Role of (3Z,6Z,8E)-dodecatrien-1-ol in trail following, feeding, and mating behavior of Reticulitermes hesperus

Article abstract of DOI:10.1007/s10886-006-9229-2

Trail pheromones mediate communication among western subterranean termites, Reticulitermes hesperus Banks. Repetitive passages of ≥28 termites were required to establish a pheromone trail and trails needed to be reinforced because they lasted <48 hr. The minimal threshold concentration for inducing responses from termite workers and secondary reproductives was between 0.01 and 0.1 fg/cm of (3Z,6Z,8E)-dodecatrien-1-ol (henceforth, dodecatrienol). Workers showed optimal trail-following behavior to dodecatrienol at a concentration of 10 fg/cm. Trails with concentrations >10 pg/cm were repellent to workers. Workers did not detect pheromone gradients, responding equally to increasing or decreasing gradients of dodecatrienol, and termite workers were not able to differentiate between different concentrations of dodecatrienol. Termites preferred dodecatrienol trails to 2-phenoxyethanol trails. Antennae played a key role in trail pheromone perception. Dodecatrienol acted as an arrestant for worker termites (10 fg/cm²) and male alates (5 ng/cm²), whereas sternal gland extracts from females attracted male alates. Workers and alates, upon contact with filter paper disks treated with higher doses (10 fg/cm² and 5 ng/cm², respectively) of dodecatrienol, were highly excited (increased antennation and palpation) and repeatedly returned to the treated disks. Dodecatrienol did not act as a phagostimulant when offered on a paper towel disk. Reticulitermes hesperus is highly responsive to dodecatrienol, and it may play an important role in orientation of workers and alates.

Full text of DOI:10.1007/s10886-006-9229-2

J Chem Ecol (2007) 33:369–389
DOI 10.1007/s10886-006-9229-2
Role of (3Z,6Z,8E)-Dodecatrien-1-ol in Trail Following,
Feeding, and Mating Behavior of Reticulitermes hesperus
Raj K. Saran & Jocelyn G. Millar & Michael K. Rust
Received: 22 November 2005 /Revised: 26 October 2006 /Accepted: 9 November 2006 /
Published online: 3 January 2007
#
Springer Science + Business Media, LLC 2007
Abstract Trail pheromones mediate communication among western subterranean termites,
Reticulitermes hesperus Banks. Repetitive passages of ≥28 termites were required to establish
a pheromone trail and trails needed to be reinforced because they lasted <48 hr. The minimal
threshold concentration for inducing responses from termite workers and secondary
reproductives was between 0.01 and 0.1 fg/cm of (3Z,6Z,8E)-dodecatrien-1-ol (henceforth,
dodecatrienol). Workers showed optimal trail-following behavior to dodecatrienol at a
concentration of 10 fg/cm. Trails with concentrations >10 pg/cm were repellent to workers.
Workers did not detect pheromone gradients, responding equally to increasing or decreasing
gradients of dodecatrienol, and termite workers were not able to differentiate between
different concentrations of dodecatrienol. Termites preferred dodecatrienol trails to 2-
phenoxyethanol trails. Antennae played a key role in trail pheromone perception.
Dodecatrienol acted as an arrestant for worker termites (10 fg/cm2) and male alates
(5 ng/cm2), whereas sternal gland extracts from females attracted male alates. Workers and
alates, upon contact with filter paper disks treated with higher doses (10 fg/cm2 and 5 ng/cm2,
respectively) of dodecatrienol, were highly excited (increased antennation and palpation) and
repeatedly returned to the treated disks. Dodecatrienol did not act as a phagostimulant when
offered on a paper towel disk. Reticulitermes hesperus is highly responsive to dodecatrienol,
and it may play an important role in orientation of workers and alates.
.
.
.
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Keywords Antennae Arrestant (3Z,6Z,8E)-Dodecatrien-1-ol Phagostimulant
Trail pheromone
Introduction
Semiochemicals play important roles in termite colonies, where they mediate caste regulation,
reproduction, nestmate recognition, and behaviors associated with defense and foraging
(Prestwich, 1988; Pasteels and Bordereau, 1998). In the subterranean termites Reticulitermes
:
:
R. K. Saran (*) J. G. Millar M. K. Rust
Department of Entomology, University of California–Riverside, Riverside, CA 92521, USA
e-mail: raj.saran@ucr.edu

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virginicus Banks, Reticulitermes lucifugus grassei Clement, Reticulitermes speratus Kolbe,
Reticulitermes santonensis Feytaud, and Coptotermes formosanus Shiraki, the trail
pheromone or at least one of its components has been identified as (3Z,6Z,8E)-dodecatrien-
1-ol (henceforth, dodecatrienol) (Matsumura et al., 1968, 1969; Honda et al., 1975; Laduguie
et al., 1994a; Tokoro et al., 1994; Wobst et al., 1999). In addition, wood degraded by the
fungus Gloeophyllum trabeum Persoon is reported to be attractive to subterranean termites
(Watanabe and Casida, 1963; Matsumura et al., 1969; Rust et al., 1996; Su, 2005), and
extracts of the infested wood were found to contain dodecatrienol (Matsumura et al., 1969).
Termites other than Reticulitermes spp. display trail-following behavior in response to
dodecatrienol (Matsumura et al., 1972; Howard et al., 1976; Yamaoka et al., 1987; Tokoro et
al., 1989, 1994; Bordereau et al., 1991; Laduguie et al., 1994a, b; Batista-Pereira et al., 2004)
and its aromatic analogs (Tai et al., 1971; Prestwich et al., 1984). Coptotermes formosanus
and Reticulitermes spp. also exhibit trail-following behavior toward 2-phenoxyethanol (Chen
et al., 1998), a component in ballpoint pen ink. However, the importance of this compound to
termites in their natural habitats remains unknown.
After fractionation of extracts of the fourth and fifth sternites of Reticulitermes hesperus
workers, Grace et al. (1995) demonstrated that a single major component was responsible
both for induction of trail following and for orientation of termites on the trail. Studies
suggest that other minor components in trail pheromones may lead to qualitative and
quantitative differences in the actual trail laid down by worker termites (Howard et al.,
1976; Kaib et al., 1982; Runcie, 1987; Tokoro et al., 1994; Cornelius and Bland, 2001;
Arab et al., 2004). It has been suggested that time, number of termites laying the trail, and
presence or absence of a food source also has an effect on trail quality and longevity
(Runcie, 1987; Cornelius and Bland, 2001). Different aspects of termite trail-following
behavior have been studied using trails made from natural pheromone (sternal gland
extracts [SGE]), synthetic dodecatrienol, and 2-phenoxyethanol (Traniello, 1982; Grace et
al., 1988, 1995; Grace, 1991; Chen et al., 1998; Appel and Hu, 2005; Fe et al., 2005).
Minor components might also provide the basis for species- and caste-specific responses
(Affolter and Leuthold, 2000; Gessner and Leuthold, 2001), and quantitative differences in
the concentration of trail pheromone were proposed for differential response in the
recruitment behavior of soldiers and worker termites in Nasutitermes costalis Holmgren
(Traniello and Busher, 1985).
Trail pheromones have been reported to have secondary roles as sex pheromones in
various termite species. In R. santonensis Feytaud (Laduguie et al., 1994b) and Cornitermes
bequaerti Emerson (Bordereau et al., 2002), it has been suggested that dodecatrienol is a
component of the alate female’s sex pheromone. The analogous alcohol, (3Z,6Z)-
dodecadien-1-ol, has been identified as a sex and trail pheromone in Ancistrotermes
pakistanicus Ahmad (Robert et al., 2004). So far, dodecatrienol has not been shown to be
either a trail or sex pheromone of R. hesperus, and we did not detect dodecatrienol by GC/
mass spectroscopy (MS) analysis of solid phase microextraction extracts of workers and
female alates of R. hesperus (unpublished data). However, R. hesperus has been shown to
be very responsive to dodecatrienol, which has been reported to elicit trail-following
behavior at a threshold of 6 fg/cm (Matsumura et al., 1972). Grace (1986) was able to
isolate trail pheromone from excised sternites by sequential high-performance liquid
chromatography fractionation. Based on a GC and subsequent MS analysis of the active
fraction and synthetic standards [(6E, 8Z- and 6E,8E)-dodecadien-1-ol], Grace (1986)
concluded that an unsaturated dodecatrienol was present in the active fraction. However, its
structure could not be elucidated because of the minute quantities. We also were unable to

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detect dodecatrienol in solvent extracts of sternal glands of workers by GC-MS analysis,
almost certainly because of the minute quantities of dodecatrienol produced by and present
in sternal glands. However, previous studies across several species of subterranean termites
indicate that dodecatrienol elicits trail-following behavior and is likely a trail pheromone
component. Thus, we tested behavioral responses of workers and males to dodecatrienol
with the goal of investigating the role of dodecatrienol as a possible pheromonal signal for
R. hesperus. Our specific objectives were: (1) to study the preferences and minimal
thresholds for trails of natural pheromone (SGE), synthetic trail pheromone (dodecatrienol),
and 2-phenoxyethanol; (2) to determine whether termites could detect increasing or
decreasing gradients of dodecatrienol; (3) to determine the potential role of dodecatrienol as
a feeding stimulant and arrestant; and (4) to assess the responses of alate and dealate adults
to synthetic dodecatrienol and to SGE from females.
Methods and Materials
Termites
Western subterranean termites, R. hesperus Banks, were collected on the University of
California, Riverside Campus in polyvinyl chloride (PVC) traps buried in the ground and
provisioned with rolls of corrugated cardboard. Termites were gently removed and
maintained in the laboratory in a plastic container (43.3×29.9×17.7 cm, Rubbermaid
Inc., Wooster, OH, USA) at 24°C and 98% RH. Pieces of brown paper towel (Fort James
Corp., Deerfield, IL, USA) were regularly provided as a food source. Undifferentiated
larval termites [mean weight 1.6 0.44 mg (N=50)] were used for bioassays (Thorne,
1996). All worker and soldier termites used in bioassays were from the same colony.
Reticulitermes hesperus alates used in the study were collected from the PVC traps before
they swarmed and during swarms. The wings of alates collected from the traps 2–3 d before a
swarming event were still wet, grayish-white in color, and the alates were unable to fly. Alates
collected from active swarms were kept in the dark inside a plastic petri dish provisioned with
a moistened brown paper towel to avoid desiccation. Alates were allowed to acclimate for
1 hr, and individuals that shed their wings (dealates) were used for bioassays.
Termites were starved by confining them inside a plastic petri dish (5.0×0.9 cm,
Falcon, BD Biosciences, Franklin Lakes, NJ, USA) containing 5 g of sand moistened
with 1 ml of deionized water. One hundred termites were transferred to each of five
dishes. Termites were held without food for 24, 48, 72, 96, and 120 hr. Control dishes
were provisioned with brown paper towel disks (3.0 cm diam.) as a food source. All
the petri dishes were transferred to a plastic container and held at 100% RH. Humidity
indicator strips (Sude-Cheme Performance Packaging, Colton, CA, USA) were taped to
the walls of the container to ensure that RH remained at 100% throughout the studies.
To ensure that termites did not feed on dead individuals, we removed dead or moribund
termites regularly during the starvation period.
Sternal Gland Extracts
Fifty worker termites were frozen at −20°C for 5 hr. Sternal glands were dissected from the
fourth and fifth abdominal sternites under a binocular stereoscopic microscope (Grace et al.,
1988). Dissected glands was transferred to a 2-ml amber glass vial with a Teflon-lined cap

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(Wheaton, Millville, NJ, USA) kept at 0°C. The combined glands were extracted overnight
in 1 ml n-hexane. The supernatant was used for bioassays, with 20 μl considered one
termite SGE equivalent. Sternal gland extract from the female alates (N=50) was obtained
following the same procedure.
Production of Natural Trails
To determine how many termites were required to establish a pheromone trail, individual
termites were placed at one end (release end) of a Y-maze (described under “Choice
Bioassays”) and were allowed to walk on one arm of the maze. At the intersection, a
surgical blade was inserted halfway (0.5 mm) inside one of the two Y-arms to ensure that
termites walked only on one arm. To test for trail pheromone, the surgical blade was
removed and a termite was placed at one end of the Y-maze and allowed to walk up the
trail. Thirty termites were tested, recording the arm chosen by each individual. The test was
repeated using trails laid by 5, 10, 15, 20, 25, and 30 termites on the treatment arm. The
passage of 30 termites generated a significant response. To narrow the range, we repeated
the tests by increasing the number of termites in increments of 1 after 25 termites. The
procedure was repeated on the other arm of a new Y-maze to avoid any directional biases.
All Y-mazes treated with different treatments and naturally laid trails were stored
separately in sealed plastic containers at room temperature to avoid any direct effects of light
on the laid trails. The mazes were tested when the trails were 0.1, 24, 48, and 60 hr old.
Straight-line Bioassays
Straight-line bioassays were conducted to determine the concentration (fg/cm) of
dodecatrienol to which termites exhibited optimal trail-following responses. Lines were
drawn (27 cm long, <1 mm wide) with a 0.5-mm mechanical pencil on a piece of white
printer paper (21.6×27.9 cm, Boise™ X-9™, Boise, ID, USA). The line was lightly
marked with a pencil 1 cm from each end of the 27-cm line. This 25-cm-long section was
used as the walking distance for each bioassay. These lines were drawn very lightly, and
preliminary runs were conducted to ensure that termites did not use them as guidelines. A
10-μl syringe (Hamilton Company, Reno, NV, USA) was used to treat these lines (27 cm)
with hexane solutions of test treatments at the desired concentration (wt/cm) of a particular
treatment.
For each test run, 100 termites were transferred to a plastic petri dish (3.5×1.0 cm)
provisioned with a moistened brown paper towel disk. Termites were allowed to acclimate
for ∼30 min. A small acrylic paintbrush (no. 3/0 Round Red Sable, M. Grumbacher, NJ,
USA) was used to gently put a termite at one end of the treated line. A stopwatch was used
to record the time taken by termites to reach the other end of the trail. Timing began as soon
as the termite’s abdomen crossed the marked starting point of the 25-cm treated section of
the trail, and ended as soon as it crossed the opposite end point. The number of termites that
did not walk to the end of the trail, left the trail, or reversed their direction was recorded.
Time recordings of these termites were not included in the rate of movement analysis.
Seven concentrations of dodecatrienol (0.01 fg/cm, 0.1 fg/cm, 1 fg/cm, 10 fg/cm, 100 fg/cm,
1 pg/cm, and 10 pg/cm) were tested with 30 termites per concentration. To ensure that termites
did not detect any natural pheromone from a previous run, a new sheet of paper and a
dodecatrienol trail were used for every run.

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373
Gradient Bioassays
To determine whether termites responded to increasing or decreasing dodecatrienol
concentration gradients, trails were drawn on printer paper as described above. The
25-cm long section was further divided into five equal sections. We selected a range of
concentrations between 1 and 10 fg/cm to construct the gradient trail. Each 5-cm section
was treated with an increasing concentration of dodecatrienol (1, 2, 5, 8, and 10 fg/cm). A
single termite was placed at either the low concentration (increasing gradient) or the high
concentration end (decreasing gradient). The termite’s walking speed, pauses, and reversals
in direction and the number of termites leaving the trail were observed. A new gradient trail
was constructed on a new sheet of paper for each run. Thirty termites were tested on
increasing and decreasing gradient trails.
Choice Bioassays
To study different aspects of trail-following behavior of termites, natural trails (laid down
by worker termites), SGE trails, and artificial trails with dodecatrienol were used. A Y-maze
was designed from a cardboard sheet (1 mm thick), with each arm of the Y-maze being
120° apart, 5 cm in length, and 3 mm in width (Fig. 1). All the Y-mazes were cut on a die-
cutting machine such that each maze had a well-perforated margin. A no. 7 insect mounting
pin (Ward’s Natural Science, Rochester, NY, USA) was gently inserted in the Y-maze at the
center at the junction of the three arms. The other end of the pin was inserted into a Styrofoam
block. The Styrofoam served as a base to hold the Y-maze stable so that the arms were level.
A 10-μl syringe was used to treat the surface of one arm with solutions of the desired
concentration (wt/cm) of a particular treatment. The other arm of the maze was treated with
acetone or hexane only. Three concentrations of SGE (TE = termite equivalent) (0.002, 0.02,
and 0.2 TE/cm) and seven concentrations of dodecatrienol (0.01 fg/cm, 0.1 fg/cm, 1 fg/cm,
1 pg/cm, 0.01 ng/cm, 0.1 ng/cm, and 1 ng/cm) were tested. In other studies, different
concentrations of dodecatrienol were applied to the two arms of the Y-maze to determine
termites’ ability to discriminate between different concentrations. Similarly, 2-phenoxyethanol
Fig. 1 Diagram of the Y-maze
used for choice bioassays. Each
arm is 120° apart, 5 cm long, and
3 mm wide
5 cm
120°
3 mm
5 cm

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[ethylene glycol phenyl ether, (90+ percent purity), Aldrich Chemical Company, Inc.,
Milwaukee, WI, USA] trails were laid down using three concentrations (0.02 ng/cm,
0.2 ng/cm, and 10 μg/cm) on one arm and compared with the untreated arm as well as
with different dodecatrienol concentrations.
The same acrylic paintbrush described above was used to gently put a termite at the end
of one of the untreated arms, and the worker walked up to the intersection and chose
between the treated or untreated arm. The run was considered complete when the termite
walked to the end of one 5-cm arm. A new termite was used for each run, and 30 runs were
made for each test. To control for directional biases, 15 runs were conducted with the left
arm treated and 15 runs with the right arm treated. A new Y-maze was used for each run.
All bioassays were performed in a room (6.70×3.65×3.35 m) illuminated by four 32-W
white fluorescent tubes. We conducted preliminary runs in the absence of light and
determined that light did not influence the trail following ability of R. hesperus workers
(without functional eyes) and male and female dealates.
Detection of Trail Pheromone
To determine how termites detect trail pheromone, termites were tested on a bilevel Y-maze
arena. A Y-maze with one arm treated as described above was inverted and rested upon
three insect-mounting pins inserted in a Styrofoam sheet so that the Y-maze was level
(Fig. 2). The arms of another Y-maze were cut such that each arm measured 4 cm. A no. 7
pin (cut to 3.7 cm in length) was inserted through a cork such that the pointed end was
about 2 cm out on the other side. The pin and cork assembly was carefully pressed onto the
center of the shorter Y-maze so that it securely held the shorter Y-maze. The shorter Y-maze
was put directly under the inverted Y-maze (one arm treated) described above (Fig. 2). The
distance between the untreated surface of the arms of the shorter Y-maze and the inverted
Y-maze above it was ∼1.0 cm. A single termite was placed on the end of the bottom Y-
maze. The termite’s behaviors were observed when it came to the intersection, specifically
looking for any antennal response toward the treated arm above its head. The run was
terminated when a termite walked to the end of the 4-cm arm of the shorter Y-maze (Fig. 2).
For the series of assays, the distance (4 to 10 mm) between the treated inverted Y-maze and
untreated smaller Y-maze surfaces was adjusted by inserting 1-mm-thick disks (2.5 cm
diam.) of cardboard under the cork holding the smaller Y-maze.
Fig. 2 A bilevel Y-maze used to
determine whether the antennae
or palpi detected the trail phero-
mone. One arm of the top Y-maze
(each arm 5 cm long) was treated
with acetone solutions of dodec-
atrienol and the other with ace-
tone only. The arms of the shorter
Y-maze were not treated. The
distance between the top and
bottom Y-mazes was adjusted by
putting 1-mm-thick cardboard
disks under the wooden cork

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375
Arrestant Bioassays
To determine whether dodecatrienol or SGE arrested termite movement, four separate bioassays
were conducted with workers and male dealates. The bottom of a plastic petri dish (9.0 cm
diam.) was completely covered with a filter paper disk (Whatman no. 1, Whatman International
Ltd., Maidstone, UK). For the first bioassay, a small filter paper disk (2.1 cm diam., Whatman
no. 1) was treated with 10 μl of a solution of dodecatrienol in acetone (3.46 pg/ml) using a 10-μl
syringe (Hamilton). The resulting concentration of dodecatrienol on the treated disk was
10 fg/cm2. Another filter paper disk (2.1 cm diam.) was treated with 10 μl of acetone only.
Both disks were allowed to dry for 5 min under a laminar flow hood and then glued to the
bottom of the larger disk (9.0 cm diam.) so that they were 5 cm apart (between their center
points). Individual workers were released in the center of the arena. The amount of time each
termite (N=15) spent contacting each disk was recorded for 5 min. Their gnawing and
palpation behaviors on the treated disk were also observed. New disks were used for each run.
In the second bioassay, the attractiveness of dodecatrienol to male dealates (N=15) was
tested. Preliminary bioassays at different concentrations ranging from 10 fg/cm2 to 10 ng/cm2
were tested, and 5 ng/cm2 was selected for a full experiment. The amount of time spent by
male dealates contacting each disk was recorded for 5 min. Their palpation, excitement, and
gnawing behaviors on treated vs. untreated disks were visually observed. The third bioassay
was carried out using SGE from female dealates (1 female alate equivalent) on one disk and
hexane (control) on the other disk. Male dealates (N=15) were released in the arena one at a
time as described above. The amount of time each male spent contacting each disk was
recorded for 5 min. Their palpation, excitement, and gnawing behaviors on treated vs.
untreated discs were visually observed. The fourth bioassay was conducted to compare
attractiveness of dodecatrienol with that of female SGE, for male dealates (N=15). The
amount of time each male spent contacting each disk was recorded for 5 min. Their palpation,
excitement, and gnawing behaviors on treated vs. untreated discs were visually observed.
Termite workers used in the first bioassay were taken from the lab colony in cohorts of 50
termites, and were transferred to a plastic petri dish (3.5 cm diam.). The disk was provisioned
with a moistened brown paper towel disk. Termites were allowed to acclimate for 30 min.
Termites were used only once. The alates used were collected from an active swarm. They
were immediately transferred to a plastic container provisioned with moistened brown paper
towel, and dealates separated by sex were used for bioassays.
Feeding Bioassays
To determine whether dodecatrienol acted as a phagostimulant, workers were tested in a
feeding choice bioassay. Small disks of brown paper towel (2.5 cm diam.) were cut in half,
and both halves were weighed. One half was designated as treated (T) and the other as control
(C). A calculated volume of dodecatrienol solution in acetone was applied to the treated half
disk with an Eppendorf micropipetter to provide the desired concentration (pg/cm2). The
untreated half was treated with the same volume of acetone. Both disk halves were dried
under a laminar flow hood for 5 min and were then weighed again. Concentrations of 0.01,
0.1, 1, and 10 pg/cm2 and 1 ng/cm2 were tested. After drying, treated and untreated half
disks were stuck back together with a small strip of Scotch tape (3M®, St. Paul, MN,
USA). The intact disk was placed in a plastic petri dish (3.5 cm diam.×1 cm, Falcon, BD
Biosciences) with the taped side facing down. All the petri dishes with disks were then
transferred to a plastic container (43.3×29.9×17.7 cm) and were maintained at 100% RH.

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Forty termites of similar size and weight were transferred to each petri dish, the dishes were
covered with lids to keep moisture high, and the dishes were held in the container for 5 d.
Appropriate controls were used to check for mortality and amount of feeding on disks
treated with acetone only. Five replicates were conducted for each concentration. At day 5,
the termites were removed, and the two halves of the disk were separated, cleaned by gently
brushing the fecal material and dirt from them, and dried in a laminar flow hood for 24 hr.
The half disks were weighed again. Net feeding was calculated after subtracting amount of
feeding on control halves from the feeding on treated halves.
Statistical Analyses
Statistical analyses of choice tests on Y-mazes were based on a binomial distribution, using
a chi-square test (P<0.05) (SAS Institute, 1999). Results of termite feeding and straight-line
bioassays were analyzed with the general linear model analysis of variance (ANOVA)
procedure (SAS Institute, 1999). Means were separated using Tukey’s honestly significant
difference (HSD) test for significance at P<0.05. Arrestant bioassay data were analyzed by
paired t-tests.
Synthesis of (3Z,6Z,8E)-Dodecatrien-1-ol (Fig. 3)
Unless otherwise stated, reactions were run under argon atmosphere. Tetrahydrofuran
(THF) was dried by distillation from sodium-benzophenone ketyl. Solutions were dried
over anhydrous Na2SO4 and concentrated with rotary evaporation under reduced pressure,
unless otherwise stated. NMR spectra were taken on a Varian INOVA 400 NMR
spectrometer, and mass spectra were taken with a Hewlett-Packard 6890 gas chromatograph
interfaced to a Hewlett-Packard 5973 mass selective detector, using electron impact
ionization at 70 eV. Reaction yields have not been optimized.
(E)-1-Iodo-1-pentene (3) Diisobutylaluminum hydride in hexanes (1 M, 100 ml, 100 mmol)
was added dropwise to a solution of 1-pentyne 2 (7.5 g, 110 mmol) in hexanes chilled to
−40°C. The resulting mixture was stirred 30 min, then slowly warmed to room temp over
3 hr, and stirred overnight. The mixture was warmed to ∼50°C for 4 hr, then cooled to
b,c
OH
a
I
+
2
3
4
RO
d
OTHP
+
5: R = H
6: R = Tosyl
7
e,f
THPO
RO
9: R = THP
1: R = H
8
Fig. 3 Synthesis of dodecatrienol

J Chem Ecol (2007) 33:369–389
377
−40°C, and a solution of iodine (25.4 g, 100 mmol) in THF (100 ml) was added dropwise
over 30 min. The resulting dark brown suspension was allowed to warm to room temperature
with stirring overnight, producing a clear pale yellow solution. The solution was cooled in an
ice bath, and slowly quenched by dropwise addition of ice-cold H2SO4 (20%, 50 ml)
(CAUTION: EXOTHERMIC) with vigorous stirring. After stirring for 30 min, the layers
were separated, and the organic layer was washed with dilute NaHSO3 solution and brine,
and dried over MgSO4. The resulting pale yellow solution was filtered through a 2.5-cm pad
of silica, rinsing with pentane, then concentrated by rotary evaporation without heating. The
resulting pale orange oil 3 (28.9 g, 88.8% pure) was contaminated with isobutyl iodide
(8.7%), 1-iodo-1-pentyne (0.8%), and iodopentane (1.8%), but appeared to be isomerically
pure. The crude material was used without further purification. MS: m/z (%): 196 (M+, 40),
167 (22), 154 (28), 127 (13), 69 (20), and 41 (100).
(E)-Oct-4-en-2-yn-1-ol (4) A 250-ml three-neck flask fitted with a thermometer was
flushed with Ar and loaded with bis(triphenylphosphine)palladium (II) dichloride (1 g,
1.4 mmol), 0.5 g CuI (2.6 mmol), and pyrrolidine (50 ml). The mixture was stirred and
cooled in an ice bath, and crude (E)-1-iodo-1-pentene 3 (14.5 g, ∼50 mmol) was added
dropwise, followed by dropwise addition of propargyl alcohol 4 (3.5 ml, 60 mmol). The
mixture was slowly warmed to room temperature, at which point a rapid exothermic
reaction commenced, necessitating external cooling with an ice bath. After cooling the
mixture to room temperature, checking the mixture revealed a clean reaction with complete
consumption of iodoalkene 3 and production of a single product. The reaction mixture was
worked up by pouring into an ice-cold 1:1 mixture of saturated aqueous NH4Cl and 2 M
HCl (300 ml), and extracting several times with ether. The combined ether extracts were
washed with a small portion of the NH4Cl and HCl mixture and brine, dried and
decolorized with a mixture of anhydrous Na2SO4 and decolorizing charcoal, filtered
through a plug of celite, and concentrated. The resulting viscous orange oil was Kugelrohr-
distilled (oven temp ∼70°C, 1 mmHg), yielding 3.5 g of clear oil (56%). MS: 124 (M+, 96),
109 (15), 95 (42), 91 (40), 81 (100), 79 (40), 77 (34), 67 (40), 65 (41), 55 (28), 54 (26), 53
(31), 41 (48). 1H NMR: 6.14 (dt, 1H, J=15.97, 7.04 Hz, H-5), 5.48 (dtt, 1H, J=15.97, 2.05,
1.5 Hz, H-4), 4.35 (d, 2H, J=2.05 Hz, H-1), 2.09 (overlapped tdd, 2H, J∼7.5, 7.1, 1.5 Hz,
H-6), 1.91 (br s, 1H, OH), 1.41 (overlapped qt, 2H, J∼7.5, 7.3 Hz, H-7), and 0.89 (t, 3H, J
=7.3 Hz, H-8). 13C NMR: 145.8, 109.1, 85.8, 84.8, 51.8, 35.3, 22.1, and 13.8.
(E)-Oct-4-en-2-yn-1-ol Tosylate (6) Enynol 5 (3.1 g, 25 mmol) and p-toluenesulfonyl
chloride (5.4 g, 28 mmol) were dissolved in 100 ml of ether, and the solution was cooled to
∼−10°C in an ice–salt bath. With vigorous stirring, powdered KOH (16.8 g, 300 mmol) was
added in portions over 30 min, and the resulting mixture was warmed to ∼ 0°C and stirred
until the starting alcohol had been completely consumed (~1 hr). The mixture was then
poured into 100 ml of ice water, stirred for 10 min, and the layers were separated. The
aqueous layer was extracted again with ether, and the combined ether extracts were washed
with brine, dried, concentrated, and then pumped under high vacuum (∼0.2 mmHg) for 1 hr
to remove traces of solvent, yielding the tosylate as a gold oil (6.32 g). This was used
immediately in the next step without further purification.
Isopropyl magnesium chloride (2 M, 16 ml, 32 mmol) was added dropwise at room
temperature to a solution of THP-protected 3-butyn-1-ol 7 (4.62 g, 30 mmol) in 75 ml THF.
The mixture was stirred for 2 hr at room temperature, then cooled to 0°C, and CuI (0.5 g,

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2.6 mmol) was added. After stirring for 15 min, crude tosylate 6 (3.2 g, ∼12.5 mmol) in
20 ml of THF was added dropwise. The mixture was warmed slowly to room temperature
overnight in an insulated bath, then worked up by addition of saturated NH4Cl and
extraction with hexane. The hexane extract was washed with brine and dried over Na2SO4,
filtered, concentrated, and Kugelrohr-distilled (oven temperature <90°C, 0.2 mmHg) to
remove the excess THP-protected butynol. The residue was then purified by vacuum flash
chromatography (7.5% ethyl acetate [EtOAc] in hexanes), yielding the protected endiynol
8 (1.06 g), which was used immediately in the next step.
A solution of dicyclohexylborane was prepared by addition of cyclohexene (2.43 ml,
24 mmol) dropwise to a THF solution (20 ml) of borane-dimethylsulfide complex (2 M, 6 ml,
12 mmol) maintained at <−10°C in an ice–salt bath. After the addition was complete, the
mixture was slowly warmed to room temperature over 3 hr, yielding a white slurry. The mixture
was cooled to <−10°C again, and the protected enediynol (0.78 g, 3 mmol) in 10 ml THF was
added dropwise. The solution was slowly warmed to 0°C over 3 hr, and stirred at 0°C for a
further 3 hr, yielding a cloudy orange solution. Acetic acid (3 ml, 50 mmol) was then added
dropwise, and the solution was warmed to room temperature overnight. The next morning, the
mixture was cooled to 0°C again, and a solution of 4 g NaOH in 20 ml H2O was added over
10 min, followed by dropwise addition of 1.4 ml of 30% H2O2 (EXOTHERMIC). The
resulting mixture was warmed to room temperature and stirred for 30 min, poured into water,
and extracted with hexane. After washing with brine, drying over Na2SO4, and concentrating,
the residue was purified by vacuum flash chromatography (5% EtOAc in hexane). The
resulting yellow oil 9 was dissolved in MeOH (15 ml) containing ∼50 mg p-toluenesulfonic
acid, and stirred at room temperature for 3 hr. The mixture was then poured into aqueous
NaHCO3 and extracted twice with hexane. The combined hexane extracts were washed with
saturated NaHCO3 and brine, then dried over a mixture of anhydrous Na2CO3 and Na2SO4.
After filtration and concentration, the residue was purified by flash chromatography (22%
EtOAc in hexane). The purified trienol 1 was Kugelrohr-distilled (oven temperature ∼80–
90°C, 0.2 mmHg), yielding 280 mg of trienol 1, >99.5% pure by GC. MS: 180 (29), 137 (8),
133 (10), 119 (25), 105 (48), 93 (41), 91 (100), 79 (73), 77 (37), 67 (40), 55 (28), and 41
(30). 1H NMR: 6.31 (ddq, 1H, J=15.1, 11.0, 1.3 Hz, H-8), 5.97 (br t, 1 H, J=11.1 Hz, H-7),
5.69 (dt, 1H, J=15.1, 7.0 Hz, H-9), 5.56 (ddt, 1H, J=10.7, 7.3, 1.5 Hz, H-4), 5.43 (ddt, 1H,
J=10.7, 7.3, 1.5 Hz, H-3), 5.25 (dd, 1H, J=10.8, 7.5 Hz, H-6), 3.66 (br q, 2H, J=6.3 Hz,
H-1), 2.95 (br t, 2H, J=7.3 Hz, H-5), 2.37 (br q, 2H, J=6.8 Hz, H-2), 2.09 (br q, 2H, J∼
7.1 Hz, H-10), 1.42 (overlapped tq, 2H, J∼7.3, 7 Hz, H-11), and 0.91 (t, 3H, J=7.3 Hz, H-
12). All spectra were in accord with literature values (Eya et al., 1990; Argenti et al., 1994).
Results
Termites (N=30) walked on the Y-maze at an average speed of 0.73 0.12 cm/sec on the
untreated arm of the Y-maze where they were released. Once the termites reached the
intersection of the maze and turned toward the dodecatrienol treated arm (1 fg/cm), they
traveled significantly faster (t=8.08, df=29, P<0.05) with fewer delays toward the end than
on nontreated controls. The average speed on the treated arm was 1.05 0.18 cm/sec (N=30).
Production of Natural Trails
Individual termites laid trails that could be detected by other termites in significant numbers
only after about 28 termites walked along the given Y-maze treatment arm compared to the

J Chem Ecol (2007) 33:369–389
379
other control arm (χ2=13.34, df=1, P<0.001). This trail lasted for 1 d when the maze was
held inside a sealed container at room temperature (Table 1). By day 2, the trail no longer
elicited a significant response.
Straight-line Bioassays
Termites showed optimal responses to trails of 0.1 to 10 fg/cm dodecatrienol in our
bioassays. When exposed to trails made with the various concentrations of dodecatrienol,
they did not show significant differences in their mean walking speed on the trail except for
the 10 fg/cm trail (F=6.91, df=5, 29, P<0.001; Table 2), on which they walked more
quickly (1.15 cm/sec) than they did with most of the other concentrations. The percentage
of termites that walked the entire distance of the treatment trails was lower at higher
concentrations of dodecatrienol (F=17.91, df=5, 29, P<0.001; Table 2) compared with
lower concentrations (≤10 fg/cm).
Gradient Bioassays
There was no difference in trail-following behavior of the termites when they were exposed
to increasing or decreasing gradients of dodecatrienol. Their speed walking up or down
gradients was not significantly different, with termites covering the distance in about 24 sec
(increasing gradient 24.7 5.0 sec and decreasing gradient 23.7 4.5 sec). Similarly, no
differences were observed in the percentages of termites (88 to 90%) that completed the
gradient trails compared to nongradient trails.
Choice Bioassays
A minimum of 0.02 TE/cm (worker SGE) was required for trail following (Table 3). The
threshold concentration of 2-phenoxyethanol to elicit significant levels of trail following on
a Y-maze was 0.2 ng/cm. For dodecatrienol the threshold concentration was between 0.01
Table 1 The number of R. hesperus walking on the Y-maze arm before other termites followed the trail
No. of termitesa
Control/Treated
Chi-squareb
5
10
15
20
25
26
27
28
18/12
16/14
13/17
14/16
19/11
10/20
10/20
5/25
3/27
6/24
9/21
13/17
1.2
0.13
0.54
0.13
2.13
3.34
3.34
13.3
19.2
10.8
4.8
29
30
28 (24 hr)
28 (48 hr)
0.54
a Deposits fresh unless indicated by the number of hours later that the bioassay maze was tested after
deposition.
b Chi-square (χ2 ) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference between
treated (traveled by termites) and control arms, and χ2 values less than 3.84 are not significant at the 0.05 level of
significance.

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Table 2 Reticulitermes hesperus worker responses to different concentrations of dodecatrienol on a 25-cm-
long trail
Concentration
Percent of Termites Completing the Trail
Time (sec) Mean SD
0.01 fg/cm
0.1 fg/cm
1 fg/cm
10 fg/cm
100 fg/cm
1 pg/cm
0.0a
75.0b
90.9b
93.0b
51.7c
48.4cd
38.5d
0.0
28.7 6.5a
26.0 6.3ab
21.7 3.8b
29.5 6.2a
26.3 5.8a
27.4 5.4a
10 pg/cm
Mean values with the same letter in a column are not significantly different (Tukey’s HSD test, P<0.05).
and 0.1 fg/cm. Thus, R. hesperus workers were 107 times more responsive to dodecatrienol
than they were to 2-phenoxyethanol (Table 3).
Sternal gland extracts at 0.2 TE/cm elicited significant trail following for only 24 hr after
application (Table 3), whereas a 2-phenoxyethanol trail at 0.2 ng/cm on a Y-maze lasted for
24 hr, and a 1 fg/cm trail of dodecatrienol was active for at least 48 hr.
Threshold concentrations for trail following on dodecatrienol trails were similar for
workers and secondary reproductives (Table 4). Soldiers and alate males of R. hesperus were
not able to follow trails at 0.1 fg/cm dodecatrienol (Table 4). Both workers and soldiers
followed the 2-phenoxyethanol trails at equivalent threshold levels (0.2 ng/cm). Workers,
Table 3 Responses of R. hesperus workers to trails of 2-phenoxyethanol, SGE, and synthetic dodecatrienol
on a Y-maze
Compound
SGE
Concentration
Age (hr)
Control/Treated
Chi-squarea
0.002 TE/cm
0.02 TE/cm
0.2 TE/cm
0.1
0.1
0.1
24
13/17
3/27
5/25
7/23
14/16
17/13
4/26
7/23
10/20
6/24
14/16
8/22
1/29
5/25
4/26
20/10
6/24
23/7
22/8
24/6
0.53
19.2
13.34
8.54
0.13
0.53
16.13
8.54
3.34
10.8
0.13
6.54
26.1
13.3
16.13
3.34
10.8
8.54
6.54
10.8
48
2-Phenoxyethanol
Dodecatrienol
0.02 ng/cm
0.2 ng/cm
0.2 ng/cm
0.2 ng/cm
10 μg/cm
0.01 fg/cm
0.1 fg/cm
1 fg/cm
1 fg/cm
1 fg/cm
1 fg/cm
1 pg/cm
0.1
0.1
24
48
0.1
0.1
0.1
0.1
24
48
60
0.1
0.1
0.1
0.1
0.01 ng/cm
0.1 ng/cm
1.0 ng/cm
TE = termite equivalent.
a Chi-square (χ2 ) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference
between treated and control arms, and χ2 values less than 3.84 are not significant at the 0.05 level of
significance.

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381
Table 4 Responses of different castes of R. hesperus to SGE, 2-phenoxyethanol, and dodecatrienol on a
Y-maze
Treatment
Caste
Control/Treatment
Chi-squarea
SGE (Worker)
0.2 TE/cm
Worker
Soldier
SR
Worker
Soldier
SR
Worker
Soldier
SR
Alate (M)
Alate (M)
Worker
Soldier
Worker
Soldier
Worker
Soldier
SR
5/25
3/27
4/26
7/23
9/21
7/23
13/17
16/14
14/16
3/27
17/13
4/26
13.34
19.2
16.13
8.54
4.8
0.02 TE/cm
8.54
0.53
0.13
0.13
19.2
0.53
16.13
13.34
0.13
0.53
26.13
19.2
22.53
13.34
3.34
4.8
0.002 TE/cm
SGE alate (F) (0.2 TE/cm)
SGE alate (F) (0.02 TE/cm)
2-Phenoxyethanol (0.2 ng/cm)
5/25
2-Phenoxyethanol (0.02 ng/cm)
16/14
13/17
1/29
3/27
2/28
5/25
10/20
9/21
Dodecatrienol
1 fg/cm
Alate (M)
Soldier
SR
0.1 fg/cm
Alate
Soldier
SR
12/18
13/17
16/14
12/18
1.2
0.01 fg/cm
0.53
0.13
1.2
Alate
SGE = sternal gland extract, TE = termite equivalent, SGE alate (F) = sternal gland extract of female alates,
SR = secondary reproductive
a Chi-square (χ2 ) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference
between the control and treated arms, and χ2 values less than 3.84 are not significant at the 0.05 level of
significance.
secondary reproductives, and soldiers readily followed the trails of 0.02 TE of SGE/cm of
worker termites. Male alates followed the trails of 0.2 female alate SGE/cm (Table 4).
Starvation appeared to have no effect on the sensitivity of termites to dodecatrienol.
Those starved for 120 hr were still capable of following 0.1 fg/cm dodecatrienol trails on
the Y-maze (χ2=6.54, df=1, P<0.013; Table 5). However, those starved up to 120 hr were
not able to differentiate between 1 fg/cm vs. 104 fg/cm dodecatrienol trails (χ2=2.13, df=1,
P<0.18; Table 5).
Termites were not able to differentiate between different concentrations of fresh trails of
dodecatrienol or SGE when offered simultaneously on each of the two arms of the Y-maze
(Table 6). Even when the differences in concentrations of dodecatrienol were as great as
104, they did not show any preference. However, significantly more followed the higher
concentration trail after it aged for 48 hr.
When given a choice between 2-phenoxyethanol and dodecatrienol, workers generally
preferred the latter (Table 7). Only when the concentration of 2-phenoxyethanol was 1011
(0.1 mg/cm) or 1012 (1 mg/cm) times greater than dodecatrienol (1 fg/cm) did they show
significantly higher preference for the 2-phenoxyethanol trails (Table 7). Unlike

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Table 5 Trail-following behavior of R. hesperus workers starved for different durations
Starvation Period (hr)
24
Conc. (fg/cm)
Control/Treatment
Chi-squarea
1.0
0.1
0.01
1.0 vs. 104
1.0
0.1
0.01
1.0 vs. 104
5/25
7/23
16/14
12/18
4/26
8/22
14/16
11/19
13.34
8.54
0.13
1.2
16.13
6.54
0.13
2.13
24
120
120
a Chi-square (χ2 ) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference
between treated and control arms, and χ2 values less than 3.84 are not significant at the 0.05 level of
significance.
dodecatrienol (0.01 to 1 ng/cm, Table 3), at a higher concentration 2-phenoxyethanol
(1 mg/cm, Table 7) was not repellent to termite workers.
Detection of Trail Pheromone
In this experiment, a treated Y-maze was placed directly above the untreated Y-maze upon
which the test animal walked. Neither the antennae nor the mouthparts of the termites could
touch the treated surface of the Y-maze above them. When the distance between the treated
and untreated surfaces of the two Y-mazes was reduced to 4 mm, termites were able to
detect and follow the treated arm (Table 7). When the distance between the two mazes was
reduced to less than 4 mm, they were able to touch the upper Y-maze surface and in few
cases were able to climb up the maze over their heads. Dodecatrienol has relatively low
vapor pressure (estimated as 3.73×10−4 Torr at 25°C; Chemical Abstracts, 2005), and
termites were only able to detect it when their antennae were about 1–2 mm from the trail.
Increasing the concentration of dodecatrienol did not alter the distance over which it was
perceived by the antennae (Table 8). Concentrations as high as 1 ng/cm were not repellent
to workers in this setup.
Table 6 Ability of R. hesperus workers to differentiate between different concentrations of synthetic
dodecatrienol and SGE on the Y-maze
Concentration/cma of Arm A vs. Arm B
1 fg/cm vs. SGE 0.2 TE/cm
Time (hr)
A/B
Chi-squareb
0.2
24
48
0.2
24
48
0.2
24
48
13/17
14/16
12/18
16/14
11/19
8/22
13/17
12/18
9/21
0.53
0.13
1.2
0.13
2.13
6.53
0.53
1.2
1 vs. 100 fg/cm
1 vs. 104 fg/cm
4.8
a TE = termite equivalent, Arm A treated with 1 fg/cm dodecatrienol
b Chi-square (χ2 ) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference
between two treated arms, and χ2 values less than 3.84 are not significant at the 0.05 level of significance.

J Chem Ecol (2007) 33:369–389
383
Table 7 Ability of R. hesperus workers to differentiate between synthetic dodecatrienol and 2-
phenoxyethanol trails
Concentration (fg/cm)
Dodecatrienol
Chi-squarea
2-Phenoxyethanol
Dodecatrienol/2-P
1
1
1
1
1
1
1
1
1
28/2
24/6
27/3
24/6
23/7
16/14
6/24
8/22
22.53
10.8
19.2
10.8
8.54
0.13
10.8
6.54
103
105
107
109
1010
1011
1012
2-P = 2-phenoxyethanol.
a Chi-square (χ2 ) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference
between two treated arms, and χ2 values less than 3.84 are not significant at the 0.05 level of significance.
Arrestant Bioassays
Both worker termites and male dealates spent more time on disks treated with 10 fg/cm2
and 5 ng/cm2 dodecatrienol, respectively, than on control disks (Fig. 4A, t=4.45, df=14, P
<0.05; Fig. 4B, t=3.61, df=14, P<0.05). Male dealates also spent more time (t=10.62, df=
14, P<0.05) on disks treated with female alate extract (SGE, 0.28 TE/cm2) than on control
disks (Fig. 4C). When female alate SGE was directly compared to dodecatrienol, male
dealates spent more time (t=6.10, df=14, P<0.05) on SGE-treated disks (0.28 termite
equivalent/cm2) than disks treated with dodecatrienol (5 ng/cm2, Fig. 4D).
Feeding Bioassays
The total amount of feeding on treated and untreated paper combined at various
dodecatrienol concentrations was not significantly different (F=2.45, df=5, 24, P=0.06).
Total feeding on the treated and control halves combined ranged from 5.9 to 3.9 mg 40
termites−1 5 d−1. Net feeding on the treatments was determined by subtracting the amount
of feeding on the control half of the disk from feeding on the treated half. Net feeding on
treated halves ranged from −1.6 to +1.9 mg 40 termites−1 5 d−1. No consistent trend of
feeding on either the control or treated half was observed. There were no significant
differences in the amount of net feeding on paper towel disk halves treated with different
concentrations of dodecatrienol (F=1.92, df=5, 20, P=0.15).
Table 8 Detection of trail pheromone by R. hesperus workers on the double Y-maze arms
Weight/cm on Y-maze Arm
Distance Between Arms (mm)
Control/Treatment
Chi-squarea
1 fg
1 fg
1 fg
1 ng
1 ng
10
5
4
5
4
12/18
11/19
6/24
10/20
7/23
1.2
2.13
10.8
3.34
8.54
a Chi-square (χ2 ) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference
between two treated arms, and χ2 values less than 3.84 are not significant at the 0.05 level of significance.

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J Chem Ecol (2007) 33:369–389
70
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
Worker
a
b
Dealate (Male)
*
*
Control Dodecatrienol
Dodecatrienol
Control
160
140
120
100
80
160
140
120
100
80
c
d
Dealate (Male)
Dealate (Male)
*
*
60
60
40
40
20
20
0
0
Female (SGE)
Female (SGE)
Control
Dodecatrienol
Fig. 4 Time spent (sec) on disks treated with dodecatrienol or female alate SGE by workers (a) and male
dealates (b–d). Asterisk denotes means that are significantly different at P<0.05 (paired t-test)
Discussion
Individual termites, when allowed to walk on the Y-maze substrate, did not establish a trail
that was recognized by conspecifics. Instead, it took a minimum of 28 termites walking
over the same trail to establish a reliably detectable pheromone trail. This suggests that
during its routine movements, a new recruit worker will prefer trails that have had
considerable traffic, and these trails must be reinforced to remain effective. Grace et al.
(1988) also suggested that R. hesperus termites are preferentially recruited to more well
traveled trails.

J Chem Ecol (2007) 33:369–389
385
Similar observations were made by Runcie (1987) concerning the trail-following
behavior of R. flavipes, where trails in tubes with heavier termite traffic lasted longer than
trails in tubes that were used less frequently. Single termite workers were allowed to lay
trails in glass tubes and superimposed trails of as many as 50 workers remained active for
only 5 min, whereas in our study trails superimposed by 28 termites remained active for
24 hr. These differences may be attributed to the properties of the substrate over which the
trail was laid. In our study, the Y-maze was made of cardboard instead of a glass surface.
The concentrations of the trails did not affect the speed at which termites completed the
trails. However, the concentration did affect the percentage that completed the trail
bioassay. It took more than 30 termites to achieve 30 replicates in which the termite walked
the entire length of the 25 cm trail because some veered off the trail before reaching the
end. Termites exposed to 0.01 and 0.1 fg/cm trails walked slowly and stopped frequently,
trying to sense the trail by actively waving their antennae (sampling substrate) on both sides
of the trail. There was a limited range of dodecatrienol concentrations (0.1–10 fg/cm) to
which the termites showed optimal trail-following behavior. The threshold concentration
determined in our study for R. hesperus was ∼60–600-fold lower than previously reported
by Matsumura et al. (1972). The difference may be due to the purity of the synthetic
dodecatrienol used in the two studies, or to the different substrates on which the trails were
laid. In another study, the reported threshold concentration of dodecatrienol for R. flavipes
was <0.01 pg/cm (Matsumura et al., 1968). On trails of higher concentration (>10 fg/cm),
more termites failed to complete the trail or stopped occasionally and reversed direction,
resulting in delays to reach the end of the treated line, than did termites on trails with
between 1 and <10 fg/cm. Similar results have been reported by other researchers using
different termite species (Grace, 1986; Laduguie et al., 1994b; Wobst et al., 1999; Peppuy et
al., 2001; Arab et al., 2004; Robert et al., 2004). In our study, this effect manifested itself
through a lower percentage of termites completing the trails at higher concentrations within
the bioassay time period. Consequently, the walking speed of termites completing the trail
was significantly greater for only one concentration (10 fg/cm), and there were no
differences in the times taken to complete the trail among the other concentrations. As
suggested by Prestwich et al. (1984), such differences may be related to the optimal
response behavior of termites to the trail and have less to do with discriminatory ability.
On the Y-maze and the 25-cm long trail, significantly higher numbers of termites
responded to 0.1 and 1 fg/cm treatments compared to untreated controls. The minimal
threshold concentration of dodecatrienol eliciting trail following was between 0.01 and
0.1 fg/cm. The 1 fg/cm trail remained active for 48 hr. This may be because of the fact that
lower concentrations volatilized from the substrate to the point where they were no longer
detectable in the bioassay; higher concentrations probably still had some material “loaded”
on the substrate. The natural trails and SGE trails (0.2 TE/cm) were active only for 24 hr.
Thus, trails have to be reinforced by termites if they are to remain active. Runcie (1987)
showed that R. flavipes trails leading to a food source were highly persistent compared to
exploration trails. Affolter and Leuthold (2000) and Gessner and Leuthold (2001) reached a
similar conclusion for trail-following behavior of Macrotermes subhyalinus Rambur. For
another species, Nasutitermes costalis, extracts of 4-yr-old paves, aboveground guidelines
formed by feces and debris by foragers, elicited orientation behavior (Traniello, 1982).
However, M. subhyalinus and N. costalis have considerably different foraging and nesting
behavior compared to Reticulitermes species, and observations on behavior of different
species cannot be readily generalized. For example, termites starved for 120 hr were not
able to discriminate between different concentrations of dodecatrienol, suggesting that R.

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J Chem Ecol (2007) 33:369–389
hesperus does not use concentration to differentiate between trails leading to food sources
or for exploration. Under natural conditions, it seems more likely that the most highly used
trails from the colony are those either leading directly to a food source, or foraging trails in
a conducive soil environment.
Reticulitermes spp. rarely forage in the open, instead spending most of their time
foraging underground in galleries constructed with a number of termites working on each
tunnel. In nature, other factors such as tactile stimuli from workers returning from a food
source, salivary secretions used to construct tunnels, moisture levels, and the age of the
tunnel may provide additional signals.
Reinhard and Kaib (2001) showed that soldiers of R. santonensis responded only to
trails of higher concentration from SGE of worker termites. In N. costalis, worker termites
were recruited at higher concentrations compared to soldiers and third instar workers had
larger sternal glands compared to soldiers, considered to be responsible for differential
recruiting effects (Traniello and Busher, 1985). In our study, different castes of R. hesperus
(worker, soldier, secondary reproductives, and male alates) responded equally well to
dodecatrienol trails at 1 fg/cm. However, at 0.1 fg/cm, soldiers and male alates did not
respond significantly more toward the treated arm, and 25% of workers tested failed to
complete the 25-cm 0.1 fg/cm dodecatrienol trail.
Differences in threshold concentrations for various trail following chemicals have been
reported in various subterranean termite genera and species (Matsumura et al., 1972; Arab et
al., 2004). As suggested by other researchers (Howard et al., 1976; Kaib et al., 1982; Tokoro
et al., 1994; Cornelius and Bland, 2001), termites of different genera and species may respond
to dodecatrienol as a common component of a trail following signal, and there also may be
other minor components involved, which may be responsible for species-specific signals.
We chose dodecatrienol concentrations of 1 fg/cm to compare with higher concentrations
of dodecatrienol because >90% of the termites were able to follow 1 fg/cm trails. The fact
that termites were not able to discriminate between different dodecatrienol concentrations
on the two arms of the Y-maze suggests that in nature, trails contain largely qualitative
information. Dodecatrienol appeared to act as a repellent at higher concentrations
(≥0.01 ng/cm, Table 3). However, when the 0.01-ng/cm concentration was offered
simultaneously with 1 fg/cm in a choice assay, termites did not discriminate between
either concentration. Furthermore, when offered a choice between dodecatrienol (1 fg/cm)
and SGE (1 TE/cm), termites were not able to discriminate between them. This suggests
that dodecatrienol is a key component of the SGE signal.
Grace et al. (1988) reported that R. hesperus showed a differential response to a
concentration gradient of SGE. In contrast, our studies indicated that R. hesperus did not
respond to a concentration gradient and simply followed the trail. The concentrations
selected for our gradient trails were based upon those used in the preliminary straight-line
bioassays. Grace (1986) reported that to discriminate between increasing and decreasing
gradients the difference on the treated sections must be a little over 10 times. The results
suggested that termites showed optimal trail-following behavior at concentrations of
between 1 and 10 fg/cm. This range seems more realistic and closer to the concentrations of
trails laid by termites in nature. The ability of individual termites to either increase the
production of trail pheromone or make it more concentrated so as to provide ≥100-fold
differences in deposition on trails in nature seems to be unlikely. In insects such as pharaoh
ants, Monomorium pharaonis, where trail pheromone is used for trail following, it has been
demonstrated that trail geometry rather than a concentration gradient provides polarity to

J Chem Ecol (2007) 33:369–389
387
ant foraging networks (Duncan et al., 2004). Their results showed that bifurcation angles
similar to those found in natural networks (∼60°) give the best reorientation ratios.
Termites were able to detect the dodecatrienol trails only when their antennae were very
close to the trail (about 1–2 mm). The mouthparts apparently did not play a role in detecting
the trail and initiating trail following. During normal trail following in termites, maxillary
palpi are closer to the substrate than the antennae. When termites were allowed to walk on
trail deposits as high as 1 ng/cm of dodecatrienol, they were repelled. In contrast, con-
centrations as high as 1 ng/cm were not repellent to termites in the bilevel Y-maze in which
the antennae and palpi were not directly touching the treated surface. Together, these results
suggest that the maxillary palpi may be involved in detecting repellent concentrations of
dodecatrienol or perhaps the absence of repellency in the bilevel maze is just because of the
additional distance of the antennae from the odor source.
In contrast, arrestant assays showed that male dealates were not repelled by dodecatrienol at
relatively high concentrations, with dealates spending more time on treated disks than on
controls. In other studies (Laduguie et al., 1994a, b; Robert et al., 2004), it has been suggested
that at higher concentrations, dodecatrienol may have another role as a sex pheromone.
However, male dealates spent more time on disks treated with extracts of female sternal
glands than on disks treated with dodecatrienol, suggesting that other components are
probably involved, which may act as synergists with dodecatrienol. On the basis of chemical
analysis of extracts of R. santonensis (Laduguie et al., 1994a, b), female alates produced 10 to
100 pg of dodecatrienol, approximately 10 times more than the worker termites. Thus,
dodecatrienol may be present as one of the components in female sex pheromone, serving to
attract male alates for mating at higher concentrations, whereas at lower concentrations, it
may act as a trail-following signal, helping in forming tandem pairs during mating.
Earlier studies (Matsumura et al., 1968; Esenther and Beal, 1979; Rust et al., 1996)
suggested that wood degraded by G. trabeum was attractive to Reticulitermes spp., and
dodecatrienol was isolated from fungus-degraded wood. Extracts of fungus-degraded wood
can be used as arrestants in termite baits and have potential to increase bait station fidelity
(Rust et al., 1996). In our feeding studies, we did not observe any phagostimulant or
attractant properties of dodecatrienol applied to brown paper towel disks. When termites
were transferred to petri dishes in which one half of the disk had been treated with
dodecatrienol, they showed greater excitement and movement than termites in untreated
controls, and it took longer for them to settle down. Thus, the attractive properties and
increased feeding on wood degraded by G. trabeum may be due more to the effect of the
degradation process on the wood composition or to other as yet unidentified compounds
rather than the production of dodecatrienol.
Overall, our studies indicated that the context in which dodecatrienol was presented was
critically important. When worker termites were offered a paper disk treated with a spot of
dodecatrienol much larger than a termite rather than as a thin line, workers became excited
and showed random movements, retouched the spot, and remained in contact with and
around the treated disk. Workers perceived the stimulus, but they were not completely
arrested by the stimulus. Termites exhibited normal trail-following behavior only when
dodecatrienol was presented to them in its normal context, as a thin line. Concentration did
not seem to play an important role in routine trail following but may have a role in mating
behavior of alates.
Acknowledgments We thank the Carl/Strom Western Exterminator Scholarships for partially funding this
research.

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