diaperinus males plus vermiculite. (R)ꢀlimonene (1); (E)ꢀocimene (2); 2ꢀnonanone (3);
(S)ꢀlinalool (4); (R)ꢀdaucene (5) and (E,E)ꢀαꢀfarnesene (6).
Figure 2. First choice and residence time of female (A and B) and male (C and D)
Alphitobius diaperinus in Yꢀtube olfactometer bioassays with the odor of live insects
against air as control. Analyses were carried out using chiꢀsquare test for first choice
and Wilcoxon’s matchedꢀpairs test for residence time. Bars indicate the number of
responsive insects and the residence time (%) to each olfactometer arm. *P<0.05 and
***P<0.001. Numbers in brackets in the figure represent the number of insects that did
not respond to the treatment tested.
545
546
547
548
549
550
551
552
553
Figure 3. First choice and residence time of female (A and B) and male (C and D)
Alphitobius diaperinus in Yꢀtube olfactometer bioassays in response to collected male
volatile extracts at 1 Insect Equivalent (IE)/µL, and synthetic solutions (SS) containing
all male specific compounds [(R)ꢀlimonene, (E)ꢀocimene, 2ꢀnonanone, (S)ꢀlinalool, (R)ꢀ
daucene and (E, E)ꢀαꢀfarnesene] at 0.1, 1 and 10 IE/ µL against nꢀhexane as control.
Analyses were carried out using chiꢀsquare test for first choice and Wilcoxon’s
matchedꢀpairs test for residence time. Bars indicate the number of responsive insects
and the residence time (%) to each olfactometer arm. * P<0.05 and **P<0.01. Numbers
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 31
554
555
in brackets in the figure represent the number of insects that did not respond to the
treatment tested.
556
557
558
559
560
561
562
563
564
Figure 4. First choice and residence time of female (A and B) and male (C and D)
Alphitobius diaperinus in Yꢀtube olfactometer bioassays in response to individual
components of maleꢀproduced aggregation pheromone at 1 Insect Equivalent (IE)/ µL
against nꢀhexane as control. Analyses were carried out using chiꢀsquare test for first
choice and Wilcoxon’s matchedꢀpairs test for residence time. Bars indicate the number
of responsive insects and the residence time (%) to each olfactometer arm. **P<0.01
and *P<0.05. Numbers in brackets in the figure represent the number of insects that did
not respond to the treatment tested.
565
566
567
568
569
570
571
572
573
Figure 5. First choice and residence time of female (A and C) and male (B and D)
Alphitobius diaperinus in Yꢀtube olfactometer bioassays in response to synthetic
solutions (SS) of aggregation pheromone minus (w/o) a single component, at 1 Insect
Equivalent (IE)/ µL against nꢀhexane as control. Analyses were carried out using chiꢀ
square test for first choice and Wilcoxon’s matchedꢀpairs test for residence time. Bars
indicate the number of responsive insects and the residence time (%) to each
olfactometer arm. n.s. = no significance. Numbers in brackets in the figure represent the
number of insects that did not respond to the treatment tested.
574
575
576
577
578
579
580
581
582
583
Figure 6. First choice and residence time of female (A and B) and male (C and D)
Alphitobius diaperinus in Yꢀtube olfactometer bioassays in response to synthetic
solutions (SS) of aggregation pheromone, prepared with one component present as an
incorrect isomer, at 1 Insect Equivalent (IE)/ µL against nꢀhexane as control. Analyses
were carried out using chiꢀsquare test for first choice and Wilcoxon’s matchedꢀpairs test
for residence time. Bars indicate the number of responsive insects and the residence
time (%) to each olfactometer arm. Numbers in brackets in the figure represent the
number of insects that did not respond to the treatment tested.
20
ACS Paragon Plus Environment

Page 21 of 31
Journal of Agricultural and Food Chemistry
TABLES
Table 1.
Quantity ± SE
ng/insect/day
Retention index
DBꢀ5MS
Retention index
DBꢀWAX
Compounds
(R)ꢀlimonene
(E)ꢀocimene
2ꢀnonanone
49.0 ± 10.4
31.3 ± 6.9
7.0 ± 1.4
1030
1050
1091
1103
1378
1508
1188
1249
1386
1551
1487
1735
(S)ꢀlinalool
50.0 ± 12.9
18.4 ± 1.2
44.5 ± 10.8
(R)ꢀdaucene
(E,E)ꢀαꢀfarnesene
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 31
Table 2.
Males response
Females response
Amount
(IE/µL)
Residence
Time
First choice
First choice Residence Time
Synthetic solution (SS) composition
χ2 = 0.07
P = 0.79
χ2 = 3.48
P = 0.03*
χ2 = 0.29
P = 0.59
χ2 = 1.14,
(R)ꢀlimonene, (E)ꢀocimene, 2ꢀnonanone, (S)ꢀlinalool,
(R)ꢀdaucene, (E,E)ꢀαꢀfarnesene
W = 250,
W = 251.5
0.1
1
P = 0.48
P = 0.46
P = 0.28
χ2 = 7.49
P = 0.003**
χ2 = 1.14
P = 0.28
(R)ꢀlimonene, (E)ꢀocimene, 2ꢀnonanone, (S)ꢀlinalool,
(R)ꢀdaucene, (E,E)ꢀαꢀfarnesene
W = 689
W = 688
P < 0.001***
P < 0.001***
(R)ꢀlimonene, (E)ꢀocimene, 2ꢀnonanone, (S)ꢀlinalool,
(R)ꢀdaucene, (E,E)ꢀαꢀfarnesene
W = 140
W = 290
10
P = 0.66
P = 0.12
Individual compounds
χ2 = 0.28
P = 0.59
χ2 = 0.78
P = 0.38
χ2 = 0.08
P = 0.78
χ2 = 0.06
P = 0.79
χ2 = 0.08
P = 0.77
χ2 = 3.00
P = 0.08
W = 220.5
P = 0.95
W = 131
P = 0.83
W = 235
P = 0.71
W = 97
(R)ꢀlimonene
1
1
1
(E)ꢀocimene
P = 0.13
W = 16
W = 189
2ꢀnonanone
P = 0.90
P = 0.48
22
ACS Paragon Plus Environment

Page 23 of 31
Journal of Agricultural and Food Chemistry
χ2 = 2.57
W = 284.5
χ2 = 4.57
P = 0.03*
χ2 = 4.57
P = 0.03*
χ2 = 1.20
P = 0.27
W = 251.5
(S)ꢀlinalool
1
P = 0.15
P = 0.46
P = 0.11
χ2 = 0.06
W = 328.5
P = 0.01**
W = 93
W = 282
P = 0.16
W = 68
(R)ꢀdaucene
1
P = 0.79
χ2 = 3.33
(E,E)ꢀαꢀfarnesene
1
P = 0.06
P = 0.65
P = 0.07
SS minus one component
χ2 = 1.80
W = 420
χ2 = 0.20
P = 0.65
χ2 = 1.80
P = 0.18
χ2 = 0.20
P = 0.65
χ2 = 0.20
P = 0.65
χ2 = 0.20
P = 0.65
χ2 = 0.80
W = 440
SS w/o (R)ꢀlimonene
1
P = 0.68
P = 0.68
P = 0.18
χ2 = 3.20
W = 414
P = 0.96
W = 352
P = 0.99
W = 470.5
P = 0.42
W = 475
P = 0.24
SS w/o (E)ꢀocimene
SS w/o 2ꢀnonanone
SS w/o (S)ꢀlinalool
1
P = 0.07
χ2 = 0.80
W = 496.5
P = 0.39
1
P = 0.37
χ2 = 1.80
W = 441.5
P = 0.30
1
P = 0.18
χ2 = 0.20
W = 431.5
W = 422
SS w/o (R)ꢀdaucene
1
P = 0.77
P = 0.87
P = 0.65
χ2 = 0.20
SS minus (E,E)ꢀαꢀfarnesene
1
W = 447.5
W = 461.5
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 31
P = 0.65
P = 0.62
P = 0.37
P = 0.69
SS with incorrect isomers
(S)ꢀlimonene, (E)ꢀocimene, 2ꢀnonanone, (S)ꢀlinalool,
(R)ꢀdaucene, (E,E)ꢀαꢀfarnesene
χ2 = 3.20
P = 0.07
χ2 = 0.20
P = 0.65
χ2 = 1.80
P = 0.18
χ2 = 3.20
P = 0.07
W = 131
χ2 = 0.80
P = 0.37
χ2 = 3.20
P = 0.07
χ2 = 3.20
P = 0.07
χ2 = 1.80
P = 0.18
W = 102
1
1
1
1
P = 0.33
P = 0.78
(R)ꢀlimonene, (Z)ꢀocimene, 2ꢀnonanone, (S)ꢀlinalool,
(R)ꢀdaucene, (E,E)ꢀαꢀfarnesene
W = 142.5
P = 0.16
W = 99.5
P = 0.86
W = 106
P = 0.97
(R)ꢀlimonene, (E)ꢀocimene, 2ꢀnonanone, (R)ꢀlinalool,
(R)ꢀdaucene, (E,E)ꢀαꢀfarnesene
W = 112.5
P = 0.78
(R)ꢀlimonene, (E)ꢀocimene, 2ꢀnonanone, (S)ꢀlinalool,
(R)ꢀdaucene, farnesene isomers
W = 110.5
W = 133
P = 0.84
P = 0.29
24
ACS Paragon Plus Environment

Page 25 of 31
Journal of Agricultural and Food Chemistry
FIGURES
Figure 1.
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 31
Figure 2.
26
ACS Paragon Plus Environment

Page 27 of 31
Journal of Agricultural and Food Chemistry
Figure 3.
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 31
Figure 4.
28
ACS Paragon Plus Environment

Page 29 of 31
Journal of Agricultural and Food Chemistry
Figure 5.
29
ACS Paragon Plus Environment