Evaluation of repellents for the redbay ambrosia beetle, Xyleborus glabratus , vector of the laurel wilt pathogen

Abstract

The redbay ambrosia beetle, Xyleborus glabratus, is the vector of the laurel wilt disease fungal pathogen, Raffaelea lauricola. Since the vector's initial detection in the USA in the early 2000s, laurel wilt has killed millions of redbay, Persea borbonia, trees and other members of the plant family Lauraceae. To protect host trees from beetle attack and laurel wilt infection, we tested the efficacy of host- and non-host-derived and commercial compounds as X. glabratus repellents in field experiments. In our first trial, the major constituents of the non-host tree, longleaf pine, Pinus palustris, and SPLAT Verb (verbenone 10%) were paired with manuka oil attractants and beetle captures were counted. Verbenone and a 1 : 1 blend of myrcene and camphene were intermediate to both the manuka positive and blank negative controls. Subsequently, we tested different blends of methyl salicylate (MeSA), a host defence and signalling compound, and verbenone in SPLAT dispensers using freshly cut redbay bolts as an attractant. All treatments reduced X. glabratus captures and boring holes as compared to the redbay (-) repellent positive control; however, SPLAT Verb and SPLAT MeSA-Verb (5% each) achieved the highest repellency, with results comparable to that of the non-host (laurel oak). These trials establish that host-derived and commercially available repellent compounds can reduce X. glabratus attacks and therefore have potential as part of an integrated management strategy against laurel wilt and its vector.

Full text

ORIGINAL CONTRIBUTION
Evaluation of repellents for the redbay ambrosia beetle,
Xyleborus glabratus, vector of the laurel wilt pathogen
M. A. Hughes
1,a
, X. Martini
2,3,a
, E. Kuhns
2,
*, J. Colee
4
, A. Mafra-Neto
5
, L. L. Stelinski
2
& J. A. Smith
1
1 School of Forest Resources and Conservation, University of Florida, Gainesville, FL, USA
2 Department of Entomology and Nematology, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, USA
3 Department of Entomology and Nematology, North Florida Research and Education Center, University of Florida, Quincy, FL, USA
4 IFAS Statistical Consulting Unit, University of Florida, Gainesville, FL, USA
5 ISCA Technologies, Inc., Riverside, CA, USA
Keywords
forest pests, laurel wilt, methyl salicylate,
redbay ambrosia beetle, repellents,
verbenone, Xyleborus glabratus
Correspondence
Marc A. Hughes (corresponding author),
School of Forest Resources and Conservation,
University of Florida, PO Box 110410,
Gainesville, FL 32611, USA.
E-mail: Mhughes741@ufl.edu
*Present Address: Scotts Miracle-Gro
Company, Marysville, OH, USA
Received: October 14, 2016; accepted:
December 5, 2016.
doi: 10.1111/jen.12387
a
These authors contributed equally.
Abstract
The redbay ambrosia beetle, Xyleborus glabratus, is the vector of the laurel
wilt disease fungal pathogen, Raffaelea lauricola. Since the vector’s initial
detection in the USA in the early 2000s, laurel wilt has killed millions of
redbay, Persea borbonia, trees and other members of the plant family Lau-
raceae. To protect host trees from beetle attack and laurel wilt infection,
we tested the efficacy of host- and non-host-derived and commercial com-
pounds as X. glabratus repellents in field experiments. In our first trial, the
major constituents of the non-host tree, longleaf pine, Pinus palustris, and
SPLAT Verb (verbenone 10%) were paired with manuka oil attractants
and beetle captures were counted. Verbenone and a 1 : 1 blend of myr-
cene and camphene were intermediate to both the manuka positive and
blank negative controls. Subsequently, we tested different blends of
methyl salicylate (MeSA), a host defence and signalling compound, and
verbenone in SPLAT dispensers using freshly cut redbay bolts as an attrac-
tant. All treatments reduced X. glabratus captures and boring holes as com-
pared to the redbay (-) repellent positive control; however, SPLAT Verb
and SPLAT MeSA-Verb (5% each) achieved the highest repellency, with
results comparable to that of the non-host (laurel oak). These trials estab-
lish that host-derived and commercially available repellent compounds
can reduce X. glabratus attacks and therefore have potential as part of an
integrated management strategy against laurel wilt and its vector.
Introduction
Since establishment by European settlers, over 450
invasive insects and several serious tree pathogens
have colonized the urban and rural forests of the Uni-
ted States (Aukema et al. 2010). Of these accidental
introductions, some species have become important
forest pests, causing widespread tree mortality and
severe ecological disturbances (Wingfield et al. 2016).
Wood borers, in particular, can be an especially dam-
aging group, with introduced insects such as the
emerald ash borer (Agrilus planipennis Fairmaire) and
the native mountain pine beetle (Dendroctonus pon-
derosae Hopkins) killing billions of susceptible host
trees (Romme et al. 1986; Herms and McCullough
2014). The term ‘ambrosia beetle’ is an ecological clas-
sification that combines different phylogenetic groups
of minute wood-boring beetles belonging to the Cur-
culionidae (subfamilies Scolytinae and Platypodinae).
Unlike bark beetles, which feed on host phloem,
ambrosia beetles are completely reliant on their sym-
biotic fungi for nutrition (Batra 1985). Ambrosia bee-
tles commonly attack and colonize stressed and
wounded trees and are, therefore, usually considered
benign or beneficial to ecosystem health as they accel-
erate forest nutrient recycling (Rudinsky 1962). How-
ever, there are exceptions; non-native ambrosia
beetle species may attack susceptible healthy trees
©2016 Blackwell Verlag GmbH 1
J. Appl. Entomol.

and become detrimental pests in forestry, agriculture
and the nursery trade (Hulcr and Dunn 2011; Ploetz
et al. 2013).
Among the ambrosia beetles that attack live and
apparently healthy trees, the redbay ambrosia beetle,
Xyleborus glabratus Eichhoff, was first detected near
Savannah, Georgia, in monitoring traps maintained
by the US Forest Service in 2002 (Haack 2006). Native
to Asia (Rabaglia et al. 2006), X. glabratus was likely
introduced to the USA through untreated wood pack-
ing material (e.g. crates and pallets). Soon after initial
detection, X. glabratus and its pathogenic fungal sym-
biont, Raffaelea lauricola T.C. Harr., Fraedrich &
Aghayeva, were associated with attacks of healthy
redbay (Persea borbonia [L.] Spreng.) trees and the
newly described, lethal vascular disease named laurel
wilt (Fraedrich et al. 2008; Harrington et al. 2008).
Laurel wilt disease affects most USA native and some
non-native members of the Lauraceae family (Kendra
et al. 2014; Hughes et al. 2015a). Laurel mortality has
been most severe in forests of the south-eastern
Atlantic and Gulf coastal plains where, in total, mil-
lions of mature redbay, swamp bay (Persea palustris
[Raf.] Sarg.) and silk bay (P. humilis Nash) trees have
been attacked by the beetle, infected and killed (Frae-
drich et al. 2008; Shields et al. 2011; Kendra et al.
2012a; Spiegel and Leege 2013; Cameron et al. 2015).
Xyleborus glabratus attacks apparently healthy Lau-
raceae, introducing the laurel wilt pathogen into the
xylem, disrupting vascular function and causing the
leaves to wilt in a matter of weeks to a few months
(Fraedrich et al. 2008; Hughes et al. 2015a,b).
Due to the wide distribution of Lauraceae in the
Americas, laurel wilt threatens a diversity of habitats,
including the central and western forests, where sas-
safras (Sassafras albidum [Nutt.] Nees) and California
laurel (Umbellularia californica [Hook. & Arn.] Nutt.)
are native, respectively (Fraedrich 2008; Fraedrich
et al. 2015). In 2012, laurel wilt was found in another
Lauraceae member, avocado (P. americana Mill.),
damaging commercial production (Mosquera et al.
2015). However, in south Florida, other ambrosia
beetle species may also be involved in R. lauricola
transmission in avocado (Carrillo et al. 2012, 2014),
requiring unique considerations for the management
of the disease in that system (Ploetz et al. 2011; Crane
et al. 2015).Given its rapid spread, laurel wilt repre-
sents a threat to the commercial avocado growers of
California and, to the world’s largest producer,
Mexico (Pisani et al. 2015).
Current laurel wilt management relies on rapid san-
itation and chemical treatments of susceptible host
trees (Mayfield et al. 2008; Spence et al. 2013;
Hughes et al. 2015a). As with the Dutch elm and oak
wilt diseases in America, proper sanitation consists of
tree felling and covering logs or chips in plastic sheets
to vector visitation, development and/or escape
(Bruhn and Heyd 1992; Haugen 2001; Spence et al.
2013). Although effective in reducing vector popula-
tions, this treatment is only appropriate for diseased
trees and may not be practical across a large area. A
prophylactic application of the fungicide propicona-
zole can offer protection (Mayfield et al. 2008); how-
ever, the longevity of the treatment is variable, likely
requiring frequent re-applications. Propiconazole
applications, although cost prohibitive in forest set-
tings, is an option currently used by avocado growers
to limit the spread of laurel wilt within a grove. Topi-
cal and systemic insecticides are relatively ineffective
in X. glabratus suppression due to their limited dura-
tion of efficacy in host trees (Pe~
na et al. 2011; Carrillo
et al. 2013). Entomopathogenic fungi have been
described that infect X. glabratus, but they kill the bee-
tles after they bore into the wood, not preventing the
inoculation with R. lauricola (Carrillo et al. 2015). For
avocado, pre-symptomatic aerial detection of laurel
wilt diseased plants using spectral imagery is in devel-
opment (Sankaran et al. 2012; de Castro et al. 2015),
but not yet operational. Overall, there is no reliable
tool available to prevent R. lauricola infection; conse-
quently, there is a crucial need for management tools
to prevent X. glabratus attack and to slow the spread
of laurel wilt disease.
The host selection process of X. glabratus is largely
driven by their chemical ecology, which is also impor-
tant to the epidemiology of laurel wilt. Xyleborus
glabratus does not respond to ethanol, a chemical indi-
cator of plant stress and a common lure component
for most ambrosia beetle species investigated to date
(Hanula and Sullivan 2008; Johnson et al. 2014), but
instead responds to other host volatiles (Niogret et al.
2011; Kendra et al. 2012a,b, 2014). Wood, leaf and
fungal odours, in addition to visual cues such as stem
silhouette, can direct X. glabratus attack, suggesting
that a complex combination of cues may be utilized
by the beetle for host selection (Hulcr et al. 2011;
Mayfield and Brownie 2013; Kuhns et al. 2014a,b;
Martini et al. 2015). Additionally, lure development
has focused on the sourcing and enrichment of the
attractant a-copaene (Kendra et al. 2016a,b), a plant-
derived sesquiterpene common in laurel wilt hosts
(Hanula and Sullivan 2008; Kendra et al. 2011;
Niogret et al. 2011).
The study of insect repellents for tree protection,
specifically against X. glabratus and laurel wilt, is just
starting; however, trials with other ambrosia beetles
©2016 Blackwell Verlag GmbH2
Evaluation of X. glabratus repellents M. A. Hughes et al.

show the potential of the use of repellents as part of
an integrated pest management programme (Borden
et al. 2001; Burbano et al. 2012; Ranger et al. 2013).
To expand laurel wilt management options, we inves-
tigated the efficacy of a commercial repellent formula-
tion and of additional host and non-host volatiles to
deter visitation and attack by X. glabratus and other
ambrosia beetle species on traps and host bolts in field
experiments.
Methods and Materials
Experimental locations
Field sites were selected based on the presence of lau-
rel wilt and a mixture of diseased and asymptomatic
redbays still onsite. Experiments were conducted at
Wekiwa Springs State Park (WSSP, Apopka, FL –
28°42040.93″N, 81°27045.85″W) in 2012 and in the
Historic Haile Homestead (HHH, Gainesville, FL –
29°35040″N82°2607″W) and Ichetucknee Springs
State Park (ISSP, Fort White, FL –29°5802.47″N
82°46033.82″W) in 2015. WSSP was a pine flatwoods
forest dominated by redbay, slash pine (Pinus elliottii
Engelm.) and red maple (Acer rubrum L.). HHH con-
sisted of a mixed pine–hardwood forest dominated by
live oak (Quercus virginiana Mill.), redbay, southern
magnolia (Magnolia grandiflora L.), cabbage palmetto
(Sabal palmetto [Walter] Lodd. ex Schult. & Schult. f.)
and slash pine. ISSP’s vegetation consisted of an oak–
hickory overstory forest dominated by laurel oak
(Quercus laurifolia Michx.), scrub hickory (Carya flori-
dana Sarg.), redbay, southern sugar maple (Acer flori-
danum [Chapm.] Pax) and cabbage palmetto.
Chemicals and formulations
Manuka oil lures were purchased from Synergy
Semiochemicals Corp. (Burnaby, BC, Canada), and
the chemicals eucalyptol (C80601), myrcene
(M100005), limonene (62122), a-pinene (147524),
b-pinene (W290300) and ocimene (W353901) were
purchased from Sigma-Aldrich (St. Louis, MO).
SPLAT
â
(ISCA Technologies, Riverside, CA) is an
inert, wax-based matrix that allows for the infusion
and subsequent slow release of volatile compounds.
ISCA Technologies supplied SPLAT Verb (active ingre-
dient verbenone at 10%) (Mafra-Neto et al. 2014a;
Fettig et al. 2015) and formulated SPLAT MeSA
(methyl salicylate at 10%) and a SPLAT MeSA/Verb
combination (5% each). Verbenone is a ketonic, anti-
aggregation semiochemical produced by the mycan-
gial fungi of certain bark beetles (Brand et al. 1976)
and is commonly deployed as spatial repellent
(Amman et al. 1989, 1991; Gibson et al. 1991; Miller
et al. 1995; Mafra-Neto et al. 2014b; Fettig et al.
2015; Perkins et al. 2015). Methyl salicylate is a
plant-derived volatile derivative of salicylic acid
(Loake and Grant 2007) and has been found to be
repellent against X. glabratus in laboratory experi-
ments (X. Martini and M. A. Hughes, unpublished).
Non-host plant volatile collection and analysis
The needles of the non-host tree, longleaf pine (Pinus
palustris Mill.), were found to be significantly repel-
lent to X. glabratus in laboratory olfactometer bioas-
says (data not shown). Therefore, the major chemical
constituents of longleaf pine were analysed by gas
chromatography–mass spectrometry (GC-MS) as
described below, and combinations of volatile compo-
nents were used for repellency in field trials. Longleaf
pine needle samples of approximately 1.0–1.5 g were
cut into small pieces and were placed in a clean 40-ml
glass vial and sealed with a lid and septa for volatile
equilibration. After equilibrating for at least 15 min at
21°C, a triphase 50/30 lm DVB/Carboxen/PDMS Sta-
bleFlex
TM
solid page microextraction (SPME) fibre
(Supelco, Bellefonte, PA) was inserted through the
septum and exposed to the leaf odours for 30 s. The
SPME was desorbed onto a GC-MS wax column, and
the odour constituents were separated over 40 min
on a Restek Stabilwax capillary column using a tem-
perature gradient from 40 to 240°at 7°/min. Identifi-
cation of the compounds was performed using a
PerkinElmer Clarus 500 quadrupole mass spectrome-
ter and Turbo Mass software (GC-MS). Linear reten-
tion times of authentic standards, when available, and
matching mass spectra to the NIST database were used
to identify components. The per cent abundance of
major constituents is provided in table 1.
Treatment repellents and experimental design
Experiment 1: Test of repellents with manuka oil as attractant
The goal of this field trial was to provide an initial
screening of non-host volatiles and verbenone for
potential repellency to X. glabratus. At WSSP, Elm
Bark Beetle Sticky Traps (Great Lakes IPM, Vestaburg,
MI) were prepared as two sticky cards (46 932 cm)
that were affixed to a wooden post at 1 m in height
(see Kuhns et al. 2014a). Treatments consisted of the
pairing of two separate components: a manuka oil
lure (attractant) (Hanula and Sullivan 2008) and
either a non-host synthetic volatile blend or SPLAT
Verb (potential repellents). Manuka lures were
©2016 Blackwell Verlag GmbH 3
M. A. Hughes et al. Evaluation of X. glabratus repellents

constructed from an internal matrix soaked in man-
uka oil, enclosed in a plastic pouch (Synergy Semio-
chemicals Corp.). The putative X. glabratus repellents
consisted of the following synthetic blends: ‘pine 10
was a 2 : 1 mixture of a-pinene and b-pinene by vol-
ume, ‘Pine 2’ was a 1 : 1 mixture of myrcene and
camphene by volume, ‘Pine 3’ was a 1 : 1 mixture of
limonene and ocimene by volume (table 1), and
SPLAT Verb contained 10% verbenone by volume.
Synthetic liquid blends (5 ml) of each artificial plant
odour were injected directly into the manuka lure
using a 21-gauge hypodermic needle and glass syr-
inge. The hole was covered with a small piece of cello-
phane tape. Control manuka lures were also
punctured and patched in a similar manner, but noth-
ing was injected into the lure. For the SPLAT Verb
treatment, one dollop (17.5 g deployed as 1 cm diam-
eter sphere) of SPLAT was wrapped in medical gauze
and attached with a paper clip to the sticky trap in
close proximity to the manuka lure. Traps baited with
manuka oil only served as positive controls, and non-
baited traps served as negative controls. Traps were
arranged in a randomized complete block design, con-
sisting of four blocks of six beetle traps (n =24).
Within-block traps were placed no less than 6 m
apart, with blocks no less than 60 m apart. Sticky
cards were collected weekly, and X. glabratus captured
were identified and counted. The mean temperature
was 25.5 0.2°C and relative humidity
78.0% 1.3%.
Experiment 2: Test of repellents with cut logs as attractant
The goal of this field trial was to test the repellency of
verbenone and MeSA, the only volatiles that showed
repellency against X. glabratus in the initial field
experiment (see Results section) and in laboratory
tests (X. Martini and M. A. Hughes, unpublished) as
compared with natural tree host tissue. Field trials
were conducted at HHH and ISSP in 2015. To increase
the number of X. glabratus captured on controls, we
utilized freshly cut redbay bolts as the attractant (posi-
tive control) instead of manuka lures, and non-host,
laurel oak bolts as a negative control. To obtain the
fresh wood tissue, a single uninfected redbay and lau-
rel oak were cut down and the main stem sectioned
into segments (30 cm length, 10 cm diam.). A large
construction nail was hammered into the bottom of a
cut end, and the bolt was then placed into the top of a
hollow metal pole (3 cm dia.) that was previously dri-
ven into the ground 1.5 m in height, an appropriate
location for capture of X. glabratus (Brar et al. 2012).
Two (28 923 cm) sticky cards (Wing trap sticky liner
–Scentry Biologicals Inc., Billings, MT) were stapled
to the lower portion of the treatment bolt on opposite
sides (Figure S1). Repellent treatments consisted of
the redbay bolts plus SPLAT MeSA, SPLAT Verb or a
MeSA/Verb mixture (1 : 1). Dollops (17.5 g) of SPLAT
compounds were applied directly to the bolts with a
pre-calibrated caulking gun and allowed to cure for
24 h before field deployment (Figure S1). Positive and
negative control bolts were left untreated.
Traps were arranged in a randomized complete
block design. A block consisted of a linear transect
with treatments spaced at 10 m and blocks 30 m
apart. Treatments were randomly positioned within
blocks and were rotated to the next position every
trapping period to minimize positional effects. To fur-
ther reduce the potential of a positional bias, no traps
were placed within 3 m of symptomatic redbays.
Traps were inspected and rotated every 14 days for
HHH and ISSP. At each trapping period, two circular
wounds (2 cm dia.) were made with a cork borer to
expose fresh redbay xylem and thus refresh the bolts’
attractiveness. At the end of every trapping period,
sticky cards were replaced and brought to the labora-
tory where beetles were identified and counted under
a dissection microscope. The trial was terminated once
each treatment completed a full positional rotation
cycle along the linear transect (five rotations/
10 weeks). At the end of the trial, trap bolts were
transported to the laboratory (University of Florida,
Gainesville), where they were debarked and
X. glabratus entrance holes were counted as described
in Hanula et al. (2008). A subsample of bolts with
beetle entrance holes was further dissected using a
band saw and examined for the presence of X. glabra-
tus and the extent of gallery formation. The mean
temperature was 22.9 0.3°C, and relative humidity,
87.0% 0.7%.
Table 1 GC-MS analysis of volatiles emitted from longleaf pine (Pinus
palustris) needles and the synthetic volatile blends used in field repel-
lent experiment 1. Mixtures were made by volume
Linear
retention
time Identification
Relative
abundance (%)
Synthetic blends for field
testing (% by volume)
Longleaf Pine Pine 1 Pine 2 Pine 3
1038 a-Pinene 23.1 67 ––
1097 Camphene 1.1 –50 –
1131 b-Pinene 51.8 33 ––
1135 Sabinene 2.2 –––
1189 Myrcene 1.8 –50 –
1220 Limonene 1.6 –– 50
1248 Ocimene 2.0 –– 50
Total 83.6 100 100 100
©2016 Blackwell Verlag GmbH4
Evaluation of X. glabratus repellents M. A. Hughes et al.

Statistical analysis
The reported means SE and sums were calculated
in Microsoft Excel (2010). Statistical analyses of beetle
captures and X. glabratus entrance holes were calcu-
lated using generalized linear mixed models with a
negative binomial distribution using the GLIMMIX
procedure on SAS 9.4 (SAS Institute, Cary, NC). For
boring activity analysis, laurel oak data were omitted
due to the lack of X. glabratus entrance holes on this
host. Weekly capture data were analysed using a lin-
ear mixed model where WSSP was square root-trans-
formed to meet the assumptions of the model, while
counts at HHH and ISSP were not transformed. At
WSSP, the violation of the model assumptions was
probably due to low beetle counts. Block and site
locations were treated as random effects in all models.
Type III tests were used to determine significance of
the fixed effects. Multiple comparisons of least squares
means were analysed and adjusted for using Tukey’s
multiple comparison procedure. Standard errors were
calculated as [std dev/√n], where n =number of repli-
cates. Figures were prepared using GraphPad Prism v.
7.0 (GraphPad Software, La Jolla, CA) and edited in
Adobe Illustrator CS6 (Adobe System Inc., San Jose,
CA). All mean temperature and relative humidity
data were collected and analysed from the Florida
Automated Weather Network (FAWN) provided by
the University of Florida.
Results
Non-host plant synthetic blends
Volatile analysis of longleaf pine needles revealed the
predominant constituents as aand bpinene; addition-
ally, myrcene, camphene, limonene and ocimene
comprised the less abundant components of the vola-
tile profile (table 1). All of these components except
ocimene are present in redbay wood odours (Kuhns
et al. 2014a; Martini et al. 2015); however, long leaf
pine does not contain eucalyptol, a known attractant
of redbay ambrosia beetle (Kuhns et al. 2014a). Com-
binations of components were selected for field testing
based on their relative abundance in longleaf pine
and are shown in table 1.
Experiment 1: Test of repellents with manuka oil as
attractant
Manuka oil attracted significantly more X. glabratus
beetles than the non-baited control, with most of the
treatment repellents showing intermediate trap
catches (fig. 1). Captures decreased for all treatments
after 4 weeks (fig. 1). Mean weekly X. glabratus cap-
tures were statistically higher (P =0.007) for
manuka +pine 1 (4.4 1.0), manuka +pine 3
(5.1 1.2) and manuka alone (5.6 1.0) than the
negative control treatment (0.3 0.1) (table 2).
Mean beetle captures with manuka +pine 2
(2.9 1.0), and manuka +SPLAT Verb (2.1 0.5)
did not differ significantly to all other treatments
(table 2). The manuka positive control captured the
most total X. glabratus throughout the duration of the
experiment (90), and SPLAT Verb was the repellent
with the fewest (34) total captures (table 2). Non-
X. glabratus ambrosia beetle captures were low and
statistically similar (P =0.42) among all treatments,
with means ranging from 0.9 to 1.9 weekly catches
and total sums 14–30 beetles (table 2). Non-target
ambrosia beetles comprised 17%–53% of all total cap-
tures per treatment (table 2) and included members
of the genera Xyleborus and Xylosandrus (data not
shown).
Experiment 2: Test of repellents with cut logs as
attractant
The effects of block and site location (ISSP and HHH)
were not statistically significant (at P >0.05, data not
shown); thus, data from both locations were com-
bined. For X. glabratus, all repellent treatments and
the laurel oak negative control captured significantly
fewer beetles than the redbay positive control in our
14-day trapping periods (P =0.0001) (fig. 2, table 3).
Both the SPLAT MeSA-Verb (3.5 0.6) and SPLAT
Verb (2.2 0.6) repelled the most beetles, and mean
trap captures were statistically similar to the non-host
laurel oak (5.8 0.8 beetles) (table 3). Initially,
SPLAT MeSA had a relatively high level of repellency,
similar to the other best performing potential repel-
lents; however, after four weeks, the effectiveness of
this repellent treatment decreased, resulting in more
mean captures in SPLAT MeSA (14.6 2.4 beetles/
biweekly) than the treatments containing verbenone
(fig. 2, table 3). The redbay bolts without SPLAT cap-
tured the most beetles per trapping period
(39.8 3.9) (table 3). A total of 1992 X. glabratus
beetles were captured with the redbay positive con-
trols during the experiment. SPLAT MeSA-Verb and
SPLAT Verb treatments captured fewer beetles than
the laurel oak control (290 total X. glabratus), with a
total of 177 and 108, respectively (table 3). Compared
to the positive control, SPLAT MeSA-Verb reduced
total captures by 91% and SPLAT Verb by 94.5%,
indicating strong levels of repellency. Captures of
©2016 Blackwell Verlag GmbH 5
M. A. Hughes et al. Evaluation of X. glabratus repellents

other bark and ambrosia beetles were low (0.4–1.6
beetles/biweekly, 22–79 total sum) and seemed not to
be affected by treatment (P =0.48) (table 3). Non-tar-
get ambrosia beetles comprised 3%–27% of all total
captures per treatment (table 3) and included mem-
bers of the genera Xyleborus,Xylosandrus,Hypothene-
mus and Ambrosiodmus (data not shown).
Xyleborus glabratus entrance-hole counts were
higher for the redbay positive control with a mean of
16.6 3.7 per bolt (table 3). Significantly fewer
mean boring holes occurred on redbay bolts treated
with SPLAT MeSA (4.3 1.0) as compared with the
positive control. SPLAT Verb was the most effective
repellent with 1.2 boring holes per bolt at experimen-
tal conclusion. SPLAT MeSA-Verb (3.3 1.1 boring
holes) was statistically similar to the treatments con-
taining methyl salicylate and verbenone alone. SPLAT
MeSA-Verb and SPLAT Verb suppressed X. glabratus
bolt boring by over 80% and 92%, respectively, com-
pared to the control with untreated redbay bolts
(table 3). No beetle boring was detected on the non-
host laurel oak. To explore the extent of X. glabratus
Fig. 1 Trapping experiment at Wekiwa
Springs State Park (WSSP) in Florida. Mean
Xyleborus glabratus captures SE over four
weeks. Traps consisted of sticky panels
(46 932 cm) affixed to wooden posts baited
with manuka oil. Putative repellent synthetic
blends were labelled as pine 1–3 (see table 1)
and SPLAT Verb. Traps baited with only man-
uka oil were positive controls, and negative
controls were unbaited traps. Separations of
means are listed at weekly intervals. Columns
denoted with different letters are statistically
different at a=0.05 according to Tukey’s mul-
tiple comparisons procedure of treatment
least squares means. n =24.
Treatment*
Xyleborus glabratus Non-target ambrosia beetle spp.
Mean weekly
trap captures
†
Sum of
trap
captures
Mean weekly
trap captures
†
Sum
of trap
captures
Per cent
of total
captures
Manuka (pos. control) 5.6 1.0 A 90 1.2 0.4 A 19 17.4
Manuka +pine 1 4.4 1.0 A 75 1.9 0.4 A 30 28.6
Manuka +pine 2 2.9 1.0 AB 46 0.9 0.3 A 14 23.3
Manuka +pine 3 5.1 1.2 A 81 0.9 0.2 A 14 14.7
Manuka +SPLAT Verb 2.1 0.5 AB 34 1.2 0.2 A 19 35.8
Blank (neg. control) 0.3 0.1 B 16 1.2 0.4 A 18 52.9
P value 0.007 0.42
Weekly means +SE and total sums of X. glabratus and non-target ambrosia beetles captured dur-
ing four week experiment (four replicates per treatment, n =24).
*Treatments consisted of manuka lures (attractant) paired with a test repellent. Positive and nega-
tive controls were manuka lure only and non-baited traps, respectively. See table 1 for con-
stituents of synthetic blends.
†
Columns denoted with different letters are statistically different at a=0.05 according to Tukey’s
multiple comparisons procedure of treatment least squares means.
Table 2 Summary results of X. glabratus repel-
lency experiment at Wekiwa Springs State Park
(WSSP)
©2016 Blackwell Verlag GmbH6
Evaluation of X. glabratus repellents M. A. Hughes et al.

colonization, a subset of bolts were further dissected
by sectioning them into thin slices with a band saw.
Most boring activity consisted of shallow tunnels
extending only 1 cm into the sapwood, lacking
signs of significant gallery formation or beetle
reproduction (e.g. eggs or callow beetles).
Discussion
Here, we evaluated volatile compounds for their
potential repellency against X. glabratus, the primary
vector of the laurel wilt pathogen (Raffaelea lauricola)
that has been causing high levels of mortality in
native Lauraceae in the eastern USA. The volatile
blends chosen were based on chemical analyses of
non-host volatiles, and damage-induced volatiles
known to repel this species, as well as known repel-
lents for other bark beetle species. In our first experi-
ment, we paired manuka lures with three synthetic
volatile blends and SPLAT Verb (verbenone 10%). A
50 : 50 blend of camphene and myrcene, as well as a
commercially available formulation of verbenone
(SPLAT Verb), was intermediate to all treatments.
Further testing with fresh host bolts or improved lures
would likely results in the higher capture rates needed
to truly discriminate between these treatments.
In our subsequent experiments, we abandoned the
use of manuka oil lures as an attractant to investigate
repellency in the field and instead used fresh redbay
wood due to its superiority as an attractant (Kendra
et al. 2011) and relevance to our overall goal of
protecting live host trees. We selected verbenone due
to the relatively low number of beetles captured when
it was included in the initial experiment and methyl
salicylate (MeSA) for its repellency to X. glabratus
under laboratory conditions (X. Martini and M. A.
Hughes, unpublished). Methyl salicylate is a plant-
derived volatile ester released following stress events,
such as herbivory, pathogen infection and associated
disease-resistance pathways (Shulaev et al. 1997).
The compound is normally absent in healthy plant tis-
sue; however, upon herbivore and pathogen attack,
MeSA acts as a volatile signalling molecule that
induces host systemic acquired resistance (SAR) and
salicylic acid defence response pathways to other
areas of the plant and neighbouring plants (Shulaev
et al. 1997; Loake and Grant 2007). Increased MeSA
levels induced by insect feeding have been linked to
repellency in certain herbivores (Hardie et al. 1994;
Losel et al. 1996) and the attraction of natural ene-
mies (Shimoda et al. 2002; James 2003; Mallinger
et al. 2011), suggesting that this volatile has a com-
plex and variable role in host signalling and defence.
Borden et al. (2001) found that MeSA reduced striped
ambrosia beetle (Trypodendron lineatum Oliver) densi-
ties in traps baited with an aggregation pheromone in
British Columbia. MeSA (10%) alone reduced cap-
tures and boring holes of X. glabratus for the duration
of the experiment; however, longevity of effectiveness
was lower as compared with verbenone from the
release devices tested here. It is possible that optimiz-
ing the release rate and duration of effective release of
Fig. 2 Trapping of combined experiments at
Historic Haile Homestead (HHH) and Ichetuck-
nee Springs State Park (ISSP) in Florida. Mean
Xyleborus glabratus captures SE over
10 weeks. Traps consisted of a redbay bolt
with two sticky cards attached. Repellents con-
sisted of SPLAT +methyl salicylate (MeSA),
SPLAT Verb and a blend of SPLAT
Verb +MeSA. Traps baited only with redbay
bolts were positive controls and with laurel
oak (non-host) bolts were negative controls.
Separations of means are listed at 14-day
intervals. Columns denoted with different let-
ters are statistically different at a=0.05
according to Tukey’s multiple comparisons
procedure of treatment least squares means.
n=50.
©2016 Blackwell Verlag GmbH 7
M. A. Hughes et al. Evaluation of X. glabratus repellents

MeSA from SPLAT or other semiochemical dispensers
may further improve the effectiveness of MeSA as a
repellent against X. glabratus.
In our experiments, X. glabratus was repelled most
effectively by the commercial SPLAT Verb formula-
tion of verbenone (10%) alone and SPLAT formulated
with a 1 : 1 mixture of verbenone and MeSA (5%
each). These two repellent treatments reduced cap-
ture of X. glabratus to levels observed with the laurel
oak negative control. Boring by X. glabratus was
reduced most effectively by the SPLAT MeSA-Verb
and SPLAT Verb treatments over 10 weeks, decreas-
ing entrance holes by fivefold and over 10-fold,
respectively, compared to the untreated redbay bolts.
Interestingly, beyond the initial entrance cavities
(Brar et al. 2013), no galleries, eggs or larva were
observed in any redbay transverse sections, regardless
of treatment. This observation is congruent with Frae-
drich et al. (2008) who suggest that redbay host trees
are not suitable for X. glabratus colonization and
reproduction until the advanced stages of laurel wilt
and mortality induced by R. lauricola.
Verbenone has been widely tested as an anti-aggre-
gant to protect pine trees in the USA against several
Dendroctonus bark beetle species, including the moun-
tain (D. ponderosae Hopkins) (Lindgren et al. 1989;
Shea et al. 1992; Fettig et al. 2015), southern
(D. frontalis Zimmerman) (Payne and Billings 1989)
and western (D. brevicomis) pine beetles (Fettig et al.
2008). When examined with ambrosia beetles, ver-
benone reduced captures of Xylosandrus compactus
Eichhoff, X. crassiusculus Motschulsky and Xyleborinus
saxesenii Ratzeburg in Hawaii (Burbano et al. 2012)
and Euwallacea validus Eichoff, Xylosandrus germanus
Blandford and two other taxa in Ohio in ethanol-bai-
ted traps (Ranger et al. 2013). Collectively, these data
suggest that verbenone-based compounds should
have potential as an effective X. glabratus repellent
and management tool, especially in conjunction with
additional components of integrated management
(i.e. sanitation and fungicides).
Although these results are promising, further test-
ing is required to assess some of the unique difficulties
of protecting host trees against X. glabratus and laurel
wilt. Most notable is the extreme virulence of the fun-
gal pathogen, R. lauricola, to host Lauraceae. Unlike
other pestiferous bark and ambrosia beetles that
require a high density of attacks in a very short time
to overwhelm and kill their hosts (Lee et al. 2011;
Tarno et al. 2011; Gitau et al. 2013), even very few
attacks of X. glabratus and a low load of fungal propag-
ules are sufficient to inoculate and kill healthy redbay
trees (Fraedrich et al. 2008; Hughes et al. 2013,
2015b). As our tests utilized cut bolts as attractants,
which likely differed in terms of volatile release pro-
file from non-wounded intact stems, further tests on
healthy, standing forest and landscape trees are
required to fully evaluate the practical utility of these
compounds. Bolts treated with verbenone or the
MeSA-verbenone mixture had very few successful
boring attacks, indicating that larger-scale testing is
warranted. The verbenone alone and 1 : 1 MeSA and
verbenone blend treatments were similar in effective-
ness. MeSA is less expensive than verbenone; the
Table 3 Summary results of X. glabratus repellency experiment at Historic Haile Homestead (HHH) and Ichetucknee Springs State Park (ISSP)
combined.
Treatment*
Xyleborus glabratus Non-target ambrosia beetle spp.
Mean biweekly
trap captures
†
Sum
of trap
captures
Mean boring
holes per bolt
†
Mean biweekly
trap captures
†
Sum
of trap
captures
Per cent
of total
captures
Redbay (pos. control) 39.8 3.9 A 1992 16.6 3.7 A 1.4 0.2 A 70 3.4
Redbay +SPLAT MeSA 14.6 2.4 B 724 4.3 1.0 B 0.9 0.2 A 44 5.7
Redbay +SPLAT MeSA-Verb 3.5 0.6 CD 177 3.3 1.1 BC 0.4 0.1 A 22 11.1
Redbay +SPLAT Verb 2.2 0.6 D 108 1.2 0.6 C 0.8 0.2 A 39 26.5
Laurel oak (neg. control)
‡
5.8 0.8 CD 290 0 0.0 1.6 0.3 A 79 21.4
P value <0.0001 <0.0001 0.48
Biweekly (14 days) means +SE and total sums of X. glabratus and non-target bark and ambrosia beetle species captured during 10-week experiment.
n=50.
*Treatments consisted of a cut redbay bolt attractant with a 17.5 g dollop of SPLAT test repellent. Positive and negative controls were untreated red-
bay and a laurel oak bolts, respectively.
†
Columns denoted with different letters are statistically different at a=0.05 according to Tukey’s multiple comparisons procedure of treatment least
squares means.
‡
Due to the lack of boring holes in the non-host laurel oak, these data were omitted from statistical analysis.
©2016 Blackwell Verlag GmbH8
Evaluation of X. glabratus repellents M. A. Hughes et al.

1 : 1 blend of MeSA and Verbenone is ca. 45% less
expensive than verbenone alone. Further research is
needed to determine whether this blend could allow
for a more cost-effective tool to manage X. glabratus in
residential landscapes, forests or avocado groves than
verbenone alone in SPLAT.
This initial investigation did not address dosage and
environmental factors affecting the longevity of dis-
penser effectiveness. It will also be important to deter-
mine whether these repellents affect other Xyleborus
species in Florida. Xyleborus volvulus Fabricius and Xyle-
borus ferrugineus Fabricius also transmit R. lauricola to
avocado in no-choice experimental tests (Carrillo et al.
2014). If verbenone and/or MeSA act as general repel-
lents against Xyleborus species, it is possible these repel-
lents could be used against multiple ambrosia species
currently affecting south Florida avocado groves.
Furthermore, a ‘push–pull’ system (Cook et al.
2007; Gillette et al. 2012) combining the best repel-
lents identified in this study to ‘push’ the vector away
from susceptible hosts, in combination with the best
available attractant (i.e. 50% a-copaene lure; Kendra
et al. 2016a,b) to ‘pull’ them to areas of non-hosts,
may increase the effectiveness of these semiochemi-
cals than if used alone. Also, combining repellents
with application of fungicides may improve manage-
ment. For example, well-timed application of repel-
lents in concert with application of propiconazole
could extend the duration of tree protection. Integra-
tion of several tactics will be necessary for manage-
ment of X. glabratus and laurel wilt in the
south-eastern USA, and the application of behaviour
modifying chemicals for the vector warrants further
investigation.
Acknowledgements
We thank Wekiwa and Ichetucknee Springs State
Parks (FL) and the Historic Haile Plantation (FL) for
permission to utilize their property as experimental
locations. The authors would also like to thank Patrick
James, Ode Akpoji and Adam Black (University of
Florida, Gainesville) for their assistance in the field
collections. Funding for this work included a coopera-
tive agreement with the USDA-Forest Service, Forest
Health Protection, Region 8.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Trap design utilized in X. glabratus
repellency experiments at Historic Haile Homestead
(HHH) and Ichetucknee Springs State Park (ISSP).
©2016 Blackwell Verlag GmbH12
Evaluation of X. glabratus repellents M. A. Hughes et al.

Supporting Information
Figure S1. Trap design utilized in X. glabratus repellency experiments at Historic Haile Homestead (HHH)
and Ichetucknee Springs State Park (ISSP). A) healthy redbay bolt (attractant), B) SPLAT repellent, C)
sticky cards (on bolt front and back) and D) metal support pole.