Journal of Chemical Ecology, Vol. 25, No. 6, 1999

(Z,Z,E)-3,6,8-DODECATRIEN-1-OL, A MAJORCOMPONENT OF TRAIL-FOLLOWING PHEROMONE

IN TWO SYMPATRIC TERMITE SPECIES Reticulitermeslucifugus grassei AND R. santonensis

BIRGIT WOBST,1 JEAN-PIERRE FARINE,1 CHRISTIAN GINIES,2

ETIENNE SEMON,2 ALAIN ROBERT,1 ODILE BONNARD,1

SOPHIE CONNETABLE,1 and CHRISTIAN BORDEREAU1-*

1 Universite de Bourgogne, Laboratoire de ZoologieC.N.R.S. UMR 5548 " Developpement, Communication Chimique"

6 Bd. Gabriel, 21000 Dijon, France2I.N.R.A. Laboratoire de Recherches sur les Aromes

17 Rue Sully, 21034 Dijon, France

(Received May 4, 1998; accepted February 5, 1999)

Abstract—Trail-following bioassays show that trails of the two subterraneantermites, Reticulitermes lucifugus grassei and R. santonensis, which are sym-patric in some areas of southwestern France, are not species specific. Even whentested just above threshold level, trails from pentane extracts of whole workers ofR. santonensis are always preferred by both species. If the R. santonensis extractis progressively diluted, the preference is lost. In purified extracts of workers ofR. lucifugus grassei that elicited a trail-following response, only one compoundis active. It was identified by GC-MS as the same major compound of the trail-following pheromone of R. santonensis: (Z,Z,E)-3,6,8-dodecatrien-1-ol (DTE-OH). The threshold concentration of activity of synthetic DTE-OH was deter-mined to be 10-3 ng/cm of trail; optimal activity was obtained at 10-2 ng/cmof trail. The increase of trail-following activity of worker extracts of R. lucifugusgrassei after hydrolysis by potassium hydroxide suggests that DTE-OH also isbound to other components in sternal gland secretions. DTE-OH was also iden-tified in alates of R. lucifugus grassei, suggesting that the compound functionsboth as a trail-following and a sex pheromone, as has been shown to be the casein R. santonensis. This demonstrates the high economy developed by termites intheir strategies of chemical communication.

Key Words—Trail-following pheromones, (Z,Z,E)-3,6,8-dodecatrien-1-ol,specificity, termites, Reticulitermes lucifugus grassei, Reticulitermes santonensis,GC-MS.

*To whom correspondence should be addressed.

0098-0331/99/0600-1305$16.00/0 © 1999 Plenum Publishing Corporation

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INTRODUCTION

In termites, chemical communication plays a key role in the regulation and theintegration of most of their activities. However, only a few pheromones havebeen identified in these social insects (Pasteels and Bordereau, 1998). Since theend of the nineteenth century termites have been known to forage along odor-ous trails (Dudley and Beaumont, 1889), but only two compounds have beenidentified as trail-following pheromones. (Z,Z,E)-3,6,8-Dodecatrien-l-ol (DTE-OH) was first identified in Reticulitermes virginicus (Matsumura et al., 1968;Tai et al., 1969) and then in other Rhinotermitidae, R. speratus (Yamaoka etal., 1987) and Coptotermes formosanus (Tokoro et al., 1989).(1E,5E,9E)-Neo-cembrene is the trail-following pheromone of Nasutitermes exitiosus (Birch etal., 1972; Kodama et al., 1975) and Trinervitermes bettonianus (McDowell andOloo, 1984). These results raise the question of how specific termite pheromonesare and how termite societies remain isolated.

In our study we were interested in the two species of European Rhinoter-mitidae that are sympatric in some areas in France, Reticulitermes santonen-sis Feytaud and R. lucifugus grassei Clement. R. santonensis is distributed inwestern France and R. lucifugus grassei in southwestern France. These speciescoexist around Rochefort and Bordeaux (Clement, 1977). Although the specificstatus of these termites was controversial in the past (Lash, 1952; Buchli, 1958),different studies have shown separation between R. lucifugus, which originallylived in southern France, and R. santonensis, which may have been introducedinto western France from the United States (Feytaud, 1924, 1925; Clement, 1978,1979; Bagneres et al., 1990, 1991; Vieau, 1993). From a biometrical study onEuropean species of R. lucifugus, Clement (1978) distinguished four subspeciesand named R. lucifugus grassei the subspecies living in southwestern France.

The aim of our study was to compare the trail-following pheromones ofthe two species R. santonensis and R. lucifugus grassei. The trail pheromone ofR. santonensis was previously identified as DTE-OH (Laduguie et al., 1994a).Here we report the identification of the trail-following pheromone of R. lucifugusgrassei.

METHODS AND MATERIALS

Biological Material. Species determinations were made according toClement (1978) and Vieau (1993). Colonies of R. lucifugus grassei Clementwere collected in stumps of pine wood and infested abandoned huts in southwest-ern France (Bordeaux, Carcans, Lacanau). Colonies of R. santonensis were col-lected in western and southwestern France (Les Sables d'Olonne, He d'Oleron,Saint Jean de Monts, Saint Medard). Colonies of both species were collected in

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November 1995 and May 1996 and reared in the laboratory at 25°C, 80% humid-ity, and a 12L: 12D period. Alates were collected from laboratory colonies andin the fields as mentioned above.

Preparation of Termite Extracts. Workers were separated from colonies andheld for two days on moistened filter paper to remove intestinal contents. Indi-viduals were counted and weighed before extraction. The average weight of theextracted workers was 3.09 mg for R. lucifugus grassei and 3.60 mg for R. san-tonensis.

For bioassays 50 workers of R. lucifugus grassei or R. santonensis wereextracted for 30 min in 1 ml purified pentane at room temperature. For chemicalanalyses workers were extracted using a Soxhlet apparatus for 48 hr in purifiedpentane. The extracts were concentrated to about 1 ml by the Kuderna-Danishmethod (Marquardt and Luce, 1961) and stored at -20°C for further use. Wholealates were extracted for 48 hr at 4°C with purified pentane.

Treatment with KOH. Because the trail pheromone could be bound or com-plexed to other compounds, methanolic extracts of R. lucifugus grassei werehydrolyzed with KOH before testing. Workers (500) were extracted for 24 hrwith methanol in a Soxhlet apparatus. Then the extract was divided in two parts.KOH (2% w/v) was added to one part and 2% (v/v) distilled water was addedto the other part as a control. After an 8-hr incubation at room temperature,the methanolic samples were extracted three times with purified pentane. Theextracts were pooled, concentrated, and tested for activity in releasing trail-fol-lowing in a choice bioassay.

Purification and Fractionation of Termite Extracts. Pentane extracts ofwhole individuals were purified by using either silica gel columns (workers) orhigh-performance liquid chromatography (HPLC) (alates). In the first method,3.5 g of purified silica gel (SiO2 60, 230-400 mesh ASTM, Merck) was addedto a glass column (diameter: 10 mm) and successively washed with pentane,dichloromethane, and methanol until no peaks could be detected in the lasteluted fraction of each solvent when analyzed by GC. The concentrated workerextract (1.5 ml), corresponding to about 1000 individuals, was loaded onto thetop of the silica gel column and developed with 4 x 2 ml pentane, 8 x 2 mldichloromethane, and 4 x 2 ml methanol. In the second method the extractswere purified by HPLC (Waters apparatus, Waters SiO2 column, 25 cm x 4mm ID, 5-um particle size Lichrospher Si60). An isocratic elution was madeusing distilled dichloromethane containing 1 % (v/v) methanol at a flow rate of1 ml/min. The UV detector was calibrated at 240 nm. Fractions eluted werecollected every 2 min. The signal was recorded and integrated on a computerfitted with PC software (Millenium Waters). The potential biological activity ofeach fraction obtained by separation on the SiO2 column or HPLC was testedin a Y-choice bioassay as described below.

Chemical Analysis. GC analyses were carried out with a Chrompack CP-

TERMITE TRAIL-FOLLOWING PHEROMONE 1307

9002 instrument fitted with a split-splitless injector and a flame-ionization detec-tor. A CP Wax 58 CB (25 m x 0.25 mm ID, 0.22 um film thickness, Chrompack)fused silica capillary column was used for analyses with temperature program-ming from 40°C to 240°C, at 2°C/min. Helium was used as carrier gas at a flowrate of 5 ml/min. The temperature of injector and detector was set to 250°C and270°C, respectively. Collection of the data was done automatically by PC soft-ware (Maestro, Chrompack).

GC-MS was conducted with a Nermag R 10-10 C quadrupole mass spec-trometer coupled with a Hewlett Packard HP 5890 gas chromatograph. The GCconditions were the same as those described above. The column was connecteddirectly to the ion source of the spectrometer through a heated transfer line main-tained at 260°C. Electron impact mass spectra were obtained at 70 eV with a0.8-sec cycle, and with the instrument scanning from 25 to 300 amu with asource temperature of 150°C.

(Z,Z,E)-3,6,8-Dodecatrien-1-ol (DTE-OH) was identified by comparing itsspectrum to previously published spectra (Yamaoka et al., 1987; Bordereau etal., 1991). Correct identification was checked by analyzing the biological peakand its corresponding synthetic compound under the same conditions on a CPWax 58 CB column. Synthetic DTE-OH was a gift from Professor Yamaoka,Institute of Technology, Kyoto, Japan. It was purified by preparative capillarygas chromatography as described by Laduguie et al. (1994a).

Y-Choice Bioassay. Trail-following assays were performed by Y-choicebioassay on Whatman No. 1 filter paper, with a 120° angle between each branch.On the base of the Y (3 cm) and on one of the branches (7 cm), an artificialtrail was drawn with a microliter Hamilton syringe containing 1 ul of extractper centimeter of trail. Pure solvent as a control for an activity test or anotherextract for a choice test was deposited in the same conditions on the Y base andon the other branch. One termite was gently deposited from a small perspex vialonto the Y base. For an activity test, the distance covered by each individual wasmeasured. The maximal response was 10 cm distance, and the activity thresholdwas defined as a trail followed longer than 3 cm. Results were analyzed with a ttest (P < 0.05). For a choice test, the number of termites that chose one trail wasrecorded and the data were compared by a Monte Carlo test procedure (Vaillantand Derridj, 1992) based upon chi-square at level P = 0.05. Before each testseries, concentrated solvent was tested to exclude any trail-following activity ofthe solvent. For every test a new individual and a new filter paper were used toprevent behavioral conditioning or trail reinforcement.

RESULTS

Trail-Following of R. lucifugus grassei and R. santonensis Workers. Thethreshold and maximum activity was first determined for homospecific extracts

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TERMITE TRAIL-FOLLOWING PHEROMONE

FIG. 1. Trail-following of workers of Reticulitermes lucifugus grassei (open bars) and R.santonensis (hatched bars) on homospecific trails made from pentane extracts of wholeworkers. Results are similar in both species, the threshold level being at 5 x 10- 3 workerequivalent/cm of trail (mean ± SE); N = 20 for 10-3 and 10-1 concentrations, N = 30for 5 x 10-3 and 10-2 concentrations.

of workers (Figure 1). For both species, the activity threshold of whole workerextracts was 5 x 10-3 worker equivalent/cm (WE/cm) of trail, and the maximumactivity occurs at 10-1 WE/cm of trail.

Choice bioassays between R. lucifugus grassei and R. santonensis trailswere then performed from the threshold concentration to the optimal concen-tration. At each concentration, workers of R. lucifugus grassei (Figure 2A) pre-ferred trails from R. santonensis extracts. Results were significantly different for5 x 10-3 and 10-2 WE/cm of trail, not for 10-1 WE/cm. Workers of R. santo-nensis (Figure 2B) also preferred trails from R. santonensis. The results for R.santonensis were statistically significant at each concentration.

The same bioassays were performed after progressive dilution of theextracts of R. santonensis. Figure 3A shows that at 5 x 10-2 WE/cm of trail,trail-following of R. lucifugus grassei workers was equal on trails made withextracts of R. lucifugus grassei or R. santonensis. Further dilutions to 3 x 10-2

and 10-2 WE/cm of trail resulted in trail-following of 72% and 100%, respec-tively, for the R. lucifugus grassei extracts. Figure 3B shows that after dilutionof the extracts of R. santonensis to 5 x 10-2 WE/cm of trail, only 56% of theirown extracts were followed by R. santonensis workers. After further dilution,29% of R. santonensis workers followed their own trail at 3 x 10-2 WE/cm.At 10-2 WE/cm of trail of R. santonensis extract, only the trails of R. lucifugusgrassei extract were followed, i.e., the preference for the R. santonensis trail was

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FIG. 2. (A) Choice bioassays for Reticulitermes lucifugus grassei workers betweenequally concentrated trails of R. lucifugus grassei (open bars) and R. santonensis (hatchedbars). Black bars represent the percentage of no choice. Workers of R. lucifugus gras-sei have a significant preference for R. santonensis trails, except at the concentration of10-1 WE/cm of trail. *Significant difference at P < 0.05, n.s.: no significant difference(Monte Carlo test). N = 30 for 5 x 10-3 and 10-2 concentrations, N = 84 for 10-1 con-centration. (B) Choice bioassays for Reticulitermes santonensis workers between equallyconcentrated trails of R. lucifugus grassei (open bars) and R. santonensis (hatched bars).Black bars represent the percentage of no choice. Workers of R. santonensis have a sig-nificant preference for their own trails at each concentration. *Significant difference atP < 0.05, n.s.: no significant difference (Monte Carlo test). N= 30 for 5 x 10-3 and 10-2

concentrations, N = 62 for 10-1 concentration.

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TERMITE TRAIL-FOLLOWING PHEROMONE

FIG. 3. (A) Choice bioassays for Reticulitermes lucifugus grassei workers between trailsof R. lucifugus grassei (open bars) and R. santonensis (hatched bars) after progressivedilution of R. santonensis trails. Trails of R. lucifugus grassei are significantly preferredwhen R. santonensis trails are 3-10 times less concentrated. *Significant difference atP < 0.05, n.s.: no significant difference (Monte Carlo test). N= 84 for concentration 10-1,

N = 30 for the other concentrations. (B) Choice bioassays for Reticulitermes santonensisworkers between trails of R. lucifugus grassei (open bars) and R. santonensis (hatchedbars) after progressive dilution of R. santonensis trails. Preference for R. santonensis trailsis replaced by preference for R. lucifugus grassei when R. santonensis trails are diluted3-10 times. *Significant difference at P < 0.05, n.s.: no significant difference (MonteCarlo test). N = 62 for 10-1 concentration, N = 30 for the other concentrations.

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changed after diluting the extracts. Both species preferred trails of R. lucifugusgrassei extract when a 10-times diluted extract of R. santonensis was used. Fromthe data of Figure 3B, the trail-following activity of the R. santonensis extractsappeared twice as high as that of R. lucifugus grassei.

Trail-Following Pheromone of R. lucifugus grassei Workers. The crudeextract of 1400 workers was purified and fractionated with a silica gel col-umn. Only the last three fractions eluted with dichloromethane were active intrail-following bioassays, and these fractions were investigated by GC-MS. Themajor peaks were organic acids and alcohols (Figure 4A): octanol, heptadec-anoic acid, 9-octadecenoic acid, 9,12-octadecadienoic acid, and two carbonicacid esters—tetradecanoic acid-methylester and octadecanoic acid-methylester.Bioassays of trail-following performed with corresponding synthetic compoundsshowed that none of these substances elicited trail-following. MS analysis of thesmall peak that has the same retention time as the synthetic DTE-OH (peak 2,Figure 4B) gave a molecular ion of m/z 180 and fragment ion peaks character-istic for (Z,Z,E)-3,6,8-dodecatrien-1-ol.

Synthetic DTE-OH was highly active on workers of R. lucifugus grassei andR. santonensis in eliciting trail-following, and both species have similar sensi-tivity to this alcohol (Figure 5). The threshold concentration was determined at10-3 ng/cm of trail and the optimum at 10-2 ng/cm. Higher concentration ledto lower trail-following activity.

To find out whether in termites DTE-OH was bound to proteins or com-plexed to another molecule, extracts of workers were hydrolyzed by KOH,extracted with pentane, and tested in a Y-choice bioassay at the concentration of1 WE/cm of trail. Results obtained clearly showed that extracts were much moreactive in eliciting trail-following after hydrolysis. In nearly all cases (29 of 30bioassays) workers of R. lucifugus grassei preferred the trails of the hydrolyzedextracts over nonhydrolyzed extracts.

Trail-Following Activity of Alate Extracts of R. lucifugus grassei. Crudeextracts of 1600 males and 1600 females of R. lucifugus grassei were fraction-ated by HPLC. The resulting fractions were eluted with dichloromethane andtested in a trail-following bioassay with R. lucifugus grassei workers. For maleand female extracts, only the fraction eluting between 32 and 38 min in theHPLC run was found to be active for trail-following. DTE-OH was identified inthis fraction by GC-MS.

DISCUSSION

GC-MS analysis showed the presence of (Z,Z,E)-3,6,8-dodecatrien-1-ol(DTE-OH) in Reticulitermes lucifugus grassei workers exclusively fed on filterpaper for 48 hr. Of all compounds identified in purified fractions, only DTE-

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FIG. 5. Trail-following activity of synthetic (3Z,6Z,8E)-3,6,8-dodecatrien-l-ol in work-ers of R. lucifugus grassei (open bars) and R. santonensis (hatched bars). No significantdifference is observed between R. santonensis and R. lucifugus grassei responses; bothspecies show a similar sensitivity to this compound. The threshold level is observed at10-3 ng/cm, the optimal activity at 10-2 ng/cm. Higher concentrations reduce trail-fol-lowing. Values are mean ± SE, N = 30 for each concentration.

OH was active in eliciting trail-following. Workers of R. lucifugus grassei weresensitive to synthetic DTE-OH at pheromonal concentrations. Thus, DTE-OHcould be considered the major compound of the trail-following pheromone in R.lucifugus grassei.

The same compound was identified as the trail pheromone in several speciesof Rhinotermitidae; R. virginicus (Matsumura et al., 1968, 1969), R. speratus(Yamaoka et al., 1987), and Coptotermes formosanus (Tokoro et al., 1989). Inter-estingly, DTE-OH is also the major component of the trail-following pheromoneof R. santonensis (Laduguie et al., 1994a), which is sympatric with R. lucifu-gus grassei in some areas of France. This could explain why no specificity wasobserved in choice bioassays of trail-following between both species, even whenextracts were tested just above threshold level. Lack of trail-following specificitywas also found by Oloo and McDowell (1982) in several species of African ter-mites of the genus Trinivervitermes in which the major compound of the trailpheromone was identified as neocembrene (McDowell and Oloo, 1984).

In contrast, Howard et al. (1976) obtained species-specific responses inthree species of the subterranean termite Reticulitermes. Kaib et al. (1982)showed specific trail-following in Trinervitermes bettonianus and T. gratiosus.

In some cases, however, the choice of trails could be easily explained byquantitative differences of secreted pheromone between each species. In R. san-tonensis and R. lucifugus grassei, for example, the preference for R. santonen-

WOBST ET AL.

sis extracts was reduced progressively to that of nonpreference and then prefer-ence for R. lucifugus grassei following progressive dilution of the R. santonen-sis extract. According to our bioassays, workers of R. santonensis secrete abouttwice as much DTE-OH as do R. lucifugus grassei. Grace et al. (1995) alsoshowed concentration-mediated trail-following responses to extracts containingtrail pheromone in different colonies of R. hesperus.

Some data suggest, however, that the trail pheromone of Reticulitermesspecies probably is a multicomponent system, as previously claimed from behav-ioral observations by Kaib et al. (1982) and Traniello (1982) for other species.Although only the fraction containing DTE-OH was active in eliciting trail-fol-lowing, some of the other compounds in this fraction could not be identified andtested. The estimated quantities of DTE-OH in termites from chemical analy-sis (1-10 pg) were 2-100 times lower than those eliciting trail-following in thebioassays (20-100 pg, from Figures 1 and 5), which suggests synergistic com-ponents. R. santonensis workers appeared to be more reactive to R. santonensistrails than R. lucifugus grassei. This could be due not only to higher pheromonalconcentrations, but might hint at additional species-specific components. Finally,the increase in trail-following activity of the worker extracts of R. lucifugus gras-sei after KOH hydrolysis also suggests storage of esterified precursors of DTE-OH in the sternal gland, as previously shown in Coptotermes formosanus byTokoro et al. (1992).

R. santonensis and R. lucifugus grassei were considered in the past as twosubspecies of R. lucifugus (Lash, 1952; Buchli, 1958) and the nonspecificity oftheir trail pheromones may underline the close phylogenetic relationship of thetwo species. DTE-OH and neocembrene are used by several species of termites,however, which demonstrates that chemical communication of termites is unusu-ally nonspecific compared to other social insects. Contrary to trail pheromones ofants, which are secreted by many glands and are chemically variable (Holldoblerand Wilson, 1990), trail pheromones of termites are secreted from only one glan-dular source, the sternal gland. In termite species in which the workers do notgo outside the nest to collect food (species with one-piece life type; Abe, 1987),secretions of their sternal gland probably allows them to mark and recognize theirenvironment and to coordinate their activities of excavating. In species that for-age outside the nest (species with separate life type; Abe, 1987), termites veryoften move along subterranean tunnels or galleries on the surface, which may beconsidered as expansions of the nest and serve as physical guides. In this case,the orientation role of the trail-following pheromone is limited to the first stepsin building the galleries. In fact, specific trails only appear useful for termitespecies foraging above ground in open air and far from the nest. This is infre-quent, but does occur in harvester termites such as Trinervitermes bettonianusand T. gratiosus, in which species-specific trails were demonstrated by Kaib etal. (1982).

TERMITE TRAIL-FOLLOWING PHEROMONE 1315

DTE-OH was also found in alates of R. lucifugus grassei. Probably it isused as a trail-following pheromone in these individuals during the nuptial prom-enade. Although it could not be tested for its sexual activity, it might repre-sent a major component of the female sex pheromone of this species as previ-ously shown for the sympatric species R. santonensis (Laduguie et al., 1994a).Clement (1982) noted that the reproductive isolation of both species would notarise from sex pheromone specificity since males of R. santonensis were attractedby females of R. santonensis and R. lucifugus grassei. The main factor of isola-tion is the time lag between swarming periods of the two species. In their areaof sympatry, the dispersal flights of R. santonensis occur at the beginning ofApril and those of R. lucifugus grassei at the beginning of May. These resultsare similar to those observed in two African fungus-growing termites Pseuda-canthotermes spiniger and P. militaris (Bordereau et al., 1991, 1993; Laduguie etal., 1994b) and show again the economy developed by termites in their strategiesof chemical communication.

Acknowledgments—This work was supported by a grant from the Conseil Regional deBourgogne. The authors wish to thank Professor R. Yamaoka, from the Kyoto Institute of Technol-ogy for a generous gift of a sample of DTE-OH, and F. Vieau (University of Nantes) for providingmany samples of Reticulitermes species. We are also grateful to Professor Ch. Noirot (Dijon) forhis critical manuscript review and to Barbara Sommerville for editorial help.

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