Sex Pheromone of the Invasive Mealybug Citrus Pest, Delottococcus aberiae (Hemiptera: Pseudococcidae). A New Monoterpenoid with a Necrodane Skeleton

Article abstract of DOI:10.1021/acs.jafc.9b01443

Native to sub-Saharan Africa, Delottococcus aberiae De Lotto (Hemiptera: Pseudoccidae) is an invasive mealybug that has been recently reported in Europe, seriously damaging citrus production in eastern Spain. In this study, we isolated and determined the structure of the sex pheromone of D. aberiae, to provide a highly specific and effective lure for detecting, monitoring, and potentially controlling this pest. The volatile profile of D. aberiae virgin and mated females was studied by aeration and collection of effluvia in Porapak-Q. The resulting extracts were analyzed by gas chromatography coupled to mass spectrometry (GC-MS), revealing a candidate compound specific of virgin females. GC-MS and nuclear magnetic resonance spectroscopy data evidenced a new compound, (4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl acetate, with an unusual β-necrodol skeleton. This compound was synthesized and shown to be attractive to male D. aberiae in both laboratory and field experiments. A GC analysis using an enantioselective stationary phase and polarimetry analyses of the synthetic enantiomers showed the natural compound emitted by virgin females to be the-enantiomer.

Full text of DOI:10.1021/acs.jafc.9b01443

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Agricultural and Environmental Chemistry
Sex pheromone of the invasive mealybug citrus pest,
Delottococcus aberiae (Hemiptera:Pseudococcidae).
A new monoterpenoid with a necrodane skeleton
Sandra Vacas, Ismael Navarro Fuertes, Javier Marzo, Vicente Navarro-Llopis, and Jaime Primo
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01443 • Publication Date (Web): 05 Aug 2019
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Page 1 of 37
Journal of Agricultural and Food Chemistry
Sex pheromone of the invasive mealybug citrus pest, Delottococcus aberiae
(Hemiptera:Pseudococcidae). A new monoterpenoid with a necrodane skeleton
1
2
2
1
Sandra Vacas, Ismael Navarro, Javier Marzo, Vicente Navarro-Llopis, Jaime
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Primo1
1CEQA-Instituto Agroforestal del Mediterráneo, Universitat Politècnica de València,
Camino de Vera s/n, edificio 6C-5ª planta, 46022 Valencia (Valencia), Spain.
2Ecología y Protección Agrícola SL, Pol. Ind. Ciutat de Carlet, 46240 Carlet (Valencia),
Spain.
1
Correspondence to: Sandra Vacas. CEQA-Instituto Agroforestal del Mediterráneo,
Universitat Politècnica de València. Camino de Vera s/n, edificio 6C-5ª planta, 46022-
Valencia, Spain. Email: sanvagon@ceqa.upv.es; phone: +34963879058; fax:
+34963879059
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ABSTRACT
Native to sub-Saharan Africa, Delottococcus aberiae De Lotto (Hemiptera:
Pseudoccidae) is an invasive mealybug that has been recently reported in Europe
seriously damaging citrus production in eastern Spain. In this study, we isolated and
determined the structure of the sex pheromone of D. aberiae, to provide a highly
specific and effective lure for detecting, monitoring and potentially controlling this pest.
The volatile profile of D. aberiae virgin and mated females was studied by aeration and
collection of effluvia in Porapak-Q. The resulting extracts were analyzed by gas
chromatography coupled to mass spectrometry (GC-MS), revealing a candidate
compound specific of virgin females. GC-MS and nuclear magnetic resonance
spectroscopy data evidenced a new compound, (4,5,5-trimethyl-3-methylenecyclopent-
2
2
3
3
0
5
0
5
1
-en-1-yl)methyl acetate, with an unusual β-necrodol skeleton. This compound was
synthesized and shown to be attractive to male D. aberiae in both laboratory and field
experiments. A GC analysis using an enantioselective stationary phase and polarimetry
analyses of the synthetic enantiomers showed the natural compound emitted by virgin
females to be the (-)-enantiomer.
KEYWORDS
semiochemical; chemical ecology; attractant; lure; volatile
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INTRODUCTION
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Delottococcus aberiae (De Lotto) (Hemiptera: Pseudococcidae) is an invasive
mealybug that has been recently reported to cause severe damage to citrus in the
1
2
Mediterranean area. Native to sub-Saharan Africa, the presence of D. aberiae has been
reported in several countries in central and southern Africa, such as Kenya,
3
Mozambique, Swaziland, Tanzania and Zimbabwe. It is commonly found in South
4
5
5
Africa on wild olive trees, the roots of the flowering shrub Chrysanthemoides
monilifera (L.) T. Norl., and is irregularly distributed in citrus orchards north of the
4
country. Its recent introduction into eastern Spain has been demonstrated to match
mealybug populations on citrus from the Limpopo Province (South Africa) by
5
molecular techniques, and has probably come via the international trade of citrus plants
6
or fruits, which is the main pathway to disperse scale insects to Europe. Like most
mealybugs, D. aberiae is multivoltine and completes several generations per year under
7
Mediterranean conditions and remains active even in winter. After hatching,
individuals undergo two nymphal stages (first and second) and then, females and males
start to develop differently. Males had two pupal stages (pre-pupa and pupa) inside a
cottony cocoon before developing into winged adult males. Females have one more
nymphal stage to become adult females, and changes very slightly in appearance
(wingless) except the size they grow to.
The population trends of D. aberiae in citrus have been recently investigated in Spain
by visual examination of plant material, sampling developmental stages in corrugated
7
,8
6
0
cardboard band traps and detecting male flights in adapted sticky traps. The seasonal
trend revealed that D. aberiae density increases in spring, and reaches its first
significant maximum in May and June, to coincide with fruit development in the
Mediterranean Region, before developing on fruits until the end of August, and then
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7
decreasing and remaining at low levels for the rest of the year. In that work, visual
examinations and samplings of developmental stages revealed several overlapping
generations (two to four). Nonetheless, monitoring mealybugs by visual inspections is a
time-consuming process because it is necessary to count the live insects present on plant
material. However, two main male peak flights were also well-defined by using adapted
sticky traps baited with virgin females, which confirmed the two main generations
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detected by inspections. Thus, Martinez-Blay et al. provided evidence for a substance
emitted by D. aberiae virgin females to attract males. The identification of the female
sex pheromone would allow pheromone traps to be used to detect and monitor D.
aberiae populations, and to develop control methods to be included in Integrated Pest
Management strategies.
7
5
In the present work, we isolated and characterized the sex pheromone from the airborne
volatiles emitted by D. aberiae virgin females by gas chromatography coupled to mass
spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectrometry. Having
elucidated the structure, the candidate compound was synthesized to confirm its
structure and activity in both laboratory and field tests.
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MATERIALS AND METHODS
Mealybug stock colony
The colony of D. aberiae was established in our facilities at the Universitat Politècnica
de València (UPV, Valencia, Spain) using specimens from the Laboratory of
Entomology, based at Instituto Valenciano de Investigaciones Agrarias (IVIA, Valencia,
Spain). Mealybugs were reared on organic green lemons, which were previously
covered with paraffin around the mid-section to delay their desiccation. Starting from
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gravid females and ovisacs deposited on lemons, newly hatched individuals establish on
the surface of fruit and follow the developmental cycle. Mated females are allowed to
oviposit. When ovisacs are laid, they are gently transferred with an entomological
needle to new lemons. Insects were maintained in a rearing chamber, in the dark at 24±2
ºC, with 40-60% relative humidity.
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0
After the second instar stage, males produce a distinguishable cottony cocoon that
covers their bodies to pupate and transform into winged adults. Lemons were visually
inspected every 3-4 days for the presence of cocoons, which were manually removed
with an entomological needle to leave lemons infested only with virgin females for
volatile collection and profiling purposes. Mated females were sampled on the lemons
from the main stock colony after checking for the presence of ovisacs.
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00
Collection of volatiles
Five to six lemons infested with approx. 200 D. aberiae females (virgin or mated
separately) were placed in 5-L glass reactors: 25-cm high × 17.5-cm diameter flask,
with a 10-cm open mouth and a ground glass flange to fit the cover with a clamp. The
cover had a 29/32 neck on top to fit the head of a gas washing bottle and to connect
downstream a glass cartridge to trap effluents in 3 g Porapak-Q (Supelco Inc., Torrance,
CA, USA) adsorbent. Samples were collected continuously for 9 days under 14:10
1
05
(L:D) light-darkness conditions by using an ultrapurified-air stream, provided by an air
compressor (Jun-air Intl. A/S, Norresundby, Denmark) coupled with an AZ 2020 air
purifier system (Claind Srl, Lenno, Italy) to provide ultrapure air (amount of total
hydrocarbons < 0.1 ppm). In front of each glass reactor, an ELL-FLOW digital
flowmeter (Bronkhorst High-Tech BV, Ruurlo, The Netherlands) was fitted to provide
an air push flow of 400 mL/min during sampling. Trapped volatiles were then extracted
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with 20 mL pentane (Chromasolv®, Sigma-Aldrich, Madrid, Spain) and extracts were
concentrated to 500 µL under a nitrogen stream prior to the chromatographic analysis.
Fifty rounds of virgin female collections were performed to obtain approximately
1
15
9
0,000 female-day equivalents (FDE), a sufficient quantity for the nuclear magnetic
resonance (NMR) analysis.
All the resulting pentane extracts were analyzed by GC-MS in a Clarus 600 GC-MS
(PerkinElmer Inc, Waltham, MA). The GC was equipped with a ZB-5MS fused silica
1
20
capillary column (30 m × 0.25 mm i.d. × 0.25 μm; Phenomenex Inc., Torrance, CA) and
the oven was held at 40 °C for 2 min and then programmed at 5 °C/min to 180 °C,
before being raised to 280 °C at 10 °C/min and maintained at 280 °C for 1 min. Helium
was used as the carrier gas at a flow of 1 mL/min. Detection was performed in the EI
mode (70 eV) with the ionization source set at 180ºC. Spectrum acquisition was carried
out in full scan mode (mass range m/z 35-500) and chromatograms and spectra were
recorded by means of GC-MS Turbomass software v. 5.4 (PerkinElmer Inc., Waltham,
MA).
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Isolation of the candidate compound
Having compared the GC-MS volatile profiles of the virgin and mated samples (lemons
infested with virgin and mated females, respectively), the first step to isolate the virgin-
specific compound was the fractionation of the Porapak-Q pentane extracts by gravity
column (300 mm × 15 mm i.d.). Composite samples of ca. 30,000 FDE in 1 mL pentane
were loaded in each column and fractionation was performed using 9.5 g of silica gel as
the stationary phase (40-60 µm) and 20 mL mixtures of pentane:diethyl ether as the
eluents (100:0, 95:5, 80:20, 0:100). Thirty fractions were collected and they were all
analyzed by GC-MS to identify those that contained the candidate compound, by the
methods described in the previous section. These enriched fractions were gathered to
1
30
1
35
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finally isolate the pheromonal compound by preparative-GC, using a modified Clarus
5
00 GC (Perkin Elmer Inc., Wellesley, PA) equipped with a flame ionization detector
1
1
1
1
40
(FID) that was set at 250ºC and a TRB-1 fused-silica capillary column (30 m × 0.53 mm
i.d. × 0.5 μm; Teknokroma Analítica SA, Sant Cugat del Vallès, Barcelona, Spain). The
GC oven was programmed at 40 °C for 2 min and then raised at 3 °C/min to 100 °C
and, when reached, raised to 280 °C at 30 °C/min and maintained at 280 °C for 12 min.
The end of the column was connected to a 1/16-inch stainless steel tube (10 cm long)
which was then connected to a T-splitter to divide the column effluent towards the FID
detector (a 180 cm long empty fused-silica capillary line) and a handmade fraction
collection system. The collector consisted of a 12-gauge stainless steel needle (30 cm
long) connected to the T-splitter on one side, while the opposite passed through an
insulated thermostatted port set at 270º C (Syntech temperature controller TC-02,
Ockenfels SYNTECH GmbH, Kirchzarten, Germany) to cross the GC left-side panel
leaving an accessible luer-lock connector. Fractions were collected in 100 µL stainless
steel HPLC loops connected to the aforementioned luer-lock connector and cooled by a
dry ice–acetone bath for volatile condensation purposes. Compounds were then eluted
from the loop with 2 × 100 µL pentane or deuterobenzene (C D ). Thirty injections, 5
45
50
55
6
6
µL/each, were performed to collect the required quantity for the NMR analysis (ca. 30
1
µg). Then, the H NMR spectrum of the natural isolated compound was recorded by a
Bruker 600 Ultrashield Plus spectrometer (Bruker, Billerica, MA) at a frequency of 600
MHz, using C D as the solvent with tetramethylsilane (TMS) as the internal standard.
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Microreaction: hydrolysis of the natural (4,5,5-trimethyl-3-methylenecyclopent-1-
en-1-yl)methyl acetate (1)
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The pentane extract of an aeration sample of ca. 1,000 FDE (~ 300 ng of pheromone)
9
was hydrolyzed following related procedures. The extract was dried under a gentle
nitrogen stream in a 2-mL GC glass vial. The residue was treated with a 0.5 M solution
of KOH in methanol (150 µL) and stirred for 45 min at room temperature. After this
time, water (0.5 mL) was added and the solution was extracted with hexane (0.5 mL
twice). The combined organic phases were concentrated under nitrogen to ca. 100 µL
volume before being submitted to GC-MS analysis.
1
65
1
70
Synthesis of (4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl acetate (1)
(Figure 1)
General procedures. The H and 13C spectra were recorded on a Bruker AC-300
1
spectrometer (Bruker, Billerica, MA) using deuterated chloroform (CDCl ) or C D as
3
6
6
the solvents and TMS as the internal standard. Chemical shift values are reported in δ
1
13
1
1
1
75
80
85
(ppm) relative to chloroform ( H NMR: 7.26 ppm and C NMR: 77.0 ppm) or benzene
(1
H NMR: 7.16 ppm and C NMR: 128.4 ppm). High resolution mass spectra (ESI-
13
HRMS) were measured on a Waters Xevo Q-TOF spectrometer (Waters Corp., Milford,
MA) coupled with an Acquity UPLC-PDA system (Waters Corp., Milford, MA) using
ionization by electrospray (ESI). The ESI source operated in the positive ionization
+
mode using leucine-enkephalin as the reference mass ([M+H] ion m/z 556.2771). The
sample (2 μL) was injected into a Waters Acquity BEH column (50 x 2.1 mm i.d., 1.7
μm) using MeOH as isocratic eluent. The GC-MS analyses were performed with the
aforementioned equipment (apparatus and column) and the following oven temperature
program: 55 °C for 3 minutes, raised at 15 °C/min up to 180 °C, and then at 35 °C/min
up to 280 °C, held for 6 minutes. A helium flow of 1 mL/min and an injection volume
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of 1 μL were employed. Detection and spectra acquisition were performed as indicated
above.
All the reagents and solvents (reagent grade) were purchased from Sigma-Aldrich
(Madrid, Spain) and employed with not further purification unless otherwise stated. All
1
90
the organic solvents employed in these experiments were dried with appropriate drying
agents and distilled before use. Unless otherwise stated, all the reactions sensitive to
moisture and/or air were carried out under nitrogen atmosphere using anhydrous
solvents. The solvent extracts of the reaction mixtures were dried over MgSO and
4
concentrated by rotary evaporation under reduced pressure. Crude products were
purified by column flash chromatography using silica gel Merck 9385 (230-400 mesh).
Thin-layer chromatography (TLC) was performed using Macherey-Nagel silica gel 60
F254 plates with a fluorescent indicator and UV light of 254 nm wavelength as the
visualizing agent. Ceric ammonium molybdate and p-anisaldehyde were used as stains.
1
95
3
,4,4-trimethylcyclopent-2-en-1-one (3). Isobutyl crotonate 2 (40 g, 0.28 mol) was
2
2
2
00
05
10
slowly added to polyphosphoric acid (200 g) at 95 ºC for 3 h. After continuous stirring
at this temperature for 3 h, the solution was cooled down and poured over water (350
mL) with stirring. The mixture was extracted with diethyl ether (2 × 120 mL) and the
organic layers were successively washed with NaHCO sat. (50 mL), brine (50 mL),
3
dried over Mg SO and concentrated under reduced pressure. The crude material was
4
distilled under reduced pressure to give 21 g of 3 as yellow oil. The spectroscopic data
were fully coincident with those described in the literature.10
3
-(bromomethyl)-4,4-dimethylcyclopent-2-en-1-one (4). A 1-L Pyrex round-bottom
flask containing a solution of 3 (5 g, 0.04 mol) and NBS (11.4 g, 0.064 mol, 1.6 eq.) in
DCM (200 mL) was placed inside an irradiation chamber equipped with a 400 W visible
lamp (HPI Plus, Koninklijke Philips N.V., Amsterdam, The Netherlands) and an air
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cooling system. The flask was irradiated with stirring for 4.5 h, and during this time, the
chamber’s temperature remained below 30 ºC. After this period, the solution was
poured in hexane (200 mL) and filtered. The organic solution was concentrated under
vacuum and the crude residue (ca. 6.0 g) was used in the next step with no further
purification. A small portion of the crude (200 mg) product was purified by flash
2
2
2
2
2
15
20
25
30
35
column chromatography (silica gel, 10% Et O/hexane) to give 140 mg of 4 (65 %) as a
2
1
yellow oil. H NMR (CDCl , 300 MHz) δ 6.19 (1H, s), 4.17 (s, 2H), 2.41 (s, 2H), 1.30
3
1
3
(
s, 6H). C NMR (75 MHz, CDCl ) 206.4, 181.0, 132.5, 52.6, 43.2, 27.4 (2C), 24.3.
3
EM (70 eV) m/z: 41 (40), 67 (100), 79 (78), 95 (44), 109 (32), 123 (23), 187 (30), 189
(28), 202 (17), 204 (M+, 18).
(5,5-dimethyl-3-oxocyclopent-1-en-1-yl)methyl acetate (5). KOAc (39 mmol, 3.83 g, 1.5
eq.) was added in portions during the 45-min period to a solution of crude 4 (ca. 6 g, 26
mmol) in DMSO (40 mL). The suspension was stirred for an additional 20-min period
and poured into water (60 mL). The mixture was extracted with AcOEt (2 × 70 mL) and
the combined organic layers were successively washed with brine (2 × 30 mL), dried
over MgSO and concentrated under reduced pressure. The crude residue was purified
4
by flash column chromatography (silica gel, 10% Et O/hexane) to give 4.45 g of 5 (60%
2
1
starting from ketone 3) as a yellow oil. H NMR (CDCl , 300 MHz) δ 5.99 (1 H, t, J
3
H
1
3
1
.8 Hz), 4.91 (2 H, d, J 1.8 Hz), 2.36 (2 H, s), 2.15 (3 H, s), 1.28 (6 H, s). C NMR (75
MHz, CDCl ) δ 206.7, 181.2, 170.5, 127.6, 60.3, 52.2, 41.9, 27.4 (2C), 20.8. EM (70
3
eV) m/z: 43 (100), 67 (23), 79 (42), 95 (12), 110 (18), 125 (63), 140 (52), 167 (10), 182
(12, M+).
(4,5,5-trimethyl-3-oxocyclopent-1-en-1-yl)methyl acetate (6). A solution of 5 (3.0 g,
1
6.4 mmol) in THF (15 ml) was dropwise added over a solution of LDA (47.6 ml, 0.5
M, 23.8 mmol) at -78 ºC. After 20 min, the solution was warmed to -30 ºC and MeI (85
1
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mmol, 5.3 mL, 5 eq.) was added. The mixture was kept at this temperature for 1 h and
then, gradually warmed to room temperature. The reaction was quenched with NH Cl
4
sat. (6 ml), poured into water (15 mL) and extracted with AcOEt (2 × 60 mL). The
combined organic layers were successively washed with HCl (1M, 25 mL), NaHCO3
2
2
2
2
2
40
sat. (30 mL), brine (30 mL), dried over MgSO and concentrated under reduced
4
pressure. The crude residue was purified by flash column chromatography (silica gel,
1
5
% Et O/hexane) to give 1.4 g of 6 (43 %) as a yellow oil. H NMR (C D , 300 MHz) δ
2
6
6
6
.11 (1 H, s), 4.70 – 4.55 (2 H, m), 1.97 (1 H, qt, J 7.1, 2.5 Hz), 1.72 (3 H, s), 1.03 (3
H
1
3
H, d, J 7.1 Hz), 0.75 (3 H, s), 0.68 (3 H, s). C NMR (75 MHz, C D ) δ 206.8, 178.5,
6
6
45
50
55
60
169.5, 126.4, 60.2, 53.6, 44.3, 25.6, 23.4, 20.1, 9.7. EM (70 eV) m/z: 43 (100), 55 (23),
6
7 (33), 77 (14), 93 (38), 108 (20), 121 (14), 139 (57), 154 (32), 181 (22), 196 (8, M+).
+
HRMS (ESI) m/z calculated for C H O ([M +H]) 197.1172, found 197.1170.
1
1
16
3
(4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl acetate (1). Methyl magnesium
chloride (8.1 mL, 3 M, 24.3 mmol) was added over a suspension of
bis(cyclopentadienyl)titanium(IV) dichloride (3.03 g, 12.2 mmol) in toluene (110 mL)
at 0 ºC. After 20 min of continuous stirring, the solution was gradually warmed to room
temperature and a solution of ketone (1.2 g, 6.1 mmol) in dry toluene was added. The
solution was warmed to 60 ºC for 24 h and was then cooled down to room temperature
and poured into water (25 mL). The mixture was extracted with Et O (2 × 40 mL). The
2
combined organic layers were successively washed with NaHCO sat. (20 mL), brine (2
3
×
20 mL), dried over MgSO and concentrated under reduced pressure. The crude
4
residue was purified by flash column chromatography (silica gel, 5% Et O/hexane) to
2
1
give 615 mg of 1 (52 %) as a yellow oil. H NMR (300 MHz, Benzene-d ) δ 6.06 (1
6
H
H, s), 4.94 (1 H, d, J 2.5 Hz), 4.74 (1 H, d, J 2.4 Hz), 4.70 – 4.55 (2 H, m), 2.36 (1 H, qt,
1
3
J 7.1, 2.5 Hz), 1.69 (3 H, s), 0.93 (3 H, d, J 7.1 Hz), 0.88 (3 H, s), 0.75 (3 H, s). C
1
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NMR (75 MHz, C D ) δ 169.8, 156.7, 153.4, 129.2, 103.1, 60.8, 49.4, 47.5, 25.9, 22.1,
6
6
2
0.4, 12.4. EM (70 eV) m/z: 43 (72), 53 (12), 65 (14), 77 (21), 79 (19), 91 (40), 105
(20), 119 (100), 121 (72), 134 (22), 194 (10, M+). HRMS (ESI) m/z calculated for
+
C H O ([M +H]) 195.1380, found 195.1375.
1
2
18
2
2
65
Assignment of enantiomeric series to the natural compound
The enantiomers contained in the synthetic sample of deacetylated 1, following a
standard procedure (Figure S2), were separated by means of preparative chiral liquid
chromatography (LC) using a Sigma LP-1100 preparative system (VWR International,
Radnor, PE), equipped with a DAICEL 19335 Chiralpak AD-H (1 × 25 cm) chiral
column (Chiral Technologies Europe SAS, Illkirch, France). A hexane:isopropanol
2
2
2
70
75
80
(99:1) solvent mixture was used as the eluent. Six injections, each containing 1 mg each
of the racemic material dissolved in 0.5 ml of a 10:1 hexane:isopropanol mixture, were
carried out. The pure fractions of each enantiomer were collected, and ca. 1.3 mg were
recovered of each one after the solvent underwent rotary evaporation. The assignment
of the specific optical rotation to each enantiomer was done after re-acetylation of the
obtained alcohols under standard conditions using a polarimeter (Perkin Elmer Inc.
Model 343) equipped with a sodium lamp (linea D, 589 nm) and a 1-dm glass cell at 20
ºC, using chloroform as the solvent (approx. 0.13 g/100 ml). Then, assigned levo (-) and
dextro (+) enantiomers were injected into a InertCap CHIRAMIX chiral capillary
column (30 m × 0.25 mm i.d. × 0.25 μm; GL Sciences Inc., Tokyo, Japan) installed in a
Clarus 500 GC (Perkin Elmer Inc., Wellesley, PA) equipped with FID detector set at
2
2
50 ºC. The oven temperature was raised at 0.6 ºC/min from 50 ºC to 115ºC, and then at
5 ºC/min to 150ºC, which were finally held for 10 min. The assignment of the
1
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enantiomeric series to the natural compound was done according to the matching
retention times and coeluted samples (Figure S1).
Laboratory bioassay
The activity of the Porapak-Q extracts of the volatile collections containing the
pheromone or samples of the synthetic 4,5,5-trimethyl-3-methylenecyclopent-1-en-1-
yl)methyl acetate enantiomers were tested by the methodology described by Arai et al.11
2
2
3
90
95
00
2
In each test, two pieces of 1 cm filter paper were placed in a 30 cm-diameter glass Petri
dish. One was treated with the extract or the enantiomers and the other was taken as the
reference filter paper treated with the control extract or solvent (pentane). The control
extract refers to Porapak-Q pentane extract of the volatile sampling of lemons infested
with immature instars (not containing adult females). The filter paper pieces were
placed opposite one another in the Petri dish and 5-7 D. aberie males were released at
the center of the dish. Ten minutes later, the number of males that had come into contact
with each paper was revised. The filter paper pieces were loaded with 20 μL of pentane
solutions containing the substances to be tested. Twenty-μL of the crude pentane extract
of the volatile sampling means approximately 1.25 female-hour equivalents (FHE),
whereas 10 ng of a synthetic sample represents ca. 1.5 FHE. The reference filter paper
pieces were loaded with the same volume of solvent (20 μL pentane). The position of
the stimulus was rotated and the glass Petri dish was washed after each test.
3
05
The employed males were collected from the above-described stock colony. Recently
formed cocoons were gently transferred from lemons using an entomological needle to
the filter paper pieces inside the plastic Petri dishes, and remained there until
emergence. After leaving the cocoons, emerged males were observed under a
stereomicroscope to check their fitness (no missing legs or waxy caudal filaments, and
1
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10
unfolded undamaged wings) before transferring them to the above-described glass Petri
dishes for testing. All the assays were conducted under laboratory conditions, with
artificial light, at 25 ± 2 °C and with 40-50% relative humidity. After each test, insects
were discarded, so each test employed different males.
The null hypothesis that D. aberiae showed no preference for either treated filter paper
(response equal to 50:50 for stimulus:solvent) was analyzed with a Chi-square goodness
of fit test with the SPSS 16.0.1 statistical package (SPSS Inc., Chicago, IL).
3
15
Field experiments
The male D. aberiae response to synthetic 4,5,5-trimethyl-3-methylenecyclopent-1-en-
1-yl)methyl acetate was also tested under field conditions. Substances were emitted
from rubber septa (Ecología y Protección Agrícola SL, Carlet, Spain), which were
loaded by impregnation with a hexane solution of the separated (-) or (+) enantiomers
3
3
3
20
25
30
(100 µg/each), or 100 µg of the racemate and allowing the solvent to evaporate. The
traps employed were 95 × 150 mm white sticky boards (Ecología y Protección Agrícola
SL, Valencia, Spain). Trials were carried out in a Citrus reticulata Blanco orchard var.
Marisol, located in the municipality of Sagunto (Valencia, Spain) (coordinates:
3
9.689765, -0.289564). Four blocks of four traps were installed to test the attractiveness
of the synthetic samples, also including a blank trap (only with the solvent employed for
rubber septa impregnation). Traps were placed in the field in October 2018, rotated
within each block and revised 6 times (every 7-15 days). Traps were hung at a height of
1
.5 m and were spaced 20 m apart, with each block at least 50 m apart. The number of
captured males was counted under a stereomicroscope (Stemi 508; Zeiss, Oberkochen,
Germany) at 50X magnification.
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The total number of D. aberiae males captured per trap was accumulated after one
complete trap rotation inside each block, in such a way that two periods were used to
avoid the dependence of catches as a factor of time. Then, one-way analysis of variance
3
35
(ANOVA) was applied to these data without transformation, as they fulfilled the
homocedasticity requirements and the residuals of the ANOVA fitted a normal
distribution. The Fisher’s least significant difference [LSD] test at P < 0.05 was
employed for the multiple comparisons among the attractants. These analyses were
conducted using the Statgraphics Centurion XVI package (StatPoint Technologies,
Warrenton, VA).
3
40
RESULTS
3
45
Chemical analysis and structure elucidation
The chromatographic volatile profiles of the samples of lemons infested with either
virgin or mated D. aberiae females showed a virgin-specific compound at 24.24 min
(KI = 1322 on the ZB-5 column) (Figure 2). By sampling 90,000 FDE, ca. 30 µg of this
compound were collected, which allowed us to calculate that a single D. aberiae female
emitted approximately 0.3 ng on average. The mass spectrum of this compound had two
main peaks at m/z 119 and 121, and significant fragments at m/z 134, 105, 93, 91 and
3
50
4
3, assuming the peak at m/z 194 to be the molecular ion (Figure 3). The fragmentation
pattern suggested a monoterpenoid structure with an acetate ester indicated mainly by
the characteristic loss of 60 amu to give m/z 134 and also supported by the presence of
the ion at m/z 43. After performing the alkaline hydrolysis of one sample, the former sex
pheromone peak completely disappeared, supporting the presence of an ester. The
molecular ion at 194, and the demonstrated presence of an acetate, would lead to a
3
55
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possible molecular formula of C H O , which in turn would require 4 sites of
1
2
18
2
unsaturation in total.
1
3
3
3
3
3
60
65
70
75
80
The H NMR spectrum (Figure 4) of the pure collected 1 by preparative GC (ca. 30 g)
showed some defined signals such as 6.07, 4.95 (d, J=2.4 Hz) and 4.75 (d, J=2.4 Hz)
ppm. Given the small coupling constant shown, these three signals were initially
assigned to a conjugated vinylic proton and two geminal methylenic protons
respectively. The signal centered at 4.64 ppm was compatible with the presence of two
isolated protons coupled to one another (AB system). The strong downfield chemical
shift of the AB system suggested the vicinity of the former with the acetate moiety,
which was, in turn, consistent with the singlet present at 1.68 ppm. Finally, the signals
at 0.935, 0.87 and 0.75 ppm were tentatively assigned to three methyl groups. The first
methyl group, a doublet with a coupling constant of J=7.1 Hz, was clearly coupled to
1
1
the multiplet centered at 2.35 ppm according to the H- H COSY spectrum, whereas the
signals at 0.87 and 0.75 ppm probably corresponded to a geminal dimethyl group or two
1
quaternary carbons. The three vinylic signals observed in H NMR spectra probably
indicated a combination of two double bounds and a ring to fulfill the aforementioned
three unsaturations. This allowed us to suggest a necrodol carbon framework with an
extra unsaturation as a plausible structure for this pheromone. In particular, a -necrodol
1
skeleton was compatible with the exo-methylenic group observed in the H NMR
spectra. Although the precise positions of the geminal and coupled methyl groups, as
well as that of the acetate moiety, were not deducible a priori with the data in hand,
(
4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl acetate 1 was tentatively
proposed to be the target structure based on biosynthetic precedents of this core. Then,
the synthesis was carried out to confirm the identity of the sex pheromone.
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Synthesis and identification of the pheromone
4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl acetate was synthesized in the
(
3
3
3
4
85
laboratory (Figure 1). The synthesis commences with the cyclization of isobutyl (E)-
but-2-enoate I polyphosphoric acid to afford cyclopentyl enone 3, under the previous
1
0
conditions reported by Conia and Leviredend. The functionalization of the enone
methyl group via the allylic bromination of ketone 3 using NBS in DCM under
irradiation,12,13 gave bromo-ketone 4 as the main product with a 65 % yield (in some
experiments, the -keto di brominated product was also identified by the GC-MS
analysis in variable amounts as a byproduct, which proved unstable after isolation by
column chromatography, and decomposed in a few hours). The crude bromo-ketone 4
was treated with potassium acetate in DMSO14,15 to afford acetate 5 with an overall 60
90
95
00
%
yield starting from ketone 3. The alkylation of the allylic acetate, with methyl iodide
1
6
using LDA and THF as base , provided ketone 6 with a low yield of 43 %. Any attempt
to change the base to HMDSLi or solvent did not improve the result, and was probably
due to the presence of the acetate group. It was possible to finish the structure with the
methylenation of ketone 6, which was initially attempted under Wittig conditions, but in
our hands, no conversion of the product was observed. The Lombardo methylenation
using titanium tetrachloride and zinc metal, resulted in a total sample decomposition.
1
7
Finally, a modest yield of 52 % of 1 was obtained by using the Petasis reagent in situ
generated with bis(cyclopentadienyl)titanium(IV) dichloride and methyl magnesium
chloride in toluene.
1
The H NMR analysis of the sample in C D fully coincided with the signals observed in
6
6
4
05
the natural sample (Figure 4). The identification of the pheromone was also fully
supported by the coincidence of the GC retention time (24.24 min; Figure 2) and the MS
1
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fragmentation pattern which was virtually identical to that of the natural virgin female
volatile sampling.
Unfortunately, the direct separation of enantiomers of racemic 1 by employing
preparative chiral LC was not possible under any of the assayed conditions. However,
the separation of the corresponding racemic alcohol of 1, after deacetylation with
4
4
4
10
15
20
1
8
potassium carbonate in methanol, by chiral LC afforded a small amount of pure
enantiomers. The specific optic rotation of each enantiomer of (4,5,5-trimethyl-3-
methylenecyclopent-1-en-1-yl)methyl acetate 1 was then measured after re-acetylation
under standard conditions by using triethyl amine as a base and acetic anhydride in
1
9
DCM. The retention times obtained by chiral GC and the co-injection of samples
demonstrated that the natural (4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl
acetate emitted by D. aberie virgin females matched the previously assigned (-)-
enantiomer (Figure S1). Given the small amount initially obtained of each enantiomer,
no experiments were carried out to determine the absolute configuration of the
pheromone and the enantiomeric purity of both enantiomers could not be accurately
measured.
Laboratory and field assays
4
25
In the laboratory assays, the pentane extracts of virgin female volatile sampling had a
2
significant effect on D. aberie males’ behavior, as they significantly preferred (χ =
2
0.57, P < 0.0001) the filter paper treated with this sample to a pentane extract of the
volatile sampling of lemons infested with immature instars (Figure 5A).
Having synthesized the candidate compound and obtained the separated enantiomers,
the laboratory tests showed that males significantly moved directly towards the filter
4
30
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2
paper treated either with the (-)-enantiomer (Figure 5B; χ = 31.17, P < 0.0001) or the
2
(
+)-enantiomer (Figure 5C; χ = 7.60, P = 0.006), when they were separately presented
against a blank filter paper piece loaded with pentane. Males significantly preferred the
2
(
-)-enantiomer when simultaneously offered both substances (Figure 5D; χ = 18.90, P <
4
35
0.0001).
In the field, blank traps captured only one male throughout the trial, whereas 1,644 and
9
31 males were captured in the traps baited with the (-) and (+) enantiomers,
respectively, and 1,164 males were recorded with the racemate. Accordingly, trapping
efficacy of the (-)-enantiomer obtained significantly more captures than the unnatural
(+)-enantiomer (F3,28 = 17.61; P < 0.0001; Table 1), which confirmed the preference
observed in the laboratory assays and, consequently, the identity of the natural sex
pheromone compound previously determined by the analytical procedures. Even though
the attractiveness observed for the unnatural enantiomer is not uncommon in this insect
4
40
2
0
family, a small contamination of the natural enantiomer in the unnatural one cannot be
discarded, which might be responsible for the activity recorded. Racemate was also
effective in trapping D. aberiae males but their trap counts were significantly lower than
those of the (-)-enantiomer (Table 1).
4
45
DISCUSSION
4
50
Necrodols having 1,1,2,2-pentamethyl cyclopentane (necrodane) as core structural unit,
2
1
have rarely been found in the plant kingdom and only a few examples are reported in
2
2
insects. Eisner and Meinwald described the isolation of two novel irregular
monoterpenoids,  and -necrodols, from the defensive spray of the red line carrion
beetle Necrodes surinamensis (Fabricius) (Coleoptera: Silphidae). Later, Figadere et
1
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2
3
4
55
al. identified the female-produced sex pheromone of the grape mealybug
Pseudococcus maritimus (Ehrhorn) (Hemiptera: Pseudococcidae) as trans--necrodyl
isobutyrate. Given its very uncommon irregular monoterpenoid structure, only a few
synthetic routes have been developed for trans-necrodol and its isomers. As far as we
know, the biosynthesis of this class of monoterpenes has not yet been studied. As
2
3
4
60
suggested by Figadere et al., the necrodol skeleton could be generated from geranyl
diphosphate that comes from the typical head-to-tail 1’-4 linkage of DMAPP and IPP
(Figure 6) via the isocamphane skeleton, but assembling two isoprenoids units via 2-3’
2
’-4 cyclization was also hypothesized. The sex pheromones of other mealybug species
are mainly irregular terpenoids that display non head-to-tail 1’-2 linkages between the
isoprene units. Of these, lavandulyl esters are certainly the simplest structural examples.
Based on this fact, we suggest an alternative biosynthetic pathway to the necrodol
skeleton via lavandulyl cation (Figure 7), followed by cyclization and the usual methyl
rearrangement.
4
65
The diversity of relations between the chirality and bioactivity of pheromones has been
2
0
4
70
thoroughly reviewed by Mori, including many examples in which the unnatural
stereoisomer(s) showed bioactivity. This can be stated of some pheromones belonging
to the Coccoidea species. The primary component of the maritime pine scale
(
Matsucoccus feytaudi Ducasse (Hemiptera: Margarodidae)) pheromone, (8E,10E)-
,7,9-trimethyl-8,10-dodecadien-6-one, was determined as (3S,7R); however, the
(3R,7R) isomer showed similar activity and, albeit weakly, males also responded to the
3
4
75
2
4
others (3R,7S) and (3S,7S). A closest example can be found in pink hibiscus
mealybug (Maconellicoccus hirsutus (Green) (Hemiptera: Pseudococcidae) males,
which slightly responded to the 1:5 mixture of the SS esters of the natural pheromone
described as a 1:5 mixture of (R)-lavandulyl (S)-2-methylbutanoate and (R)-maconelliyl
2
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9
4
4
4
4
5
80
(S)-2-methylbutanoate. In other cases, and according to the classification of Mori,20
only a single enantiomer of the pheromone is bioactive but the presence of other
enantiomers in a mixture does not inhibit the response, which is the most common case
in species of the family Pseudococcidae; e.g. the vine mealybug (Planococcus ficus
2
5
26
Signoret), Comstock mealybug (Pseudococcus comstocki Kuwana) or citrus
2
7
85
90
95
00
mealybug (Pseudococcus cryptus Hempel), etc. Thus, mealybugs appear to generally
insensitive or not negatively affected by the presence of stereoisomers or other analogs
2
8
of their pheromones. This has been hypothetically attributed by Millar et al. to the
unique irregular terpenoid structures of the mealybug pheromones that create singular
communication channels, free of any possible interference with, or competing for, the
pheromone channel. Nonetheless, many examples can be found for the importance of
enantiomeric specificity for different insect orders, and indeed the opposite enantiomer
2
0
of the sex pheromone could be a strong behavioral antagonist in some cases. As
shown in the results of the field trial, enantiomeric purity was not crucial for D. aberiae
attractiveness given that the racemate was also active. The substantial field
attractiveness observed for the unnatural (+)-enantiomer reinforces this statement but,
being these species strongly sensitive to small pheromone amounts, an effect of a small
contamination with the natural enantiomer cannot be disregarded. Although absolute
configuration of 1 needs further confirmation, trapping efficacy of the synthetic
racemate is advantageous to further implement this sex pheromone as a management
tool.
The convenient monitoring of D. aberiae with pheromone-baited traps will help
growers to know pest pressure and when to apply insecticide treatments or to release
natural enemies to optimize the efficacy of control methods. This species causes direct
damage involving marked fruit distortions and size reduction, which are similar to those
2
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5
05
symptoms caused by infestations of a close mealybug species Delottococcus elisabethae
2
9
(
Brain) (Hemiptera: Pseudoccidae), which is reported only in South Africa. In this
1
case, D. elisabethae is a quarantine pest in USA, Israel and South Korea and it is,
therefore, quite likely that D. aberiae can become a quarantine pest in the near future. In
line with this, the availability of the D. aberiae pheromone will provide an
unambiguous detection method to identify new infested areas, or to even detect infested
fruits as part of shipments. The identification of this new pheromone opens up a new
possibility to develop control methods that are already being used to fight against other
Pseudococcidae species, such as mating disruption for Planococcus ficus (Signoret),30-31
mass trapping or lure and kill.32
5
10
5
15
ACKNOWLEDGEMENTS
Authors are grateful to Martina Palomino-Schätzlein and Ruben Lamas (Servicio de
Resonancia Magnética Nuclear, Centro de Investigación Príncipe Felipe, Valencia,
Spain) for NMR support. Thanks to Helen L. Warburton for language revision and
editing.
5
20
FUNDING SOURCES
This work received funding from Universitat Politècnica de València (post-doctoral
contract for SV) and Conselleria de Agricultura, Medio Ambiente, Cambio Climático y
Desarrollo Rural (Generalitat Valenciana) under grant agreement (S8456000).
5
25
SUPPORTING INFORMATION
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Journal of Agricultural and Food Chemistry
Figure S1. Chiral GC chromatogram of single (-) and (+)-enantiomers of (4,5,5-
trimethyl-3-methylenecyclopent-1-en-1-yl)methyl acetate, natural pheromone and
coeluted samples ((+)-enantiomer and natural pheromone).
5
30
Figure S2. Deacetylation of 1.
Figure S3. Chiral HPLC chromatogram of synthetic alcohol 1a.
1
13
Figure S4. (A) H and (B) C NMR spectra of 4.
1
13
Figure S5. (A) H and (B) C NMR spectra of 5.
1
13
5
35
Figure S6. (A) H and (B) C NMR spectra of 6.
1
13
Figure S7. (A) H and (B) C NMR spectra of 1.
1
1
Figure S8. H- H COSY NMR spectrum of natural 1.
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27) Nakahata, T.; Itagaki, N.; Arai, T.; Sugie, H.; Kuwahara, S. Synthesis of the sex
pheromone of the citrus mealybug, Pseudococcus cryptus. Biosci. Biotechnol.
Biochem. 2003, 67, 2627-2631.
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28) Millar, J. G.; Midland, S. L.; McElfresh, J. S.; Daane, K. M. (2, 3, 4, 4-
Tetramethylcyclopentyl) methyl acetate, a sex pheromone from the obscure
mealybug: first example of a new structural class of monoterpenes. J. Chem. Ecol.
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005, 31, 2999-3005.
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29) García-Morales, M.; Denno, B, D.; Miller, D.R.; Miller, G. L.; Ben-Dov, Y.;
Hardy, N. B. ScaleNet: A literature-based model of scale insect biology and
systematics. 2016. URL
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(http://scalenet.info/catalogue/Delottococcus%20elisabethae/) (2 May 2019)
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30) Walton, V. M.; Daane, K. M.; Bentley, W. J.; Millar, J. G.; Larsen, T. E.; Malakar-
Kuenen, R. Pheromone-based mating disruption of Planococcus ficus (Hemiptera:
Pseudococcidae) in California vineyards. J. Econ. Entomol. 2006, 99, 1280-1290.
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Marchesini, E.; Conte, G.; Benelli, G. Managing the vine mealybug, Planococcus
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32) Silva, E. B.; Mouco, J.; Antunes, R.; Mendel, Z.; Franco, J. C. Mate location and
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Page 28 of 37
Table 1. Total number of D. aberiae males captured during the field trial with each
enantiomer of (4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl acetate and the
racemate.
period* block (-)-enantiomer (+)-enantiomer
racemate
281
180
101
111
149
179
74
blank
1
2
3
4
1
2
3
4
285
302
161
175
296
192
86
193
153
68
0
1
0
0
0
0
0
0
1
2
83
130
163
54
147
87
89
mean ± SE** 205.5 ± 30.2 a 116.4 ± 19.0 b 145.5 ± 25.6 b 0.1 ± 0.1 c
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6
45
50
* Each period accumulates total captures of one complete trap rotation.
*
* Mean number of male catches ± standard error. Values with different letters are significantly
different according to Fisher’s least significant difference [LSD] test (F3,28 = 17.61; P <
.0001).
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Journal of Agricultural and Food Chemistry
FIGURE CAPTIONS
Figure 1. Synthesis of (4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl acetate
: a) Polyphosphoric acid, 95 ºC, 60 %; b) NBS, DCM, hv, 65 %; c) DMSO, KOAc, 95
; d) LDA, MeI, THF, 43 %; d) Petasis reagent, PhMe, 55 ºC, 55 %.
1
%
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Figure 2. GC-MS chromatogram of the volatile collections of lemons infested with
virgin (A) and mated (B) Delottococcus aberiae females, showing a virgin-specific peak
at 24.24 min, which matches the synthetic sample of (4,5,5-trimethyl-3-
methylenecyclopent-1-en-1-yl)methyl acetate (C).
Figure 3. Mass spectrum of (4,5,5-trimethyl-3-methylenecyclopent-1-en-1-yl)methyl
acetate.
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1
Figure 4. H-NMR (600 MHz) spectrum of the natural pheromone sample (ca. 30 µg)
isolated from the virgin females volatile sampling and of the synthetic sample.
Figure 5. Behavioral response of male Delottococcus aberiae in the two-choice Petri
dish laboratory bioassays: (A) pentane extract of the volatile sampling of lemons
infested with virgin females (VF) vs. those infested with immature instars (I) (n = 36);
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(B) (-)-enantiomer vs. pentane (n = 34); (C) (+)-enantiomer vs. pentane (n = 36); (D) (-)
vs. (+)-enantiomer (n = 42). Percentages were calculated according the total number of
males employed in the bioassays. ***For each pair, differences were significant by Chi-
square goodness of fit test (P < 0.001).
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Figure 6. Proposed biosynthetic pathways from the regular monoterpenoid linkage and
direct cyclization.
Figure 7. Alternative biosynthetic pathway from the irregular monoterpenoid linkage.
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