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Evaluation of Terpene-Volatile Compounds Repellent to the Coffee
Berry Borer, Hypothenemus hampei (Ferrari)
(Coleoptera: Curculionidae)
Carmenza E. Góngora
1
&Johanna Tapias
1
&Jorge Jaramillo
1
&Ruben Medina
2
&Sebastian Gonzalez
3
&
Herley Casanova
3
&Aristófeles Ortiz
4
&Pablo Benavides
1
Received: 15 January 2020 /Revised: 2 June 2020 / Accepted: 20 July 2020
#Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The coffee berry borer (CBB) is one of the main coffee pests in the world including Colombia. This pest is difficult to manage
because of its cryptic habits and the continuous availability of coffee fruits. Among the new management strategies being tested is
the use of volatile compounds as insect repellents. In this work, the behavioral response of female adult CBBs to terpenes
previously identified in the CBB-repellent plant species Lantana camara was evaluated. α-Terpinene, (R)-limonene, farnesene
and β-caryophyllene terpenes were tested via a Y-tube olfactometer in which ripe coffee fruits were accompanied by terpenes at
concentrations between 25 and 200 ppm. Only β-caryophyllene induced a significant and consistent CBB repellent effect at all
tested doses. The protective effect of microencapsulated β-caryophyllene was then determined under laboratory conditions by
incorporating the terpene in a colloidosome-gel system at 2.8 × 10
5
ng/h in the middle of coffee fruits with adult CBBs. The
coffee fruits in turn presented a decrease in fruit infestation. Furthermore, the protection of coffee fruits when β-caryophyllene
gels were hung in coffee trees was evaluated in the field; infestations were artificially induced by the use of raisins (CBB-infested
old coffee fruits) placed on the ground. Compared with unprotected trees, the trees treated with caryophyllene gels exhibited a 33
to 45% lower degree of infestation. Taken together, the results show that β-caryophyllene is a promising compound for an
integrated pest management (IPM) program in commercial coffee plantations.
Keywords β-caryophyllene .repellency .olfactometry .volatiles .coffee berry borer .agroecological control
Introduction
Coffee is the principal crop species in Colombia, with a plant-
ing area of nearly 900,000 ha. The coffee berry borer (CBB),
Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae,
Scolytinae), continues to be the main pest of coffee in the
country because it attacks coffee fruits, reducing their
commercial value (Federación Nacional de Cafeteros 2018).
The CBB is difficult to control because of the continuous
availability of coffee fruits that are suitable for attack and the
development of the insect throughout the year, as well as the
life cycle of the insect occurring entirely within the coffee seed
(Benavides et al. 2012).
Adult CBB females attack only members of the genus
Coffea. A fertile female or female founder enters coffee fruits
by boring through the pericarp tissue into the seed endosperm,
where it lays eggs. The hatched larvae subsequently eat the
endosperm, damaging the seeds; afterward, they become pu-
pae and then adults, the females mate with their male siblings,
and then they emerge from the fruits to search for new fruits.
Volatiles emitted by coffee fruits during their development
allow CBBs to locate host plants (Bustillo Pardey 2006).
Integrated CBB management is based on the timely harvest
of mature coffee fruits. Once the ripe coffee fruits of the main
crop have been fully harvested, the Colombian National
Coffee Research Center - National Federation of Coffee
Growers (Cenicafé) recommends removing all the coffee
*Carmenza E. Góngora
carmenza.gongora@cafedecolombia.com
1
Department of Entomology, National Coffee Research Center,
Cenicafé, Manizales, Colombia
2
Department of Biometrics, National Coffee Research Center,,
Cenicafé, Manizales, Colombia
3
Colloidosomes Group, Faculty of Chemistry, University of
Antioquia, Medellín, Colombia
4
Department of Plant Physiology, Coffee Research Center, Cenicafé,
Manizales, Colombia
Journal of Chemical Ecology
https://doi.org/10.1007/s10886-020-01202-5
fruits (green, overripe and shriveled fruits) that remain on the
trees and the ground to avoid creating reservoirs for CBB
insects, thus disrupting their life cycle in the crop. The control
of CBBs via chemical or biological insecticides has become
necessary during the critical period of the coffee fruits, which
occurs when they are 90 to 120 days old, are still green but
also attractive to CBBs and are suitable for insect develop-
ment. These control measures should be applied when the
percentage of insect infestation in the coffee fruits is equal to
or greater than 2% and when more than 50% of the CBB
adults are infesting the coffee fruits in the A/B position (bee-
tles are present in the fruit pericarp but have not yet bored into
the coffee seed) (Benavides et al. 2012).
Among the strategies that are beginning to be investigated
for the control of CBBs is the use of volatile compounds that
are emitted either by the coffee trees themselves (Mendesil
et al. 2009;Dufouretal.2013; Jaramillo et al. 2013)orby
companion plants (Castro et al. 2017). Some of these com-
pounds can attract or repel CBBs. In the Coffea genus, volatile
compounds that attract CBBs have been identified and include
mainly alcohols emitted during the ripening process
(Mendesil et al. 2009; Ortiz et al. 2004a,b). These compounds
have been used as lures in field traps to capture and monitor
CBB populations (Borbón Martínez et al. 2000; Cárdenas
et al. 2000; Mora 1991; Pereira et al. 2012). Additionally,
Jaramillo et al. (2013) identified the compounds conophtorin
and chalcogran, which are emitted by coffee fruits and some
coniferous plant species and seem to be key to CBB attraction
and colonization in coffee plantations. Some compounds,
such as 2-heptanone, 2-heptanol, 3-ethyl-4-methylpentanol,
2-phenylethanol, methyl salicylate and α-copaene, have been
shown to elicit an electrophysiological response in female
CBBs (Cruz Roblero and Malo 2013). In addition, Idárraga
et al. (2012) reported that an isoprene precursor and the en-
zyme isoprene synthase are highly expressed in coffee fruits
under attack by CBBs, possibly inducing the emission of vol-
atiles that exert allelopathy. Cruz-López et al. (2016)showed
that both CBB females and their parasitoids were attracted to
methyl salicylate.
However, few studies have identified secondary metabo-
lites that are repellent to members of the genus
Hypothenemus. Within various Coffea species, the following
secondary metabolites have been reported: cis-3-hexanol and
trans-2-hexenol (Borbón Martínez et al. 2000;Dufouretal.
2013), cis-3-hexenyl acetate (Borbón Martínez et al. 2000),
spiroacetal frontalin (1,5-dimethyl-6,8-dioxabicyclo [3.2.1]
octane) (Njihia et al. 2014), camphene (Dufour et al. 2013),
verbenone (Jaramillo et al. 2013;Mafra-Netoetal.2018)and
(E,E)-α-farnesene (Vega et al. 2017). In many cases, the in-
sect response depends on the concentration of the compound,
as in the case of 1,6-dioxaspiro, which induces attraction at
low doses but repellency at high doses (Njihia et al. 2014).
Among plant species other than coffee, extracts of Capsicum
spp., Allium sativum (Benavides and Góngora 2018), and
tropical plants such as Piper spp. (Henao 2008; Santos et al.
2010), Moringa oleífera (Santoro et al. 2011)andTilesia
baccata (Niño et al. 2007) have shown different degrees of
CBB repellency.
Using laboratory olfactometry bioassays, Castro et al.
(2017) recently reported the olfactory response of CBBs for
coffee fruits of Coffea arabica L. (Rubiaceae) compared with
coffee fruits accompanied by materials of the plant species
Crotalaria micans,Lantana camara,Artemisia vulgare,
Nicotiana tabacum,Calendula officinalis,Stevia rebaudiana
and Emilia sonchifolia. In these studies, N. tabacum L.
(Solanaceae), L. camara L. (Verbenaceae) and C. officinalis
were repellents; when these plant species were added, the
preference of the insects for coffee trees decreased when in-
sects were offered a choice between coffee with and without
these additional species. Controlled field experiments corrob-
orated the findings that N. tabacum and L. camara repelled
CBBs (Castro et al. 2017,2018). Furthermore, the composi-
tion of the volatile compounds emitted by these repellent
plants was determined, and several monoterpenes and sesqui-
terpenes were identified.
In insect-plant interaction studies, monoterpenes and ses-
quiterpenes have been identified as insect repellents or attrac-
tants. The chemical structure of these compounds consists of
chains of isoprene units (C
5
H
8
) that can be both cyclic and
acyclic; in addition, many are volatile oils that interact with
the olfactory receptors of phytophagous insects (Moore et al.
2006).
To identify new compounds that can diffuse into the air as
CBB repellents, the behavioral response of CBBs to the ter-
penes previously identified in the CBB-repellent plant spe-
cies L. camara was evaluated. For this, mature coffee fruits
accompanied by the terpenes α-terpinene, (R)-limonene,
farnesene and β-caryophyllene were tested via a Y-tube ol-
factometer. Furthermore, the behavior and infestation of
CBBs were evaluated in the presence of coffee fruits accom-
panied by the sesquiterpene β-caryophyllene in the labora-
tory, and the protective effect of microcapsulated β-
caryophyllene gels against the insects was assessed under
field conditions.
Methods and Materials
Olfactometer Bioassay Laboratory tests were conducted at
Cenicafé (Manizales, Caldas, Colombia) in the olfactometry
laboratory of the Department of Entomology in a room under
the following controlled conditions: a relative humidity (RH)
of 75 ± 5% and a temperature of 25 ± 2 °C, with diffuse uni-
form fluorescent light (58 W) and an extra 13-W lamp. The
evaluations were performed during the hours of greatest insect
activity—from 13:00 h to 16:00 h.
JChemEcol
The behavioral responses of female adult CBBs to different
volatiles were tested with the use of a Y-tube olfactometer as
described by Castro et al. (2017). Two Teflon® hoses 20 cm
in length were connected to the olfactometer, and the ends of
the hoses were attached to polypropylene bag compartments
(30 × 18 cm) that contained either untreated coffee fruits or
coffee fruits together with filter paper treated with volatiles. At
the opposite end, each compartment was then attached to a ¼-
inch Y-shaped glass tube 10 cm in length, and the entire sys-
tem was fitted with a vacuum pump and an air delivery system
with six carbon filters (ARS, Gainesville, FL, USA). The air
speed was controlled by pressure regulators such that the flow
was a constant 100 ml/s according to the recommendations of
Sengonca and Kranz (2001), and the air flowed from the pump
through the two compartments to the Y-tube. Each compart-
mentwaspreparedwith25fresh-pickedripecoffeefruitsthat
had been under development for 200 to 220 days. One of the
compartments contained only coffee fruits, and the other
contained a single 25-mm diameter Whatman No. 1 filter pa-
per (Whatman International, Maidstone, UK) treated with one
of each compound to be evaluated, in addition to coffee fruits.
The compounds to be evaluated were added to the filter papers
by a 50-µl Hamilton microsyringe (Hamilton Company,
Reno, Nevada, USA), and 10 µl of each compound was
applied.
The compound treatments included α-terpinene (85%;
223182 Sigma), (R)-(+)-limonene (97%, 98%; 183164
Sigma), a mixture of farnesene isomers (383902 Sigma), β-
caryophyllene (≥80%; 225205 Sigma) at concentrations be-
tween 25 and 200 ppm, and β-caryophyllene (≥98%; 22075
Sigma) at 50 ppm and 200 ppm. Because of the polarity of the
volatiles, 0.13% aqueous acetone was used to prepare or dilute
each concentration.
In addition to the volatiles, the following treatments were
evaluated: 1. coffee fruits in both compartments of the olfac-
tometer, which corresponded to the absolute control; 2. un-
treated coffee fruits vs. coffee fruits with 0.13% acetone as a
relative control; and 3. untreated coffee fruits vs. coffee fruits
with a mixture of methanol:ethanol at a 3:1 ratio (Barrera et al.
2006), which corresponded to the attractant control.
An independent bioassay was carried out with each volatile
compound concentration. Four groups of 50 female CBBs
were used each on different days, for a total sample of 200
insects. For all experiments, newly emerged female CBBs
were used. Each individual female CBB was initially intro-
duced at the end of the Y-tube and tested individually by being
placed at the entrance of the Y-tube by a brush, and each insect
was given five minutes to make a choice. The direction of the
Y-tube was changed after every 10 insects, and the Y-tube
was changed after every 25 insects. New filter papers contain-
ing fresh volatile sources were prepared and placed together
with the fruits as described above. CBBs that did not choose
within five minutes after release were considered
nonresponding individuals and were excluded from the statis-
tical analysis. To eliminate directional bias, volatile source
positions were alternated every five releases. After an inde-
pendent bioassay was performed, the Y-tube glassware was
cleaned by rinsing with warm water followed by ethanol
(Fisher Scientific, Leicestershire, UK) and then sterilized, af-
ter which it was heated in a glassware oven at 120 °C for
60 min.
The preference percentage for each treatment was estimat-
ed on the basis of the number of insect visits to one of the
arms.
Before evaluating the volatiles and identifying their ability
to attract or repel the insects at different concentrations, we
first determined that the percentage of insects reaching one
end of the Y-tube was 50% when coffee fruits were placed
at both ends. Similarly, to verify that the solvent used with the
volatiles did not affect the outcome (i.e., repel or attract), the
solvent was placed in one of the ends of the olfactometer, and
the mean percentage of CBBs at both ends was 50% according
to a z-test at the 5% level of significance.
The Y-tube olfactometer bioassay data were analyzed by
calculating the mean percentage of CBBs reaching the com-
partment containing the treatment for each volatile, and on the
basis of those results, it was determined whether the mean
percentage of CBBs selecting the treatment was greater than
50% for each concentration; if this was the case, attraction was
the conclusion. Otherwise, if the mean percentage was less
than 50%, it was concluded that the treatment repelled the
CBBs.
The statistical analyses were performed using SAS® soft-
ware. The means and standard errors (SEs) of the response
variables were obtained, and a z-test (95%, P < 0.05) was ap-
plied. The z-test was selected because, according to the central
limit theorem, the data were normally distributed, since the
distribution of the number of CBBs was binomial and since
the quantity of observations used in the aforementioned trials
is considerable.
Behavioral Bioassay To evaluate the repellent effect of β-
caryophyllene on CBBs under laboratory conditions, a
colloidosome system consisting of a gel with β-
caryophyllene microcapsules supplied by the company
Nexentia-Sumicol S.A. (Medellín, Colombia) was used. A
0.1-g piece of the colloidosome system (gel consisting of
6.5% microencapsulated β-caryophyllene) emitting β-
caryophyllene at 2.8 × 10
5
ng/h was placed among 30 coffee
fruits within an open methacrylate box (20 × 12 × 3 cm). The
coffee fruits were then infested with 60 adult female insects
(Fig. 1).
The experimental unit (EU) constituted the open box into
which the coffee fruits and CBBs were placed. In each exper-
iment, the treatments were compared with a control treatment
JChemEcol
that involved coffee fruits without volatiles, and 10 replica-
tions were included per treatment. The treatments were
assigned to the EUs in accordance with a completely random-
ized design. After the insects were added, the boxes were
observed for 1 h, and the insects that flew away from the boxes
into the air or otherwise left the boxes were counted. The
number of CBBs that did not fly away or remained in the
boxes were counted along with the number of perforations
on the coffee fruits 24 h after the insects were added. The
number of CBBs that flew away out of the total number of
insects was used to estimate the escape percentage, and the
percentage of infested coffee fruits was calculated. Means and
SEs of the response variables were obtained, and analysis of
variance (ANOVA) and Fisher's least significant difference
(LSD) tests were applied.
Caged Field Experiment Caged field experiment field trials
were carried out in a commercial plantation of three-year-old
Castillo® variety coffee trees in a 1 × 1.5-m spatial arrange-
ment at the Paraguaicito-Cenicafé experimental station in the
municipality of Buenavista, Quindío, Colombia, at a temper-
ature of 22.1 °C, a RH of 75.3% and an annual precipitation of
2,728 mm.
In the plots, two trees located one in front of the other in
adjacent furrows were selected as EUs. Prior to the establish-
ment of the treatments, previously infested coffee fruits were
removed from each tree, and the total number of coffee fruits
was determined. The two trees were enclosed in an entomo-
logical mesh cage constructed with polyvinyl chloride (PVC)
pipes (1.5 m wide, 3.8 m long and 1.9 m high), and two
treatments with 20 replications (pairs of trees) each were ran-
domly assigned. In the first treatment, three 45-g pieces of
6.5% β-caryophyllene (≥80%) microencapsulated gels were
wrapped in Vinipel® paper to prevent desiccation and were
distributed throughout the productive branches of one of the
two trees in the pair; gels were not added to the other tree
(control tree), as shown in Fig. 2. In the second treatment,
none of the trees that constituted the EU received β-
caryophyllene gels (control).
After the treatments were established, artificial infestation
of the trees in each EU was induced by the use of raisins
(CBB-infested old coffee fruits) previously infested at a 4:1
(CBBs:coffee fruits) ratio with adult insects; the raisins were
placed onto the soil directly between each pair of trees.
By dissecting a sample of coffee fruits, we determined that,
on average, 8.6 adult insects emerged from each raisin. The
amount of raisins deposited on the soil for each EU relative to
the total amount of fruit on the trees was estimated. It was
expected that the adult insects emerging from the raisins
would achieve an infestation rate of 12%.
For both treatments, the total number of newly infested
coffee fruits in each pair of trees from each EU was quantified
after seven, 14 and 21 days. The mean infestation percentage
(response variables) by treatment per tree was calculated along
with the respective confidence interval. The infestation levels
of the trees in each EU were compared between and among
treatments.
ANOVA was performed in accordance with a randomized
complete block design. The means and SEs of the response
variables were obtained, and Fisher's LSD tests were applied.
Statistical analyses were performed by SAS® software.
Results
Olfactometer Bioassay In the control with coffee fruits in both
olfactometer compartments, CBB selection was 50% for each
compartment (P > 0.05), indicating no preference for a partic-
ular compartment or directional bias (Fig. 3). In the evaluation
of coffee fruits vs. coffee fruits + 0.13% acetone, no prefer-
ence for either arm was detected (P > 0.05), so CBB selection
was 50:50 and comparable to that of the control (coffee fruits
vs. coffee fruits). With respect to the mixture of coffee fruits
vs. coffee fruits + alcohol (methanol:ethanol at a 3:1 ratio),
74% of the insects chose the arm containing the coffee fruit
with alcohol, which significantly differed from 50%
(P<0.001).
With respect to CBB preference for coffee fruits vs. coffee
fruits accompanied by limonene (Fig. 4), attraction was ob-
served only at 25 ppm (P < 0.001), and repellence was ob-
served at 100 and 200 ppm (P = 0.011 and P = 0.016, respec-
tively). With respect to α-terpinene (Fig. 5) and farnesene
(Fig. 6), there was no preference at any of the evaluated con-
centrations (P > 0.05).
At concentrations of 25 ppm to 200 ppm β-caryophyllene
(≥80%) in combination with coffee fruit exhibited signifi-
cant repellency (P < 0.001) (Fig. 7). Only between 16 and
Fig. 1 Colloidosome system with β-caryophyllene for 30 green coffee
fruits and CBBs
JChemEcol
34% of the insects selected coffee fruits accompanied by β-
caryophyllene (≥80%) at the four concentrations evaluated,
while between 66 and 84% chose coffee fruits alone. When
evaluated at 50 ppm and 200 ppm (Fig. 7), β-caryophyllene
(≥98%) showed the same repellency as β-caryophyllene (>
80%) showed at similar concentrations (P < 0.001).
Behavioral Bioassay In the experiment, a greater number of
CBB adults flew away, and a lower CBB infestation rate was
observed in the coffee fruits in the presence of β-
caryophyllene than under the control after 24 h, according to
the LSD test at 5% (Fig. 8). The number of CBB that flew
away from the coffee fruits in the control was 22%, while in
the presence of the volatile, this number was almost three
times greater than in the control (62%), showing a significant
difference (P < 0.001).
In addition, only 39% of the treated coffee fruits were
infested, whereas 73% of the coffee fruits in the control
showed infestation (Fig. 8), indicating the repellent effect of
β-caryophyllene.
Caged Field Experiment The infestation percentage increased
over time in both the control and treated trees. There were
significant differences in infestation between the trees with
the β-caryophyllene gels and the control trees at seven, 14
and 21 days after the dried coffee fruits were placed onto the
ground (P < 0.001), indicating a repellent effect on the CBBs.
In contrast, the percentages of infested coffee fruits in the
control treatment were equal among the pairs of trees that
constituted the EU at all evaluated dates, indicating that the
insects did not prefer either tree (Fig. 9).
The difference in the infestation percentage between the
trees that formed the treatment pairs corroborated the response
Fig. 2 Colloidosome system
(gels) experimental design. aThe
two trees were enclosed by an
entomological mesh cage. Pieces
of β-caryophyllene
colloidosomes were placed on the
first tree. The other tree did not
have gels (control tree). Artificial
infestation of the trees was
induced by the use of previously
infested raisins placed on the soil
in the middle of each pair of trees.
bTree showing the distribution of
the β-caryophyllene
colloidosome system
Fig. 3 Behavioral response of H. hampei females in a Y-tube
olfactometer (percentage ± SE; n = 200 per volatile). CBBs were given
the choice between two volatile sources in three different experiments
setups: coffee fruits vs. coffee fruits, coffee fruits vs. coffee fruits +
solvent control (0.13% acetone) and coffee fruits vs. coffee fruits + an
attractant control (3:1 methanol : ethanol mixture). The asterisks
indicate significant differences from a 50% response (95% z-test: *P <
0.05, *** = P < 0.001)
Fig. 4 Behavioral response of H. hampei females to two sources of
volatiles in a Y-tube olfactometer. Coffee fruits vs. coffee fruits +
limonene volatiles (percentage ± SE; n = 200). The asterisks indicate
significant differences from a 50% response (95% z-test: * P < 0.05,
*** = P < 0.001)
JChemEcol
of the CBBs to the β-caryophyllene volatile. Under field con-
ditions, caryophyllene reduced the infestation percentages in
the trees by 33 to 45% compared with that of the control trees.
With respect to the control trees without gels, the difference in
the infestation percentage did not exceed 10%. In addition, the
analysis revealed no differences among treatments with β-
caryophyllene over time (7, 14 and 21 days) (P > 0.05), which
means that the volatiles had repellent activity against the in-
sects at least until day 21.
Discussion
The results of this work showed the complex processes in-
volved in chemical communication and host plant recognition
between CBBs and coffee trees. Depending on their concen-
tration, the tested compounds showed attractant or repellent
effects, even in the presence of ripe coffee fruits. Some of the
evaluated terpenes are part of the allelochemicals emitted by
the coffee fruits, which would explain their effects on the
insects.
According to the initial olfactometry results, neither the
CBBs in the coffee fruit vs. coffee fruit control treatment nor
the CBBs in the coffee fruit vs. coffee fruit + 0.13% aqueous
acetone treatment preferred one compartment over another,
and acetone did not elicit a response, which allowed it to be
used for preparing the different concentrations of the treat-
ments. As previously reported, the 3:1 methanol:ethanol mix-
ture was confirmed as an attractant control ( Cárdenas et al.
2000; Green et al. 2015; Mathieu et al. 1998;Mora1991).
Some of the volatile terpenoids identified by (Castro et al.
2017) in CBB-repellent plant species were reported to be asso-
ciated with the chemical ecology of several different species of
bark beetles, where they have a critical role in host location
identification and have been used in management strategies
(Byers 1995; Byers et al. 2004; Kandasamy et al. 2016). CBB
adults can detect host and nonhost signals as do other
Scolytinae species that not only identify their aggregation pher-
omones and host volatiles but also are able to perceive and
avoid signals from nonhost plants (Jaramillo et al. 2013).
Therefore, the monoterpenes α-terpinene and limonene and
Fig. 7 Behavioral response of H. hampei females to two sources of
volatile in a Y-tube olfactometer. Coffee fruits vs. coffee fruits + β-
caryophyllene (≥80% and ≥98 purity) (percentage ± SE; n = 200). The
asterisks indicate significant differences from a 50% response (95% z-
test: * P < 0.05, *** = P < 0.001)
Fig. 8 Escape percentage of H. hampei (mean ± SE; n = 60 for each
replication) and percentage of infested coffee fruits (mean ± SE; n = 30
for each replication). The asterisks indicate significant differences
between the control and the treatment (95% LSD test: * P < 0.05, *** =
P < 0.001)
Fig. 5 Behavioral response of H. hampei females to two sources of
volatiles in a Y-tube olfactometer. Coffee fruits vs. coffee fruits + α-
terpinene volatiles (percentage ± SE; n = 200). The asterisks indicate
significant differences from a 50% response (95% z-test: * P < 0.05,
*** = P < 0.001)
Fig. 6 Behavioral response of H. hampei females to two sources of
volatiles in a Y-tube olfactometer. Coffee fruits vs. coffee fruits +
farnesene volatiles (percentage ± SE; n = 200). The asterisks indicate
significant differences from a 50% response (95% z-test: * P < 0.05,
*** = P < 0.001)
JChemEcol
the sesquiterpenes farnesene and β-caryophyllene were select-
ed for evaluation at concentrations between 25 and 200 ppm, as
these compounds were emitted mainly by repellent plants and
were commercially available for laboratory evaluation.
Limonene is a monoterpene that is emitted mainly by citrus
species, but it has also been detected in coffee fruits and is
associated with CBB interactions (Cruz-López et al. 2016;
Mathieu et al. 1996,1998; Ortiz et al. 2004b). Its effect on
CBBs had previously been evaluated via electroantennography,
albeit with inconclusive results (Mendesil et al. 2009).
Limonene showed mixed effects, inducing a concentration-
mediated attraction response that reflects the differences in the
detection thresholds for the individual components of the mix-
ture of volatiles emitted by a species and can determine whether
a host is attractive or not, depending on the proportions of the
components (Njihia et al. 2014). Additionally, as the com-
pounds evaluated here are not specific to a particular species,
the recognition and response of CBBs will not be due to a
particular compound but rather will be due to the mixture
(Castro et al. 2017;Mendesiletal.2009).
In the case of α-terpinene, the results showed that no re-
sponse was elicited in CBBs. This monoterpene had previous-
ly shown no repellency of CBB, although it is one of the major
components of the essential oils of Aeollanthus pubescens and
Chenopodium ambrosioides, which have an insecticidal activ-
ity against these insect (Mawussi et al. 2009;Mendesiletal.
2009).
With respect to (E,E)-α-farnesene, this sesquiterpene is
closely associated with the communication processes of dif-
ferent insect species. This compound has been reported to be a
repellent of Pityophthorus pubescens (Coleoptera:
Curculionidae: Scolytinae) (López et al. 2013), an attractant
and oviposition stimulant of Cydia pomonella (Lepidoptera:
Tortricidae) (Sutherland et al. 1977), and an alarm pheromone
of Prorhinotermes canalifrons (Isoptera: Rhinotermitidae)
(Šobotník et al. 2008); in addition, (E,E)-α-farnesene is one
of the components of an aggregation pheromone in
Anoplophora glabripennis (Coleoptera: Cerambycidae)
(Crook et al. 2014). In the current study, the use of farnesene
at concentrations between 50 and 200 ppm did not elicit a
response in the CBBs. Similarly (Blassioli-Moraes et al.
2019)reportedthat(E,E)-α-farnesene, either at a high purity
or as a component of a mixture of isomers, is not a repellent to
CBB females, but it can affect the foraging behavior of CBBs
by reducing the attractiveness of volatiles from noninfested
coffee fruits. However, (Vega et al. 2017) reported a high
repellence of farnesene, albeit only in combination with a
3:1 methanol:ethanol mixture, in attractant traps at a concen-
tration of 80 µg/µl, which is much greater than the concentra-
tions evaluated in this study. Additionally, the purity of the
compound evaluated by Vega et al. is unclear.
β-Caryophyllene is present in various plant species, such
as clove, pepper, wormwood and rosemary, and is associated
with the defense response against insect herbivory.
Additionally, β-caryophyllene is one of the most widespread
sesquiterpene floral volatiles; this compound occurs in floral
scents of more than 50% of angiosperm family members and
is one of the mostcommon volatile compounds in floral scents
(Knudsen et al. 2006). (E)-β‐Caryophyllene thus appears to
serve as a defensive agent against pathogens in floral tissues
(Huang et al. 2012) and may play multiple roles in plant de-
fense. This compound also serves as an antifeedant against
herbivorous insects when it occurs in nonfloral tissues
(Junker et al. 2010). For example, in Pityogenes bidentatus,
β-caryophyllene reduced the attraction to pheromones in the
field and elicited electroantennographic responses (Byers et al.
2004). In addition, this compound has been shown to act as a
powerful repellent against Tribolium castaneum (Coleoptera:
Tenebrionidae) (Chaubey 2012), causing the death of adults
and larvae at 1.438 µl/ml (1438 ppm) while inhibiting ovipo-
sition and development. It is believed that the lethal effect of
β-caryophyllene is due to inhibition of biosynthesis processes
(Don-Pedro 1989) and neurotoxic action (Kostyukovsky et al.
2002).
Among Coffea species, β-caryophyllene has been reported
to be a volatile emitted by healthy, ripe coffee fruits as well as
mechanically damaged coffee fruits and by CBB-infested
Coffea canephora (Cruz-López et al. 2016). β-
Caryophyllene was reported by (Mathieu et al. 1998)ina
study of volatiles of both C.arabica and C.canephora but
Fig. 9 Infestation percentage of
H. hampei in coffee trees (mean ±
SE; n = 20 for each replication).
The asterisk indicates a
significant difference within a
treatment (* P < 0.05)
JChemEcol
was detected only in C. canephora, and its presence has also
been evidenced in C. arabica var. Catimore at low concentra-
tions but not in other varieties of C. arabica (Mathieu et al.
1996). In C. canephora, the concentration of β-caryophyllene
changes depending on the ripeness of the fruit (Mathieu et al.
1998). It is known that mature coffee fruits attract colonizing
insect females, so the components of mixtures of specific vol-
atile compounds emitted by both C. canephora and C. arabica
must be involved in this response. However, there is no dif-
ference in CBB preference between these two varieties, both
of which are equally susceptible to insect attack, which im-
plies that β-caryophyllene is not involved in the recognition of
the host coffee tree by CBBs. On the other hand, β-
caryophyllene is not the main volatile emitted by
C. canephora; in fact, this compound corresponds only to
8% of these volatiles. Until now, the action of β-
caryophyllene as an allelochemical agent against CBBs had
not been established.
High repellency of CBB caused by β-caryophyllene was
observed in response to the evaluated concentrations between
25 and 200 ppm when the ≥80% and ≥98% purity β-
caryophyllene samples were used, indicating that the repellen-
cy was due to the β-caryophyllene present in the sample and
not to possible contaminants.
In the laboratory, the gel exhibited a repellent effect on the
CBBs when the fruit infestation was evaluated, and it was
demonstrated that volatile emissions from the gel continued
after 24 h. These experiments involving the slow-release–β-
caryophyllene gel system demonstrated protection of the cof-
fee fruits, since only half of the coffee fruits (39%) were
infested compared with those of the control (73% infestation)
because of the repellency and greater CBB escape rate.
In the case of the CBB controls under field conditions, we
previously established an experiment without the use of an
entomological mesh cage, and although we detected large
variability among the data, the coffee trees containing the
microencapsulated β-caryophyllene gel pieces displayed low-
er infestation than did the reference trees. Establishing the
experiment with cages allowed us to demonstrate the repellent
effect of the compound clearly.
Most of the relevant previous work has focused on the use
and improvement of methanol:ethanol attractant traps at ratios
of 1:1 (Dufour and Frérot 2008) or 3:1 (Mora 1991; Pereira
et al. 2012;Vegaetal.2015), but few field studies have
evaluated individual repellent compounds.
In general, the methods used for evaluating CBB behavior-
al responses in previous studies have involved the use of at-
traction traps involving a mixture of alcohols with the addition
of repellent compounds such that if the compound is effective,
the attraction trap would capture a lower number of CBBs. On
the basis of this premise, the attractant effect of brocain (1,6-
dioxaspiro [4,5] decane) and frontalin, two spiroacetals emit-
ted by coffee beans, was evaluated in the field alone and in
combination with the methanol:ethanol mixture. Compared
with the other treatments, frontalin alone or in combination
with methanol:ethanol resulted in the capture of the fewest
number of insects, thus confirming the repellent effect
(Njihia et al. 2014). Under field conditions, traps containing
(E,E)-α-farnesene plus 3:1 methanol:ethanol captured be-
tween 40 and 80% fewer CBBs than did traps containing only
the attractant (Vega et al. 2017).
This work is the first experimental study under field con-
ditions to reveal the repellent effects of compounds on CBBs
when trees with and without the repellent and without the
intervention of an attractant such as a methanol:ethanol mix-
ture were compared. The use of entomological cages allowed
the respective comparisons to be made without the interven-
tion of an attractant trap. However, the range of diffusion of
the compound is still unknown, and in the future, this should
be determined.
Onthebasisoftheresults,itisexpectedthatanemitter
device containing β-caryophyllene, when used in the field,
would provide stability under environmental conditions and
would slowlycontrol the emission of the compound; it was for
these reasons that this particular volatile was incorporated into
microcapsules that were then distributed in a gel system in this
study.
There is concern about the environmental impacts of the
use of synthetic insecticides. Together with the problem of
pest resistance to insecticides and the effects on human health
and the environment, there is interest in the use of other types
of compounds for insect control (Oliveira et al. 2014;LeGoff
and Giraudo 2019).Theidentifiedvolatile,βcaryophyllene,
showed repellency over a wide range of concentrations, im-
plying stability within the volatile system, which would facil-
itate its use in the field and ensure greater effectiveness over
time. This characteristic makes this terpene a good candidate
for use as a repellent against CBBs within an integrated pest
management (IPM) plan. This work shows for the first time
evidence of the repellent effect of β-caryophyllene on CBBs;
thus, the use of β-caryophyllene in the field as a practical
alternative to protect coffee trees is promising. Other emitter
devices should be evaluated, and the range of their action must
also be determined.
Acknowledgements The authors thank the insect breeding unit Biocafé
for providing CBBs and engineer Myriam Cañon of the Experimental
Station Paraguaicito for support during field evaluations.
Author Contribution Carmenza E. Góngora: Conceived and designed the
research and wrote the manuscript. Johanna Tapias: Conducted lab ex-
periments and helped write the manuscript. Jorge Jaramillo: Conducted
field experiments and helped write the manuscript. Ruben Medina:
Statistically analyzed all the data. Sebastian González and Herley
Casanova: Developed and provided the colloidosome-gels. Aristófeles
Ortiz: Provided GC/GC-MS support. Pablo Benavides-Machado:
Codesigned the research. All authors have read and approved the
manuscript.
JChemEcol
Funding Information This research was financed by the National
Federation of Coffee Growers of Colombia, the National Coffee
Research Center (Cenicafé), Colciencias and the company Nexentia-
Sumicol S.A.
Compliance with Ethical Standards
All the procedures performed in studies involving insects were in accor-
dance with the ethical standards of the institution or practice at which the
studies were conducted.
This article does not contain any studies with human participants or
vertebrate animals performed by any of the authors.
Conflict of Interest None.
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