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Journal of Chemical Ecology, Vol. 27, No. 3, 2001
0098-0331
/
00
/
0300-0523$19.50
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0 2001 Plenum Publishing Corporation
523
NOOTKATONE IS A REPELLENT FOR FORMOSAN
SUBTERRANEAN TERMITE (Coptotermes formosanus)
BETTY C. R. ZHU,
1
GREGG HENDERSON,
2,
*, FENG CHEN,
2
LARA MAISTRELLO,
2
and ROGER A. LAINE
1
1
Department of Biological Sciences
2
Department of Entomology
Louisiana State University Agricultural Center
Louisiana Agricultural Experiment Station
Louisiana State University
Baton Rouge, Louisiana
70803
(Received July 31, 2000; accepted November 14, 2000)
Abstract—We examined the behavior of Formosan subterranean termites
toward one of the components of vetiver grass oil, the roots of which
manufacture insect repellents. We found nootkatone, a sesquiterpene ketone,
isolated from vetiver oil is a strong repellent and toxicant to Formosan
subterranean termites. The lowest effective concentration tested was
10 mg
/
g
substrate. This is the first report of nootkatone being a repellent to insects.
Key Words—Vetiveria zizanioides, vetiver oil, sesquiterpenes, ketones.
INTRODUCTION
The Formosan subterranean termite is the most destructive termite species
wherever it occurs in the world. These termites search for and find cellulose
largely using chemical cues from the wood itself (Amburgey and Smythe,
1977;
Reinhard et al.,
1997), and they exhibit similar chemoresponses to a variety of
chemicals. For example, trail-following behavior has been shown to naphtha-
lene, a well-known insect toxicant (Chen et al.,
1998b,c), 2-naphthalenemethanol
(Henderson et al.,
1996), and 2-phenoxyethanol, a component of some ballpoint
inks (Chen et al.,
1998a). Formosan subterranean termites live in a miasma of
chemicals present in their nests, many of which appear to be plant derived (Chen
et al.,
1998b; Henderson et al., 1999). Our recent investigations have focused
*To whom correspondence should be addressed.
ZHU ET AL.524
on natural plant extracts that may disrupt this insect’s ability to recruit food
sources.
Vetiver grass (Vetiveria zizanioides, Linn Nash), a fast-growing native plant
of India, is a distant relative of maize, sorghum, sugarcane and lemongrass
(National Research Council,
1993). The plant has been used in India for weaving
mats, baskets, fans, sachets, and ornaments that have insect repellent properties.
Clothes moths, head lice, and bedbugs are thought to be repelled by vetiver grass
so fashioned (National Research Council,
1993). The Formosan subterranean
termite, Coptotermes formosanus Shiraki, readily consumes sugarcane, causing
agricultural damage to the crop in some countries (Chen and Henderson,
1996;
National Research Council,
1993). Therefore, it was not readily apparent that
termites would be repelled by components of vetiver grass. The compounds in
vetiver oil that repel cockroaches and flies are believed to include a- and b-
vetivone, khusimone, khusistone, zizanal, and epizizanal (Jain et al.,
1982).
Preliminary studies using dried vetiver root in a sand substrate (
1 : 10 ratio)
indicated that Formosan subterranean termites were effectively repelled from
tunneling to a food source (Henderson et al., unpublished data). Gas chro-
matographic and mass spectrometric analysis of vetiver oil separated on silica
columns, along with behavioral bioassays, allowed us to identify nootkatone as
a repellent to Formosan subterranean termites.
METHODS AND MATERIALS
Extraction of Vetiver Oil from Roots
We examined the extracts from the roots to determine which components of
the oil were repellent. The roots of fresh Louisiana-grown vetiver grass Vetiveria
zizanioides (provided by the Donald O. Heumann Greenhouse and Laboratory,
Poydras, Louisiana) were cleaned, dried at room temperature, and ground with
a blender. Vetiver oil was obtained by petroleum ether extraction of the dried
roots. The components of vetiver oil were isolated by silica column (
2.5 × 20
cm) and eluted with hexane and increasing amounts of methylene chloride. Five
fractions were obtained by eluting with hexane–CH
2
Cl
2
: 80 : 20 for fraction 1,
70 : 30 for fraction 2, 60 : 40 for fraction 3; 40 : 60 for fraction 4, and 20 : 80
for fraction 5. Examination of sesquiterpenic ketones were visualized for their
intrinsic fluorescence under UV light (Andersen,
1970) since they are potential
insect repellents. The fractions detected by UV were further isolated by prepar-
ative TLC (Analtech, Newark, Delaware) using CHCl
3
as a mobile phase. The
bands were visualized by charring the plate at
120 C after spraying with 50%
sulfuric acid.
Gas chromatography–mass spectrometry (GC-MS) was performed on a
Finnigan GCQ (Trace GC
2000 coupled with Polaris MSD). A silica capillary
NOOTKATONE IS TERMITE REPELLENT 525
MS column, DB-5MS (30 m × 0.25 mm × 0.25 mm; J&W Scientific, Folsom,
California), was operated at
60 C for 1 min, increased to 150 C at 2.5 C
/
min
held for
15 min at this temperature and increased to 260 C at 5 C
/
min, where
is was finally held for
10 min. The injector port was operated in splitless mode
at
250 C, and helium was used as carrier gas at 0.8 m
/
min. The mass spectral
detector (MSD) was set on full-scan mode (m
/
z 41–400). The authentic stan-
dard of nootkatone (crystalline,
97%) was purchased from Lancaster Synthesis
Inc. (Windham, New Hampshire). Both a- and b-vetivone were kindly provided
by Professor Ekkhard Winterfeldt (Institut f
¨
ur Organische Chemie, Technische
Universit
¨
at, Berlin).
Termite Bioassay
Experiment
1. A three-compartment plastic container (18 × 18 × 4 cm;
Pioneer Packaging Co., North Dixon, Kentucky) was used for the termite bioas-
says. A small hole (
0.5 cm diameter) was melted at the bottom of each of two
inner walls, connecting the bioassay chamber compartments. For testing,
50 mg
of nootkatone was dissolved in
10 ml ethanol as a stock solution. Five concentra-
tions of nootkatone were placed into a sand substrate in the middle compartment
of the biossay chamber:
0 (control), 10, 20, 100, and 200 mg
/
g sand. Four repli-
cates were conducted for each concentration. For each concentration,
500 g of
blasting sand was mixed with a series of dilutions of the stock solution (except
for
0 mg
/
g) in 25 ml of ethanol in a glass pan and dried in a hood for 2 hr.
The following day,
115 g of untreated blasting sand was added to one end of
each compartment (home compartment), and
115 g of treated sand at one of
five concentrations was added to the middle compartment. The
0-mg concen-
tration was treated only with
25 ml of ethanol. Filter paper (Whatman No. 1,
2.3 cm diameter) was dried at 70 C for 3 hr and cooled to room temperature
for
30 min before weighing. A weighed filter paper with 200 ml distilled water
added was placed in the third compartment farthest from the introduction end.
Ten milliliters of distilled water was added to the treated and untreated sand in
the other compartments just prior to the introduction of termites. Fifty workers
and five soldiers of Formosan subterranean termites from a large colony col-
lected in Algiers, Louisiana, on November
18, 1997, were placed in the home
compartment. The containers were covered with lids and kept in a dark incu-
bator at
29 C. On day 16, each apparatus was dismantled, living termites were
counted, and filter papers were cleaned, dried at
70 C for 3 hr, cooled for 30
min, and weighed. Consumption was calculated as the difference between the
weight of filter paper before and after the
16-day incubation. The tunnels ter-
mites constructed in the sand were copied using a scanner for measurement of
total tunnel lengths.
Experiment
2. The materials and methods were the same as described for
ZHU ET AL.526
experiment 1, except: (a) the concentrations of nootkatone used to treat the sand
in the middle compartment were slightly different; (b) observations of tunneling
were recorded several times during the experiment; and (c)
50 g sand was placed
in the third compartment under the filter paper disk and was moistened every
two days with
0.3 ml of distilled water. For this experiment the concentrations
of nootkatone were
0 (control), 5, 10, 20, 40, and 100 mg
/
g sand. Four repli-
cates were performed for each concentration. Every two days all containers were
checked for tunneling activity and the evaluating criterion was:
0 termites on
the surface (no tunneling);
1 tunneling in the first chamber; 2 tunneling in
the first and in the middle (treated) chamber;
3 tunneling in all chambers.
Statistical Analysis
For experiment
1, the differences among treatments in filter paper consump-
tion, tunnel length, and percentage mortality were analyzed by ANOVA. For
experiment
2, we used a nonparametric analysis of variance (Kruskal-Wallis
ANOVA) in order to analyze trends in tunneling differences between days of
observation. At the end of the experiment, differences among treatments were
analyzed as in experiment
1. For both experiments, differences between treat-
ments were analyzed by Tukey’s studentized range test (HSD). Although per-
centage values were transformed to arc-sin of the square root for data analysis,
nontransformed means are reported.
RESULTS
Identification of Nootkatone as Active Fraction from Vetiver Oil
Three fractions (
3–5) contained autofluorescing bands detected by UV. Each
fraction was further separated by preparative TCL with CHCl
3
as a mobile phase.
In fraction
4, bands 1 and 2 autofluoresced, and were examined by GC-MS.
Nootkatone was a major constituent of band
2 (Figure 1A). The chromatograph
of authentic nootkatone is shown in Figure
1B. The nootkatone identification
was verified by comparing the mass spectrum of its GC peak (Figure
2A) with
authentic nootkatone (Figure
2B).
Termite Bioactivity of Nootkatone
Experiment
1. The mean consumption of filter paper decreased significantly
in the presence of nootkatone; the decrease in consumption was concentration
dependent (Table
1). Even at the lowest concentration tested (10 mg
/
g sand),
nootkatone significantly decreased feeding as compared with the control. Little
consumption of filter paper was found when concentrations of nootkatone were
higher than
20 mg
/
g. Termite mortality increased as nootkatone concentrations
NOOTKATONE IS TERMITE REPELLENT 527
FIG. 1. Gas chromatography of band 2 (A) and the standard of nootkatone (B). The reten-
tion time of nootkatone in band
2 is 53.22 min (first peak) and in the standard is 53.35
min.
increased (Table 1). All concentrations of nootkatone had a significant impact on
mortality when compared with the control. Ninety percent mortality or greater
was observed in all chambers when nootkatone concentrations were ≥
100 mg
/
g
sand. The presence of nootkatone substantially decreased the tunneling activity
even at the lowest concentration of
10 mg
/
g sand (Table 1). At higher concentra-
tions of nootkatone (
20 mg
/
g and higher), no tunneling was visible in the middle
chamber.
Experiment
2. Units treated with different concentration of nootkatone
showed significantly different levels of tunneling activity. On day two, signifi-
cant differences were detected among treatments (H
20.47, df 5, P 0.001,
Kruskal-Wallis ANOVA); almost all groups treated with lowest concentrations
were able to tunnel in the first and even in the middle (treated) chamber, while
termites faced with the highest concentration remained on the surface. The same
trend is confirmed on day
6 (H 17.39, df 5, P 0.0038), where some ter-
mite groups (control and concentration
3) tunneled into the feeding chamber,
ZHU ET AL.528
FIG. 2. Mass spectrum of the nootkatone peak in band 2 (A) and authentic nootkatone (B).
NOOTKATONE IS TERMITE REPELLENT 529
TABLE 1.MEAN (±SD) OF PAPER CONSUMPTION, PERCENT MORTALITY, AND TUNNELING
LENGTH OF FORMOSAN SUBTERRANEAN TERMITE AFTER 16-DAY EXPOSURE
(EXPERIMENT 1)
a
Concentration of Weight loss of Termite
nootkatone filter paper mortality Tunneling length
(mg
/
g sand) (mg) (%) (cm)
010.500 ± 2.867A 58.50 ± 8.72A 27.000 ± 6.072A
10 1.900 ± 0.641B 88.50 ± 10.12B 7.225 ± 2.155B
20 0.525 ± 0.709B 87.00 ± 18.22B 0.000 ± 0.000B
100 0.025 ± 0.050B 90.50 ± 11.72B 0.000 ± 0.000B
200 0.275 ± 0.050B 95.00 ± 8.718B 0.000 ± 0.000B
a
Four replicates were included for each treatment. Means within a column followed by the same
letter are not significantly different (ANOVA: F
9.39, df 4 and P < 0.0005 for paper
consumption; F
4.22, df 4, P < 0.0174 for percentage mortality and F 43.1, df 4, P < 0.0001
for tunneling length.)
and on day 11 (H 14.52, df 5, P 0.0126) (Table 2). As in experiment
1, significant differences were detected in filter paper consumption, where only
the controls and groups treated with
20 and 40 mg
/
g sand of nootkatone could
eat some filter paper. Differences were also detected in tunnel length: only the
controls and units treated with the lowest concentration of nootkatone showed
tunneling behavior. Due to high variability between units belonging to the same
treatment group, no significant differences were detected on termite mortality.
TABLE 2.MEAN (±SD) OF PAPER CONSUMPTION, PERCENT MORTALITY, AND TUNNELING
LENGTH OF FORMOSAN SUBTERRANEAN TERMITES AFTER 16-DAY EXPOSURE
(EXPERIMENT 2)
a
Concentration of Weight loss of
nootkatone filter paper % termite Tunneling length
(mg
/
g sand) (mg) mortality (cm)
03.675 ± 3.500 A 27.727 ± 10.111 A 29.450 ± 8.065 A
50.150 ± 0.300 B 36.364 ± 7.120 A 24.975 ± 14.558 A
10 0.650 ± 1.300 AB 52.273 ± 45.281 A 11.000 ± 16.818 AB
20 0.300 ± 0.600 AB 48.182 ± 36.980 A 2.250 ± 2.872 B
40 0 ± 0B 39.091 ± 32.710 A 0 ± 0 B
100 0 ± 0B 58.636 ± 33.008 A 0 ± 0 B
a
Four replicates were included for each treatment. Means within a column followed by the same
letter are not significantly different (ANOVA: F
3.416, df 5, P 0.024 for paper consumption;
F
0.580, df 5, P 0.715 for percentage mortality and F 7.215, df 5, P 0.0007 for tunneling
length.)
ZHU ET AL.530
DISCUSSION
Vetiver oil is a complex essential oil containing several hundred individual
compounds. Of them,
92 compounds have been characterized and can be divided
into
33 esters, 36 sesquiterpenic hydrocarbons, 5 bi- and tricyclic sesquiterpenic
alcohols,
9 ketones, 3 aldehydes, and 6 acids (Cazaussus et al., 1989). Nootka-
tone,
4,4a,5,6,7,8-hexahydro-6-isopropenyl-4,4a-dimethyl-2(3H)-naphthalone, is
a mildly pungent sesquiterpene ketone found in the oil of Alaska yellow cedar,
Chamaecyparis nootkatensis (Lamb) Spach (Erdtman and Hirose,
1962) and in
a great number of citrus oils, especially oil from grapefruit, Citrus pavadisi Calli
(MacLeod and Buigues,
1964). Nootkatone is widely used in the perfume and
flavor industries, being essentially nontoxic to humans (National Research Coun-
cil,
1993).
Our studies determined that nootkatone has potential as a termite repel-
lent barrier. Considering tunneling behavior as a measurement of termite activ-
ity, these results confirmed that nootkatone effects termite vigor. Termites from
colonies treated with lower concentrations of nootkatone were able to dig tun-
nels in the home and in the treated chamber, and this ability was negatively
correlated with the concentration of nootkatone. Termites treated with the high-
est concentrations of nootkatone were not able to dig tunnels, even in the home
chamber, possibly because of the nootkatone vapors acting as an inhibitor of
termite mobility. We believe that a concentration of nootkatone bewteen
10 and
1000 mg
/
g, preferably between 10 and 200 mg
/
g, may be useful in repelling
and killing termites. We are also evaluating nootkatone as a chemical that could
be of value against termites in structural wood treatments or cellulose mulch
applications (Henderson et al., unpublished data).
A barrier created by plants that manufacture a termite repellent could poten-
tially provide long-lasting repellency. Vetiver grass is a fast growing plant with
a huge spongy mass of roots. The roots grow to depths of
3 m, and with a little
attention the plant could live for
50–60 years (National Research Council, 1993).
Since the oil occurs primarily in the roots, a low-cost way to prevent invasion by
subterranean termites and other insidious underground insects may be to plant a
solid band of vetiver grass around a house. Recently, we found that some of the
insect repellents found in the roots also occur in the soil surrounding the plant
(Henderson et al., in preparation).
Vetiver oil is one of the most complex essential oils (Cazaussus et al.,
1989).
In addition to nootkatone, we also have found that vetiver oil itself and its major
components a- and b-vetivone were powerful repellents and toxicants against
Formosan subterranean termite (Zhu et al., in preparation). Jain et al. (
1982)
reported six substances in vetiver oil that have potent topical irritant activity on
cockroaches and flies.
NOOTKATONE IS TERMITE REPELLENT 531
Acknowledgments—This project was approved for publication by the Louisiana State Univer-
sity Agricultural Center and Louisiana Agricultural Experiment Station as manuscript
00-17-0304.
Partial funding was provided through a specific cooperative agreement with the USDA
/
ARS-New
Orleans (
58-6435-8-084).
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