Learning, odour preference and flower foraging in moths
1 Department of Zoology and Entomology, University of Queensland, St Lucia,
Brisbane 4072, Australia
2 Department of Primary Industries, Yeerongpilly, Brisbane 4105,
Australia
3 I.C.A.P.B., University of Edinburgh, Edinburgh EH9 3JT, UK
* Author for correspondence (e-mail: p.cunningham{at}uq.edu.au)
Accepted 30 September 2003
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Summary |
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We tested whether moths could discriminate between flowers that differed in
a single volatile component. Moths were trained to feed on flowers that were
odour-enhanced using either phenylacetaldehyde or -pinene. Choice tests
were then carried out in an outdoor flight cage, using flowers enhanced with
either volatile. Moths showed a significant preference for the flower type on
which they were trained. Moths that were conditioned on flowers that were not
odour-enhanced showed no preference for either of the odour-enhanced flower
types. The results imply that moths may be discriminating among odour profiles
of individual flowers from the same species. We discuss this behaviour within
the context of nectar foraging in moths and odour signalling by flowering
plants.
Key words: Lepidoptera, phenylacetaldehyde, -pinene, wind tunnel, volatiles, insect, preference, feeding
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Introduction |
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Pre-dating these techniques is a wealth of studies that focus on the
ecological and evolutionary importance of insect learning in foraging
behaviour (Marler and Terrace,
1984; Papaj and Lewis,
1993
; Papaj and Prokopy,
1989
). In Lepidoptera, learning has been shown to influence the
nectar-foraging behaviour of both butterflies (Lewis,
1989
,
1993
) and moths
(Cunningham et al., 1998
;
Lewis, 1989
;
Weiss, 1997
). Odour learning
in feeding behaviour has been elegantly demonstrated using the insect
proboscis extension reflex (PER), a technique pioneered by studies on bees
(Menzel and Bitterman, 1983
;
Smith, 1993
) and adapted for
moths (Daly et al., 2001
;
Fan et al., 1997
;
Hartlieb, 1996
). In PER
studies, the insect is restrained and the feeding response to an odour
stimulus can be measured before and after conditioning
(Smith, 1993
). In moths,
proboscis extension in response to an odour stimulus is strongly influenced by
associative learning (Daly et al.,
2001
; Fan et al.,
1997
; Hartlieb,
1996
). However, the influence of learning on preferences for
individual volatiles in free-flying moths remains to be demonstrated. If
learning strongly influences a moth's responses to floral volatiles during
foraging, the study of responses in naive moths may tell us little about the
extent to which certain odours attract moths in nature.
We investigated whether learning influences innate preferences for two
volatiles, phenylacetaldehyde and -pinene, in the nectar-feeding
noctuid moth Helicoverpa armigera. These volatile compounds are
common to many flowers that act as hosts for both nectar-feeding and
egg-laying Helicoverpa moths
(Bruce and Cork, 2001
;
Burguiere et al., 2001
). We
studied preferences for odours using a dual-choice flight test within a wind
tunnel. We tested (1) innate preferences, (2) preference immediately after a
conditioning treatment and (3) preferences 24 h after conditioning. We used
male moths to prevent interactions between oviposition and feeding behaviours
from influencing the preferences for odours.
Our wind tunnel experiment used a simplistic environment with few natural stimuli and controlled air flow. Learning to distinguish odours in a wind tunnel may tell us little about the importance of learning individual odour components in nature. In a second study, we integrated learning of single odours into the context of flower-visiting behaviour in foraging moths. Here, we tested whether moths could distinguish between flowers of the same species that emitted odour blends that differed by a single volatile. To achieve this we artificially manipulated the odour profiles of tobacco flowers by adding selected volatiles. Moths were conditioned to a particular flower type, and flower preferences were tested against a natural odour background in an outdoor flight cage.
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Materials and methods |
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In Experiment 1a and 1b, adult moths were kept in a laboratory at 25°C under ambient light conditions. In Experiment 2, moths were transferred to new holding cages and placed outdoors, under shelter. To prevent the insects from dehydrating in Experiment 2, cages were sprayed lightly with water (using a hand-held sprayer) at noon each day.
Tobacco (Nicotiana tabacum) was cultivated from seed under glasshouse conditions. To maintain new floral growth, maturing fruits were removed, preventing seed production.
Volatiles
The odours phenylacetaldehyde and -pinene (obtained from
Sigma-Aldrich, Sydney, NSW, Australia) were used in the conditioning
experiments. We used (racemic)
-pinene, which is a mixture of two (+
and ) enantiomers. Previous electroantennogram (EAG) studies have
demonstrated that these compounds elicit a peripheral olfactory response in
H. armigera (Bruce and Cork,
2001
; Burguiere et al.,
2001
).
Experiment 1: Odour preference in conditioned and unconditioned
moths
Dual-choice preference tests were carried out in a wind tunnel with a
Perspex flight chamber measuring 1600 mmx650 mmx650 mm. Air was
circulated through the flight chamber at 0.7 m s1 (as
measured at the centre of the chamber) using a fan system. A clean airstream
was maintained by passing the circulated air through an activated charcoal
filter and a dust filter before allowing it to enter the chamber. A laminar
airflow was obtained by directing air through a honeycomb of soda straws and
then a fine stainless steel screen (1.25 mm aperture) prior to entering the
chamber.
Adult Helicoverpa moths show a characteristic surge in activity
commencing at sunset, which corresponds with location of feeding sites
(Topper, 1987). Feeding
activity subsides around 90 min later
(Beerwinkle et al., 1993
). In
all experiments, trials commenced 15 min after sunset and were confined to a
90 min testing period. Moths were exposed to changing ambient light conditions
associated with sunset in order to instigate and maintain a regular pattern of
behaviour. Additional lighting (for observation) was supplied using a diffuse
light source, with the light intensity in the flight chamber measuring less
than 1 lux throughout the experiment. The temperature inside the wind tunnel
during the experiment was 24.4±0.12°C (mean ±
S.E.M.).
Three- and four-day-old moths that had been held in individual plastic containers (120 mm diameter) without access to food or water were used in experiments. One antenna of the moth was gently touched with a cotton wool bud that had been soaked in 25% (w/v) sucrose solution in order to test feeding responsiveness. Only moths that extended their proboscis once the cotton wool bud had made contact with the antenna were used in conditioning trials. Each moth was only used once.
Conditioning trials
Moths were randomly allocated to one of three treatment groups: (1)
`conditioned'; moths exposed to a volatile (phenylacetaldehyde or
-pinene) whilst feeding on sucrose solution; (2) `exposed'; moths
exposed to volatiles without allowing feeding; or (3) `no exposure'; moths
given no exposure to volatiles and left unfed. The groups were constructed in
order to ascertain whether feeding was required to initiate any changes in
preferences and whether any innate odour preferences existed. We did not look
in detail at the precise nature of the pairing between the unconditioned
stimulus (sucrose) and the conditioned stimulus (volatile) involved in odour
conditioning. This has been covered by previous studies on
Helicoverpa species using proboscis extension tests
(Hartlieb, 1996
;
Hartlieb et al., 1999
).
Treatment 1: conditioned
Odour sources (referred to as `lures' hereafter) were created by inserting
a 15 mm absorbent cotton wool plug to a depth of 25 mm below the wide end (5
mm diameter) of a 145 mm glass pipette. 2 µl of either phenylacetaldehyde
or -pinene were pipetted onto the cotton wool no more than 15 min
before the start of each experiment. The narrow end of the pipette was
inserted into a 40 mmx50 mmx50 mm block of floral foam
(Smithers-Oasis Ltd, South Australia), positioning the odour source at a
height of 145 mm above the floor of the wind tunnel. Feeding sites were
constructed similarly by plugging the end of a glass pipette with a cotton
wool wick soaked in 25% (w/v) sucrose solution. This second pipette was
positioned such that the sucrose wick was situated 2 cmdownwind of the lure.
New feeding sites and lures were used in each experiment.
Conditioning trials commenced by placing an individual moth on the sucrose wick and allowing a 30 s feeding bout. Feeding was identified as contact of the extended proboscis with the sucrose wick. In this way, the moth fed approximately 2 cm downwind from the lure. After 30 s, the moth was removed with a wooden toothpick and placed 400 mm directly downwind from the lure/feeding site. Moths were then allowed to fly freely back to the feeding source. Upon contact with the sucrose wick, the moth was allowed to feed for a further 20 s and was then returned to the downwind starting position. This process was repeated until moths had been given a total of four feeding visits in the presence of the volatile; one initial 30 s feed and 3x20 s return feeds.
Treatment 2: exposed
Moths were exposed to either phenylacetaldehyde or -pinene without
being allowed to feed in order to test whether any differences in response
between treatments may have occurred through exposure to the volatile,
irrespective of feeding. Each insect was placed into a 50 mmx50 mm black
mesh bag clipped (using a fold back clip) to a wooden skewer at a height of
130 mm. To expose the insect to the odour, the base of the skewer was inserted
into floral foam immediately downwind from the odour source, holding the
insect at the same height and position relative to the lures as insects used
in the conditioning trials. To match the exposure time in this treatment group
with that of the conditioning trials, each moth was placed in this downwind
position for four bouts (1x30 s and 3x20 s). Intervals of 1 min
were allowed between each exposure bout. During this interval, the moth was
placed 30 cm upwind of the lure in the centre of the wind tunnel.
Treatment 3: no exposure
We used unfed male moths with no previous exposure to either volatile to
determine the innate odour preferences. Adult moths were placed into
individual sealed (120 mm diameter) plastic pots upon emergence and kept until
testing at 34 days old. Preference for either -pinene or
phenylacetaldehyde was determined using the dual-choice testing procedure
described below.
Preference testing
Each preference test comprised a dual-choice test using one -pinene
and one phenylacetaldehyde lure, the same procedure being employed for all
three treatments. The lures were placed 300 mm apart at the upwind end of the
wind tunnel. Smoke tests (titanium tetrachloride) showed that, at a wind speed
of 0.7 m s1, these plumes remained separate within the wind
tunnel. Two 200 mmx150 mmx150 mm Perspex wedges were placed at the
downwind end of the wind tunnel, bringing odour plumes together at a distance
of 800 mm from the lures and leaving a 200 mm gap through which the odours
were directed into the rear 350 mm portion of the flight chamber.
Experiment 1a: preference testing (immediate)
Immediately following the conditioning treatment, the lure and feeding
source were removed. The two odour lures were placed in position only when
moths were in the 350 mm-long section at the downwind end of the wind tunnel,
where both the plumes had merged. This method was used in preference to
catching moths and placing them at the downwind end; disturbing moths in this
way often instigated avoidance behaviours and erratic looping movements. In
the absence of an odour plume, moths generally relocated to the downwind end
of the wind tunnel, making it easy to position the lures. If a moth remained
in the upwind end of the tunnel after a 3 min period it was caught and
released downwind once the lures were in position. In the exposed and no
exposure treatments, moths could be released directly into the downwind end of
the wind tunnel.
Preference for a volatile was seen as a characteristic upwind flight pattern in the odour plume to within 100 mm of a lure. Once a lure had been approached, the odour source (lure type) was recorded and the test was terminated. If moths failed to approach either lure within a 5 min period, the test was terminated. The position of the feeding lure and odour source in the conditioning trials (centrally placed; 325 mm from either wall) differed from the position of either lure in the preference trials (200 mm from either the right- or left-hand side wall) so that learning the position of the feeding lure would not influence the choice of lure in the test. The position of each lure (nearest to the right- or left-hand wall of the chamber) was allocated randomly throughout the experiment to avoid positional biases. The volatile used in conditioning treatments was alternated throughout the experiment.
Experiment 1b: preference testing (24 h after conditioning)
Moths were conditioned to either odour source as in Experiment 1a. Once the
conditioning treatment was completed, the moth was placed into a 120
mm-diameter airtight plastic container where it was held in the laboratory at
25°C under ambient light conditions for 24 h. The following night,
preference tests were carried out as in Experiment 1a. Moths were released
individually into the downwind end of the flight chamber and the lure
approached was recorded.
Experiment 2: odour learning using odour-enhanced flowers
Tobacco flowers attract feeding adult H. armigera
(Cunningham et al., 1998). We
used standard volatiles-trapping techniques followed by GCMS (gas
chromatographymass spectrometry) analysis to establish that no
phenylacetaldehyde or
-pinene was present among the volatile odour
compounds of cut tobacco flowers. This is consistent with published data
(Loughrin et al., 1990
). Our
choice of plant and test volatiles was directed in part by the desire to
augment the natural flower odour with compounds that were normally absent.
Odour profiles of tobacco flowers were augmented by adding either
phenylacetaldehyde or -pinene into the base of the corolla tube. In
this way, two types of flower were created, these flowers being identical in
visual cues but differing in specific olfactory components detected by
foraging moths. Tobacco flowers were picked one hour before dusk from plants
reared in the glasshouse. Using a micropipette, 2 µl of either
phenylacetaldehyde or
-pinene were added into the inside base of the
corolla tube. A third group of flowers, to which neither volatile was added,
was prepared. The corolla tube was partially plugged using a small absorbent
cotton wool plug that was lodged between the stamens at the lip of the corolla
(Fig. 1A). The cotton wool plug
was moistened with three drops (
0.1 ml) of 25% (w/v) sucrose solution
administered from a pipette. This procedure provided sufficient sucrose
solution for the duration of the conditioning experiments and prevented the
insects from contacting either the floral nectar or added volatiles. Previous
results have shown that moths that are fed from the top of the corolla will
not attempt to enter the corolla tube in order to probe deeper
(Cunningham et al., 1998
).
|
To construct a standardised inflorescence, five flowers (from the same treatment group) were inserted, to the depth of the calyx, into a block of `Oasis' floral foam (80 mmx60 mmx40 mm) such that a single flower protruded from each of five faces of the block (Fig. 1B).
Conditioning experiments
On any one night, moths were conditioned using a single treatment group of
flowers: (1) odour enhanced using phenylacetaldehyde; (2) odour enhanced using
-pinene or (3) flowers with no added volatiles (non-enhanced). The
standardised inflorescence was positioned at a height of 1 m on a bamboo cane
in the centre of an outdoor flight cage (1.8 mx1.8 mx1.8 m).
To begin each conditioning trial, a single male was removed from the
holding cage and encouraged to commence feeding using a cotton wool bud
moistened with 25% (w/v) sucrose. Once proboscis extension was observed, the
moth was placed onto the corolla lip of one of the tobacco flowers where it
was allowed to feed on the sucrose wick for 30 s. The moth was then removed
using a wooden toothpick and held at a distance of 200 mm from the flower
head. Insects were allowed three return visits to the flowers, with 20 s
feeding at each return. After the third return visit (insects having had a
total of 90 s of feeding), the moth was caught and held in a plastic
container. Moths were conditioned in this manner until feeding activity
subsided (90 min after the experiment commenced).
Preference testing
Preference testing of flower-conditioned moths was carried out on the
following night. Two standardised inflorescences were constructed as
previously described; one inflorescence using odour-enhanced flowers augmented
with phenylacetaldehyde, the other with flowers augmented with -pinene.
Flower corolla tubes were partially blocked with cotton wool, as in
conditioning trials, but no sucrose solution was added to the cotton wool.
Conditioned moths were released into the flight cage one hour before sunset.
Fifteen minutes before sunset, the inflorescences were placed on 1 m canes at
a distance of 1 m apart.
Moths that approached and landed on flowers were captured and the treatment
group of the inflorescence visited was recorded. Once captured, moths were not
re-released. The experiment was continued until all moths had been captured or
until flight activity ceased, 90 min later.
The experiment was repeated over 22 nights (seven trials for each odour-enhanced flower treatment and eight trials for the non-enhanced flower treatment). On each night of conditioning, the treatment group was assigned randomly. In preference tests, the position of the flower head within the cage was assigned randomly using a grid. Each insect was only used once.
Statistical analysis
Data were analysed using generalised linear modelling techniques
(McCullagh and Nelder, 1989)
in the GLIM statistical package (Crawley,
1993
). Choice test outcomes were analysed as proportions, with the
number of moths selecting a particular odour as the response variable and the
total number of moths selecting either host as the binomial denominator.
Binomially distributed error variances were assumed and a logit link function
employed. In all cases, we initially fitted a maximal model to the data, with
all explanatory variables and experimental treatments. We then used the
process of stepwise deletion (see Crawley,
1993
) to remove terms from the model until a minimal model was
obtained. Hypothesis testing was carried out using a Gtest on
differences in deviance. Differences in treatments were assessed by testing
whether grouping them caused a significant change in the deviance
explained.
In Experiments 1a and 1b, due to the low number of moths tested each night (46 moths), data were pooled over the 27 nights of testing. Treatment order was randomised to prevent any biasing that may have resulted from the night of testing. In Experiment 2, night of testing was included in the analysis to avoid pseudo-replication.
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Results |
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The innate preferences of the adult male moths for either
phenylacetaldehyde or -pinene were determined by testing moths that had
been given no experience of volatiles and no feeding experience before testing
(no exposure treatment). These moths showed a significant preference for the
phenylacetaldehyde lure (G(1)=7.312,
P<0.01).
All moths (N=40) tested on the same night as conditioning (Experiment 1a) flew to the lure emitting the volatile on which they had been conditioned. Feeding experience in the presence of a volatile therefore led to significant differences in odour choice (G(1)=55.45, P<0.001). No differences were found between moths given no experience of volatiles or feeding (no exposure group) and moths exposed to volatiles for the same amount of time as in conditioning trials but without pairing this with feeding (exposed group; G(2)=1.09). Thus, changes in preference were attributed to classical conditioning; pairing of odour with feeding.
When moths were tested 24 h after conditioning (Experiment 1b), preference
for the conditioned odour was significantly lower than in Experiment 1a moths
(G(1)=4.40, P<0.025). When the proportion of
`errors' (moths choosing the non-conditioned odour) per night was examined for
Experiment 1b moths, night of testing was not found to be significant
(G(6)=5.288); therefore, the decrease in preference in
this group was not attributed to greater error on any one night. Moths
conditioned on -pinene showed a significantly higher preference for the
-pinene lure compared with moths conditioned on phenylacetaldehyde
(G(1)=21.64, P<0.001) and moths without
associative conditioning (unconditioned moths and exposed moths;
G(1)=17.98, P<0.001). Preferences of
Experiment 1b moths conditioned on phenylacetaldehyde were not significantly
different from moths without associative conditioning
(G(1)=2.97, P>0.05).
Experiment 2: odour learning using odour-enhanced flowers
We carried out 22 trials using 111 adult male H. armigera. Moths
trained on -pinene- and phenylacetaldehyde-enhanced flowers (41 moths
per treatment) were trained over 14 trials (seven trials for each flower type,
5.86±0.48 moths per trial). Moths trained on non-enhanced flowers
(N=29 moths) were trained over eight nights (3.63±0.56 moths
per trial). Treatment groups showed significant differences in preference for
flowers (G(2)=19.5, P<0.001).
Moths conditioned on the -pinene-enhanced flowers showed a greater
preference for these flowers when compared with moths trained on
phenylacetaldehyde-enhanced flowers (G(1)=18.8,
P<0.001) and moths trained on non-enhanced flowers
(G(1)=8.0, P<0.005;
Fig. 2). Moths trained on
phenylacetaldehyde showed a preference for the flowers containing
phenylacetaldehyde but this was not significantly different from the moths
trained on non-enhanced flowers (G(1)=1.41,
P>0.05). Moths trained on non-enhanced flowers showed no
difference in preference for either flower type
(G(1)=0.07, P>0.05).
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Discussion |
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Moths flew upwind to both odours in the absence of conditioning, which
implies that an innate attraction to these odours exists. An innate preference
for phenylacetaldehyde over -pinene was demonstrated in this treatment
group, which suggests that attraction to odours is hierarchical, with certain
odours being more attractive than others. These innate preferences then become
modified through experience. Strictly speaking, conditioning to the odours in
this form is termed an
-response, as a prior response to the
conditioning stimulus (odour) already exists
(Menzel et al., 1993
).
Following a 24 h period without reinforcement, a strong preference for the
odour on which the moths were conditioned the previous night was still
evident. This suggests that foraging decisions that occur during one night of
feeding influence behaviour on the following night. The fidelity to the
learned odour after 24 h was lower than on the initial night of conditioning.
A decline in the strength of the conditioned stimulus in eliciting a response
with the absence of reinforcement is typical of classical conditioning
(Papaj and Prokopy, 1989).
Differences between the immediate test and the 24 h test may also be related
to the changes associated with short- and long-term learning and memory
(Menzel et al., 1993
).
When the odour of tobacco flowers was enhanced with either
phenylacetaldehyde or -pinene, feeding experience again led to
significant differences in flower visiting. Moths preferred to visit flowers
enhanced with the same odour as the flowers on which they were trained. Moths
could therefore discriminate between flowers that differed in a single
volatile compound. We therefore show that the discrimination and learning of
odours is not solely a product of a `timulus-deficient' wind tunnel
environment, where only a single conditioning stimulus (odour) is present.
Moths can detect differences in odours that may exist between flowers with
many common visual and olfactory stimuli. These differences are learned
associatively with feeding. Moths with experience of the non-enhanced flowers
show no preference for either enhanced flower type. Here, differences in
preference may reflect natural variations in odour output of individual
flowers.
The odour profile of flowers within a single species can vary in the
presence, concentration and relative proportions of their constituents at
different times of day (Baldwin et al.,
1997; De Moraes et al.,
2001
; Heath et al.,
1992
; Shaver et al.,
1997
). Such variations in odour output have been linked to the
attraction of pollinators and deterrence of pests
(De Moraes et al., 2001
;
Heath et al., 1992
). Other
variables, such as insect damage (De Moraes
et al., 2001
; Kessler and
Baldwin, 2001
; McCall et al.,
1994
) and the onset of pollination
(Schiestl and Ayasse, 2001
),
can lead to variations in odour among plants of the same species. Where such
odour signals are consistent with changes in nectar rewards from flowers,
recognising such correlations between odour and reward will have fitness
benefits to foraging insects. Associative learning of these subtle differences
in odour would be advantageous to the generalist forager.
In conclusion, we demonstrate that both innate and learned behaviours are
playing important roles in attraction to the individual volatile components of
a floral blend. Innate responses to odours predict the expected environment
and will have a strong influence on floral choice in newly emerged adult
insects. Learning shapes the insect's response to odours in its local
environment, increasing the response to odours that have previously led to
successful foraging. Thus, the role of odours in plantinsect
communication cannot be determined by concentrating solely on the behavioural
responses of naive moths. The `attractiveness' of volatiles to moths in nature
is likely to depend as much on ecological factors such as host abundance as on
inherited odour preferences (Cunningham et
al., 2001; West and
Cunningham, 2002
). Where learning has a strong influence on the
preference for floral odours, the volatiles emitted from the most frequently
visited rewarding host species will be those towards which the insect will be
the most attracted. Clearly, response to odour is a dynamic system that is as
dependent on an ever-changing environmental and behavioural context as it is
on a highly evolved system of odour recognition and response.
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Acknowledgments |
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