Chem. Senses 28: 545-549,
2003
© Oxford University Press 2003
Parasitic Wasps Learn and Report Diverse Chemicals with Unique Conditionable Behaviors
? Crop Management and Research Laboratory, Agricultural Research Service, United States Department of Agriculture, PO Box 748, Tifton, GA 31793 1 Department of Biological and Agricultural Engineering, University of Georgia, Tifton, GA 31793, USA 2 Institut für Biologie, Angewandte Zoologie/Oekologie der Tiere, Freie Universität Berlin Haderslebener Strasse 9, D-12163, Germany 3 Faculty of Agriculture, Kyushu University, Fukuoka 812–8581, Japan 4 Center for Medical, Agricultural, and Veterinary Entomology, PO Box 14565, Gainesville, FL 32604, USA 5 Netherlands Institute of Ecology, NIOO-CTO, PO Box 40, 6666 ZG Heteren, The Netherlands
Correspondence to be sent to: W. Joe Lewis, Crop Management and Research Laboratory, Agricultural Research Service, United States Department of Agriculture, PO Box 748, Tifton, GA 31793, USA. e-mail: wjl{at}tifton.usda.gov
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Parasitoids exploit numerous chemical cues to locate hosts and food. Whether they detect and learn chemicals foreign to their natural history has not been explored. We show that the parasitoid Microplitis croceipes can associate, with food or hosts, widely different chemicals outside their natural foraging encounters. When learned chemicals are subsequently detected, this parasitoid manifests distinct behaviors characteristic with expectations of food or host, commensurate with prior training. This flexibility of parasitoids to rapidly link diverse chemicals to resource needs and subsequently report them with recognizable behaviors offers new insights into their foraging adaptability, and provides a model for further dissection of olfactory learning related processes.
Key words: behavior, learning, Microplitis croceipes, olfaction
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Historically, we have marveled at the capacity of organisms to sense chemical cues and learn to utilize them for foraging and other forms of biotic interactions (von Frisch, 1915
,
1950;
Muller-Schwarze and Silverman,
1980; Menzel,
1985; Vet et al.,
1995; Eisner and Meinwald,
1999). We have been able to harness the chemical detection ability
in dogs for use in, for example, search and rescue, and detection of a
multitude of illegal chemical substances. Despite the abundant information on
olfaction and odor learning in organisms, the mechanisms or processes involved
in the use of odor cues in biotic interactions are poorly understood
(Alkasab et al.,
1999). Parasitoid species may provide good models for olfaction
studies, because of their extraordinary capacity to utilize odors and learning
to increase foraging success [reviews in
(Vet et al., 1995;
Takasu and Lewis, 1996;
DeMoraes et al.,
1998)].
Microplitis croceipes, a relatively specialized parasitoid of
three highly polyphagous larval hosts, Heliocoverpa zea, Heliothis
virescens and Heliothis subflexa (Lepidoptera: Noctidae) can
detect their hosts through associative learning of hosts, host byproducts and
host plant-related chemicals (Lewis and
Tumlinson, 1988; Turlings
et al., 1989; DeMoraes
et al., 1998). This wasp species has also demonstrated
the ability to learn and subsequently fly to the novel odors chocolate and
vanilla, and these parasitoids can learn these odors in association with both
food and hosts and subsequently respond in accordance to their physiological
state (Takasu and Lewis,
1993). However, encounters with plant-related compounds, such as
those associated with chocolate and vanilla, are expected within the scope of
typical foraging activities.
To further explore the range of odors that female M.croceipes can
detect and learn, they were conditioned to associate several structurally
diverse chemicals that they would not encounter in their natural foraging.
These compounds were cyclohexanone (a cyclic ketone), 3-octanone (an aliphatic
ketone), octanal (an aliphatic aldehyde), diisopropyl aminoethanol (an
aliphatic alcohol), and 2,4- and 3,4-dinitrotoluene (DNT) (aromatic
hydrocarbons with methyl and nitrogen side chains). These compounds were
presented with adult food as the unconditioned stimulus in flight bioassays.
Behavioral responses to cyclohexanone for food-associated odor conditioned
Manduca sexta (Daly et
al., 2001), and electroantennogram responses to cyclohexanone
and diissopropyl aminoethanol for unconditioned male and female M.
croceipes (Ochieng et al.,
2000; Park et al.,
2001) have been previously documented.
In addition to oriented flight, chemical cues also mediate a sequential
array of parasitoid foraging behaviors, including hovering, landing,
antennation and ovipositor probing (Lewis
et al., 1976). As the wasps get closer to the resource,
their behavior is progressively more intense, distinctly characteristic and
specific to the respective resource (Lewis
et al., 1976;
Wäckers et al.,
2002). Thus, we also examined whether `foreign' chemicals can
mediate these characteristic close-range responses, which historically have
been considered `genetically fixed' to cues strongly associated with the
subject resource.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Larvae of Heliocoverpa zea Boddie (Lepidoptera: Noctuidae) were reared on pinto bean artificial diet (Burton, 1969
). Microplitis
croceipes were reared on H.zea larvae at 28°C, 60–70%
relative humidity, and a 16 h/8 h light/dark cycle. All M.croceipes
females were 2–4 days old when tested, and food-conditioned females had
been provided water for 26–30 h at the time of the bioassays. A 50%
sucrose solution by weight (99% pure) in distilled water was the unconditioned
stimulus for all food conditioning, and feces from diet-fed third instar
H.zea larvae was the unconditioned stimulus for hosts. Food
conditioned females were fed for 5–10 s three times, with 30 s intervals
between feeding (Takasu and Lewis,
1996). For conditioning, odors were delivered to females while
they were feeding or antennating feces.
A structurally diverse group of compounds with very different volatilities
was chosen to probe the limits of the wasps' behavioral responses. For this
reason a variety of formulation methods were required to deliver the different
odors to the wasps at constant rates. No attempt was made to determine the
limits of responses to any of the compounds or to compare responses to equal
quantities of different compounds by the wasps since this was beyond the
objectives of this study. Cyclohexanone and diisopropyl aminoethanol, which
are highly volatile, were formulated in capillaries (0.7 mm i.d.), and the
length of the vapor column above the meniscus controlled release rate
(Weatherston et al.,
1985). Both 2,4- and 3,4-DNT, which have low volatility, were
dispensed from filter paper, and 3-octanone and octanal were dispensed from
membrane formulations (Heath et
al., 1996). The release rate of each compound from its
respective formulation was measured by collecting the volatized compound on a
Super Q (Alltech, Deerfield, IL) filter trap and analyzing by capillary GC-MS
(Heath et al., 1993).
All chemicals were obtained from Sigma and had chemical purity >99%.
Flight tests
Odor conditioning
Pure cyclohexanone and diisopropyl aminoethanol were loaded into 200 µl
capillary tubes (diam. 1.68 mm) with one end sealed; the length of the vapor
column above the meniscus was 1–2 mm. The capillary tube was held
upright within a horizontal glass tube, and air was pushed through the larger
tube at 
10 ml/min. Wasps fed while the odor was blown over their
antennae. Control females fed while air was blown over their antennae. Females
fed on sucrose and water at 2–3 mm from 3,4-DNT on filter paper (release
rate 11 ng/min). Control females were conditioned with sucrose and water
only. Both control and test females fed on sucrose and water while exposed to
membranes (Heath, 1996) loaded
with pure 3-octanone (release rate 33 ng/min).
Test
The flight tunnel was previously described
(Drost et al., 1986).
Females were placed on a stand (13 cm high), 80 cm downwind of the chemical
source(s). The air speed in the wind tunnel was 60 cm/s. Pure cyclohexanone
(300 ng/min) and diisopropyl aminoethanol (0.5 ng/min) were dispensed from
capillary tubes placed vertically at the upwind end of the wind tunnel. Two
filter papers (3 x 5 cm), folded in half with one loaded with
yellow-colored 3,4-DNT (release rate 11 ng/min) and the other a blank
(filter paper only), were placed in the wind tunnel with the yellow-colored
compound side of the paper facing away from the wasp to lessen any potential
for visual attraction. Females were tested to membranes
(Heath et al., 1996)
loaded with pure 3-octanone or octanal (33 ng/min).
Statistical analysis
For test and control females, the number of landings on the target or
alternate source was recorded. Females that did not complete a flight after
three chances or that did not take flight after 5 min were scored as no
response. A total of 8–10 females per control and odor-conditioned
treatments were assayed per day over 2- 4days. The influence of conditioned
odor on the response of trained and control females were tested with
chi-square analyses (SAS Institute,
1998).
We developed separate training protocols for the close-range
food-associated and host-associated behaviors. The food-associated behavior
that we termed `seeking' is a modification of `area restricted search
behavior' and related food-specific behaviors
(Curio, 1976;
Wäckers et al.,
2002). Instead of intense local search and substrate antennation,
we trained the odor-conditioned wasp to enter a recessed hole from which the
conditioned odor emanates (Figure
1A). The host-associated behavior we termed `coiling' is the
behavior this species exhibits just prior to host attack, whereby females rise
on their hind legs with a characteristic bending of their antennae
(Figure 1B).
|
Seeking behavior: food as unconditioned stimulus
Females were conditioned to 3-octanone (20 µl, 0.176 g/µl, dichloromethane) and 2,4-DNT (20 µl, 0.05 µg/µl, dichloromethane) on filter paper (2.3 cm diam.) placed in the center of a glass Petri dish (5 cm i.d., 1.5 cm high) covered with aluminum foil. A 4 mm2 filter paper saturated with sucrose and water was placed on top and center of the aluminum foil and 9–10 holes (1 mm diam.) were punctured through the foil around the edge of the filter paper so that odors flowed upward and around the food source.
Test females were released near a recessed hole (2 mm diam) in the center of a hollowed out Teflon stand (external: 2 cm diam. x 1 cm high; internal: 1 cm i.d, 0.5 cm high). This stand was set directly on top of the compound-loaded filter paper. Females responded positively by entering the hole (at least half of their body length within the hole) within 10 s. Females responded negatively by walking over the hole and around the Teflon stand for >10 s.
Coiling behavior: host as unconditioned stimulus
Females antennated larval feces near 2,4-DNT or 3-octanone loaded on filter paper as for food conditioning. A piece of filter paper (0.7–1 cm diam.) loaded with 20 µl of 0.05 µg/µl 2,4-DNT with dichloromethane or 20 µl of 0.176 g/µl of 3-octanone with dichloromethane was presented to the wasp at the end of a pin (6 cm long). The paper was gently and continuously waved in front of the wasps while maintaining a distance of 0.5–1 cm; none of the females were allowed to touch the paper. Females responded positively by coiling within 10–30 s. Females responded negatively by showing no coiling response within 1 min.
Statistical analysis
For each odor, 3-octanone and 2,4-DNT, a total of 40 food and
odor-conditioned females, 40 host and odor-conditioned females, 40 food-only
conditioned females (control), and 40 host-only conditioned females (control)
were tested for seek and coil behavioral responses. Positive and negative
seeking and coiling behaviors were recorded for each female. For each odor,
conditioning and testing was carried out over 4 days with a total of 10
females from each of the four conditioning regimes, five of which were tested
for seeking and five tested for coiling responses. All females were
conditioned and tested a single time. The influence of day and conditioning
regime on the seeking and coiling behaviors of odor-conditioned and control
females were tested with chi-square analyses
(SAS Institute, 1998).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
There were no significant effects of the day of conditioning and testing on wasp responses for 3-octanone (
2df3 = 1.79,
P = 0.615, n = 160) and 2,4-DNT
(2df3 = 0.79, P = 0.851, n =
160). Wasps that were trained to associate an odor with the food source were
significantly more responsive to the learned chemicals in upwind flight tunnel
bioassays than their control or untrained counterparts
(Figure 2A–D). The
trained wasps not only flew upwind more readily, but they showed more
straightline or directed upwind flight compared to the controls, and they
landed at the source of the learned chemical
(Figure 2A–D).
|
After training with chemicals in association with either sugar water or host feces, the wasps successfully linked each of the chemicals to both the food and host resource as shown by significantly higher behavioral responses to each of the food- or host-associated odors as compared to untrained wasps (Figure 3A,B). We also found that wasps will express the specific behavior, seeking or coiling, exclusively dependent on the conditioned stimulus (Figure 3A,B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Our study indicates that M.croceipes can learn a very broad range of chemicals as foraging cues, including chemicals not related to their natural history. The hosts of M.croceipes are highly polyphagous, feeding on >100 species of plants from several families (Li et al., 1992
). In
addition, plants emit numerous volatiles with various functional groups and
ranging in structure from short, straight carbon chains to aromatic
hydrocarbons and complex multi-ring sesquiterpenes
(Visser, 1979;
Knudsen et al., 1993;
Paré and Tumlinson
1997). Thus, the natural volatile compounds that these insects are
exposed to are many and diverse. Responses to such a diverse group of
compounds suggest that their receptors are broadly tuned. Their ability to
detect a broad array of chemicals and to show rapid adaptation, suggested by
the speed with which they can be trained, may be related to their ecology,
where they must locate very specific hosts species within highly variable and
dynamic foraging environments.
We have successfully conditioned M.croceipes to many other
compounds (Meiners et al.,
2002) (also J. Tomberlin et al., unpublished data), but
were not able to condition M.crociepes to nonanol and decanol
(Meiners et al.,
2002), although their antennae will respond to these relatively
long aliphatic alcohols (Li et
al., 1992). There are also differences in their responses to
similar compounds that differ in the position or the type of the functional
group (Meiners et al.,
2002); although they learned all compounds, they showed stronger
responses to some of them. It may be possible to use both positive and
negative rewards in conditioning to increase the responses to the lower
affinity compounds (Vet et al.,
1998).
After training with chemicals in close association with either sugar water
or host feces, the wasps successfully linked each of the chemicals to either
the food and host resource. Untrained wasps did not show these behaviors. We
also found that trained wasps will express the specific behavior, seeking or
coiling, exclusively in accordance to the resource, food or hosts, associated
with the conditioned stimulus. Thus, food as unconditioned stimuli will elicit
seeking but not coiling behavior, whereas, host frass as unconditioned stimuli
elicits coiling but not seeking behavior. In flight biaossays, Takasu and
Lewis (Takasu and Lewis, 1993)
found that M.crociepes could discriminate between expectations of
food or hosts utilizing different odors associated with each resource. Future
studies are planned to determine if they display their expectations with the
more stereotyped behaviors of seeking and coiling. Our results indicate that,
M.croceipes is highly versatile and effective in the mechanism by
which they employ chemical cues to enhance foraging success. They are able to
associate a highly diverse array of chemicals with the resource, as well as
utilize the cue to determine resource specificity and assess its
proximity.
This finding of additional levels of plasticity of predictable responses by
parasitoids not only expands our understanding of their foraging mechanisms,
but also opens important new prospects for innovative chemical detection
technology (DARPA, 2002). The
attribute of distinct behavioral displays in response to the learned chemicals
provides observers a non-arbitrary means to monitor for positive detection of
chemicals. These demonstrated abilities by insects, and probably other
invertebrates, greatly expand our potential resources for biological detectors
beyond those previously limited primarily to dogs. The short lifecycle and
genetic diversity of these organisms, along with the apparent speed with which
they can be trained, offers potential benefits of flexibility and convenience
to the science of olfactory learning, and the science of biological
detectors.
|
Acknowledgments |
|---|
The authors thank V. Jurjevic for assistance in the flight tunnel experiments, and two anonymous reviewers for their very helpful comments, which have improved the manuscript. The financial support of the Defense Advanced Research Projects Agency (DARPA) to W.J. Lewis and J.H. Tumlinson is gratefully acknowledged.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Alkasab, T.K., Bozzo, T.C., Cleveland, T.A., Dorries, K.M, Pearce, T.C., White, J. and Kauer, J.S. (1999) Characterizing complex chemosensors; information-theoretic analysis of olfactory systems. Trends Neurosci.,22 , 102-105.[CrossRef][Web of Science][Medline]
Burton, R.L. (1969) Mass rearing the corn earworm in the laboratory. US Dept. Agric. Tech Bull. ARS Ser., 33,134 .
Daly, K.C., Durtschi, M.L. and Smith, B.H. (2001) Olfactory-based discrimination learning in the moth, Manduca sexta. J. Insect Physiol.,47 , 375-384.[CrossRef][Web of Science][Medline]
DARPA (2002) Biosensor Technologies: Controlled Biological and Biomimetic Systems. Available at http://www.darpa.mil.dso.
Curio, E. (1976) The Ethiology of Predation. Springer-Verlag, Berlin.
DeMoraes, C.M., Lewis, W.J., Paré, P.W., Alborn, H.T. and Tumlinson, J.H. (1998) Herbivore-infested plants selectively attract parasitoids. Nature,393 , 570-573.[CrossRef]
Drost, Y.C., Lewis, W.J., Zanen, P.O. and Keller, M.A. (1986) Beneficial arthropod behavior mediated by airborne semiochemicals. I. Flight behavior and influence of preflight handling of Microplitis croceipes (Cresson). J. Chem. Ecol., 12,1247 -1262.
Eisner, T. and Meinwald, J. (eds) (1999)Chemical Ecology: The Chemistry of Biotic Interaction . National Academy of Sciences. National Academy of Science Press, Wash. DC.
Heath, R.R., Epsky, N.D., Landolt, P.J. and Sivinski, J. (1993) Development of attractants for monitoring carribean fruit flies (Diptera: Tephritidae). Fla. Entomol., 76,233 -244.
Heath, R.R., Epsky, N.D., Jimenez, A., Dueben, B.D., Landolt, P.J., Meyer, W.L., Aluja, M., Rizzo, J., Camino, M. and Jeronimo, F. (1996) Improved pheromone-based trapping system to monitor Toxotrypana curvicauda (Diptera: Tephritidae).Fla. Entomol. , 79,38 -48.
Knudsen, J.T., Tollsten, L. and Bergstrom, L.G. (1993) Floral scents—a checklist of volatile compounds isolated by head-space techniques. Phytochemistry,33 , 253-280.[CrossRef][Web of Science]
Lewis, W.J. and Tumlinson, J.H. (1988) Host detection by chemically mediated associative learning in a parasitic wasp. Nature, 331,257 -259.[CrossRef]
Lewis, W.J., Jones, R.L., Gross, H.R., Jr and Nordlund, D.A. (1976) The role of Other behavioral chemicals in host finding by parasitic insects. Behav. Biology,16 , 267-289.
Li, Y., Dickens, J.C. and Steiner, W.W.M. (1992) Antennal olfactory responsiveness of Microplitis croceipes (Hymenoptera: Braconidae) to cotton plant volatiles.J. Chem. Ecol. , 18,1761 -1773.
Meiners, T., Wäckers, F. and Lewis, W.J. (2002) The effect of molecular structure on olfactory discrimination by the parasitoid Microplitis croceipes. Chem. Senses 27,811 -816.
Menzel R., (1985) Learning in honeybees in an ecological and behavioral context. In Hölldobler, B and Lindauer, M. (eds), Experimental Behavioral Ecology. Gustav Fischer, Stuttgart, pp. 55-74.
Muller-Schwarze, D. and Silverman, R.M. (1980) Chemical Signals: Vertebrates and Aquatic Invertebrates. Plenum, Washington, DC.
Ochieng, S.A., Park, K.C., Zhu, J.W. and Baker, T.C. (2000) Functional morphology of antennal chemoreceptors of the parasitoid Microplitis croceipes (Hymenoptera: Braconidae).Arthrop. Struct. Dev. , 29,231 .
Paré, P.W. and Tumlinson, J.H. (1997) De novo biosynthesis of volatiles induced by insect herbivory in cotton plants. Plant Physiol.,114 , 1161-1167.[Abstract]
Park, K.C., Zhu, J., Harris, J., Ochieng, S.A. and Baker, T.C. (2001) Electroantennogram responses of a parasitic wasp, Microplitis croceipes, to host-related volatile and anthropogenic compounds. Physiol. Entomol., 26,69 -77.[CrossRef]
SAS Institute (1998) SAS User's Guide: Statistics. SAS Institute Inc., Cary, NC.
Takasu, K. and Lewis, W.J. (1993) Host-and food-foraging of the parasitoid Microplitis croceipes: learning and physiological state effects. Biol. Control, 3,70 -74.[CrossRef]
Takasu, K. and Lewis, W.J. (1996) The role of learning in adult food location by the Larval parasitoid, Microplitis croceipes (Hymenoptera: Braconidae). J. Insect Behav., 9,265 -281.[CrossRef]
Turlings, T.C.J., Tumlinson, J.H., Lewis, W.J. and Vet, L.E.M. (1989) Beneficial arthropod behaviour mediated by airborne semiochemicals. VII. Learning of host-related odours induced by a brief contact experience with host by-products in Cotesia marginiventris (Cresson). J. Insect Behav., 2,217 -225.[CrossRef]
Vet, L.E.M., Lewis, W.J. and Cardé, R.T. (1995) Parasitoid foraging and learning. In Cardé, R.T., Bell, W.J. (eds), Chemical Ecology of Insects, Vol. 2. Chapman & Hall, New York, pp. 65-101.
Vet, L.E.M., de Jong, A.G., Franchi, E. and Papaj, D.R. (1998) The effect of complete versus incomplete information on odor discrimination in a parasitic wasp. Anim. Behav., 55,1271 -1279.[CrossRef][Web of Science][Medline]
Visser, J.H. (1979) Electroantennogram responses of the Colorado beetle (Leptinotarsa decemlineata), to plant volatiles. Entomol. Exp. Appl.,25 , 86-97.[CrossRef]
von Frisch, K. (1915) Der Farbensinn und formensinn der biene. Zool. Abt. Allg. Zool. Physiol.,35 , 1-182.
von Frisch, K. (1950) Bees, Their Vision,Chemical Senses and Language . Great Seal Books, Cornell Univeristy Press, Ithaca, NY.
Wäckers, F.L., Bonifay, C. and Lewis, W.J. (2002) Conditioning of appetitive behavior in the Hymenopteran parasitoid Microplitis croceipes. Entomol. Exp. Appl., 103,135 -138.[CrossRef]
Weatherston, I., Miller, D. and Dohse, L. (1985) Capillaries as controlled release devices for insect pheromones and other volatile substances—a reevaluation. Part I. Kinetics and development of predictive model for glass capillaries.J. Chem. Ecol. , 11,953 -965.
Accepted June 13, 2003
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J.-N. Wei and L. Kang Electrophysiological and Behavioral Responses of a Parasitic Wasp to Plant Volatiles Induced by Two Leaf Miner Species Chem Senses, June 1, 2006; 31(5): 467 - 477. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||