Crop Protection 132 (2020) 105110

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Crop Protection
journal homepage: www.elsevier.com/locate/cropro

Monitoring and discrimination of Pandemis moths in apple orchards using

semiochemicals, wing pattern morphology and DNA barcoding
Sebastian Larsson Herrera a, *, Cristina Tha a, Ramesh R. Vetukuri b, Alan Knight c, 1, Laura
J. Grenville-Briggs a, Marco Tasin a
a
Dep. of Plant Protection Biology, Swedish University of Agricultural Science, 23053, Alnarp, Sweden
b
Dep. of Plant Breeding, Swedish University of Agricultural Science, 23053, Alnarp, Sweden
c
USDA-ARS, Yakima Agricultural Research Laboratory, Wapato, WA, 98951, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Non-pheromonal insect attractants of plant and microbial origin are often classified as kairomones. They differ
Acetic acid from moth pheromones in that they can attract both sexes of several insect species. Kairomones are nowadays the
Integrated pest management object of several studies, due to their promising properties for monitoring and selective control of agricultural
Pandemis cerasana
pests. Here we report on field trapping experiments carried out in apple orchards to quantify the response of the
Pandemis heparana
two leafroller species Pandemis heparana (Denis & Schiffermuller) and Pandemis cerasana (Hübner) to potential
Pear ester
2-Phenylethanol kairomones (acetic acid (AA), 2-phenyl ethanol (2-PET), pear ester (PE) and benzyl cyanide (BC)) in apple or­
chards. Specimens of the two species were sexed and classified to the species level using a morphological key
based on wing traits. DNA barcoding was used to validate the discrimination between the two species through
the morphological key. A two-component blend of AA and 2-PET was effective in catching significant numbers of
females of P. heparana and P. cerasana. The addition of PE increased male but not female catches of only
P. heparana. For P. heparana blends containing AA and BC with or without PE caught significantly fewer males
and females than blends with PET and the AA/BC combination was as effective as PET releasing blends in
trapping P. cerasana females. Morphological identification of Pandemis by wing pattern was in agreement with
the DNA barcoding in the majority of cases. Additional studies are now required to establish an economic
threshold to correlate moth catches with fruit damage and to the possibility that attract and kill based on the
identified kairomones can be used to reduce damage.

1. Introduction
Multi-species non-pheromonal semiochemicals are volatile com­
pounds able to attract several pest insects (Suckling and El-Sayed, 2017).
In comparison with sex-pheromones, which exclusively affect males,
non-pheromonal plant and microbial volatiles can attract both sexes of
several reported species in considerable numbers. According to these
properties, such compounds are a promising tool to develop monitoring
systems in integrated pest management programs based either on
pheromone mating disruption or insecticides (Joshi et al., 2011; Knight
et al., 2019a).
In fruit orchards the exploitation of such odours has been advocated
for a long time, however practical application became relevant and
achievable rather recently. Major achievements are described for
lepidopteran species such as the codling moth (Cydia pomonella L.) and
the oriental fruit moth (Grapholita molesta [Busck]), where constitutive
and herbivory induced plant volatiles have been shown to be consis­
tently behaviourally active in the field (Knight et al., 2011, 2014,
2019b). A field-response to herbivory-induced plant or microbial vola­
tiles blended with acetic acid has recently been shown for additional
species, including the New Zealand and North American leafrollers
(Epiphyas postvittana [Walker], Spilonota ocellana [Denis & Schiffer­
müller], Choristoneura rosaceana [Harris]) (El-Sayed et al., 2016) and the
grapevine moth (Lobesia botrana [Denis & Schiffermüller]) (Tasin et al.,
2018). Within the genus Pandemis (Lepidoptera:Tortricidae), both Pan­
demis limitata (Robinson) and Pandemis pyrusana (Kearfott) have been
caught in traps loaded with blends of acetic acid and a
conspecific-induced foliage volatile, benzyl cyanide (Basoalto et al.,

* Corresponding author.
E-mail address: sebastian.larsson.herrera@slu.se (S. Larsson Herrera).
1
New adress: Instar Biologicals, 4602 Snowmountain Rd., Yakima, WA 98908.

https://doi.org/10.1016/j.cropro.2020.105110
Received 27 September 2019; Received in revised form 11 February 2020; Accepted 13 February 2020
Available online 14 February 2020
0261-2194/© 2020 Elsevier Ltd. All rights reserved.
S. Larsson Herrera et al.
2017). Although the same binary blend caught significant numbers of
P. heparana (Denis & Schiffermuller) and P. cerasana (Hübner) in Eu­
ropean orchards, a cumulated higher but not significant attraction of
these species was recorded when blending acetic acid with 2-phenyle­
thanol (Giacomuzzi et al., 2016).
In Northern European apple orchards, leafrollers such as P. heparana
can cause serious economic damage, while P. cerasana is of lower rele­
vance (Sjo €berg et al., 2015). While a new multipurpose mating disrup­
tion technique was recently developed to control a wide range of
tortricids including P. heparana (Porcel et al., 2017), reliable monitoring
tools to warn growers and advisors of possible pest attacks are still
lacking. Monitoring using sex-pheromones seems to give unreliable re­
sults, with a low catch rate even in high population plots (Porcel et al.,
2015). Although non-pheromonal volatiles provide a promising new
possibility to monitor P. heparana level in orchards, the identification of
the species itself is often undermined by side catches of sister species
with a similar phenotype. In Giacomuzzi et al. (2016) for example, the
authors reported difficulties in distinguishing the two very similar Eu­
ropean species (P. heparana and P. cerasana), due to a deterioration of
morphological traits when catching high number of moths on sticky
liners. A specific pattern on the rear wings allows discrimination be­
tween these two species (Northumberland moths, 2010). However, such
a trait is only visible in freshly caught moths and it deteriorates within a
few days, especially in congested trap sticky liners.
In the present study we carried out a field trapping experiment that
aimed to precisely quantify the response of P. heparana and P. cerasana
to non-pheromonal blends with a view to provide a reliable tool to
monitor these pests in apple orchards. The caught specimens were sexed
and classified at the species level using a morphological key based on
wing traits. DNA barcoding was used to validate the identification of the
two species through the morphological key.
2. Materials & methods
2.1. Field trapping
White delta traps with a replaceable sticky liner were purchased
from Silvandersson AB (Kn€ ared, Sweden) and deployed in commercial
apple orchards across the southern Scania region, Sweden. In fields with
mating disruption, Isomate CLS (Shin-Etsu, Tokyo, Japan) were used at
800 pieces per hectare to control the population of codling moth and
leafrollers including P. heparana (Porcel et al., 2015). No specific mating
disruption or pheromone monitoring was used for P. cerasana, although
one of the pheromone component of Isomate CLS, (Z)-11-tetradecen-l-yl
acetate, is in common with P. heparana. In experiment 1, kairomone
traps were hung in four orchards with mating disruption and checked for
7 consecutive weeks from late June 2016. Only 3 orchards with more
than 10 catches of P. cerasana and P. heparana were included in the
analysis for this year. The same experiment was repeated from middle
July 2017 for eight weeks in orchards with mating disruption with six
replicates in three orchards. All the orchards were commercial orchards
with diverse surroundings, including field crops or mixed forest.
In experiment 2 (2016), sex-pheromone traps (N ¼ 2) were placed in
three mating disruption and three control plots (Number of traps ¼ 6). In
experiment 3, sex-pheromone (N ¼ 4) and kairomone traps (N ¼ 5) were
placed in two orchards treated with mating disruption.
In each of the experiments, traps were spaced at 10 m from each
other. Three ml of neat synthetic glacial acetic acid (AA) and two ml of
2-phenylethanol (2-PET) was loaded into polyethylene vials with a 2
mm hole in the lid (Eppendorf; 5 ml). Membranes with benzyl cyanide
(3 ml, BC) and rubber septum dispensers with (E,Z)2,4-ethyl deca­
dienoate (pear ester, PE, 3.5 mg) were obtained from Tr�ec� e Inc. (Salinas,
CA, USA). Dispensers and membranes were hung into the trap through a
cardboard holder to facilitate their replacement. The different combi­
nation of lures in each treatment can be found below Fig. 2 and as ab­
breviations in Fig. 3, Tables 1 and 2. Acetic acid and 2-PET were
2
Crop Protection 132 (2020) 105110
replaced every 2 weeks, while BC and PE were not, due to slower release.
Pheromone traps were baited with a rubber septum loaded with (Z)-11-
tetradecen-l-yl acetate and (Z)-9-tetradecen-l-yl acetate (100:1, Csalo­
mon, Budapest, Hungary). Blank traps were used in all experiments.
Sticky trap bases with caught insects were collected and replaced
approximately every 7 days. Removed sticky bases were stored at 20 ̊ C
and all the caught specimens were sexed and identified through a ste­
reoscope following a morphological key (Fig. 1). A subsample of the
captured moths (46 from pheromone traps and 115 from kairomone
traps) was photographed singly and stored in a 1,5 ml Eppendorf tube at
80 ̊ C until DNA bar-coding analysis.
2.2. Statistical analysis
The R software (CoreTeam, 2019) was used to analyze the data. The
number of caught females and males was assessed using a generalized
linear mixed model with a Poisson distribution (R software package
lme4, Bates et al., 2015). The fit of the model and the chosen distribution
were assessed by checking residual dispersion. Model optimization was
achieved by comparing the Akaike’s information criterion (AIC). A
chi-square test followed by Tukey’s contrast was used to estimate the
significance of treatments and for pairwise comparison. In experiment 1,
year, orchard and replicate were set as random effects and treatment as
factor. In order to obtain a homogeneous scale in Fig. 2, catches of
experiment 1 are shown as cumulative relative catches for each repli­
cate. Relative catches are calculated as the total catches for each trap
summed up across dates divided by the total number of catches.
2.3. DNA extraction, barcoding and species identification
The whole body of each individual moth sample was used for DNA
extraction. Moth samples were harvested and snap frozen in liquid ni­
trogen, and stored at 70 � C until required. Total DNA was extracted
from all the frozen 192 specimens individually, using a DNA Tissue &
Blood Kit (Qiagen) following the manufacturer’s protocol (Qiagen,
2006). Yield and integrity of each individual DNA sample was assessed
using a NanoDrop Micro Photometer (NanoDrop Technologies), and
agarose gel electrophoresis, respectively. PCR conditions used were an
initial denaturation at 95 � C for 1 min, six cycles of 95 � C for 1 min,
annealing step at 42 � C for 30 s and extension at 72 � C for 1 min, fol­
lowed by 36 cycles of 95 � C for 1 min, 48 � C for 30 s and 72 � C for 1 min,
followed by a final extension step of 7 min at 72 � C. Each 50 μl PCR
reaction contained 25 μl premade mastermix (DreamTaq DNA poly­
merase [1.25 U], 2X DreamTaqGreen buffer, dATP, dCTP, dGTP and
dTTP, 0.4 mM each,and 4 mM MgCl2) (ThermoScientific DreamTaq
Green PCR MasterMix), 0.5 μm each of forward and reverse primers (1 μl
each), 21 μl of NucleaseFree H2O and 10 ng (2 μl) of individual moth
sample genomic DNA. LEP-F1 50 -ATTCAACCAATCATAAAGATAT-30 and
LEP-R1 50 -TAAACTTCTGGATGTCCAAAAA-30 were used as primers to
amplify the mitochondrial COI gene from each individual moth sample
(Hebert et al., 2004). When the amplification with these primers failed,
the reverse primer was changed to LEP extra-R1
50 -CTTATATTATTTATTCGTGGGAAAGC-30 as suggested in Hebert
et al. (2004). The size of the PCR products for LEP-F1/R1 and
LEP-F1/extra-R1 are around 648 bp and 350 bp, respectively. The PCR
products were electrophoresed on a 1% agarose gel and visualised under
UV light. PCR products were purified using the Qiagen QIAquick PCR
Purification Kit (Qiagen) and the yield of the individual DNA samples
were assessed using a NanoDrop Micro Photometer (NanoDrop Tech­
nologies). LEP-F1 primer was used for sequencing at the GATC Biotech
AG sequencing facility (Germany). Sequences were processed and
assembled with DNA star software (DNASTAR). The resulting sequences
with complete LEP-F1/R1 and LEP-F1/extra-R1 regions were submitted
to the National Center for Biotechnology Information (NCBI) GenBank
non-redundant nucleotide database for sequence similarity searches.
Sequences mapping to known species were determined with coverage
S. Larsson Herrera et al. Crop Protection 132 (2020) 105110

Table 1
Mean number of moths (� SE) caught per trap in experiment 1-3. While mean number of moths were calculated per trap, descriptors for generalized linear mixed model
(chi-square, p-value and residual degree of freedom) are based on catches for each observation date. *A mean of one with a SE of one was added to allow for variance
calculation, due to a total lack of catches in pheromone traps.
Experiment 1

Latin Sex AA/BC AA/BC/PE AA/2-PET AA/2-PET/PE Blank Chi-square p-value Res.DF

P. heparana Female 4.7 � 1.4 A 5.1 � 1.3 A 11.4 � 2.9 B 13.1 � 4.8 B 0� 0 93.744 <0.001 241
P. heparana Male 5.8 � 2.5 A 5.2 � 1.9 A 13.1 � 4.7 B 18.6 � 8.6 C 0� 0 55.323 <0.001 241
P. cerasana Female 2 � 0.7 B 0.4 � 0.3 A 1.5 � 0.5 AB 1.6 � 0.4 AB 0� 0 7.253 0.06 209
P. cerasana Male 1.6 � 0.9 AB 0.4 � 0.2 A 2.2 � 1 BC 3.9 � 1.1 C 0� 0 20.178 <0.001 209

Experiment 2 - Sex pheromones

Latin Sex Mating disruption (MD) Control Chi-square p-value Res.DF

P. heparana Male 0.2 � 0.2 A 4.7 � 1.8 B 8 <0.001 260

Experiment 3

Latin Sex Sex pheromone AA/2-PET AA/2-PET/PE Chi-square p-value Res.DF

P. heparana Female 1 � 1 A* 16.6 � 3.8 B 20 � 7.4 B 15.225 <0.001 93

P. heparana Male 1 � 1 a* 21.2 � 6.5 b 27.2 � 14.2 b 14.18 <0.001 93

higher than that scored in sex-pheromone traps (Fig. 3B).

Table 2
Comparison between the morphological and molecular identification of
Pandemis. 3.2. DNA barcoding

Molecular identification
The DNA barcoding of the 161 samples resulted in 108 samples being
Morphological Sex P. cerasana P. heparana A. rosana Percentage successfully PCR amplified and sequenced in the first attempt (Table 2).
identification correctly
Sequences for the rest of the samples were obtained by repeating the
identified
procedures from extraction onwards (PCRs, sequencing). The amplified
Caught in kairomone traps sequences of the samples provided nearly full barcode region coverage
P. cerasana (12) M 10 2 – 83% ranging from 97% to 100%. The majority of the species identified from
P .cerasana (19) F 13 2 4 68% the barcoding were either P. heparana or P. cerasana species. Additional
P. heparana (31) M 1 30 97%
species such as Archips rosana (L.) (N ¼ 7) were also identified via DNA
–
P. heparana (49) F – 49 – 100%
barcoding. Morphological identification of Pandemis by wing pattern is
Caught in sex pheromone traps
in agreement with the DNA barcoding in the majority of the cases
P. cerasana (31) M 30 1 – 97% (Table 2). A higher agreement for P. heparana than for P. cerasana was
P. heparana (15) M – 15 – 100% observed, especially for specimens caught in kairomone traps. The 30%
of specimens were mistakenly identified as P. cerasana belonged instead
and identity and the most similar significant NCBI accession was to P. heparana (15%, representing 5 individuals) and to Archips rosana
recorded for each sample. The resulting DNA barcoding identification (also 15% or 5 individuals).
was used to validate the morphological identification of individual moth
samples. 4. Discussion

3. Results
3.1. Field trapping
In experiment 1, a higher P. heparana female/male ratio in com­
parison to P. cerasana was observed, while no moths were trapped in
unbaited traps (Fig. 2). The blend of 2-PET and AA caught a similar
number of P. heparana females in comparison to the ternary blend with
PE. Blends including AA/BC � PE caught a lower number of females
than blends including 2-PET (Fig. 2 and Table 1). A higher number of
P. heparana males was measured in the blend with AA/2-PET/PE
compared to AA/2-PET. Similarly to females, blends including BC
caught a lower number of males than those with 2-PET. A different
situation emerged for P. cerasana, where AA/BC scored a higher number
of female than the AA/BC/PE combination. Blends including 2-PET did
not differ from AA/BC. Male P. cerasana showed the highest attraction to
the AA/2-PET/PE, which did not differ from AA/2-PET. The response to
AA/BC was comparable to that of AA/2-PET.
In experiment 2, traps baited with sex-pheromone caught a higher
number of males in plots without mating disruption, while catches were
totally inhibited in pheromone treated plots (Fig. 3A).
In the third experiment, the number of females and males captured in
traps baited with AA/2-PET or with AA/2-PET/PE was significantly
In this study we carried out field trapping experiments with the aim
to quantify the response of two apple pests, P. heparana and P. cerasana,
to blends of microbial and induced plant volatiles in apple orchards. Our
results support the potential of the tested volatiles as Pandemis kair­
omones and open the possibility to develop a new monitoring tool for
this pest. While a lower female/male ratio was observed in P. cerasana
than in P. heparana, further investigations are required to measure the
real damage of P. cerasana and its population density in apple.
P. heparana caterpillars were able to elicit a de-novo release of plant
volatiles when actively feeding on apples (Giacomuzzi et al., 2016).
Compounds emitted by the infested apple trees showed a wide range of
chemical groups, including AA, 2-PET and BC. A large majority of the
induced volatiles were reported as electrophysiologically active on the
antennae of P. heparana by Giacomuzzi et al. (2016), showing that the
adult moth could perceive what was earlier induced by the conspecific
caterpillars. In fields, Giacomuzzi et al. (2016) reported low catches of
P. heparana using AA, BC or 2-PET alone. Higher catches were measured
when AA was combined with BC or with 2-PET. In our study, besides
confirming the result of Giacomuzzi et al. (2016), we also measured a
significantly higher attraction of males to a three component blend of
AA, 2-PET and PE. The blend of AA and 2-PET was not only active in
attracting P. heparana and P. cerasana in the present study, but was also
reported to be attractive to other tortricid moths including the grapevine
moth Lobesia botrana (Tasin et al., 2018) and the obliquebanded

3
S. Larsson Herrera et al. Crop Protection 132 (2020) 105110

Fig. 1. Morphological key (circles; http://www.northumberlandmoths.org.uk) used to differentiate P. heparana from P. cerasana caught on sticky liners in delta
traps. Pictures of shown specimens were selected among those used to validate the key through DNA barcoding.

leafroller Choristoneura rosaceana (El-Sayed et al., 2016). P. pyrusana
and S. ocellana were instead caught in traps baited with AA and BC
(El-Sayed et al., 2016; Basoalto et al., 2017). In general, it appears that
AA and 2-PET are attractive to both European species, while BC plays a
role exclusively in P. cerasana females. Further trapping tests should
address the possible synergism between AA, 2-PET and BC. The addition
of PE had a significant effect exclusively on P. heparana males, although
a strong tendency for P. cerasana males was measured. Fine-tuning of the
load of each volatile as well as of the ratio between them could further
increase the attraction and thus the sensitivity and the selectivity of the
AA/2-PET lure.
Whereas P. heparana sex-pheromone traps baited with (Z)-11-tetra­
decen-l-yl acetate and (Z)-9-tetradecen-l-yl acetate were totally inhibi­
ted by mating disruption in both years (2016–2017), traps baited with
AA/2-PET attracted significant numbers of both sexes, either in
control and in disrupted orchards. Similarly to what reported in other
species (Porcel et al., 2015), the information collected through
sex-pheromone traps in mated disrupted plots does not reflect the real
population. In contrast, due to higher male attraction and to the fact that
the AA/2-PET lure attracted high numbers of P. heparana females as
well, we anticipate that this lure has the potential to be used as a
monitoring tool in both conventional and mating disrupted orchards.
The availability of a commercial lure capable of monitoring P. heparana
population under mating disruption would allow practitioners to
promptly intervene when disruption of P. heparana mating would not
perform properly. Additional studies are now required to establish an
economic threshold to correlate moth catches with fruit damage.
Whereas the addition of 2-PET increased catches of both sexes of
P. heparana, only male catches of P. cerasana were enhanced. Although
additional investigations seem to be necessary to understand such a

4
S. Larsson Herrera et al. Crop Protection 132 (2020) 105110

Fig. 2. Field catches of P. heparana and P. cerasana in delta traps (N ¼ 3 in 2016 and N ¼ 6 in 2017) baited with non-pheromonal attractants (Experiment 1). A total
of 321 P. heparana females and 65 males and 44 P. cerasana females and 384 males were caught. The significant differences between years and orchards were
accounted for in the generalized linear mixed model. Different letters are the result of pairwise comparisons. Dots in the graph represents outliers (outside a 95%
confidence interval of the median).

Fig. 3. Male catches of P. heparana in sex-pheromone traps (N ¼ 6) in mating disrupted and control plots in 2016 (A, experiment 2). Female and male catches of
P. heparana in sex-pheromone (N ¼ 4) and kairomone traps (N ¼ 5) in mating disrupted plots in 2017 (B, experiment 3).

different behaviour, we argue here that the difference may be link to the
different host range of the two species. While P. heparana develops
predominantly on Rosaceous hosts such as apple, pear, Sorbus and some
other trees, P. cerasana is reported from a wider range of plant families.
Because 2-PET was identified in apple as an induced volatile by
P. heparana larvae, it is reasonable to suppose that this compound would
be more specifically attractive for conspecific females. Perhaps more
specific compounds coming from P. cerasana larval feeding on another
host plant are required to increase female catches of this species. In
kairomone traps, a higher female/male ratio was observed for
P. heparana in comparison to P. cerasana. Because of the high host fi­
delity of P. heparana to apple and pear, this might be the result of a
higher female population resident in the orchard. The opposite can be
predicted for female P. cerasana, which may be resident in forests and
were occasionally attracted to the kairomone traps. It remains to be
understood why P. cerasana males scored a very high attraction to the
kairomone, although possibly not resident in the orchard. Perhaps they
are simply more responsive to the tested kairomone due to their higher
mobility in comparison to conspecific females. A competition between
calling P. heparana females and the kairomone traps may explain the
lower catch of P. heparana males.
A higher morphological misidentification of Pandemis moths was
observed in kairomone than in pheromone traps. This is due to both the
larger number of caught moths per week and the broader spectrum of
kairomone lures, which attracted additional species than the pheromone
one.
There are many molecular technologies that can be applied to
accurately identify insects from field samples. Such tools should be ac­
curate, practical and not too costly or time consuming. DNA barcoding,
or the use of a standardized segment of DNA for species identification
and discrimination (Hebert et al., 2003) has been used for the identifi­
cation of invasive or quarantine pests (Barcenas et al., 2005; Ball and
Armstrong, 2006; Nagoshi et al., 2011) and is highly useful when
identification to the species level is required. Although it may not be

5
S. Larsson Herrera et al.
cost-effective for large numbers of samples, the main advantage of this
method is that very similar species can be differentiated. Cheaper or
higher throughput technologies such as qPCR are not sufficient when
two closely related species need to be discriminated.
The identification of the caught species through the wing pattern was
successfully confirmed by DNA barcoding. Whereas a very high corre­
spondence between the two methods was found for P. heparana, a lower
precision was observed for P. cerasana, especially in the kairomone
traps. Due to its lighter colour in comparison to P. heparana, P. cerasana
has been confused with A. rosana in these traps, and thus the use of DNA
barcoding was highly useful as a tool to more accurately discriminate
the samples where wing pattern was not a clear indicator, due to colour
or breakdown of the insect wing samples.
4.1. Conclusions
Our result shows that a two-component blend of AA and 2-PET is a
suitable candidate tool to monitor both sexes of Pandemis in apple or­
chards. An effective discrimination between the two Pandemis species
was achieved through the selected morphological key and confirmed via
Bar-coding DNA. Further research is now needed to establish an
economical threshold to correlated trap captures with fruit injury level
and to explore the possibility to reduce fruit damage by annihilation
methods based on kairomones.
Declaration of competing interest
None.
CRediT authorship contribution statement
Sebastian Larsson Herrera: Writing - original draft, Writing - re­
view & editing, Visualization, Formal analysis, Data curation. Cristina
Tha: Methodology, Investigation. Ramesh R. Vetukuri: Writing -
original draft, Investigation, Resources, Formal analysis. Alan Knight:
Methodology, Conceptualization. Laura J. Grenville-Briggs: Investi­
gation, Funding acquisition, Project administration, Methodology,
Writing - original draft. Marco Tasin: Writing - review & editing,
Funding acquisition, Project administration, Methodology, Supervision,
Investigation, Resources, Writing - original draft.
Acknowledgements
Tr�ec�e Inc. (Salinas, CA, USA) is acknowledged for providing us with
field lures. This work was supported by the C-IPM network (API-tree
project) and by the (Swedish Farmer’s Foundation H2019). RRV and
LGB were further supported by the Swedish Foundation for Strategic
Research (FFL5) and the Swedish Research Council FORMAS (2015-
00430).
References
Ball, S.L., Armstrong, K.F., 2006. DNA barcodes for insect pest identification: a test case
with tussock moths (Lepidoptera: Lymantriidae). Can. J. For Res.-Revue Canadienne
De Recherche Forestiere 36, 337–350. https://doi.org/10.1139/x05-276.
Crop Protection 132 (2020) 105110
Barcenas, N.M., Unruh, T.R., Neven, L.G., 2005. DNA diagnostics to identify internal
feeders (Lepidoptera: Tortricidae) of pome fruits of quarantine importance. J. Econ.
Entomol. 98, 299–306. https://doi.org/10.1603/0022-0493-98.2.299.
Basoalto, E., Hilton, R., Judd, G., Knight, A., El-Sayed, A., Suckling, D., 2017. Evaluating
the use of phenylacetonitrile plus acetic acid to monitor Pandemis pyrusana and
Cydia pomonella (Lepidoptera: Tortricidae) in apple. Flor Entomol. http://www.
bioone.org/doi/full/10.1653/024.100.0424.
Bates, Douglas, M€ achler, Martin, Ben, Bolker, Walker, Steve, 2015. Fitting linear mixed-
effects models using Lme4. J. Stat. Software 67 (1). https://doi.org/10.18637/jss.
v067.i01.
CoreTeam, R., 2019. R: A Language and Environment for Statistical Computing. R
Foundation for Statistical Computing, Vienna, Austria.
El-Sayed, A.M., Knight, A.L., Byers, J.A., Judd, G.J.R., Suckling, D.M., 2016. Caterpillar-
induced plant volatiles attract conspecific adults in nature. Sci. Rep. 6 https://doi.
org/10.1038/srep37555.
Giacomuzzi, V., Cappellin, L., Khomenko, I., Biasioli, F., Schutz, S., Tasin, M., et al.,
2016. Emission of volatile compounds from apple plants infested with Pandemis
heparana larvae, antennal response of conspecific adults, and preliminary field Trial.
J. Chem. Ecol. 42 (12), 1265–1280. https://doi.org/10.1007/s10886-016-0794-8.
Hebert, P.D.N., Cywinska, A., Ball, S.L., deWaard, J.R., 2003. Biological identifications
through DNA barcodes. Proc. R. Soc. Lond. B Biol. Sci. 270, 313–322. https://doi.
org/10.1098/rspb.2002.2218.
Hebert, P.D.N., Penton, E.H., Burns, J.M., Janzen, D.H., Hallwachs, W., 2004. Ten species
in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly
astraptes fulgerator. Proc. Natl. Acad. Sci. Unit. States Am. 101 (41), 14812–14817.
https://doi.org/10.1073/pnas.0406166101.
Joshi, N.K., Hull, L.A., Rajotte, E.G., Krawczyk, G., Bohnenblust, E., 2011. Evaluating
sex-pheromone- and kairomone-based lures for attracting codling moth adults in
mating disruption versus conventionally managed apple orchards in Pennsylvania.
Pest Manag. Sci. 67 (10), 1332–1337. https://doi.org/10.1002/ps.2194.
Knight, A.L., Light, D.M., Trimble, R.M., 2011. Identifying (E)-4,8-Dimethyl-1,3,7-
Nonatriene plus acetic acid as a new lure for male and female codling moth
(Lepidoptera: Tortricidae). Environ. Entomol. 40 (2), 420–430. https://doi.org/
10.1603/en10283.
Knight, A.L., Hilton, R., Basoalto, E., Stelinski, L.L., 2014. Use of glacial acetic acid to
enhance bisexual monitoring of tortricid pests with kairomone lures in pome fruits.
Environ. Entomol. 43 (6), 1628–1640. https://doi.org/10.1603/en14153.
Knight, A.L., Mujica, V., Larsson Herrera, S., Tasin, M., 2019a. Addition of Terpenoids to
pear ester plus acetic acid increases catches of codling moth (Lepidoptera:
Tortricidae). J. Appl. Entomol. https://doi.org/10.1111/jen.12646.
Knight, A.L., Mujica, V., Larsson Herrera, S., Tasin, M., 2019b. Monitoring codling moth
(Lepidoptera: Tortricidae) with a four-component volatile blend compared to a sex
pheromone-based blend. J. Appl. Entomol. https://doi.org/10.1111/jen.12682, 0
(0).
Nagoshi, R.N., Brambila, J., Meagher, R.L., 2011. Use of DNA barcodes to identify
invasive armyworm Spodoptera species in Florida. J. Insect Sci. 11, 154. https://doi.
org/10.1673/031.011.15401.
Northumberland moths, 2010. “Four Confusing Species.” 0970. Corylana.
Porcel, M., Sjoberg, P., Swiergiel, W., Dinwiddie, R., Ramert, B., Tasin, M., 2015. Mating
disruption of Spilonota ocellana and other apple orchard tortricids using a
multispecies reservoir dispenser. Pest Manag. Sci. 71 (4), 562–570. https://doi.org/
10.1002/ps.3844.
Porcel, M., Attocchi, G., Johansson, M., Pålsson, J., Tasin, M., 2017. Refining the blend:
optimization of a multispecies pheromone formulation to control apple orchard
tortricids in Southern Sweden. IOBC-WPRS Bull. 126, 106–108.
Qiagen, A.G., 2006. DNeasy© Blood and Tissue Handbook. Qiagen AG, Hombrechtikon,
Switzerland.
Sj€
oberg, P., R€amert, B., Thierfelder, T., Hillbur, Y., 2015. Ban of a broad-spectrum
insecticide in apple orchards: effects on tortricid populations, management
strategies, and fruit damage. J. Pest. Sci. 88, 767–775. https://doi.org/10.1007/
s10340-015-0648-0.
Suckling, D.M., El-Sayed, A.M., 2017. Caterpillar-induced plant volatiles attract adult
Tortricidae. J. Chem. Ecol. 43, 487–492. https://doi.org/10.1007/s10886-017-
0847-7.
Tasin, M., Larsson Herrera, S., Knight, A.L., Barros-Parada, W., Fuentes-Contreras, E.,
Pertot, I., 2018. Volatiles of grape inoculated with microorganisms: modulation of
grapevine moth oviposition and field attraction. Microb. Ecol. https://doi.org/
10.1007/s00248-018-1164-6.