Current Biology 16, 2359–2365, December 5, 2006 ª2006 Elsevier Ltd All rights reserved
DOI 10.1016/j.cub.2006.10.031
Report
Chemoattraction in Pristionchus
Nematodes and Implications
for Insect Recognition
Ray L. Hong1,* and Ralf J. Sommer1,*
1
Max Planck Institute for Developmental Biology
Department for Evolutionary Biology
Spemannstrasse 37–39
72076 Tuebingen
Germany
Summary
Nematodes and insects are the two dominant animal
taxa in species numbers, and nematode-insect interactions constitute a significant portion of interspecies
associations in a diversity of ecosystems. It has been
speculated that most insects represent mobile microhabitats in which nematodes can obtain food, mobility, and shelter. Nematode-insect associations can
be classified as phoretic (insects used for transportation, not as food), necromenic (insect used for
transportation, then carcass as food), and entomopathogenic (insect is killed and used as food). To determine how nematodes target their hosts, we analyzed
the chemosensory response and behavioral parameters of closely related Pristionchus nematodes that
form species-specific necromenic associations with
scarab beetles and the Colorado potato beetle [1, 2].
We found that all four studied Pristionchus species
displayed unique chemoattractive profiles toward
insect pheromones and plant volatiles with links to
Pristionchus habitats. Moreover, chemoattraction in
P. pacificus differs from that of C. elegans not only in
the types of attractants, but also in its tempo, mode,
and concentration response range. We conclude that
Pristionchus olfaction is highly diverse among closely
related species and is likely to be involved in shaping
nematode-host interactions.
Results and Discussion
Pristionchus pacificus is a nematode cultivated to provide comparisons to the C. elegans model in the area
of development, genetics, and evolution [3]. Many genera in the Diplogastridae to which Pristionchus belongs
are associated with insects: Micoletzkya species associate with bark beetles, and Parasitodiplogaster species
are intimately associated with fig wasps (Agaonidae)
[4–6]. Recent studies have shown that several species
within the Pristionchus genus are necromenic and are
closely associated with scarab beetles from the United
States as well as western Europe [1, 2]. P. maupasi is
a closely related species to P. pacificus and is found primarily on Melolontha species, the European cockchafer
(Figure 1). P. entomophagus and P. uniformis are sister
species that are found primarily on Geotrupes (dung
*Correspondence: [email protected]
[email protected] (R.J.S.)
(R.L.H.),
ralf.
beetles) and Leptinotarsa decemlineata, the Colorado
potato beetle (CPB), respectively (Figure 1). Except for
the CPB, which belongs to the Chrysomelidae, Pristionchus populations so far have only been isolated extensively from beetles belonging to the Scarabaeoidea superfamily. Because odors from organisms can convey
abundant information on their location and identity, olfaction may be the favored modality for nematodes
that lack vision and have to contend with a complex gustatory environment in the soil. Hence, the orientation and
taxis of nematodes toward specific odors in chemotaxis
assays might recapitulate their behavioral response in
nature toward their preferred environments, including
their beetle hosts.
To establish species-specific attraction profiles of
four Pristionchus species, we surveyed 31 compounds
for their attractiveness to P. pacificus and C. elegans, including previously known C. elegans attractants, as well
as 11 known semiochemicals involved in insect and
plant communication on two pairs of highly related Pristionchus species (Table 1 and Figure 1). Semiochemicals are communication compounds emitted by insects
to attract mates (sex pheromone), to aggregate into
groups (aggregation pheromone), and to ward of competitors (allomone) [7]. Semiochemicals also include volatile secondary metabolites from plants in response to
insect herbivory, and such metabolites can either recruit
the natural predators of the insects as a defense mechanism or enhance the sex pheromone of the insects
(kairomone) [8]. The goal of our study was to identify attractants for Pristionchus species and to delineate their
attraction parameters as well as those semiochemicals
unique to each species.
We found that all four Pristionchus species, especially
P. maupasi, were attracted to b-caryophyllene, a ubiquitous and inducible plant defense volatile known to be
attractive to the entomopathogenic nematode Heterorhabditis megidis [9] (Figure 1). That b-caryophyllene is
a common Pristionchus attractant may likely be because of the habitat and diet of their phytophagus beetle
hosts. All Pristionchus species also showed some attraction toward the 16 carbon Z-HDA, a Lepidopteran
pheromone, but Z-HDA was not the most attractive
compound to any of these species. In the wild, Pristionchus aerivorous has been reportedly found on a Lepidopteran host, Helicoverpa zea [10].
More importantly, we found that closely related pairs
of Pristionchus species have significantly diverged chemoattraction profiles (Figure 1). P. pacificus, whose insect host has yet to be identified, was most attracted
to long-chain fatty-acid esters and acetates (E-TDA
and myristate) compared to P. maupasi. By contrast,
P. maupasi was much more attracted to other plant derived compounds such as linalool [11] and the green-leaf
alcohol ((Z)-3-hexen-1-ol) [8]. Similarly, P. entomophagus, the most frequent Pristionchus species found on
dung beetles in Europe [1], displayed the highest attraction to isopentylamine. Isopentylamine smells of
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Figure 1. Semiochemical Attraction Profiles of Pristionchus Species
Pristionchus attraction profiles to semiochemicals at 10% concentration (w/v or v/v). Verbenol and leucine methyl ester were at 100 mM. 10-fold
dilutions of these compounds resulted in similar profiles (data not shown). The left side shows the phylogenetic relationship of four Pristionchus
species based on 18S ribosomal DNA sequences, with Koerneria as the most basal species, along with known beetle associations: P. maupasi
Chemical Ecology of Pristionchus Nematodes
2361
decaying material and can be used to trap Geotrupes
beetles. Interestingly, isopentylamine has a branched
carbon group similar to the nonattractive leucine methyl
ester, a sex pheromone of June beetles, Phyllophaga
lanceolata [12]. P. uniformis, which has the highest fidelity to CPB, showed the strongest attraction to verbenol,
a known aggregation pheromone of bark beetles (Ips)
made from plant monoterpenoid precursors. Taken together, the attraction profiles to plant- and insectderived compounds provide sufficient resolution for discerning four closely related, morphologically similar
nematode species. These results suggest that species-specific olfaction profiles are likely to be involved
in host recognition and attraction. However, the choice
of stimuli we tested can be further refined according to
their relevant ecological contexts, with the goal of understanding the precise involvement of each compound
in specific insect-nematode interactions.
Because the analyzed Pristionchus species have species-specific chemoattraction profiles, we wondered
whether closely related Caenorhabditis species also
show such diversity in chemotaxis behavior. Wild
C. elegans isolates are primarily found in decaying organic matter, whereas the sister species C. briggsae
and C. remanei have been found to associate casually
with snails and isopods, as well as to live freely in composts [13, 14]. We proceeded to test known C. elegans
N2 attractants on C. elegans Hawaii, C. briggsae, and
C. remanei (Figure 2A). When compared to C. elegans
N2, we observed statistically significant quantitative differences in the chemoattraction of Caenorhabditis species toward isoamyl alcohol, benzaldehyde, pentanedione, diacetyl, and pyrazine but not for 2-butanone.
However, these compounds were attractive to all Caenorhabditis species to some extent, with no qualitative
differences in which chemoattraction was completely
absent or repulsive. Similarly, we found that Caenorhabditis species were repulsed by 10% b-caryophyllene,
and all but C. elegans Hawaii was repulsed by 1% myristate and E-TDA (see Figures S1A–S1C in the Supplemental Data available with this article online). Thus, not
only is the difference in semiochemicals attraction conserved between other Caenorhabditis species and
P. pacificus, the closely related Pristionchus species
but not Caenorhabditis species have distinguishing chemotaxis profiles. Nevertheless, other semiochemicals
not tested here might potentially distinguish related
Caenorhabditis species once specific invertebrate associations can be identified.
We wondered whether P. pacificus, in order to be able
to proliferate under the same standard laboratory conditions as C. elegans, is also attracted to some of the simple organic compounds as C. elegans is. We tested
seven of the most attractive compounds and found
that P. pacificus was attracted to high concentrations
of pentanedione, pyrazine, and diacetyl (Figure 2B).
P. pacificus was weakly attracted to a low concentration
of 2,4,5-trimethylthiazole TMT (1%) as well and repulsed
by high concentrations of undiluted TMT and benzaldehyde. Furthermore, P. pacificus showed no remarkable
attractions toward isoamyl alcohol, benzaldehyde, and
2-butanone, except an extremely weak attraction for
10% butanone (CI w0.3). C. elegans, as expected, was
highly attracted to the same compounds at the same
1–1003 dilutions; thus, only the 1% profile is shown
for comparison to P. pacificus (Figure 2B). These results
suggest that the recognition of high concentrations of
diacetyl, pentanedione, and pyrazine are only partially
conserved between P. pacificus and C. elegans or that
downstream signaling events are more tempered than
they are in C. elegans.
In nature, organisms must be able to discriminate one
odor from other, more abundant odors. We utilized the
discrimination assay to demonstrate that separate signaling pathways in P. pacificus can perceive two simultaneous odors, one of which is saturating [15](Figure 2C).
We tested eight attractants on plain, diacetyl-, and bcaryophyllene-saturated agar plates in a modified chemotaxis assay. Diacetyl represented the common attractant for all Caenorhabditis and Pristionchus species
that we have tested; hence, there may be a conserved
receptor and signaling pathway for diacetyl (Figure S1D).
Meanwhile, b-caryophyllene represents a basic Pristionchus odor that is likely to be present in most Pristionchus habitats. We found that P. pacificus attraction to
the four shared P. pacificus-C. elegans attractants—
diacetyl, pyrazine, isobutanol, and pentanedione—
were not reduced on saturated plates as compared to
plain plates. Similarly, four P. pacificus-specific attractants—b-caryophyllene, mono-olein, E-TDA, and myristate—were also not compromised on saturated plates.
We conclude that P. pacificus, like C. elegans, can discriminate simultaneous odors.
In general, P. pacificus has the slowest population
chemoattraction response among the four Pristionchus
species tested, with the fastest response to diacetyl and
b-caryophyllene peaking in 2–3 hr and the slowest response to myristate and E-TDA peaking after 9 hr.
C. elegans populations, by contrast, attained peak attraction to all attractive odors within 1 hr. Although observations of single worms indicated that P. pacificus
can reach attractant sources within 2 hr, attraction at the
population level was only apparent after much longer
periods. To describe this population behavior in more
detail, we conducted time-course assays for attraction
toward myristate and E-TDA over a period of 22 hr (Figure 3). CI for both myristate and E-TDA (10% and 1%) increased steadily over this time span, reaching a climax
in 9–22 hr. The long incubation time required to attain
peak CI is a Pristionchus-specific chemoattraction
phenomenon not observed in C. elegans because all
C. elegans attractants tested require 30–45 min to
attain peak CI (this study and [16]) and greater than
9 hr was also required to achieve peak CI for myristate
and E-TDA in other Pristionchus species tested (Figure 1). The slower response of P. pacificus compared
to C. elegans at the individual level is primarily due to
the relatively slower locomotion of P. pacificus, as well
as their more frequent turning and reversal behavior
both in the presence and absence of food (J. Srinivasan
(Melolontha sp., European Cockchafers), P. entomophagus (Geotrupes sp., dung beetles), and P. uniformis (Leptinotarsa decemlineata, Colorado potato beetle) (modified from [1]). The natural insect host for P. pacificus remains unidentified. All data points were derived from at least two
separate experiments with 3–6 replicates each. Error bars denote 95% confidence intervals.
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Table 1. A List of Semiochemical Compounds Tested in Four Pristionchus Species
Compound
Known Insect or
Plant Source
Known Function
(E)-b-caryophyllene
Maize and other plants
Plant defense volatile [9]
(S)-verbenol
Ips paraconfusus
Aggregation pheromone [26]
(R)-(-)-linalool
Many plants
Plant volatile which enhances
sex pheromone [11]
isopentylamine
Phyllophaga lanceolata
Phyllophaga and Geotrupes
attractant [12]
Toluquinone
Melolontha melolontha,
M. hippocastani
Sex pheromone [27, 28]
(Z)-3-hexen-1-ol(Greenleaf alcohol)
Many plants
Plant volatile that enhances
sex pheromone [8]
2-(E)-nonenol
Melolontha melolontha,
M. hippocastani
Sex pheromone [29]
L-Leucine methyl ester
Phyllophaga lanceolata
Sex pheromone [12]
(E)-11-tetradecenyl
acetate (E-TDA)
Lepidoptera; i.e., Spodoptera
and Heliothis species
Sex pheromone [30, 31]
(Z)-11-hexadecenal(Z-HDA)
Lepidoptera; i.e.,
Helicoverpa zea
Sex pheromone [32, 33]
methyl tetradecanoate
(myristate)
Philanthus and Musca species
Allomone [34–36]
Reviewed in [7, 25].
Structure
Chemical Ecology of Pristionchus Nematodes
2363
Figure 2. Differences between C. elegans
and P. pacificus Chemoattraction
Error bars denote 95% confidence intervals.
All assays for different species were conducted concurrently during multiple sessions
with each value representing at least six experiments.
(A) The strongest C. elegans N2 attractants
elicited quantitatively but not qualitatively
different attraction for C. elegans Hawaii,
C. briggsae, and C. remanei.
(B) P. pacificus share a set of attractants with
C. elegans. C. elegans attraction profiles at
1% concentration are shown as positive controls (white bars). P. pacificus attraction profiles 100%–1% are shown for all compounds
except for pyrazine (*), for which the 10%–
0.1% profile is shown (filled and shaded
bars). P. pacificus was attracted to 100% diacetyl and pentanedione as well as weakly attracted to 10% pyrazine and 1% 2,4,5-trimethylthiazole (2,4,5-TMT). P. pacificus was
repulsed by high concentrations of benzaldehyde and TMT and showed no attraction to
isoamyl alcohol, benzaldehyde, and 2-butanone. The response to 10% 2-butanone is
considered borderline attraction (CI w0.3).
(C) Discrimination assays show that P. pacificus discriminated conserved attractants to
C. elegans (100% diacetyl, 10% pyrazine,
10% isobutanol, and 100% 2,3-pentanedione) from 1:10,000 dilution of b-caryophyllene in NGM agar. P. pacificus also discriminated P. pacificus-specific attractants (10%
b-caryophyllene, 10mM 1-mono-olein, 1%
myristate, and 10% E-TDA) from 1:10,000 dilution of diacetyl in NGM agar. As controls
(asterisks), diacetyl and b-caryophyllene chemoattraction were abolished in the presence
of the same scent in the agar.
and P. Sternberg, personal communication). More precise tracking of P. pacificus velocity as well as orientation would be required for determining whether P. pacificus modulates its locomotion behavior according to the
type or strength of attractants. We have tentatively referred to these attraction modes requiring greater than
9 hr as ‘‘long-term attraction’’ based solely on the incremental increase in CI as a roaming population and not on
the behavioral mechanisms involved. Taken together,
chemoattraction in Pristionchus species represent
more complex olfactory responses than previously described for C. elegans, and these differences may be important for Pristionchus’ necromenic lifestyle.
Although P. pacificus and C. elegans share a common
set of attractants, P. pacificus was attracted to diacetyl,
pentanedione, pyrazine, and isobutanol only at the
10%–100% concentration range (Figure 2B). Even
strong P. pacificus attractants, such as E-TDA and
myristate, elicited attraction only within a 100-fold concentration range (1%–100%). Such limited sensitivity
range may be conserved in all Pristionchus species in
light of the fact that the semiochemicals profiles and
the response to diacetyl showed attraction only within
a 10-fold dilution range (Figures 1 and 2). This is in stark
contrast to the seven strong C. elegans attractants we
tested; several of these attractants can elicit C. elegans
attraction in the range of 10,000-fold, with diacetyl
having the greatest attraction range of a million fold
(0.0001%–100%) [16]. Although it is possible that
some of the Pristionchus attractants are not the optimal
ligands, it is most likely that certain blends or sequence
medley of odors are required for eliciting more sensitive responses, as may be the situations encountered
in nature.
Finally, we asked whether chemoattraction behavior
also differs between isolates of the same P. pacificus
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Figure 3. Time-Course Comparisons of
P. pacificus Attractions to 1% Myristate and
E-TDA
The chemoattraction indices were taken at
multiple time intervals for attraction to myristate at 1% (A) and 10% (C) as well as E-TDA at
1% (B) and 10% (D). Except for 10% myristate
(three replicates each), each data point is
an average of 6–12 replicates from three
experiments.
species. To our surprise, we observed significant chemoattraction differences between the two standard laboratory strains [17]. Specifically, the California strain
was not attracted to E-TDA at all, compared to the
Washington strain, although both strains showed comparable attraction toward b-caryophyllene and diacetyl
(Figure S1E and data not shown). To determine the extent of this behavioral polymorphism in P. pacificus,
we tested two additional strains. Previous studies by
AFLP analysis (Amplified Fragment Length Polymorphism) indicated that the Hawaii strain is more similar
to the Washington than the California strain, whereas
the California and Poland strains share almost identical
band patterns [18]. In our olfaction studies, the Hawaii
strain was similarly attracted to 1% E-TDA as the Washington strain, but with slightly lower attraction to 10%
E-TDA. More surprisingly, the Poland strain was also
attracted to E-TDA. Thus, a small number of genetic differences below the threshold detectable by AFLP may
be responsible for divergence in P. pacificus chemosensory behavior, although it is not yet clear whether this
difference was adapted or accumulated during the cultivation of P. pacificus.
In conclusion, we found that all four studied Pristionchus species displayed unique chemoattractive profiles
toward insect pheromones and plant volatiles with connections to Pristionchus habitats. Moreover, chemoattraction in P. pacificus differs from that of C. elegans
not only in the types of attractants but also in its tempo,
mode, and concentration response range at the population level. Although past studies on entomopathogenic nematodes have shown that plant defense volatiles and odors from insect hosts are important for
attraction to insect hosts [9, 19–23], the species-specificity of these attractions were not addressed nor
were the contributions of individual compounds identified. Owing to the enormous contributions by researchers in the C. elegans community, we already
hold some key concepts on nematode olfactory behavior, such as discrimination, adaptation, and associative
learning [15, 16, 24]. Given that P. pacificus does not
have strong attraction toward any of the key seven
C. elegans attractants (Figure 2B), it is likely that the
functions of the homologous AWA and AWC neurons
differs significantly from those in C. elegans. Interestingly, the current predicted number of seven transmembrane receptor genes in the P. pacificus genome
is substantially less than those in the C. elegans genome (www.pristionchus.org). Our long-term goal will
include investigations into the neuroanatomy and receptor-neuron mapping of P. pacificus olfaction. Identifying the molecular changes underlying the difference
between P. pacificus and C. elegans will be the first
step toward understanding how dramatically new chemosensations can arise, how these changes alter
downstream intracellular signaling pathways, and how
these molecular changes feed back to the organismal
interactions in their ecological habitats.
Chemical Ecology of Pristionchus Nematodes
2365
Supplemental Data
Supplemental Data include Experimental Procedures, one figure,
and one table and can be found with this article online at http://
www.current-biology.com/cgi/content/full/16/23/2359/DC1/.
Acknowledgments
We thank H. Witte for outstanding technical assistance. We are also
grateful for the comments of J. Srinivasan and David Rudel as well as
J. Srinivasan, C. Dieterich, and M. Herrmann for communication of
their unpublished results. R.L.H. was supported by the Alexander
von Humboldt Fellowship.
Received: July 25, 2006
Revised: September 20, 2006
Accepted: October 11, 2006
Published: December 4, 2006
References
1. Herrmann, M., Mayer, W.E., and Sommer, R.J. (2006). Nematodes of the genus Pristionchus are closely associated with
scarab beetles and the Colorado potato beetle in Western
Europe. Zoology 109, 96–108.
2. Herrmann, M., Mayer, W.E., and Sommer, R.J. (2006). Sex, bugs
and Haldanes rule: The nematode genus Pristionchus in the
United States. Frontiers in Zoology 3, 14.
3. Hong, R.L., and Sommer, R.J. (2006). Pristionchus pacificus:
A well-rounded nematode. Bioessays 28, 651–659.
4. Massey, C.L. (1960). The genus Mikoletzkya (Nematoda) in the
United States. Proceedings of the Helminthological Society of
Washington 33, 13–19.
5. Herre, E.A. (1993). Population-structure and the evolution of
virulence in Nematode parasites of fig wasps. Science 259,
1442–1445.
6. Poinar, G.O., and Herre, E.A. (1991). Speciation and adaptive
radiation of the fig wasp nematode, Parasitodiplogaster (Diplogasteridae: Rhabditida) in Panama. Revue Nematology 14, 361–
374.
7. Tillman, J.A., Seybold, S.J., Jurenka, R.A., and Blomquist, G.J.
(1999). Insect pheromones - an overview of biosynthesis and endocrine regulation. Insect Biochem. Mol. Biol. 29, 481–514.
8. Ruther, J., Reinecke, A., and Hilker, M. (2002). Plant volatiles in
the sexual communication of Melolontha hippocastani: Response towards time-dependent bouquets and novel function
of (Z)-3-hexen-1-ol as a sexual kairomone. Ecol. Entomol. 27,
76–83.
9. Rasmann, S., Kollner, T.G., Degenhardt, J., Hiltpold, I., Toepfer,
S., Kuhlmann, U., Gershenzon, J., and Turlings, T.C.J. (2005).
Recruitment of entomopathogenic nematodes by insect-damaged maize roots. Nature 434, 732–737.
10. Steinkraus, D.C., Boys, G.O., Kring, T.J., and Ruberson, J.R.
(1993). Pathogenicity of the facultative parasite, Chroniodiplogaster aerivora (Cobb) (Rhabditida, Diplogasteridae) to
corn earworm (Helicoverpa zea [Boddie]) (Lepidoptera, Noctuidae). J. Invertebr. Pathol. 61, 308–312.
11. Leal, W. (1997). Evolution of Sex Pheromone Communication in
Plant Feeding Scarab Beetles (New York: Chapman & Hall).
12. Nojima, S., Robbins, P.S., Salsbury, G.A., Morris, B.D., Roelofs,
W.L., and Villani, M.G. (2003). L-leucine methyl ester: The female-produced sex pheromone of the scarab beetle, Phyllophaga lanceolata. J. Chem. Ecol. 29, 2439–2446.
13. Baird, S.E., Fitch, D.H.A., and Emmons, S.W. (1994). Caenorhabditis-Vulgaris Sp-N (Nematoda, Rhabditidae) - a Necromenic associate of pill bugs and snails. Nematologica 40, 1–11.
14. Barriere, A., and Felix, M.A. (2005). High local genetic diversity
and low outcrossing rate in Caenorhabditis elegans natural populations. Curr. Biol. 15, 1176–1184.
15. L’Etoile, N.D., and Bargmann, C.I. (2000). Olfaction and odor discrimination are mediated by the C. elegans guanylyl cyclase
ODR-1. Neuron 25, 575–586.
16. Bargmann, C.I., Hartwieg, E., and Horvitz, H.R. (1993). Odorantselective genes and neurons mediate olfaction in C. elegans.
Cell 74, 515–527.
17. Srinivasan, J., Sinz, W., Lanz, C., Brand, A., Nandakumar, R.,
Raddatz, G., Witte, H., Keller, H., Kipping, I., Pires-daSilva, A.,
et al. (2002). A bacterial artificial chromosome-based genetic
linkage map of the nematode Pristionchus pacificus. Genetics
162, 129–134.
18. Srinivasan, J., Pires-daSilva, A., Gutierrez, A., Zheng, M., Jungblut, B., Witte, H., Schlak, I., and Sommer, R.J. (2001). Microevolutionary analysis of the nematode genus Pristionchus suggests
a recent evolution of redundant developmental mechanisms
during vulva formation. Evol. Dev. 3, 229–240.
19. Necibi, S., and Linit, M.J. (1998). Effect of Monochamus carolinensis on Bursaphelenchus xylophilus dispersal stage formation. J. Nematol. 30, 246–254.
20. Stamps, W.T., and Linit, M.J. (1998). Chemotactic response of
propagative and dispersal forms of the pinewood nematode
Bursaphelenchus xylophilus to beetle and pine derived compounds. Fundam. Appl. Nematol. 21, 243–250.
21. Stamps, W.T., and Linit, M.J. (2001). Interaction of intrinsic and
extrinsic chemical cues in the behaviour of Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae) in relation to its beetle vectors. Nematology 3, 295–301.
22. Lewis, E.E., Gaugler, R., and Harrison, R. (1992). Entomopathogenic Nematode host finding - response to host contact cues by
cruise and ambush foragers. Parasitology 105, 309–315.
23. Ishikawa, M., Shuto, Y., and Watanabe, H. (1986). Beta-Myrcene,
a potent attractant component of pine wood for the pine wood
Nematode, Bursaphelenchus-Xylophilus. Agric. Biol. Chem.
50, 1863–1866.
24. L’Etoile, N.D., Coburn, C.M., Eastham, J., Kistler, A., Gallegos,
G., and Bargmann, C.I. (2002). The cyclic GMP-dependent protein kinase EGL-4 regulates olfactory adaptation in C. elegans.
Neuron 36, 1079–1089.
25. Leal, W.S. (1998). Chemical ecology of phytophagous scarab
beetles. Annu. Rev. Entomol. 43, 39–61.
26. Renwick, J.A.A., Hughes, P.R., and Krull, I.S. (1976). Selective
production of Cis-Verbenol and Trans-Verbenol from (2)- and
(+)-Alpha-Pinene by a bark beetle. Science 191, 199–201.
27. Reinecke, A., Ruther, J., and Hilker, M. (2002). The scent of food
and defence: Green leaf volatiles and toluquinone as sex attractant mediate mate finding in the European cockchafer Melolontha melolontha. Ecol. Lett. 5, 257–263.
28. Ruther, J., Podsiadlowski, L., and Hilker, M. (2001). Quinones in
cockchafers: additional function of a sex attractant as an antimicrobial agent. Chemoecology 11, 225–229.
29. Leal, W.S., Hasegawa, M., and Sawada, M. (1992). Identification
of Anomala-Schonfeldti sex-pheromone by high-resolution
Gc-behavior bioassay. Naturwissenschaften 79, 518–519.
30. Choi, M.Y., Groot, A., and Jurenka, R.A. (2005). Pheromone biosynthetic pathways in the moths Heliothis subflexa and Heliothis
virescens. Arch. Insect Biochem. Physiol. 59, 53–58.
31. Batista-Pereira, L.G., Stein, K., de Paula, A.F., Moreira, J.A.,
Cruz, I., Figueiredo, M.D., Perri, J., and Correa, A.G. (2006). Isolation, identification, synthesis, and field evaluation of the sex
pheromone of the Brazilian population of Spodoptera frugiperda. J. Chem. Ecol. 32, 1085–1099.
32. Vetter, R.S., and Baker, T.C. (1983). Behavioral-responses of
male Heliothis-Virescens in a sustained-flight tunnel to combinations of 7 compounds identified from female sex-pheromone
glands. J. Chem. Ecol. 9, 747–759.
33. Vickers, N.J. (2002). Defining a synthetic pheromone blend attractive to male Heliothis subflexa under wind tunnel conditions.
J. Chem. Ecol. 28, 1255–1267.
34. Schmidt, J.O., Oneill, K.M., Fales, H.M., Mcdaniel, C.A., and
Howard, R.W. (1985). Volatiles from mandibular glands of male
beewolves (Hymenoptera, Sphecidae, Philanthus) and their
possible roles. J. Chem. Ecol. 11, 895–901.
35. Mcdaniel, C.A., Schmidt, J.O., and Howard, R.W. (1992). Mandibular gland secretions of the male beewolves PhilanthusCrabroniformis, P-Barbatus, and P-Pulcher (Hymenoptera,
Sphecidae). J. Chem. Ecol. 18, 27–37.
36. Henderson, G., Wells, J.D., and Jeanne, R.L. (1991). Methyl
Palmitate and Methyl Myristate repel flies. Fla. Entomol. 74,
365–368.
Please cite this article in press as: Hong and Sommer, Chemoattraction in Pristionchus Nematodes and Implications for Insect Recognition, Current Biology (2006), doi:10.1016/j.cub.2006.10.031
Supplemental Data
Chemoattraction in Pristionchus
Nematodes and Implications
for Insect Recognition
S1
Ray L. Hong and Ralf J. Sommer
Supplemental Experimental Procedures
Nematode Strains
We used standard C. elegans protocols for handling and maintenance of C. elegans strains N2 (Bristol) and CB4856 (Hawaii) as
well as C. briggsae AF16 and C. remanei EM464. Pristionchus nematodes were maintained in the same manner as C. elegans. The P.
pacificus strains used were: PS312 (California; soil), PS1843 (Washington; soil), JU138 (Hawaii; soil), RS106 (Poland; soil). Other Pristionchus species used were as follows: P. maupasi RS5015
Figure S1. Comparisons of Various Responses in Caenorhabditis and Pristionchus Species or Strains to Major P. pacificus Attractants
Caenorhabditis species are not attracted to any strong P. pacificus attractants. Error bars denote 95% confidence intervals. The incubation time
is 45–90 min for Caenorhabditis species (longer incubation periods up to 16 hr did not significantly change the indices) and 12–16 hr for Pristionchus and Poikilolaimus species.
(A–C) C. elegans Bristol (N2) and Hawaii strains, C. briggsae, and C. remanei were all not attracted to b-caryophyllene, myristate, and E-TDA. All
differences to the corresponding P. pacificus values are significant (t test, p < 0.05), with the exception of C. elegans Hawaii’s attraction to 0.1%
myristate.
(D) Diacetyl, a strong C. elegans attractant, was also a conserved attractant to all Pristionchus species. P. pacificus (Washington and California
strains) were weakly attracted to 100% but not 10% diacetyl. Other Pristionchus species and an outgroup to the Diplogastridae, Poikilolaimus
oxycercus, were all comparatively more attracted to diacetyl at both 100% and 10% (asterisks indicate t test to California strain, p < 0.05).
(E) P. pacificus California lack attraction to (E)-11-tetradecenyl acetate (E-TDA). Error bars denote 95% confidence intervals. P. pacificus California was the single strain not attracted to E-TDA compared to Washington, Hawaii, and Poland strains (t test, p < 0.05).
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Please cite this article in press as: Hong and Sommer, Chemoattraction in Pristionchus Nematodes and Implications for Insect Recognition, Current Biology (2006), doi:10.1016/j.cub.2006.10.031
S2
Table S1. A Comparison of P. pacificus and C. elegans Attraction toward Odorant Compounds
Attractions
Attractants
Attractive to P. pacificus only
b-caryophyllene, methyl tetradecanoate (myristate), ethyl tetradecanoate,
(E)-11-tetradecenyl acetate (E-TDA), and (Z)-hexadecenal (Z-HDA)
1-mono-olein, b-myrcene, a-pinene, 20-hydroxyecdysone, a-ecdysone, and dimethyl
sulfoxide (DMSO)
isoamyl alcohol, 2-butanone, 2,4,5 trimethylthiazole, benzyladehyde, 1-butanol, and
50% methoprene (CI = 0.6; Drosophila juvenile hormone III analog)
diacetyl, 2,3-pentanedione, pyrazine, isobutanol, ethyl butyrate (weak), verbenol,
isoamylamine, and green-leaf alcohol
limonene, 2-butanol, cholesterol, gibberellic acid, acetone, glycerol, and toluene
1-10% geraniol, 100% hexanol, 100% heptanol, 100% octanol, and 100% nonanol
Weakly attractive to P. pacificus only
Attractive to C. elegans only
Attractive to both P. pacificus and C. elegans
Neutral to P. pacificus or C. elegans or both
Repellent to both P. pacificus and C. elegans
No assays were performed on C. elegans for the following compounds: cholesterol, gibberellic acid, and acetone.
(Germany, Melolontha sp.), P. entomophagus RS144 (Germany;
soil), and P. uniformis RS5167 (New York, Leptinotarsa decemlineata). Although P. pacificus California is our standard lab strain,
the polymorphic Washington strain performed faster and more robustly in the chemotaxis assays and thus was used to represent
the P. pacificus species. All Caenorhabditis and Pristionchus species have a similar generation time of 3–4 days at 20 C.
Chemotaxis Assay
We defined compounds at a given concentration (10%–1%) with an
average chemotaxis index (CI) of R0.5 as attractive compounds, CI
between 0.3–0.5 as weakly attractive compounds, CI between 60.3
as neutral compounds, and CI less than 20.3 as repulsive compounds (Table S1). Because the chemotaxis assay using round petri
dishes underestimates the degree of repulsion compared to the repulsion assays using rectangular plates, we did not rank the strength
of the avoidance behavior [S1]. Also in accordance to previous studies in C. elegans, the compound concentrations are diluted in
a weight-volume or volume-volume manner, except where specifically indicated in molarity.
Population chemotaxis assays at 23 C were modified from that
used on C. elegans [S2]. The chemotaxis index was calculated as
the ratio of the number of animals at attractant minus the number
of animals at counterattractant over the total number of worms at
both attractant and counterattractant. With the strongest attractants, approximately 50%–70% of the loaded Pristionchus worms
reach in the vicinity of the point odor source. This is in contrast
to >90% C. elegans reaching the attractant after 1 hr. We used
5-day-old cultures started with 10 gravid hermaphrodites grown at
20 C to conduct the chemotaxis assays because by then most
E. coli have just been consumed and >80% of the worms were at
adult stages.
Worms were washed three times in M9 buffer, left to settle in 15 ml
polypropylene tubes by gravity, and loaded onto 8.5 cm round assay
plates containing 0.5 cm thick NGM agar. We used 1:10,000 dilutions
of diacetyl and b-caryophyllene in NGM agar for our discrimination
assays [S2]. Unlike Caenorhabditis species, we found that resuspending Pristionchus species in water before loading reduces their
chemoattractive response. Therefore, concurrent assays using Caenorhabditis and Pristionchus species were last washed in M9 buffer
before loading, whereas comparisons within Caenorhabditis and
Pristionchus species were last washed in water and M9 buffer, respectively. Loading C. elegans with M9 buffer did not seem to alter
their chemotaxis behavior. For Caenorhabditis species, 1 ml of the
attractant and counterattractant (e.g., 100% ethanol as diluent)
were used as the point odor sources, and 1 ml of 1 M sodium azide
were also added prior to each source for anaesthetization and prevention of early attracted worms from adapting to the odor and leaving the area. Pristionchus species are more resistant to azide and
therefore 1.5 ml of attractant or counterattractant and sodium azide
were used. Animals within a w0.5 cm radius circle around each point
odor source were counted at 30 and 60 min for Caenorhabditis species, and 1.5, 2.5, and greater than 12 hr intervals for Pristionchus
species. Three to five replicates containing at least ten scorable
animals were carried out for every condition tested. All attractive
responses described were summarized from at least two separate
experiments so that we could control for worm batch variations,
and only peak and consistent positive chemotaxis responses were
reported here. We interpret those compounds with 95% confidence
intervals intersecting zero as neutral because nematodes can randomly bias one side of the plate versus the other when there is no
attraction. In the experiments involving gonochoristic species
(P. uniformis and C. remanei) or P. pacificus Washington male
stocks, we did not observed sex bias in the worms scored at the
attractive sources. We used Microsoft Excel to calculate twosample equal variance Student’s t test and single-factor ANOVA
(a = 0.05). Error bars denote 95% confidence intervals.
All chemicals were purchased from Sigma-Aldrich (>97% purity;
MO) and diluted in 100% ethanol (v/v, w/v, or molar concentrations)
in most cases. b-caryophyllene does not fully dissolve in 100%
ethanol but can be applied as a suspension mix after thorough
vortexing. L-methyl leucine ester HCl salt was first dissolved in diethyl ether and then diluted in ethanol. 1-mono-olein was diluted
in hexane. Toluquinone and pyrazine crystals were diluted in 100%
ethanol to 10% (w/v) as stock solutions.
Supplemental References
S1. Troemel, E.R., Kimmel, B.E., and Bargmann, C.I. (1997). Reprogramming chemotaxis responses: Sensory neurons define olfactory preferences in C. elegans. Cell 91, 161–169.
S2. Bargmann, C.I., Hartwieg, E., and Horvitz, H.R. (1993). Odorantselective genes and neurons mediate olfaction in C. elegans.
Cell 74, 515–527.
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