Laboratory evaluation of natural and synthetic aromatic compounds as potential attractants for male mediterranean fruit fly, ceratitis capitata

Article abstract of DOI:10.3390/molecules24132409

Ceratitis capitata, the Mediterranean fruit fly, is one of the most serious agricultural pests worldwide responsible for significant reduction in fruit and vegetable yields. Eradication is expensive and often not feasible. Current control methods include

Full text of DOI:10.3390/molecules24132409

molecules
Article
Laboratory Evaluation of Natural and Synthetic
Aromatic Compounds as Potential Attractants for
†
Male Mediterranean fruit Fly, Ceratitis capitata
1
,
2
1
2
2
Nurhayat Tabanca * , Marco Masi
, Nancy D. Epsky , Paola Nocera , Alessio Cimmino
,
Paul E. Kendra 1 , Jerome Niogret 3 and Antonio Evidente *
2,
1
United States Department of Agriculture, Agricultural Research Service, Subtropical Horticulture Research
Station (SHRS), Miami, FL 33158, USA
Department of Chemical Sciences, University of Naples “Federico II”, Complesso Universitario Monte S.
Angelo, Via Cintia 4, 80126 Napoli, Italy
Niogret Ecology Consulting LLC, 13601 Old Cutler Road, Miami, FL 33158, USA
Correspondence: Nurhayat.Tabanca@ars.usda.gov (N.T.); evidente@unina.it (A.E.);
Tel.: +1-(786)-5737077 (N.T.); +39-(081)-2539178 (A.E.)
2
3
*
†
This work was produced by US government employees and is in the public domain in the US.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Jesus Simal-Gandara
Received: 30 May 2019; Accepted: 27 June 2019; Published: 29 June 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Ceratitis capitata, the Mediterranean fruit fly, is one of the most serious agricultural pests
worldwide responsible for significant reduction in fruit and vegetable yields. Eradication is expensive
and often not feasible. Current control methods include the application of conventional insecticides,
leading to pesticide resistance and unwanted environmental effects. The aim of this study was to
identify potential new attractants for incorporation into more environmentally sound management
programs for C. capitata. In initial binary choice bioassays against control, a series of naturally
occurring plant and fungal aromatic compounds and their related analogs were screened, identifying
phenyllactic acid (7), estragole (24), o-eugenol (21), and 2-allylphenol (23) as promising attractants for
male C. capitata. Subsequent binary choice tests evaluated five semisynthetic derivatives prepared
from 2-allylphenol, but none of these were as attractive as 2-allylphenol. In binary choice bioassays
with the four most attractive compounds, males were more attracted to o-eugenol (21) than to estragole
(
24), 2-allylphenol (23), or phenyllactic acid (
to quantify antennal olfactory responses to the individual compounds (
responses were elicited by 1-allyl-4-(trifluoromethyl)benzene (11), estragole (24), 4-allyltoluene (14),
trans-anethole ( ), o-eugenol (21), and 2-allylphenol (23). The compounds evaluated in the current
7
). In addition, electroantennography (EAG) was used
1
–29), and the strongest EAG
9
investigation provide insight into chemical structure–function relationships and help direct future
efforts in the development of improved attractants for the detection and control of invasive C. capitata.
Keywords: kairomone; o-eugenol; estragole; phenyllactic acid; attractants; invasive species;
electroantennography
1
. Introduction
Tropical fruit flies (Diptera: Tephritidae) are among the most economically important pests that
threaten global agriculture [
Ceratitis capitata (Wiedemann), is considered one of the most destructive agricultural pests because of
its direct damage to many varieties of fruits and vegetables [ ]. The species apparently originated in
sub-Saharan Africa; however, increased human mobility and trade in agricultural commodities have
increased the incidence of the introduction of exotic fruit flies to the United States [ ]. Medfly was first
1]. Among the exotic fruit flies, the Mediterranean fruit fly (medfly),
2–4
5
Molecules 2019, 24, 2409; doi:10.3390/molecules24132409
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2409
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detected in Hawaii in 1910 and in Florida in 1926 and later in 1956, 1962, 1963, 1967, 1981, 1990, 1997,
998, and 2010 [ ]. Eradication campaigns in response to detections are extremely laborious and
costly [ ]. For example, medfly eradication in the Tampa Bay area (Florida) in 1997 cost $US 25 million
and eradication efforts during the past 25 years in California cost nearly $US 500 million [ ]. The
embargo, loss of market, quarantine regulations, and subsequent job losses further contribute to the
overall economic impact of fruit fly incursions in many countries [ ]. In March 2015, an outbreak of
medfly was reported for the first time in the Dominican Republic and a subsequent ban imposed by
the United States resulted in losses of about $US 40 million to Dominican producers [ ]. In addition to
1
5,6
5
,
6
5
7
economic concerns, the high occurrence of pesticide residues in fruits and vegetables and the use of
postharvest fumigants like methyl bromide are not desirable by consumers [8,9].
Current management strategies for medfly include the use of trimedlure-baited traps for pest
detection and monitoring and the release of sterile male flies for population suppression [
2–4,10–12].
Trimedlure, tert-butyl 4(and 5)-chloro-2-methylcyclohexane- l-carboxylate (C H ClO ), is a synthetic
12
21
2
attractant with a mixture of different diastereomers. Of these isomers, (+)-trimedlure-C was found
to be the most attractive [12]. The development of male lures for C. capitata has a long history of
success. Angelica archangelica L. seed oil was used extensively for survey and detection during the
eradication program in Florida in 1956 [12
hydrocarbon, was identified as the main attractant in angelica seed oil and was reported to be 2–5 times
more attractive for C. capitata than trimedlure in field tests. While it is highly attractive, -copaene has
limited practical use as a field lure due to its structural complexity and difficulties with synthesis [12].
A study reported by our group also found that the concentration of -copaene in host plants was
,13]. Over a decade later, α-copaene, a natural sesquiterpene
α
α
correlated with the short-range attraction of male C. capitata but not with long-range attraction or
olfactory response, as measured by electroantennography (EAG) [14]. Previous research investigated
six essential oils—ginger root, orange, manuka, cubeb, angelica seed, and tea tree oil—for their
attractancy in laboratory and field tests and EAG response of sterile males and wild C. capitata. Results
showed that ginger root oil was the most attractive oil in field cage bioassays and elicited the greatest
EAG response [15]. These essential oils were further evaluated in short-range bioassays, and tea tree
oil, when diluted to 10 µg/µL, showed the highest attraction of sterile male medflies [16].
Although considerable progress has been made to identify food-based attractants and host-based
kairomones for exotic fruit flies, there is still a need for new and effective attractants to improve the
detection and control of invasive medflies. Here, we evaluate a series of aromatic compounds and
phenols, a group of chemicals well-known as plant and microbial bioactive metabolites [17,18].
Our ongoing studies focus on the identification of alkaloids from four native South African
Amaryllidaceae species—Crinum buphanoides (Welw. ex Baker), Crinum graminicola (I. Verd.), Cyrtanthus
mackenii (Hook. f.), and Brunsvigia grandiflora (Lindl.) [19]—and nine alkaloids belonging to
the lycorine subgroup along with the isocrabostyril, tazettine, and crinine subgroups have been
isolated [20]. Interestingly, two non-alkaloid compounds, piceol (4-hydroxyacetophenone,
and acetovanillone (apocynin, , Figure 1), were isolated from C. buphanoides, while only the former was
isolated from C. graminicola [21]. In addition, some phytotoxic phenols have been isolated recently from
phytopathogenic fungi, including tyrosol ( ) and resorcinol ( ) (Figure 1), purified from the culture
filtrates of Dothiorella vidmadera (DAR78993), a pathogen involved in the Botryosphaeria dieback of
grapevine [22] and 4-hydroxybenzaldehyde ( , Figure 1) purified from the solid cheatgrass (Bromus
1, Figure 1)
2
3
4
5
tectorum L.) culture of a Fusarium strain belonging to the F. tricinctum (Corda) species complex [23].
The findings above prompted the current collaboration to test these compounds for semiochemical
activity using a laboratory strain of C. capitata. Specifically, we (i) investigated short-range attraction of
male C. capitata to 29 structurally related natural and synthetic aromatic compounds (Figure 1) using
small cage laboratory bioassays, (ii) conducted EAG analyses to quantify olfactory chemoreception of
these compounds, and (iii) discuss the structure–activity relationships of these compounds to facilitate
identification of promising candidate attractants for future research. In addition, we describe the
synthesis of five derivatives of 2-allylphenol (23) used in this study.

Molecules 2019, 24, 2409
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O
O
O
OH
H
HO
OH
HO
HO
OH
HO
, 4-hydroxybenzaldehyde
HO
OMe
2, acetovanillone
4, resorcinol
3
, tyrosol
5
1
, piceol
O
F
HO
MeO
, trans-anethole
OH
8
, 1-allyl-4-fluorobenzene
OH
9
7
, phenyllactic acid
6
, 4-methylcatechol
1
0, allylbenzene
OH
F3C
MeO
OMe
HO
Me
1
1, 1-allyl-4-(trifluoromethyl)
12, 4-allylphenol
(chavicol)
O
1
4, 4-allyltoluene
benzene
1
3, 2-allyl-4,5-dimethoxyphenol
OH
MeO
HO
MeO
OH
O
MeO
OMe
HO
OH
OMe
7, 4-hydroxy-3-methoxymandelic acid
1
6, 3-hydroxy acetophenone
18, eugenol
4-allyl-2-methoxyphenol)
(
1
1
5, 4-allyl-2,6-
trimethoxybenzene
OH
MeO
MeO
MeO
MeO
HO
OMe
HO
2
0, 4-allyl-1,2-dimethoxybenzene
(methyl eugenol)
2
2, 4-allyl-2,6-dimethoxyphenol
OMe
1
9, 2-methoxy 4-propylphenol
21, 2-allyl-6-methoxyphenol
o-eugenol)
(
OMe
OAc
OH
MeO
4, estragole
(methyl chavicol,
2
2
3, 2-allylphenol
25, 2-allylphenyl acetate
26, 2-allylanisole
4
-allylanisole)
Br
O
S
O
3
''
4''
1''
5
''
O
O
4''
2''
5
''
3''
1''
O
2
''
6''
N3
3
'
1
1'
6
5
2
3
2'
O
O
4
3
'
1
4
1'
6
5
28, 2-allylphenyl
methanesulfonate
2
3
2'
2
5
9, 2-allylphenyl
-azidopentanoate
2
7, 2-allylphenyl
4
-bromobenzoate
Figure 1. The structures of compounds 1–29.
2
. Results and Discussion
Aromatic compounds with different functional groups isolated from plants (
and their commercially available analogs ( 22 24) and some semisynthetic derivatives (23
were investigated for potential attraction of male C. capitata. Five derivatives (25 29) (Figure 1) were
1
and
2), fungi (
3
–5),
6–
,
,
25
–
29
)
–

Molecules 2019, 24, 2409
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semisynthesized from the commercially available 2-allylphenol (23, one of the active attractants of
C. capitata in the initial bioassay; see the Materials and Methods section).
In particular, 2-allylphenol (23), by reaction with Ac O and pyridine, was converted into the
2
1
corresponding 1-O-acetylderivative (25). Its H-NMR spectrum differed from that of 23 for the presence
of the typical singlet of the acetyl group at
δ 2.32 and was very similar to that previously reported
Gresser et al. [24]. Further confirmation was given by an ESI-MS spectrum, which showed the
+
protonated form [M + H] at m/z 177.
By reaction with an ethereal solution of diazomethane, 23 was converted into the corresponding
1
methyl ether (allylanisole, 26). Its H NMR spectrum differed from that of 23 for the significant
presence of the singlet of methoxy group at
Furthermore, its ESI-MS showed the protonated form [M + H] at m/z 149.
δ
3.83 and was very similar to that previously reported [25].
+
2
-Allylphenol (23), by reaction with 4-bromobenzoic acid, yielded its corresponding
1
p-bromobenzoyl ester (27). Its H-NMR spectrum, compared with that of 23, showed the typical
signals pattern of the aromatic para-disubstituted protons, appearing as two doublets at
δ 8.08 and
+
7
.69 (J = 8.7 Hz). In addition, the ESI-MS spectrum showed the protonated form [M + H] of typical
79
81
+
+
signals due to the presence of Br and Br isotopes peaks at m/z 317 [M + H] and 319 [M + 2 + H] ,
respectively.
2
-Allylphenol (23), by reaction with mesyl chloride in pyridine, afforded the corresponding mesyl
1
ester (28). Its H-NMR spectrum was differed from 23 by the significant presence of the singlet of the
methyl group at
spectrum showed the protonated form [M + H] at m/z 213.
δ 3.20 and was very similar to that previously reported by Lei et al. [26]. Its ESI-MS
+
2
-Allylphenol (23), by reaction with 5-azidopentanoic acid, afforded the 5-azido pentanoyl
1
derivative (29). Its H-NMR spectrum differed from that of allylphenol for the presence of the signals
of 5-azidopentanoyl residue, appearing at 3.32 (t, J = 6.8 Hz, 2H, H-5”), 3.21 (d, J = 6.6 Hz, 2H, H-1 ),
0
2
.64 (t, J = 7.0 Hz, 2H, H-2”), 1.91–1.84 (m, 2H, H-3”), and 1.79
−1.74 (m, 2H, H-4”). Its ESI-MS spectrum
+
showed the protonated form [M + H] at m/z 260.
All the natural, commercially available and synthetic compounds were assayed against C. capitata
sterile males using short-range attraction bioassays and EAG analyses, as detailed in the Material and
Methods section.
Two short-range bioassays were carried out. In Experiment 1, paired t-tests found that male
response to compounds
solvent control (Table 1). Response of flies was converted to Attraction Index for comparisons among
these eight compounds There was an effect of type of chemical on Attraction Index. (F7,32 = 16.69,
p < 0.0001). The highest index was found with chemical (0.63 0.26), intermediate indexes with
chemicals 24 21, and 23 (0.41 0.19, 0.37 0.11, and 0.28 0.08, respectively), and the lowest indexes
7, 9, 11, 13, 21, 23, 24, and 27 was higher than the response to the associated
.
7
±
±
,
±
±
with chemicals 9, 11, 13, and 27 (ranging from 0.02 ± 0.01 to 0.08 ± 0.06).
These results demonstrated the importance of the functional groups and their position on the
benzene ring. In particular, the side chain of
of males in Experiment 1. The presence of the allyl group was also a critical factor, as evidenced by
little or no attraction observed with compounds lacking this moiety, as in 16, and 17. The same
result was obtained when the allylic chain was modified by isomerization of the double bond, as in
7 was the most important moiety to stimulate attraction
1–6,
9
and 18, or by the presence of a propyl residue, as in 19. However, other substitutions on the aromatic
ring of allylbenzene (10) were needed to confer activity, as 10 elicited no response. When the number
of substituents on the aromatic ring increased, those with three functional groups lost activity, as in
13, 15, and 22. Among compounds having two substituents, as in 20 and 21, only the latter, having a
substituent ortho-located with respect to the allyl group, was active. Although methyl eugenol (20) is a
male attractant for Bactrocera dorsalis (Diptera: Tephritidae) [27], male medflies showed poor response
to compound 20. Among the ortho- and para-monosubstituted allyl benzenes, only 23 and 24 displayed
activity while the others (
8, 11, 12, and 14) were inactive. This suggests that the efficacy was dependent
upon the substituents and their volatility and fragrance. Furthermore, comparing the activity of

Molecules 2019, 24, 2409
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the four esters (25
,
27–
29) and one ether of 23 with that of the parent compound, they were all less
attractive. Thus, the presence of a free hydroxyl and the ortho-allyl group was important for the activity.
It should also be noted that some of the compounds tested could be potential repellents; however, that
determination would require bioassays with a different experimental design (e.g., a known attractant
vs. a combination of attractant plus potential repellent). Further investigations are needed to assess
potential repellent properties of compounds deemed non-attractive in this study.
Table 1. Log P values and number (mean
±
std dev) of sterile male C. capitata attracted to compounds
1–29 presented in binary choice bioassays against control (Experiment 1).
Log P
Values
Number
Responding
Compound
£
phenyllactic acid (7)
estragole (methyl chavicol, 4-allylanisole) (24)
1.16
2.96
2.57
2.7
2.67
3.57
2.91
5.4
32.6 ± 12.4 *
21.0 ± 9.0 *
20.2 ± 4.3*
16.0 ± 3.8 *
10.0 ± 8.7
8.8 ± 8.3
6.0 ± 2.3 *
5.0 ± 2.3 *
3.2 ± 1.3 *
3.0 ± 3.3
3.0 ± 0.7
2.8 ± 2.6
2.6 ± 1.3
2.2 ± 1.1
2.2 ± 1.1
2.0 ± 1.6
2.0 ± 2.5
1.6 ± 2.1
1.6 ± 0.5 *
1.4 ± 0.9
1.4 ± 0.5
1.2 ± 1.3
1.2 ± 1.3
1.2 ± 0.8
1.0 ± 0.7
0.8 ± 0.8
0.6 ± 0.9
0.6 ± 0.9
0.4 ± 0.5
2
-allyl-6-methoxyphenol (o-eugenol) (21)
-allylphenol (23)
-allylphenyl acetate (25)
-allyltoluene (14)
trans-anethole (9)
-allylphenyl 4-bromobenzoate (27)
-allyl-4-(trifluoromethyl)benzene (11)
-methoxy 4-propylphenol (19)
-allylphenyl methanesulfonate (28)
-hydroxybenzaldehyde (5)
-allylphenyl 5-azidopentanoate (29)
-allylbenzene (8)
2
2
4
2
1
4.01
2.84
1.99
1.39
3.65
3.24
2.7
1.35
1.26
1.74
2.44
3.09
2.83
0.96
0.36
2.96
2.71
2.52
0.96
2.44
0.83
2
2
4
2
1
4
-allylphenol (chavicol) (12)
tyrosol (3)
resorcinol (4)
4
-methylcatechol (6)
2
-allyl-4,5-dimethoxyphenol (13)
allylbenzene (10)
4
-allyl-1,2-dimethoxybenzene (= methyl eugenol) (20)
-hydroxy acetophenone (16)
-hydroxy-3-methoxymandelic acid (17)
-allylanisole (26)
-allyl-2,6-trimethoxybenzene (15)
3
4
2
4
eugenol (4-allyl-2-methoxyphenol) (18)
piceol (1)
4
-allyl-2,6-dimethoxyphenol (22)
acetovanillone (2)
*
(
Number of flies (n = 5 replicates, 50 flies per replicate) on treated paper was greater than number on solvent control
£
paired t-test, p < 0.05). as detailed in the Material and Methods section.
Lipophilicity, expressed as the logarithm of the octanol–water partition coefficient (log P), is often
correlated with biological activity. Log P values of a series of test chemicals often follow predictable
trends in biological assays [28]. Compounds with lower log P values are classified as polar, while
those with higher log P values are considered more lipophilic with better membrane permeability [29].
However, we did not observe any correlation between Log P values for compounds
9 [31] and male response in Experiment 1 (r = 0.03037, n = 29, p = 0.8757) (Table 1).
When pairwise comparisons of the four most attractive compounds from Experiment 1 were
tested in Experiment 2, there were clear choices among most chemicals (Table 2). More males were
attracted to compound 21, and fewer males were attracted to compound in all bioassays. Attraction
to compounds 23 and 24 was intermediate, with no difference in attraction when 23 and 24 were tested
together. Phenyllactic acid ( ) has been isolated from cultures of Lactobacillus plantarum [32] and may
function as a food-based attractant. Estragole (24) is more likely a kairomone, since it occurs in a
1–28 [30] and
2
7
7

Molecules 2019, 24, 2409
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variety of essential oil-bearing plants such as basil, tarragon, chervil, fennel, clary sage, anise, and
rosemary [33], as well as in the leaves of various avocado cultivars [34], ripe apple [35] and citrus
fruits [36].
Table 2. Number (mean
of the top four compounds in Experiment 2 for 10 replicate tests with 50 flies per replicate.
±
std dev) of male C. capitata attracted to each choice in pairwise comparisons
Compounds
Tested
Number Responding to Each Compound in Bioassay
7
21
23
24
t
df
p
7
7
7
versus 21
versus 23
versus 24
2.1 ± 2.3
3.2 ± 5.7
2.4 ± 2.0
17.3 ± 4.4
10.75
5.3
7.97
4.54
4.14
0.05
16.6
22
13.7
15.6
22
<0.0001
<0.0001
< 0.0001
0.0001
0.0004
0.9809
14.2 ± 4.3
7.8 ± 2.8
10.3 ± 3.2
16.1 ± 5.6
21 versus 23
21 versus 24
23 versus 24
17.8 ± 6.0
17.1 ± 5.7
7.8 ± 5.4
10.4 ± 4.9
22
These results support that both the presence of the allyl residue, and substituents on the aromatic
ring are key structural features that confer attraction of male C. capitata.
In EAG analyses (Figure 2), there were significant differences in mean olfactory response to test
chemicals observed in all four groupings (Group 1: F6, 68 = 185.02, p < 0.001; Group 2: F6, 68 = 95.97,
p < 0.001; Group 3: F7, 76 = 375.321, p < 0.001; and Group 4: F6, 68 = 78.33, p < 0.001). In general,
strong EAG responses were elicited by compounds that were observed to be attractive in short range
bioassays (indicated by black bars in Figure 2). Of the nine highest-ranked chemicals in Experiment 1
(Table 1), only compound 7 had a low amplitude depolarization peak (Figure 2, Group 3). This may
have been due to differences in sample preparation between the bioassays and EAG analyses. The
former used 10% dilutions in acetone, whereas the latter used chemicals in their neat form. With
◦
compound
7
, the neat material at 24 C was in solid state, and the dry crystals may not have generated
significant volatiles in the headspace of the EAG sample bottle. It is also possible that compound
, when presented in bioassays, was detected by contact chemoreceptors on the tarsi rather than by
7
antennal olfactory receptors.
In addition, there were two chemicals that elicited higher than expected EAG responses. Of the
twenty low-ranked (i.e., non-attractive) chemicals from the behavioral assays (indicated by gray bars
in Figure 2), all displayed weak EAG responses except for compounds 26 and 28, which produced
relatively high amplitude EAG peaks (Figure 2, Group 3). Potential explanations for this observation are
that these two chemicals may be (i) true attractants, but the insects are not at the proper physiological
stage to respond appropriately (all test insects were sterile virgin males of the same age); (ii) synergistic
attractants, not behaviorally active alone but increasing response when combined with primary
attractants (e.g., putrescine, a synergist when combined with ammonia as a tephritid food-based
attractant [37]; (iii) repellents, which would also be detected by antennal olfactory receptors (e.g.,
ammonia, a protein feeding cue attractive at low doses but repellent at high dose) [38]; or (iv) other
biologically relevant compounds in the environment but unrelated to attraction behavior.

Molecules 2019, 24, 2409
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8
6
4
2
0
0
0
0
80
60
40
20
Group 1
Group 2
a
a
a
b
b
bc
c
b
c
c
bc
cd
d
d
0
0
0
#
1
#16
#2
#20
#18
#24
#14
#8
#9
#23
#10
#12
#25
#27
8
6
4
2
80
a
Group 3
Group 4
0
0
0
0
60
40
20
0
b
a
c
b
c
c
c
d
d
d
c
c
de
#4
e
#
28 #29 #26 #11
#6
#3
#7
#17
#5
#21
#19
#15
#13
#22
Compound
Compound
Figure 2. Mean (
saturated vapor from compounds
±
SE) electroantennogram (EAG) responses of male C. capitata to 1 mL doses of
29 (neat material). Analyses were conducted using four groupings
1–
of randomly chosen samples; responses were measured from 10 replicate males per group. All EAG
responses expressed as normalized percentages relative to the standard reference sample (tea tree
essential oil, 1 mL saturated vapor). Black bars indicate compounds found attractive in short-range
bioassays; gray bars indicate non-attractive compounds. Within each group comparison, bars topped
with the same letter are not significantly different (Tukey mean separation, p < 0.05).
3
. Materials and Methods
3
.1. General Experimental Procedures
1
H NMR spectra were recorded at 500 and 400 MHz in CDCl on Varian (Varian, Palo Alto,
3
CA, USA) and Bruker (Bruker, Karlsruhe, Germany) spectrometers. The same solvent was used as
internal standard. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on an
Agilent HPLC 1100 VL instrument (Agilent Technologies, Milan, Italy) equipped with an electrospray
ionization source (positive ion mode, ESI+). An Eclipse XBD-C18 column (150 mm
×
4.60 mm, 5 µm)
was used, adopting 0.1% formic acid: MeOH (35:65) as the eluant (flow rate: 0.4 mL/min). Analytical
and preparative TLC were performed on silica gel (Kieselgel 60, F254, 0.25 and 0.5 mm respectively)
plates. The spots were visualized by exposure to UV radiation (253 nm) or iodine vapour or by spraying
first with 10% H SO in MeOH and then with 5% phosphomolybdic acid in EtOH, followed by heating
2
4
◦
at 110 C for 10 min. Column chromatography was performed using silica gel (Merck, Kieselgel 60,
.06–0.200 mm, KgaA, Darmstadt, Germany). Log P values were computed by using ChemDraw 18.0
Ultra (1–28) [30] and ChemAxon 19.10 (29) [31].
0
3
.2. Natural and Synthetic Compounds
The compounds used for the study (Figure 1) are natural and synthetic aromatic derivatives
with different functional groups. In particular, piceol (1) and acetovanillone (2) were purified from

Molecules 2019, 24, 2409
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the organic extract of Crinum buphanoides bulbs, a native South African Amaryllidaceae plant [21].
Tyrosol ( ) and resorcinol ( ) were isolated from the culture filtrate of the fungus Dothiorella vidmadera,
a pathogen involved in the Botryosphaeria dieback of grapevine [22]. 4-Hydroxybenzaldehyde (
3
4
5
)
was isolated from the solid cheatgrass (Bromus tectorum) culture of a Fusarium strain belonging to
the F. tricinctum species complex [23]. Compound 12 (Cas# 501-92-8) was purchased from Parkway
Scientific (New York, NY, USA), 13 (Cas# 59893-87-7) was purchased from Enamine Ltd. (Monmouth
Junction, NJ, USA), and 15 (Cas# 487-11-6) was purchased from BOC Sciences (Shirley, NY, USA).
All other compounds (
(Cas# 300-57-2), 11 (Cas# 1813-97-4), 14 (Cas# 3333-13-9), 16 (Cas# 121-71-1), 17 (Cas# 55-10-7), 18
Cas# 97-53-0), 19 (Cas# 2785-87-7), 20 (Cas# 93-15-2), 21 (Cas# 579-60-2), 22 (Cas# 6627-88-9), 23 (Cas#
745-81-9), and 24 (Cas# 140-64-0) were supplied from Sigma-Aldrich Ltd. (St. Louis, MO, USA).
6 (Cas# 452-86-8), 7 (Cas# 20312-36-1), 8 (Cas# 1737-16-2), 9 (Cas# 4180-23-8),
1
0
(
1
Synthesis of 2-Allylphenol Derivatives 25–29
-allylphenil acetate (25). 2-allylphenol (23, 120
its corresponding acetyl ester (25) by acetylation with Ac O (50
2
µ
L), dissolved in pyridine (50
L). The reaction was carried out under
stirring for 12 hours at room temperature. It was stopped with MeOH, and the azeotrope formed by
addition of C H was evaporated under N stream. The residue (125.2 g) was then purified by column
µL), was converted in
µ
2
6
6
2
chromatography (CC) on silica gel eluted with n-hexane:EtOAc (9:1) yielding 25 (119.3 mg) as an
1
amorphous oil. Its H NMR data were very similar to those previously reported by Gresser et al. [24]
;
+
ESI+/MS: m/z 177 [M + H] .
2
-allylanisole (26). An ethereal solution of CH N was added to a solution of 2-allylphenol (23,
2 2
120 µL) in MeOH (120 µL) to obtain a persistent yellow color. The reaction was carried out at room
temperature under stirring and was stopped after 24 h by evaporation under an N stream. The
2
crude residue (130.1 mg) was purified by CC, using n-hexane:EtOAc (9:1) as eluent, to give 100 mg of
1
2
-allylanisole (23) as a homogeneous compound. Its H NMR data were very similar to those already
+
reported in literature [25]; ESI+/MS: m/z 149 [M + H] .
2-allylphenyl-4-bromobenzoate (27). To 2-allylphenol (23, 180
µL), dissolved in anhydrous MeCN
(
600
µL), DMAP (120.0 mg) and p-bromobenzoilchloride (120.0 mg) were added. The reaction mixture
was left under stirring for 24 h. It was then quenched with a 1 N NaHCO extracted with EtOAc
3,
and washed with H O. The residue obtained by evaporation (244.6 mg) was then purified by CC
2
eluted with n-hexane:acetone (9:1), affording the p-bromobenzoyl ester of 2-allylphenol (27, 102.2 mg).
1
Derivative 27 had H NMR (400 MHz, CDCl ,
δ, ppm);
δ
8.08 (d, J = 8.7 Hz, 2H, H-2” and H-6”), 7.69
3
(
(
1
d, J = 8.7 Hz, 2H, H-3” and H-5”), 7.32 (m, 2H, H-5 and H-3), 7.26 (td, J = 7.3 and 2.5 Hz, 1H, H-4), 7.18
0
br d, J = 7.3 Hz, 1H, H-6), 5.93 (ddt, J = 17.1, 10.6 and 6.6 Hz, 1H, H-2 ), 5.05 (dd, J = 10.6 and 2.9 Hz,
0 0 0
H, H-3 A), 5.02 (dd, J = 17.1 and 2.9 Hz, 1H, H-3 B), and 3.21 (d, J = 6.6 Hz, 2H, H -1 ); and ESI+/MS:
2
+
+
m/z 319 [M + 2 + H] , 317 [M + H] .
Mesyl ester of 2-allylphenol (28). Two hundred and fifty µL of mesyl chloride were added to a
solution of 2-allylphenol (23, 60
mixture was kept overnight and then quenched with a 1 N solution of HCO3 . The mixture was then
µ
L) in CH Cl (300
µ
L) together with 60
µ
L of pyridine. The reaction
2
2
−
extracted with EtOAc, and the resulting organic extract (63.1 mg) was purified by preparative TLC
1
eluted with CHCl , affording the methyl ester derivative of 2-allylphenol (28, 52.6 mg). H NMR data
3
+
of 28 were very similar to those previously reported by Lei et al. [26]; ESI+/MS: m/z 213 [M + H] .
Azidopentanoyl ester of 2-allylphenol (29). 2-allylphenol (23, 120.0
CH Cl (600 L) and pyridine (120 L), and DCC (N,N-dicyclohexylcarbodiimide) (40 mg) and
-azidopentanoic acid (120 L) were added. The reaction was left at room temperature for 4 days. The
µL) dissolved in anhydrous
µ
µ
2
2
5
µ
reaction was stopped by evaporation under N . The residual oil (178.2 mg) was then purified by CC
2
eluted with n-hexane:acetone (9:1), affording 5-azidopentanoyl ester of 2-allylphenol (29, 43.8 mg).
1
Derivative 29 had H NMR (400 MHz, CDCl ,
δ, ppm);
δ
7.32 (m, 2H, H-5 and H-3), 7.26 (td, J = 7.3
3
0
and 2.5 Hz, 1H, H-4), 7.18 (br d, J = 7.3 Hz, 1H, H-6), 5.92 (ddt, J = 17.1, 10.6 and 6.6 Hz, 1H, H-2 ),
.05 (dd, J = 10.6 and 2.9 Hz, 1H, H-3 A), 5.02 (dd, J = 17.1 and 2.9 Hz, 1H, H-3 B), 3.32 (t, J = 6.8 Hz,
0
0
5

Molecules 2019, 24, 2409
9 of 13
0
H, H-5”), 3.21 (d, J = 6.6 Hz, 2H, H-1 ), 2.64 (t, J = 7.0 Hz, 2H, H-2”), 1.91–1.84 (m, 2H, H-3”), and
.79–1.74 (m, 2H, H-4”).; ESI+/MS: m/z 260 [M + H] .
2
1
+
3
.3. Insects
Sterile male C. capitata were obtained from the Programa Moscamed mass rearing facility (El Pino,
Guatemala), where they were irradiated as pupae 2 d prior to emergence with 95 Gy of gamma
radiation from a Co60 source. These are the temperature-sensitive lethal strain flies [39] that are used
for the preventative release program [40] in Florida. Thus, only males were obtained, and only virgin
males were used for testing. Irradiated pupae were shipped initially to the USDA-APHIS Medfly
Project (Sarasota, FL, USA) and then to the USDA-ARS Subtropical Horticulture Research Station
◦
in Miami, FL. Holding conditions at Miami consisted of a 12/12 h L/D photoperiod, 25
30.5
±
2 C, and
7
5 ± 5% RH. Pupae were placed in collapsible cages (30.5
×
×
30.5 cm). After eclosion, adult flies
were provided with water (2% agar blocks) and food (3:1 mixture of cane sugar and yeast hydrolysate).
Flies used for all studies were 5 to 10 d-old, sexually mature sterile virgin males. Only sterile flies were
available for use in this research because there are no wild populations in Florida. Previous research,
however, has found that response of sterile males to semiochemicals is similar to response of wild
males (e.g., Reference [15]).
3
.4. Short-Range Bioassays
Small cage bioassays were used to quantify the short-range attraction of sterile male C. capitata
using a modified version of the binary choice tests [41]. All observations were carried out at room
temperature as described above in small collapsible cages (20.3 20.3 20.3 cm) into which 50 flies
×
×
were introduced 1 h prior to the start of each experiment. Tests were initiated by introducing two
Petri dishes (53 mm diameter and 12 mm height) with substrates positioned symmetrically (37 mm
apart). After 30 min, the number of flies at each dish was recorded. Experiment 1 compared the
response to each individual chemical (10
solvent control (10 L acetone). Test substrate or control was added to the center of a filter paper disk
Whatman #1, 3.5 cm diam). The filter paper disk was air-dried briefly to allow the solvent to evaporate
µL of a 10% dilution in acetone) with the response to a paired
µ
(
and was placed into the middle of a Petri dish. Bioassays were replicated five times, and the position
of substrates reversed between replicates. Flies and Petri dishes were used only once, and cages were
washed with acetone between experiments to eliminate potential residual chemicals.
Pairwise comparisons of the chemicals that elicited the highest response in the initial tests were
then conducted in Experiment 2 (compounds
chemical (10 L of a 10% dilution in acetone) was compared with the response to each other selected
chemicals in this two-choice test bioassay with all possible combinations tested ( vs. 21 vs. 23 vs.
21 vs. 23 21 vs. 24 23 vs. 24). There were ten replicates of the pairwise comparisons, with each
pair tested in separate cages at the same time.
7, 21, 23, and 24). The response to each individual
µ
7
,
7
, 7
2
4
,
,
,
3
.5. Electroantennography (EAG) Analysis
Peripheral olfactory responses were recorded from antennae of male C. capitata using a Syntech
EAG system (Syntech Original Research Instruments, Hilversum, Netherlands) and methods developed
by Kendra et al. [37 38 42]. Test substrates consisted of the 29 compounds, each 20 mg neat material.
,
,
The standard reference sample (positive control) was tea tree oil, 20 mg (Essential Oil India-SAT Group,
Kannauj, India), shown previously to elicit strong EAG responses in male medflies [15]. Each substrate
was placed into a separate 250 mL hermetic glass bottle equipped with a lid containing a short thru-hull
port (Swagelok, Solon, OH, USA) and silicone septum (Alltech, Deerfield, IL, USA). Sample bottles
◦
were sealed and equilibrated overnight at 24 C to allow for headspace saturation with volatiles.
Freshly dissected antennal preparations (whole head mounts) were secured between electrodes
using salt-free conductive gel (Spectra 360, Parker Laboratories, Fairfield, NJ, USA) and placed under
a stream of humidified air, purified with activated charcoal granules, at a flow rate of 400 mL/min.

Molecules 2019, 24, 2409
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Using gas-tight syringes (SGE Analytical Science, Victoria, Australia), samples of saturated vapor were
withdrawn from the test bottles, injected into the airstream, and presented to the antennae. In each
recording session, samples (fixed 1 mL doses) were delivered in the following order: the tea tree
standard, test chemicals in random order, a clean air injection (negative control), and a final standard
injection. There was a 2-min interval (clean air flush) between injections to prevent antennal adaptation
(diminished EAG response resulting from repeated exposure to chemical stimuli). Due to the large
number of test chemicals, EAG analyses were conducted using four groupings; each group compared
olfactory responses to seven or eight chemicals, randomly chosen, and responses were measured from
ten replicate females.
EAG responses to test substrates were measured initially in millivolts (peak height of
depolarization) and then normalized to percentages relative to the EAG response obtained with
the reference sample. Normalization corrects for time-dependent variability (gradual decline) in
antennal performance and allows for comparison of relative EAG responses obtained with different
substrates [37,43–45] and with different cohorts of insects [37,38]. Finally, any response recorded with
the negative control was subtracted from the normalized test responses to correct for “pressure shock”
caused by injection volume. All statistical analyses were performed using the corrected normalized
EAG values.
3
.6. Statistical Analysis
Pair t-tests were used to test for differences in number of males attracted to each choice in the
binary choice tests in Experiments 1 and 2 (Proc TTEST; SAS Institute, 2016) [46]. Male response was
converted to Attraction Index (number attracted to the compound minus number attracted to the
control divided by total number of males tested) [47] to compare the eight compounds that attracted
more males than the paired control in Experiment 1. One-way ANOVA and Tukey’s mean separation
tests were used to determine effect of chemical on Attraction Index in Experiment 1 and on olfactory
responses in EAG analyses. When necessary, data were transformed prior to ANOVA to satisfy
conditions of equal variance [48]; non-transformed means ± standard deviations are presented.
4
. Conclusions
In an effort to find effective new attractants for C. capitata, we investigated 29 structurally related
aromatic compounds in short range bioassays and EAG analyses. The combined results identified
phenyllactic acid ( ), estragole (24), o-eugenol (21), and 2-allylphenol (23) as promising candidates
7
for sexually mature males. Of these four compounds, o-eugenol (21) was observed to be the most
attractive in binary choice tests. The presence of the allyl residue and substituents on the aromatic ring
appear to be key structural features that confer attraction to these compounds. This study provides
insight into the attractiveness of structural variants of aromatic compounds with various substituent
groups to male C. capitata. Another promising approach could be the synthesis of estragole analogs
with allyl groups at different sites on the aromatic ring. In addition, further studies are needed to
evaluate these compounds, alone and in combination, to determine their efficacy in the field.
Author Contributions: All the authors contributed in the design of the study, the analysis of the data, and writing
of the manuscript. N.T. performed the conceptualization and wrote a draft of the manuscript; N.T., M.M., P.N.,
A.C., and A.N. conceived and designed the chemical experiments; N.T., N.D.E., and J.N. planned and performed
the bioassays; P.E.K. planned and performed the EAG analyses; N.D.E. and P.E.K. performed the statistical
analysis; N.T., P.E.K., and A.E. finalized the draft and revised the manuscript. All authors read and approved the
final manuscript.
Funding: This research was funded by Programme STAR 2017, financially supported by UniNA and Compagnia
di San Paolo grant number E62F16001250003.
Acknowledgments: The technical assistances of Micah Gill, Wayne Montgomery, Monica Blanco, Amanda
Perez-Castro, Sean Brown, and Teresa Narvaez of USDA-ARS-SHRS are greatly appreciated. The authors wish to
thank Elena Schnell of USDA-ARS-SHRS for the chemical orders and for preparing samples for the bioassays. The
authors thank Antonio Evidente, associated with “Istituto di Chimica Biomolecolare del CNR”, Pozzuoli, Italy.

Molecules 2019, 24, 2409
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We also thank the journal editors and referees for critical reviews of an earlier version of this manuscript. The
mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the
U.S. Department of Agriculture, Agricultural Research Service and does not imply its approval to the exclusion of
other products that may also be suitable.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–5 are available from the authors.
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