Elemol and amyris oil repel the ticks Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in laboratory bioassays

Abstract

The essential oil from Amyris balsamifera (Rutaceae) and elemol, a principal constituent of the essential oil of Osage orange, Maclura pomifera (Moraceae) were evaluated in in vitro and in vivo laboratory bioassays for repellent activity against host-seeking nymphs of the blacklegged tick, Ixodes scapularis, and the lone star tick, Amblyomma americanum. Both bioassays took advantage of the tendency of these host-seeking ticks to climb slender vertical surfaces. In one bioassay, the central portion of a vertical strip of filter paper was treated with test solution and ticks placed or allowed to crawl onto the untreated lower portion. In the other bioassay, a strip of organdy cloth treated with test solution was doubly wrapped (treatment on outer layer) around the middle phalanx of a forefinger and ticks released on the fingertip. Both amyris oil and elemol were repellent to both species of ticks. Elemol did not differ significantly in effectiveness against A. americanum from the widely used repellent deet. At 2 and 4 h after application to filter paper, 827 microg amyris oil/cm(2) paper repelled 80 and 55%, respectively, of A. americanum nymphs. Ixodes scapularis was repelled by lower concentrations of amyris oil and elemol than A. americanum.

Full text

Elemol and amyris oil repel the ticks Ixodes scapularis
and Amblyomma americanum (Acari: Ixodidae)
in laboratory bioassays
J. F. Carroll •G. Paluch •J. Coats •M. Kramer
Received: 30 September 2009 / Accepted: 24 November 2009 / Published online: 18 December 2009
ÓU.S. Government 2009
Abstract The essential oil from Amyris balsamifera (Rutaceae) and elemol, a principal
constituent of the essential oil of Osage orange, Maclura pomifera (Moraceae) were eval-
uated in in vitro and in vivo laboratory bioassays for repellent activity against host-seeking
nymphs of the blacklegged tick, Ixodes scapularis, and the lone star tick, Amblyomma
americanum. Both bioassays took advantage of the tendency of these host-seeking ticks to
climb slender vertical surfaces. In one bioassay, the central portion of a vertical strip of filter
paper was treated with test solution and ticks placed or allowed to crawl onto the untreated
lower portion. In the other bioassay, a strip of organdy cloth treated with test solution was
doubly wrapped (treatment on outer layer) around the middle phalanx of a forefinger and
ticks released on the fingertip. Both amyris oil and elemol were repellent to both species of
ticks. Elemol did not differ significantly in effectiveness against A. americanum from the
widely used repellent deet. At 2 and 4 h after application to filter paper, 827 lg amyris oil/
cm
2
paper repelled 80 and 55%, respectively, of A. americanum nymphs. Ixodes scapularis
was repelled by lower concentrations of amyris oil and elemol than A. americanum.
Keywords Amyris balsamifera Maclura pomifera Blacklegged tick 
Lone star tick
Introduction
Tick-borne diseases pose a serious threat to humans in most parts of the habitable world
(Sonenshine 1993; Parola and Raoult 2001). In the United States, the blacklegged tick,
J. F. Carroll (&)
USDA, ARS, Invasive Insect Behavior and Biocontrol Laboratory,
Beltsville Agricultural Research Center, BARC-East, Building 1040, Beltsville, MD 20705, USA
e-mail: john.carroll@ars.usda.gov
G. Paluch J. Coats
Department of Entomology, Iowa State University, Ames, IA 50011, USA
M. Kramer
USDA, ARS, Biometrical Consulting Service, Beltsville, MD 20705, USA
123
Exp Appl Acarol (2010) 51:383–392
DOI 10.1007/s10493-009-9329-0

Ixodes scapularis Say, and lone star tick, Amblyomma americanum (L.) are responsible for
many tick bites of humans and resultant disease transmission. The former species is the
principal vector of Borrelia burgdorferi, the pathogen causing Lyme disease in eastern and
central United States (Spielman et al. 1985), and the latter transmits Ehrlichia chaffeensis,
the causative of agent of human monocytic ehrlichiosis (Childs and Paddock 2003).
Larvae, nymphs and adults of these species bite humans. However, it is the nymphal stage
of I. scapularis that is responsible for most Lyme disease infections in humans. Where
A. americanum occurs, its aggressive host-seeking behavior tends to make it highly visible
to the public (Armstrong et al. 2001).
Recently developed area-wide tick control technologies have shown promise, but their
implementation has lagged (Fish and Childs 2009; Dolan et al. 2004; Piesman and Eisen
2008). Consequently the use of repellents is recommended for the personal protection of
people entering tick habitats (CDC 2002). Permethrin-based products applied to clothing
have proven effective in repelling A. americanum and I. scapularis (Schreck et al. 1982;
Lane and Anderson 1984). Since the 1950s, products containing deet (N,N-diethyl-3-
methyl benzamide) have been the mainstay for use on human skin. Some deet formulations
provide lasting protection from ticks (Carroll et al. 2008). With the discovery of effective
synthetic alternatives to deet-based products (e.g., picaridin, IR3535) in recent years, there
has been a growing interest in discovery of tick repellents from natural sources (e.g.,
Jaenson et al. 2005; Tuno
´n et al. 2006; Garboui et al. 2007; Carroll et al. 2007; Bissinger
et al. 2009).
Evidence of the repellent activity contained in sesquiterpene-rich essential oils and their
purified isolates and/or compounds (from heartwood, bark, leaves, etc.) has appeared in the
literature over the last 10 years, with a recent focus on sesquiterpenes containing alcohol,
aldehyde, ketone, and acid moieties from extractions of white cypress pine, Callitris
glaucophylla Thompson et Johnson, Japanese cedar, Cryptomeria japonica (L. f.) D. Don,
the American beautyberry bush, Callicarpa americana L., Alaska yellow cedar, Cha-
maecyparis nootkatensis D. Don, a Malaysian member of the custard apple family, Go-
niothalamus uvariodes King, Osage orange, Maclura pomifera (Raf.) Schneid, amyris,
Amyris balsamifera L., and Siam-wood, Fokienia hodginsii (Dunn) Henry and Thomas
(Ahmad and Jantan 2003; Watanabe et al. 2005; Wang et al. 2006; Carroll et al. 2007;
Dietrich et al. 2006; Schultz et al. 2006; Paluch et al. 2009). Availability of these essential
oils and extracts can be limited, but some are supplied by commercial sources.
Sesquiterpenes occurring in balsam torchwood, A. balsamifera (Rutaceae) and elemol, a
major constituent of the essential oil of Osage orange (Moraceae), have been shown by
Paluch et al. (2009) to repel the yellow fever mosquito, Aedes aegypti (L.), The purpose of
this study was to ascertain whether the repellent properties of Amyris essential oil and
elemol extended to ixodid ticks. To this end, we tested host-seeking A. americanum and
I. scapularis against amyris oil and elemol in in vitro and in vivo laboratory bioassays.
Materials and methods
Ticks
Larvae of I. scapularis obtained from a colony at Oklahoma State University were fed on
rats (in accordance with USDA, ARS, Beltsville Area Animal Care and Use Committee
Protocols #05-022 and #08-013). The fed larvae were held in vials at 23–24°C, &97% RH
and a photoperiod of 16:8 h (L:D). Nymphs of A. americanum were from a colony
384 Exp Appl Acarol (2010) 51:383–392
123

maintained at the USDA, ARS, Knipling-Bushland U. S. Livestock Insects Research
Laboratory, Kerrville, TX and held at 23–24°C, &97% RH and a photoperiod of 16:8 h
(L:D). The I. scapularis and A. americanum nymphs were used in bioassays 2–4 months
after eclosion.
Chemicals
Amyris (A. balsamifera) essential oil was purchased from commercial sources (Sigma–
Aldrich, St. Louis, MO; Phoenix Natural Products, Middlesex, England). Sufficient
quantities of purified elemol were isolated from technical grade materials in the laboratory
at Iowa State University. A supply of technical grade, 55% purity elemol (Augustus
Essential Oils, Hampshire, England) was further purified via column chromatography with
silica gel, 40–140 mesh (J.T. Baker, Phillipsburg, NJ), to C80% purity using hexane/
diethyl ether (9:1) mobile phase. Further column purification to C95% was achieved using
a hexane/acetone/diethyl ether (8:1:1) mobile phase system.
Purity of samples was assessed on a Hewlett Packard 5890 Series II gas chromatograph
with a 30 90.25-mm i.d. 90.25 lm film thickness DB-WAX column (Alltech, Deerfield,
IL) with flame ionization detection. The injector temperature was 250°C, and the split
valve was opened 1 min after injection. The oven initial temperature was set at 120°C for
1 min, and then increased at 4°C/min to 236°C. Confirmation of compound identity was
completed on a Hewlett Packard 5890 Series II gas chromatograph interfaced to a Hewlett
Packard 5972 Mass Selective Detector (MSD). Mass spectra were recorded from 30 to 550
a.m.u. with electron impact ionization at 70 eV. Chemical identification was assigned to
elemol and amyris essential oil compounds detected, and results confirmed by comparison
of the retention indices with reference spectra in a mass spectral library (Wiley 138 K,
John Wiley and Sons) and comparison to literature sources (Van Beek et al. 1989). Deet
was purchased from Sigma–Aldrich.
Bioassays
An in vitro and an in vivo bioassay were used to evaluate the repellency of elemol and
amyris oil. The in vitro bioassay (vertical filter paper bioassay) took advantage of the
tendency of host-seeking ticks to climb slender vertical surfaces (Carroll et al. 2004). A
497-cm rectangle of Whatman No. 4 filter paper was marked with a pencil into two
194-cm zones at either of the far ends of the paper with an intervening 4 95-cm zone.
Test solutions (165 ll) were evenly applied by pipettor to the 20-cm
2
central zone of the
filter paper strip. The strip was allowed to dry for 10–15 min and suspended from a bulldog
clip hung from a slender dowel held by an Aptex No. 10 double clip work holder (Aptex,
Bethel, CT). The vertical strip hung over a Petri dish (9 cm diameter) that had been glued
in the center of a 15-cm diameter Petri dish with water creating a moat between their walls
(1.5 cm high). A storage vial containing ticks was opened in the center of moated Petri
dishes (5.5 and 9 cm diameters). When A. americanum nymphs had crawled onto the rim
of the vial and the Petri dish, the strip was removed from the peg and held so that a total of
10 ticks crawled onto the lower untreated zone. With I. scapularis, the nymphs were
transferred to the filter paper using forceps.
Locations of the ticks were recorded at 1, 3, 5, 10, and 15 min after the tenth tick was on
the filter paper. We scored repellency based on the locations of ticks at 15 min. Ticks were
considered repelled if they remained on the lower untreated zone of the filter or if they
dropped off the strip without having crossed into the upper untreated zone. The moated
Exp Appl Acarol (2010) 51:383–392 385
123

Petri dish beneath the strip confined ticks that dropped from the paper. Ticks that climbed
to the upper untreated zone were removed to prevent their return to the lower zones.
The in vivo bioassays (fingertip bioassay with double-wrapped cloth) were conducted in
compliance with a human-use protocol (#2007-240) reviewed and approved by the Med-
Star Research Institute Institutional Review Board. This modified fingertip bioassay, was
described in detail and depicted by Carroll et al. (2005). A strip of organdy (7 97
mesh/mm) (Hancock Fabrics, Laurel, MD) was cut in the shape of a hockey stick (9 cm
long section, 4.5 cm short section, 4–4.5 cm wide) so that it could be wrapped twice
around the index finger of JFC with only the outer layer receiving test solution. The
boundary of an area of the cloth corresponding to the area between the first and second
joints of the finger was marked with a lead pencil and served as the treatment area. The
volume of the solution applied to the cloth was based on the dimensions of the left index
finger. The volume required for the desired nmoles/cm
2
cloth was calculated from the
average of the circumferences of the two finger joints multiplied by distance between the
deepest crease of each joint.
While an organdy strip was partly supported by the rim of a glass petridish, 52 ll of test
solution was evenly distributed on the treatment area with a pipettor. After allowing 10–
15 min for the cloth to dry, it was doubly wrapped around the index finger, so that the
treated portion of the cloth completely encircled the finger and covered the entire second
phalanx. An untreated portion of the cloth extended 5–6 cm beyond the first joint toward
the base of the finger. To hold the cloth in place three small dabs of beeswax were smeared
on the upper surface of the inner layer of cloth where layers overlapped and pressure from
another finger applied to adhere the layers. Because I. scapularis nymphs tended to be
slower, were more apt to drop from untreated skin, and had a far larger percent remain
immobile than A. americanum nymphs, it was necessary to screen the former for tenacity
and readiness to climb (Schreck et al. 1995). While the test solution dried on the cloth,
I. scapularis nymphs were placed on the tip of the untreated index finger held vertically.
Those ticks that climbed &0.5 were used in the bioassay. Using forceps, 10 nymphs of
I. scapularis were placed on the untreated fingertip near the base of the nail. As in the
vertical filter paper bioassay, 10 A. americanum nymphs were allowed to crawl from
the rim of an open vial and moated Petri dish onto the fingertip. Once 10 ticks were on the
fingertip, the finger was tilted slowly until vertical with the tip downward. The locations of
the ticks were recorded at 1, 3, 5, 10, and 15 min after the tenth tick was on the finger.
Ticks were considered repelled if they fell from the finger without having crossed the upper
boundary of the treated area or if they were on the untreated fingertip distal to the cloth. As
in the in vitro bioassay, repellency was scored according to the locations of the ticks at
15 min. Before each bioassay JFC washed his index finger with soap and rinsed with water.
Experimental design
In vertical filter paper bioassays, amyris oil was tested against I. scapularis at 103, 51, 26,
13 and 0 lg oil/cm
2
paper (n=30 ticks per concentration) and against A. americanum at
827, 413, 207, 103, 51, 26, and 0 lg oil/cm
2
paper to observe dose related responses
(n=40 ticks per concentration). Elemol was tested in vertical filter paper bioassays
against A. americanum at 310, 155, 78, and 0 nmoles compound/cm
2
paper (n=30 ticks
per concentration). Amyris oil at 827 lg compound/cm
2
paper, was tested against
A. americanum in vertical filter paper bioassays 2 and 4 h after application of the test
solutions ascertain duration of activity.
386 Exp Appl Acarol (2010) 51:383–392
123

In fingertip bioassays, amyris oil was tested against I. scapularis at 413, 207, 103, 51,
26, and 0 lg oil/cm
2
cloth and against A. americanum at 827, 413, 207, 103, and 0 lg
oil/cm
2
cloth to observe dose related responses (n=30 ticks per concentration). Elemol
was tested in fingertip bioassays against I. scapularis at 155, 78, 39, 19, 10 and 0 nmoles
compound/cm
2
cloth and against A. americanum at 775, 620, 310, 155, 78, 39, and
0 nmoles compound/cm
2
cloth (n=30 ticks per concentration).
For comparative purposes deet was tested in fingertip bioassays with amyris oil against
I. scapularis at 51 and 13 lg oil/cm
2
cloth and with elemol against A. americanum at 775
and 78 nmole compound/cm
2
cloth (n=30 ticks per concentration).
Statistical analysis
The data collected were binomial in nature (ticks were either repelled or not, ticks either
dropped or not) so fit in statistical framework of generalized linear models (McCullagh and
Nelder 1989). We also found that there was often a day effect (‘‘repellency’’ varied
somewhat from day-to-day), which we included in the analyses as a random effect. In
addition, for some analyses, there appeared other unknown sources of variation resulting in
over-dispersed data (accommodated by using a quasi-binomial rather than binomial dis-
tribution, with an additional over-dispersion parameter). We used the R software
(R Development Core Team 2009) with the lme4 package (Bates and Maechler 2009)to
estimate models and test compound differences.
Since the software to calculate fiducial confidence limits (inverse regression) on dose
has not been developed for generalized linear mixed models, we calculated these limits
allowing for general overdispersion (but not specific random effects) using the dose.p
function of the R MASS package (Venables and Ripley 2002) after fitting a generalized
linear model in the quasi-binomial distribution family.
We found that by taking the square root of the dose the fit to the logit of the proportion
was consistently more linear than any other transformations on dose, so we used square
root transformed dose in all analyses.
Results
Amyris oil and elemol repelled both species of ticks in all bioassays. Figure 1depicts the
dose related responses of A. americanum and I. scapularis nymphs to elemol in fingertip
and vertical filter paper bioassays and Fig. 2shows the ticks’ responses to amyris oil. In
wrapped-fingertip bioassays, all I. scapularis nymphs were repelled by 155 nmole elemol/
cm
2
cloth and 97.3 and 100% of I. scapularis were repelled by 413 and 207 lg oil/cm
2
cloth of amyris oil, respectively. Higher concentrations of elemol and amyris oil were
needed to repel A. americanum than I. scapularis. For example, 1,550 nmole elemol/cm
2
cloth were needed to repel 97.3% of A. americanum nymphs in fingertip bioassays. For
direct comparison, two concentrations of deet were tested along with amyris oil in a series
of wrapped-fingertip bioassays against I. scapularis and in a series of wrapped-fingertip
bioassays with elemol against A. americanum. Deet and elemol did not differ significantly
(P=0.558) in their effectiveness against A. americanum in the fingertip bioassays.
Against I. scapularis, deet was significantly (P=0.001), but not greatly more repellent
than amyris oil (Fig. 1). For both analyses, there was no significant interaction between
concentration and compound.
Exp Appl Acarol (2010) 51:383–392 387
123

Table 1shows the square roots of the EC
50
and EC
95
values for amyris oil in vertical
filter paper and wrapped-fingertip bioassays for both species of ticks and for elemol in
vertical filter paper bioassays against A. americanum and fingertip bioassays against both
species. The data were out of the range of the model to calculate an EC
95
for amyris oil
against A. americanum. Nymphs of A. americanum also exhibited greater variability in
within dose responses compared to I. scapularis.
Amyris oil showed some prolonged repellent activity. In vertical filter paper bioassays,
827 lg amyris oil/cm
2
paper repelled 80% of A. americanum nymphs 2 h after application
and 55% at 4 h after application.
The proclivity of A. americanum nymphs to drop off vertical surfaces treated with
repellent is obvious in Fig. 3. In contrast, the nymphs of I. scapularis were also repelled by
the elemol, but tended to remain on the untreated tip of the finger where they had been
placed at the start of the test.
Discussion
Deet is often considered the standard to which other repellents are held for comparison.
Carroll et al. (2004,2007) and Zhang et al. (2009) tested deet in the same wrapped-
fingertip and vertical filter paper bioassays using ticks from the same sources as in this
study. For fingertip bioassays using nymphal I. scapularis, Carroll et al. (2007) reported an
EC
50
and EC
95
of 23.9 and 58.4 nmole deet/cm
2
cloth, respectively, which compare to an
EC
50
and EC
95
of 26.6 and 94.34 for elemol in this study (EC values presented as square
roots in Table 1). Similarly, Zhang et al. (2009), who used the same wrapped-fingertip
010203040
0.0 0.2 0.4 0.6 0.8 1.0
sqrt (nmole/cm2)
proportion repelled
I. scapularis finger tip
A. americanum finger tip
A. americanum vertical filter paper
deet − A. americanum finger tip
Fig. 1 Responses of Ixodes scapularis and Amblyomma americanum nymphs to elemol, deet and an ethanol
control in vertical filter paper and wrapped-fingertip bioassays. Deet tested at two concentrations in fingertip
tests for purposes of comparison
388 Exp Appl Acarol (2010) 51:383–392
123

bioassay and ticks from the same source as this study, reported that 78 nmole deet/cm
2
cloth repelled all I. scapularis nymphs tested, but concentrations as high as 775 nmole
deet/cm
2
cloth did not repel more than 80% of A. americanum. In this study, the estimated
EC
50
and EC
95
for elemol against A. americanum were 218.5 and 1,121.8 nmole/cm
2
respectively, but in direct comparison, elemol did not differ in repellency from deet. Thus,
it appears that elemol is as effective a repellent of A. americanum as is deet, but is
somewhat less effective against I. scapularis than deet. Carroll et al. (2005) found that, in
fingertip bioassays with cloth wrapped once around the finger, 1.6 lmole deet/cm
2
cloth
repelled 95% of I. scapularis nymphs and 85% of A. americanum nymphs. In this study,
0.0 0.2 0.4 0.6 0.8 1.0
sqrt (µg compound/cm2)
proportion repelled
4 8 12 16 20 24 28
A. americanum fingertip
I. scapularis fingertip
A. americanum vertical filter paper
I. scapularis vertical filter paper
deet − I. scapularis fingertip
Fig. 2 Responses of Ixodes scapularis and Amblyomma americanum nymphs to amyris oil, deet and an
ethanol control in vertical filter paper and wrapped-fingertip bioassays. Deet tested at two concentrations in
fingertip tests for purposes of comparison
Table 1 Square roots of effective concentrations—of elemol and amyris oil against Ixodes scapularis and
Amblyomma americanum nymphs in in vitro (vertical filter paper) and in vivo (cloth-wrapped fingertip)
bioassays
Repellent EC Vertical filter paper bioassay Wrapped fingertip bioassay
I. scapularis A. americanum I. scapularis A. americanum
Mean lg/cm
2
Amyris oil EC
50
3.154 (0.418)
a
9.043 (1.000) 4.199 (0.791) 22.912 (2.281)
EC
95
9.061 (0.927) 23.548 (2.654) 13.768 (2.036) Outside range of data
Mean nmole/cm
2
Elemol EC
50
10.935 (1.809) 5.157 (0.399) 14.783 (1.232)
EC
95
26.107 (3.105) 9.713 (0.914) 33.494 (3.003)
a
Standard errors in parentheses
Exp Appl Acarol (2010) 51:383–392 389
123

deet repelled significantly greater proportions of I. scapularis than amyris oil in wrapped
fingertip bioassays, but as seen in Fig. 2, the difference was not great. Although we made
no direct comparison of deet and amyris oil against A. americanum in this study, the
reported EC
50
of 1.30 lmole deet/cm
2
paper for the vertical filter paper bioassay (Carroll
et al. 2004) and 775 nmole deet/cm
2
cloth for 80% repellency in the wrapped fingertip
bioassay (Zhang et al. 2009), suggest that when compared to deet amyris oil may be less
effective than elemol.
Both amyris oil and elemol repelled I. scapularis nymphs at lower concentrations than
were needed to repel A. americanum nymphs. This discrepancy between the responses of
these species has been observed in previous studies in which fingertip and vertical filter
paper bioassays were used to test deet, SS220, callicarpenal, intermedeol, and isolongif-
olenone (Carroll et al. 2004,2005,2007; Zhang et al. 2009). The responses of A. ameri-
canum to elemol and amyris oil also tended to be characterized by greater within dose
variability than those of I. scapularis. Nymphs of A. americanum, unlike I. scapularis,
often respond to repellents (e.g., deet, SS220) on vertical surfaces by dropping off them
(Carroll unpublished data). In contrast, I. scapularis tend to withdraw from repellent-
treated areas, and if the adjacent untreated surface is limited in size, as in the fingertip and
vertical filter paper bioassays, the nymphs remain on the untreated surface. Drop off and
back off behavior is well illustrated in the responses of A. americanum and I. scapularis to
elemol in fingertip bioassays (Fig. 3). In terms of human protection, it is preferable that a
tick drop off than for it to sequester itself on untreated or poorly treated skin or clothing.
Although the fingertip bioassay we used might be considered an in vivo test because a
human subject was involved, it did not involve application of repellent to human skin. The
treatment on the outer of two layers of cloth did not contact the skin. Absorption of the
repellent by the skin and interactions with epidermal chemicals could result in different
repellent activity. The double-wrapped finger bioassay does include the full array of host
01234
0.0 0.1 0.2 0.3 0.4 0.5
sqrt (nmole/cm
2
)
proportion dropped off
A. americanum
I. scapularis
Fig. 3 Proportions of Ixodes scapularis and Amblyomma americanum nymphs that dropped from finger
within 15 min, in response to three concentrations of elemol in doubly-wrapped fingertip bioassay. In
contrast to A. americanum, nearly all I. scapularis were repelled at the two highest concentrations, but high
proportions remained on the untreated fingertip
390 Exp Appl Acarol (2010) 51:383–392
123

stimuli and should elicit normal host acquisition and attachment site seeking behavior in
the tick subjects. A useful feature of the double-wrapped finger bioassay is that it protects
the human subject from dermal exposure to candidate repellents. The in vitro vertical filter
paper test results are supportive of the fingertip bioassay findings, but in the three cases, in
which both in vitro and in vivo bioassays were conducted using the same tick species and
same repellent, higher concentrations of amyris oil or elemol were needed in the fingertip
bioassay to achieve the same level of repellency as in the filter paper bioassay (Figs. 1,2).
An obvious difference between the bioassays is that ticks in the filter paper bioassay receive
chemical (CO
2
), physical and visual cues (Phillis and Cromroy 1977) from the observer, but
tactile and likely other host-associated cues are lacking. Another important difference is that
Whatman No. 4 filter paper absorbs more of the test solution than the organdy. More than
twice the volume/cm
2
of test solution was applied to the filter paper, but it is unknown how
much, if any, was absorbed too deeply into the paper to affect the ticks.
The concentrations at which elemol and amyris essential oil repelled I. scapularis and
A. americanum in our bioassays and the concentrations which Paluch et al. (2009) found
repelled A. aegypti mosquitoes are low enough to indicate that elemol and amyris oil
should be investigated more thoroughly for their potential as marketable repellents.
Acknowledgments We thank James McCrary, USDA, ARS, Invasive Insect Behavior and Biocontrol
Laboratory, Beltsville Agricultural Research Center, Beltsville, MD for performing behavioral bioassays
that were essential to this study. We are also grateful to USDA, ARS, Knipling-Bushland U. S. Livestock
Insects Research Laboratory, Kerrville, TX for providing the A.americanum used in the study.
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