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

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/cm²and 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.

.

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

J Chem Ecol (2007) 33:369–389

371

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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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

J Chem Ecol (2007) 33:369–389

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/cm²to 10 ng/cm²

were tested, and 5 ng/cm²was 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/cm²and 1 ng/cm²were 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 Na₂SO₄and 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 NaHSO₃solution 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 Na₂SO₄and 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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J Chem Ecol (2007) 33:369–389

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

NaHCO₃and extracted twice with hexane. The combined hexane extracts were washed with

saturated NaHCO₃and brine, then dried over a mixture of anhydrous Na₂CO₃and 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 termites^a

Control/Treated

Chi-square^b

5

10

15

20

25

26

27

28

18/12

16/14

13/17

14/16

19/11

10/20

5/25

3/27

6/24

9/21

13/17

1.2

0.13

0.54

0.13

2.13

3.34

13.3

19.2

10.8

4.8

29

30

28 (24 hr)

28 (48 hr)

0.54

^aDeposits fresh unless indicated by the number of hours later that the bioassay maze was tested after

deposition.

^bChi-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 χ²values 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 10⁷times 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-square^a

0.002 TE/cm

0.02 TE/cm

0.2 TE/cm

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

10 μg/cm

0.01 fg/cm

0.1 fg/cm

1 fg/cm

1 pg/cm

0.1

24

48

0.1

24

48

60

0.1

0.01 ng/cm

0.1 ng/cm

1.0 ng/cm

TE = termite equivalent.

^aChi-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 χ²values less than 3.84 are not significant at the 0.05 level of

significance.

J Chem Ecol (2007) 33:369–389

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-square^a

SGE (Worker)

0.2 TE/cm

Worker

Soldier

SR

Worker

Soldier

SR

Worker

Soldier

SR

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

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

^aChi-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 χ²values 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. 10⁴fg/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-square^a

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

^aChi-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 χ²values 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 3.73×10⁻⁴Torr 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/cm^aof Arm A vs. Arm B

1 fg/cm vs. SGE 0.2 TE/cm

Time (hr)

A/B

Chi-square^b

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. 10⁴fg/cm

4.8

^aTE = termite equivalent, Arm A treated with 1 fg/cm dodecatrienol

^bChi-square (χ²) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference

between two treated arms, and χ²values 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-square^a

2-Phenoxyethanol

Dodecatrienol/2-P

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.

^aChi-square (χ²) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference

between two treated arms, and χ²values 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/cm²dodecatrienol, 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⁻¹5 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⁻¹5 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-square^a

1 fg

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

^aChi-square (χ²) analysis is based on a binomial probability of 1:1 for a null hypothesis of no difference

between two treated arms, and χ²values less than 3.84 are not significant at the 0.05 level of significance.

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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)

*

60

40

20

0

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.

388

J Chem Ecol (2007) 33:369–389

References

AFFOLTER, J. and LEUTHOLD, R. H. 2000. Quantitative and qualitative aspects of trail pheromones in

Macrotermes subhyalinus (Isoptera, Termitidae). Insectes Soc. 47:256–262.

APPEL, A. G. and HU, X. P. 2005. Perception of trail-following substances by Reticulitermes flavipes

(Isoptera: Rhinotermitidae) using behavioral bioassays. Sociobiology 45:151–162.

ARAB, A., COSTA-LEONARDO, A. M., BATISTA-PEREIRA, L. G., DOS SANTOS, M. G., CORREA, A. G., and

BLANCO, Y. C. 2004. Trail pheromone specificity of two sympatric termites from Southeastern Brazil.

Sociobiology 43:377–387.

ARGENTI, L., BELLINA, F., CARPITA, A., ROSSI, E., and ROSSI, R. 1994. Trail-following in termites:

stereoselective syntheses of (Z)-3-dodecen-1-ol, (3Z,6Z)-3,6-dodecadien-1-ol, and (3Z,6Z,8E)-3,6,8-

dodecatrien-1-ol. Synth. Commun. 24:2281–2297.

BATISTA-PEREIRA, L. G., SANTOS, M. G., CORREA, A. G., FERNANDES, J. B., DIETRICH, C. R. R. C.,

PEREIRA, D. M., BUENO, O. C., and COSTA-LEONARDO, A. M. 2004. Electroantennographic responses of

Heterotermes tenuis (Isoptera: Rhinotermitidae) to synthetic (3Z,6Z,8E)-dodecatrienol. J. Braz. Chem.

Soc. 15:372–377.

BORDEREAU, C., ROBERT, A., BONNARD O., and LE QUERE, J.-L. 1991. (3Z,6Z,8E)-3,6,8-dodecatrien-1-ol:

sex pheromone in a higher fungus-growing termite, Pseudacanthotermes spiniger (Isoptera, Macro-

termitinae). J. Chem. Ecol. 17:2177–2191.

BORDEREAU, C., CANCELLO, E. M., SEMON, E., COURRENT, A., and QUENNEDEY, B. 2002. Sex pheromone

identified after solid phase microextraction from tergal glands of female alates in Cornitermes bequaerti

(Isoptera: Nasutitermitinae). Insectes Soc. 49:209–215.

CHEMICAL ABSTRACTS. 2005. Calculated using Advanced Chemistry Development (ACD/Labs) Software

CHEN, J., HENDERSON, G., and LAINE, R. A. 1998. Isolation and identification of 2-phenoxyethanol from

ballpoint pen ink as a trail-following substance of Coptotermes formosanus Shiraki and Reticulitermes

sp. J. Entomol. Sci. 33:97–105.

CORNELIUS, M. L. and BLAND, J. M. 2001. Trail-following behavior of Coptotermes formosanus and

Reticulitermes flavipes (Isoptera: Rhinotermitidae): is there a species specific response? Environ.

Entomol. 30:457–465.

DUNCAN, E. J., HOLCOMBE, M., and RATNIEKS, F. L. W. 2004. Trail geometry gives polarity to ant foraging

networks. Nature 432:907–909.

ESENTHER, G. R. and BEAL, R. H. 1979. Termite control: decayed wood bait. Sociobiology 4:215–222.

EYA, B. K., OTSUKA, T., KUBO, I., and WOOD, D. L. 1990. Syntheses and analyses of the eight geometric

isomers of 3,6,8-dodecatrienol, subterranean termite trail pheromone. Tetrahedron 46:2695–2706.

FE, H. X., HENDERSON, G., and LAINE, R. A. 2005. Trail-following behavior of Coptotermes formosanus

(Isoptera: Rhinotermitidae) on concentration gradients of 2-phenoxyethanol. Sociobiology 45:483–494.

GESSNER, S. and LEUTHOLD, R. H. 2001. Caste-specificity of pheromone trails in termite Macrotermes

bellicosus. Insectes Soc. 48:238–244.

GRACE, J. K. 1986. Chemically Mediated Behavior of the Western Subterranean Termite, Reticulitermes

hesperus Banks (Isoptera: Rhinotermitidae). Ph.D. thesis, University of California, Berkeley, CA, USA.

GRACE, J. K. 1991. Semiochemical mediation and manipulation of Reticulitermes behavior (Isoptera:

Rhinotermitidae). Sociobiology 19:147–162.

GRACE, J. K., WOOD, D. L., and FRANKIE, G. W. 1988. Trail following behavior of Reticulitermes hesperus

Banks (Isoptera: Rhinotermitidae). J. Chem. Ecol. 14:653–667.

GRACE, J. K., WOOD, D. L., KUBO, I., and KIM, M. 1995. Behavioral and chemical investigation of trail

pheromone from the termite Reticulitermes hesperus Banks (Isoptera, Rhinotermitidae). J. Appl.

Entomol. 119:501–505.

HONDA, R., OSHIMA, K., and YAMAMOTO, I. 1975. Chemical nature of trail-following substance from a

Japanese termite, Reticulitermes speratus Kolbe. J. Agric. Sci. Tokyo Univ. Agric. 20:121–128.

HOWARD, R., MATSUMURA, F., and COPPEL, H. C. 1976. Trail-following pheromones of the Rhinotermitidae:

approaches to their authentication and specificity. J. Chem. Ecol. 2:147–166.

KAIB, M., BRUINSMA, O., and LEUTHOLD, R. H. 1982. Trail-following in termites: evidence for a

multicomponent system. J. Chem. Ecol. 8:1193–1205.

LADUGUIE, N., ROBERT, A., SALINI, N., and BORDEREAU, C. 1994a. Les termites et le dodecatrienol. Actes

Colloq. Insectes Soc. 9:27–34.

LADUGUIE, N., ROBERT, A., BONNARD, O., VIEAU, F., LE QUERE, J.-L., SEMON, E., and BORDEREAU, C.

1994b. Isolation and identification of (3Z,6Z,8E)-3,6,8-dodecatrien-1-ol in Reticulitermes santonensis

Feytaud (Isoptera, Rhinotermitidae): roles in worker trail-following and in alate sex-attraction behavior.

J. Insect Physiol. 40:781–787.

J Chem Ecol (2007) 33:369–389

389

MATSUMURA, F., COPPEL, H. C., and TAI, A. 1968. Isolation and identification of termite trail-following

pheromone. Nature 219:963–964.

MATSUMURA, F., TAI, A., and COPPEL, H. C. 1969. Termite trail-following substance, isolation and

purification from Reticulitermes virginicus and fungus-infected wood. J. Econ. Entomol. 62:599–603.

MATSUMURA, F., JEWETT, D. M., and COPPEL, H. C. 1972. Interspecific response of termites to synthetic

trail-following substance. J. Econ. Entomol. 65:600–602.

PASTEELS, J. M. and BORDEREAU, C. 1998. Releaser pheromones in termites, pp. 193–215, in R. K. Vander

Meer, M. D. Breed, K. E. Espelie, and M. L. Winston (eds.). Pheromone Communication in Social

Insects. Westview Press, CO, USA.

PEPPUY, A., ROBERT, A., SEMON, E., GINIES, C., LETTERE, M., BONNARD, O., and BORDEREAU, C. 2001.

(Z)-dodec-3-en-1-ol, a novel trail pheromone identified after solid phase microextraction from

Macrotermes annandalei. J. Insect Physiol. 47:445–453.

PRESTWICH, G. D. 1988. The chemicals of termite societies (Isoptera). Sociobiology 14:176–191.

PRESTWICH, G. D., ENG, W.-S., DEATON, E., and WICHERN, D. 1984. Structure–activity relationships among

aromatic analogs of the trail-following pheromone of subterranean termites. J. Chem. Ecol. 10:1201–

1217.

REINHARD, J. and KAIB, M. 2001. Trail communication during foraging and recruitment in the subterranean

termite Reticulitermes santonensis De Feytaud (Isoptera, Rhinotermitidae). J. Insect Behav. 14:157–171.

ROBERT, A., PEPPUY, A., SEMON, E., BOYER, F. D., LACEY, M. J., and BORDEREAU, C. 2004. A new C12

alcohol identified as a sex pheromone and a trail-following pheromone in termites: the diene (Z,Z)-

dodeca-3,6-dien-1-ol. Naturwissenschaften 91:34–39.

RUNCIE, C. D. 1987. Behavioral evidence for multicomponent trail pheromone in the termite, Reticulitermes

flavipes (Kollar) (Isoptera: Rhinotermitidae). J. Chem. Ecol. 13:1967–1978.

RUST, M. K., HAAGSMA, K., and NYUGEN, J. 1996. Enhancing foraging of western subterranean termites

(Isoptera: Rhinotermitidae) in arid environments. Sociobiology 28:275–286.

SAS Institute Inc. 1999. SAS/STAT User’s Guide, Version 8.2. SAS Institute, Cary, NC.

SU, N.-Y. 2005. Directional change in tunneling of subterranean termites (Isoptera: Rhinotermitidae) in

response to decayed wood attractants. J. Econ. Entomol. 98:471–475.

TAI, A., MATSUMURA, F., and COPPEL, H. C. 1971. Synthetic analogues of the termite trail following

pheromone, structure and biological activity. J. Entomol. Sci. 33:97–105.

THORNE, B. L. 1996. Termite terminology. Sociobiology 28:253–263.

TOKORO, M., TAKAHASHI, M., TSUNODA, K., and YAMAOKA, R. 1989. Isolation and primary structure of

trail pheromone of the termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae). Wood Res.

76:29–38.

TOKORO, M., TAKAHASHI, M., and YAMAOKA, R. 1994. (Z, E, E)-3,6,8-dodecatrien-1-ol: a minor component

of trail pheromone of termite Coptotermes formosanus Shiraki. J. Chem. Ecol. 20:199–215.

TRANIELLO, J. F. A. 1982. Recruitment and orientation components in a termite trail pheromone.

Naturwissenschaften 69:343–345.

TRANIELLO, J. F. A. and BUSHER, C. 1985. Chemical regulation of polyethism during foraging in the

neotropical termite Nasutitermes costalis. J. Chem. Ecol. 11:318–332.

WATANABE, T. and CASIDA, J. E. 1963. Response of Reticulitermes flavipes to fractions from fungus-infected

wood and synthetic chemicals. J. Econ. Entomol. 56:300–307.

WOBST, B., FARINE, J., GINIES, C., SEMON, E., ROBERT, A., BONNARD, O., CONNETABLE, S., and

BORDEREAU, C. 1999. (Z,Z,E)-3,6,8-dodecatrien-1-ol, a major component of trail-following pheromone

in two sympatric termite species Reticulitermes lucifugus and R. santonensis. J. Chem. Ecol. 25:1305–

1318.

YAMAOKA, R., TOKORO, M., and HAYASHIYA, K. 1987. Determination of geometric configuration in minute

amounts of highly unsaturated termite trail pheromone by capillary gas chromatography in combination

with mass spectrometry and Fourier-transform infrared spectroscopy. J. Chromatogr. 339:259–267.

Article Doi

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

DOI: 10.1007/s10886-006-9229-2

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