Antennal detection of sex pheromone by female Pandemis limitata (Robinson) (Lepidoptera: Tortricidae) and its impact on their calling behaviour

2005·5.7 MB·English

by  Naomi C Delury

J.Entomol. Soc.Brit. Columbia 102,December2005 3
Antennal detection of sex pheromone by female
Pandemis limitata (Robinson) (Lepidoptera: Tortricidae)
and its impact on their calling behaviour
NAOMI C. DeLURY^ ^ GARY J.R. JUDD^
and MARK G.T. GARDINER^
ABSTRACT
Previous observations lead us to believe that femalePandemis limitata (Robinson) (0 to
24 h old) are as attractive as theirpheromone gland extract to males in clean air, but are
more attractive in an environment permeated with their major pheromone component
(Z)-11-tetradecenyl acetate. Therefore, in this study, we tested the hypothesis that fe-
males can detect and/or respond to their pheromone components. Using electroanten-
nographic detection, we found female P. limitata able to perceive both oftheir known
pheromone components, (Z)-11-tetradecenyl acetate and (Z)-9-tetradecenyl acetate. Fe-
male antennal response was found to be 46.3% weaker than that ofmales, under identi-
cal conditions, with male antennae producing significantly higher deflections to the
higher pheromone doses tested and to the plant volatile, (£)-2-hexanal. Observations of
females in clean air versus (Z)-11-tetradecenyl acetate-permeated air showed no signifi-
cant differences with respect to onset time, frequency or duration of calling. Females
moved significantly less often in a (Z)-11-tetradecenyl acetate-permeated portion of a
flighttunnel than inthe corresponding clean-airportion.
Key Words: (Z)-11-tetradecenyl acetate, (Z)-9-tetradecenyl acetate, female electroan-
tennography, flighttunnel, matingdisruption, threelined leafroller, sprayablephero-
mone, microencapsulatedpheromone, movement
INTRODUCTION
Although pheromone-based mating dis- formation of this type is lacking for most
ruption of Lepidopteran species is widely species where commercial use of mating
employed in some agricultural systems disruption is under study. During our own
(Thomson et al 2001), questions about the studies of pheromone communication dis-
behaviour of female moths in the presence ruption in the threelined leafroller, Pan-
of their own sex pheromone often remain demis limitata (Robinson) (Lepidoptera:
unanswered. In particular, while there is Tortricidae), it appeared that males were
evidence that some female moths perceive more responsive to "calling" females (0 to
(Michell et al. 1972, Birch 1977, 24 h old) in a pheromone-permeated atmos-
Palaniswamy and Seabrook 1978, Light and phere than they were to female gland ex-
Birch 1979, Barnes et al. 1992) and even tracts, although no difference in male re-
modify their behaviour (Palaniswamy and sponse was detectable between these
Seabrook 1978, 1985, Palaniswamy et al. sources in clean air (N.C.D., unpublished
1979, Sanders 1987, Weissling and Knight data). One explanation for this observed
1996, Evenden 1998) in response to their difference is that female P. limitata can
own sex pheromone, others apparently do detect their pheromone environment and
not (El-Sayed and Suckling 2005) and in- adjust their behaviour in a manner that
'AgricultureandAgri-FoodCanada,PacificAgri-FoodResearchCentre,4200Hwy97, Summerland,BC,
VOH IZO,Canada
^Towhomcorrespondenceshouldbeaddressed:Tel.: [phone];Fax: + 1 250494 0755; E-mail:
[email protected]
4 J. Entomol. Soc.Brit.Columbia 102,December2005
makes them more detectable to males than 14:Ac] and (Z)-9-tetradecenyl acetate [Z9-
the female gland extract alone. Using elec- 14:Ac] (Roelofs et al. 1976) and asked the
troantennograms (EAGs), we assessed the question, do females alter their calling be-
ability of female P. limitata to perceive haviour in the presence of microencapsu-
their two known sex pheromone compo- lated (MEC) Zll-14:Ac applied in labora-
nents, (Z)-l1-tetradecenyl acetate [Zll- tory flight-tunnel assays (Juddetal. 2005)?
MATERIALS METHODS
Laboratory cultures. Experiments Company, St. Louis, MO), Z9-14:Ac (97%
were conducted with P. limitata from a purity, Regine Gries, Simon Eraser Univer-
laboratory colony maintained at the Pacific sity, BC), and a 94:6 ratio blend of both,
Agri-Food Research Centre, Summerland, respectively. The amount of Zll-14:Ac
British Columbia (BC) since 1992 that remained equal between the treatments of
originated from wild populations collected the main component and the blend; female
in the Similkameen Valley of BC. Pan- antennae were also exposed to 100 ^ig doses
demis limitata were maintained on a modi- from both Zl1-14:Ac and the blend. Each
fiedpinto bean-based diet (Shorey and Hale antenna was exposed to each dose of each
T
1965) at 25 under a 16:8 h L:D photo- ofthe three compounds in order ofincreas-
regime. Pupae were removed from diet, ing concentration. Treatments were puffed
sexed and placed individually in 150 ml (200 ms; 10 ml per s) over each antenna,
plastic cups provided with a wet cotton beginning with the lowest dose (0.1 ng) of
wick until adults eclosed. Male and female each of the three compounds, which were
moths were isolated from each other in presented in random order at each incre-
separate environmental chambers (25 ^C, mental dose. Baseline antennal responses
65% r.h. with a 16:8 h L:D reversed photo- were established using HPLC-grade hexane
regime). (two puffs) and a puffofthe plant volatile,
Female and male perception of syn- (£)-2-hexanal in paraffin oil, before and
thetic pheromone. Perception ofsynthetic after each pheromone test concentration.
pheromone by female and male P. limitata All stimulus puffs were apphed at 30 s in-
was measured using EAGs. Our EAG sys- tervals. All chemical stimuli were dis-
tem consisted of an IDAC-02 computer- pensed in 10 |il aliquots onto folded
coupled data acquisition board, an INR-02 Whatman #5 filter paper (3 cm x 2 cm),
EAG-SSR system and AutoSpike software placed in pipette tubes and connected to the
(Syntech, Hilversum, The Netherlands). puffer. The procedure was repeated using
Antennae excised from 0 to 24 h old fe- six female antennae and four male anten-
males or males were mounted individually nae.
inside 10 \A glass capillaries containing Normalized percentage antennal deflec-
silver-coated wire recording electrodes. tions were calculated using the plant vola-
Glass tubes were pulled at 300 °C on aNar- tile response as the standard. Data were
ishige (Model PN-30, Tokyo, Japan) micro- analyzed by one-way analysis of variance
pipette puller. The distal antennal segment (ANOVA) followed by Dunnett's test to
was removed and that end ofthe remaining compare each treatment mean to the hexane
antenna inserted into the indifferent elec- control. All experimental error rates were
trode capillary. As a resuh, each antenna set at a = 0.05. Comparisons between fe-
was suspended between two glass capillary male and male antennal responses at each
electrodes filled with insect Ringer's solu- dose were conducted on individual antennal
tion (DeLury et al. 1999). Each antenna deflections (mV) by first performing a two-
was challenged with six doses, in ten-fold way ANOVA, where the factors were sex
increments from 0.1 ng to 10 |Lig, of Zll- and pheromone stimulus. A sex x phero-
14:Ac (97.5% purity, Sigma Chemical mone stimulus interaction term was in-
J.Entomol. Soc.Brit.Columbia 102,December2005 5
eluded in the model. Following a significant entered the clean-air portion of the tunnel.
ANOVA, linear contrasts using /-ratio sta- Untreated panes were shielded during all
tistics were used to make paired compari- pheromone applications and the tunnel was
sons ofmean antennal deflections for males allowed to ventilate for a minimum of 18 h
and females at each dose. Experiment-wise between replicates. Pheromone treatment
error rates for these multiple-paired com- consisted of Phase I MEC Zll-14:Ac (3M
parisons were fixed at 5% by adjusting a Canada Company, London, ON) applied at
values for individual comparisons using the a rate equivalent to 10 mg ai m"~ to the
Bonferroni inequality. All statistical tests cross-sectional area of the tunnel as de-
wereperformedwith JMP 5.1 (2003). scribed in detail by Judd etal. (2005).
Female behaviour in a pheromone- Virgin female moths (0 to 24 h old)
permeated environment. Female moths were chilled for 5 min at 2 ^C and trans-
were placed in clean air or a pheromone- ferred individually into stainless steel mesh
W
permeated environment and observed in observation chambers (4.5 cm x 3.5 cm
side-by-side comparisons. To accomphsh H X 5 cm D). Chambers were stacked verti-
this, a sheet metal divider (73.3 cm x 37.4 cally to form a series of five individually-
cm) was installed vertically in the middle of caged females. Treated and control portions
the upwind portion ofa puUing-style flight of the tunnel each received one series of
tunnel [75 cm wide x 73.3 cm high x 187 five individually-caged females ca. 2.5 h
cm long flight section] covered with a re- before scotophase, for a total of 15 females
placeable 4 ml transparent polyester in each treatment. Females were observed
'skin' (3M Canada Company, London, ON) for calling (raised wings with protruding
(Judd et al. 2005), creating two identical abdomen) and movement (physically walk-
isolated chambers (upwind area = 36.65 ing in the chamber) every 15 m until the
cm^). The upwind cross-sectional surface initiation of scotophase, when they were
was constructed ofa set ofreplaceable hori- observed continually for 3 h. Data gathered
zontal sheet metal panes (73.3 cm x 3.7 cm before scotophase was used solely for de-
with a 0.7 cm bend forstability) onto which termination of onset of calling behaviour
pheromone was applied, with an untreated and was not included in any otheranalysis.
G200 Filtrete® with 0.5 oz Coverweb* (3M Frequencies, or the number of discrete
Canada Company, London, ON) that observations, of calling and movement
smoothed airflow, sealing the tunnel ca. 20 were analyzed separately using a two-factor
cm upwind ofthe panes. Smoke tests con- ANOVA where treatment (Zll-14:Ac- and
firmed that air was drawn directly into each clean-air) and vertical position of female
chamber at the upwind end and immedi- were the factors (JMP 5.1 2003). Onset and
ately moved downwind in a linear fashion duration of calling were analyzed using a
through the tunnel. After observing females two-sided Wilcoxon Signed Ranks Test
in both chambers in clean air, one chamber (JMP 5.1 2003), as the assumption ofnor-
was randomly chosen to receive pheromone mality was not met for these data. The tun-
(10 mg ai • m'^), which was apphed to hori- nel was assessed for positional bias be-
zontal metal panes forming the upwind end tween the two sides with respect to each
of the tunnel. As a precaution, pheromone behaviour before the application of phero-
was not applied to a 1.5 cm strip immedi- mone and resulting data were analyzed as
ately adjacent to the untreated chamber, above.
creating a buffer to ensure no pheromone
RESULTS
Female and male detection of syn- = 19.40; P < 0.0001). However, antennal
thetic pheromone. EAGs confirmed that deflections significantly differed from those
aduh female P. limitata can perceive both of the hexane control only for doses of 10
components oftheir sex pheromone (F21,224 |ig or greater for Zll-14:Ac and the 94:6
6 J. Entomol. Soc. Brit.Columbia 102,December2005
ratio of Zll-14:Ac: Z9-14:Ac (Fig. lA). 94:6 ratio of Zll-14:Ac: Z9-14:Ac. Male
Response to Z9-14:Ac differed from the antennal response to the plant volatile was
hexane control at each ofthe extreme doses also significantly higher than the female's
tested, 0.1 ng and 10 [xg, but did not differ (F2o,352=25.11;/'= 0.00134).
from any ofthe doses in between (Fig. 1A). Female behaviour in a pheromone-
Antennae of male P. limitata were more permeated environment. Observations of
sensitive to the pheromone components females in clean air and Zll-14:Ac-
than female antennae (with the exception of permeated environments showed that there
0.1 ng Z9-14:Ac), responding with deflec- were no significant differences in onset of
tions significantly higher than those to hex- calling {P > 0.934), frequency of calling
ane at 1 jig for each individual compound (Fi,24=0.6859; P > 0.4157), or duration of
and as low as 100 ng for the blend (Fig. calling (P > 0.890) (Fig. 2) among females.
IB). Male response to the plant volatile was However, females moved significantly less
also significantly higher than their response in the Zll-14:Ac environment
to hexane (Fig. IB). Females had signifi- (F],i1=5.0999; P < 0.0452) than in clean air
cantly (Fi.i9= 125.85; P < 0.0001) lower (Fig. 2). There was no observed positional
antennal deflections (least squares mean ± bias in the tunnel for either chamber with
SE: -6.13mV ± 0.30mV) when compared to respect to any of the observed behaviours
males (least squares mean ± SE: -11.41mV [{P> 0.152), (Fi.]4=3.5383; P> 0.0809), (P
=t 0.36mV). Comparisons of female and > 0.193) and (Fi.io=1.1703; P > 0.3047)
male antennal responses to individual doses respectively]. No vertical positional effects
showed that females had significantly were observed for frequency of calling or
(F2o.352= 25.11; P < 0.0001) lower antennal movement [(Fi.24=0.9835; P > 0.4352) and
deflections for 10 jig Z9-14:Ac, and for (Fi,i1=0.8725;P>0.5107) respectively].
both 1 |j.g and 10 |ig ofZll-14:Ac and the
DISCUSSION
Wehave found that adult femaleP. limi- The ability of female P. limitata anten-
tata (0 to 24 h old) are able to perceiveboth nae to perceive their own pheromone com-
of their pheromone components; however, ponents, albeit at high doses, is not surpris-
significant responses to the main compo- ing as this has been shown for other tortri-
nent alone or in a blend were only detected cids. Palaniswamy and Seabrook (1978)
for doses of 10 jig or greater, in contrast to noted that female Choristoneurafumiferana
males, which responded to 1 |ig ofthe indi- (Clemens) (Lepidoptera: Tortricidae) dis-
vidual compounds and to 100 ng of the played threshold response levels corre-
blend. Interestingly, female antennae gave a sponding to pheromone concentrations
significant response to only the lowest (0.1 above which various behavioural patterns
ng) and the highest (10 |ig) doses of the became evident, such as increased walking,
minor component Z9-14:Ac. It is possible extension of the ovipositor and antennal
that as treatments were presented randomly grooming. Ross et al. (1979) examined re-
in increasing concentrations, 0.1 ng Z9- sponse offemale C.fumiferana antennae to
14:Ac would have contacted a relatively differing doses ofpheromone and with in-
'fresh' antenna compared to subsequent creasing age, finding that females have a
doses, and as Z9-14:Ac is the minor phero- higher threshold for pheromone response
mone component, occurring at approxi- than males and that female antennae are at
mately 6 to 9% in the female gland their peak responsiveness in 3 to 6 d old
(Roelofs et al. 1976), the antennae may be insects. Higher response thresholds may be
able to perceive it at a lower level than Zl1- explained by the fact that antennae of fe-
14:Ac; however, a similarphenomenon was male C. fumiferana have one-third to one-
notobserved forthe blend orforthe males. half the number of sensilla trichodea as
J.Entomol. Soc.Brit. Columbia 102,December2005 7
CL
0 100-
1ng 10ng 100ng 10ug 01ng Ing 10ng lOOng lOug lOOiig 0Ing
Z11:Z9-140Ac(94:6)
B.
m
i
hexane PV 1ng 10ng 100ng Uig 10ng Oing
Z9-14:OAc Z11-14:0Ac Z11:Z9-14:OAc(94:6)
PheromoneStimulus(10|xlvolume)
Figure 1. Mean (± SE) normalized electroantennogram responses ofantennae from 0 to 24 h old A)
female (n = 6) or B) male (n = 4) Pandemis limitata to synthetic (Z)-11-tetradecenyl acetate (Zll-
14:0Ac) or (Z)-9-tetradecenyl acetate (Z9-14:OAc), or both in a ratio of 96:4. (£)-2-hexanal was
puffed overeachantennabefore and aftereachpheromone source forthepurpose ofnormalization of
each antenna over time and HPLC-grade hexane was puffed over each antenna at the beginning and
end to establish a base response. All stimulus puffs were spaced by 30 s intervals. Normalized per-
centage deflections were calculated using the plant volatile (PV) response as the standard. Asterisks
indicate a significant difference {P < 0.05) from the hexane base response as determined by an
ANOVAfollowedbyDunnett'stest, treatmentversushexane(a=0.05).
8 J.Entomol. Soc. Brit.Columbia 102,December2005
Atmospherictreatment
Figure 2. Mean (+ SE) duration (A and C) or frequency (B and D) of0 to 24 h old female P.
limitata in either side ofthe divided permeation turmel, exposed to either clean air or (Z)-ll-
tetradecenyl acetate (Zll-14:Ac) released from 3M microcapsules applied to theupwindportion
ofone side ofthe tunnel. Individually-caged females wereplacedin the tunnel (n= 15), immedi-
ately before pheromone application ca. 2 h before scotophase. Females were observed every 15
m
from application until initiation of scotophase for onset of calling, and continuously once
scotophase began for frequency and duration of calling and frequency ofmovement. For each
behaviourobserved,barswiththe same letterarenot significantly different {P> 0.05).
J.Entomol. Soc.Brit.Columbia 102,December2005 9
male antennae (Albert and Seabrook 1973). moved less often in air permeated with
In Trichoplusia ni Hubner (Lepidoptera: Zll-14:Ac than females in the clean air
Noctuidae), Light and Birch (1979) found (Fig. 2D). It is possible that the tendency to
that antennal response in females was 25% remain still in the pheromone background
that ofmales. Our results show that anten- may translate, in time, to increased calling,
nal responses in female P. limitata over a as observed in C.fumiferana (Palaniswamy
wide range of pheromone doses is 46.7% and Seabrook 1985). Sanders (1987) found
less than that ofmales, with significant dif- that although pheromone in the background
ferences observed at high doses for all com- clearly increased flight activity ofboth vir-
pounds tested. A difference was even ob- gin and mated females, virgin females re-
served forthe plant volatile. mained inactive for 48 h after emergence,
Simultaneous observations offemales in even in the presence of the pheromone. As
clean and pheromone-permeated air, from we observed increased movement of fe-
ca. 2 h before initiation ofscotophase until males in clean air, this does not appear to
3 h into scotophase, showed no difference be happening inP. limitata, although obser-
in onset of female calling time (Fig. 2A). vations over time would determine if the
However, a larger sample size may reveal level ofactivity increases with age.
that females in environments permeated Similar to our results in P. limitata, El-
with Zl1-14:Ac do initiate calling earlier as Sayed and Suckling (2005) found that per-
there was a large variance among females. manent exposure of female Eupoecillia
No differences were detected with respect ambiguella (Hubner) (Lepidoptera: Tortri-
to duration or frequency of calling once cidae), Lobesia botrana (Denis and Schif-
scotophase began (Fig. 2B, C). We did not fermilUer) (Lepidoptera: Tortricidae), or
have the ability to measure female output of Spodoptera littoralis (Boisduval)
pheromone directly in a background of (Lepidoptera: Noctuidae) to their main
Zll-14:Ac, therefore we must rely on ob- pheromone compounds did not alter the
servations ofbehaviour to give us an indi- timing, duration or frequency ofcalling. In
cation of female response. We did not ob- addition, Weissling and Knight (1996)
serve changes in frequency or duration of found that the temporal pattern ofcalling in
calling, as one might expect iffemales were Cydia pomonella (L.) (Lepidoptera: Tortri-
ahering concentrations or outputs of their cidae) was unaffected by the major compo-
effluvia to compete with the level of com- nent oftheir pheromone; however, the like-
pounds perceived in the background lihood of calling was increased. Neverthe-
(N.C.D., unpublished data). Of course, less, other species have been found to initi-
these changes, as well as a change in onset ate calling earlier in pheromone environ-
ofcalhng, may notbe observable in the first ments, such as female C. fumiferana
3 h ofscotophase, only becoming apparent (Palaniswamy and Seabrook 1985). At the
after the female has been in the environ- other extreme, Evenden (1998) observed a
ment continuously for more than 24 h delay in calling for female Choristoneura
(Palaniswamy and Seabrook 1985). In fact, rosaceana (Harris) (Lepidoptera: Tortrici-
studies on female C.fumiferana have found dae) in field plots treated with their com-
that female EAG responses to their sex plete pheromone blend, followed by a po-
pheromone increase with age at least until tential reduction in time spent calling com-
three days after emergence (Palaniswamy pared to their counterparts in clean air. It is
and Seabrook 1978), indicating that females important to remember that our experi-
may become more sensitive to their phero- ments, as well as those of El-Sayed and
mone over time. As we only looked at re- Suckling (2005), did not use the complete
sponse in 0 to 24 h old P. limitata females pheromone blend in the background, which
for a 5 h period, we would not have ob- could have a greater impact on female re-
served such aphenomenon ifone existed. sponse.
Our observations showed females While experiments described here do
10 J. Entomol. Soc. Brit. Columbia 102,December2005
not encompass all of the potential factors respect to frequency of movement, during
that may impact females in a pheromone the first 3 h ofthe first scotophase. As such,
environment, such as background phero- alternative explanations, such as mode of
mone dose, completeness of pheromone dehvery ofthe gland extract, as well as the
blend, age offemales or changes in mating use of nonchemical cues to attract males
status, our results are relevant to previous like sound or vision (Castrovillo and Carde
work on disruption of pheromone commu- 1980), or even chemical cues not associated
nication in P. Umitata (Judd et al. 2005; with the sex pheromone gland, need to be
N.C.D., unpublished data). The presence of explored to determine what cues become
the major pheromone component (Zll- important to males as they search for fe-
14:Ac, applied at 10 mg ai • m") does not male moths in pheromone-permeated envi-
appear to cause female P. Umitata to ronments (N.C.D., unpubhsheddata).
change their calling behaviour, except with
ACKNOWLEDGEMENTS
We thank Brenda Lannard for modifica- lations; Michael Roach for providing us
tions to the permeation flight tunnel, rearing with Filtrete*^ material; Tom Lowery and
insects and other technical assistance; Maya Evenden for kindly reviewing an
Regine Gries for supplying chemicals; earlier version of this manuscript. This re-
Lynn Lashiuk for laboratory assistance; search was funded by 3M Canada and the
Don Thomson and Kent Nielsen for techni- Agriculture and Agri-Food Canada Match-
cal advice on 3M MEC pheromone formu- ing Investment Initiative.
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