Insect egg deposition induces defence responses in Pinus sylvestris: characterisation of the elicitor
1 Institute of Biology, Freie Universität Berlin, Haderslebener Str. 9,
D-12163 Berlin, Germany
2 Finnish Forest Research Institute, Vantaa Research Centre, PO Box 18,
FIN-01301, Vantaa, Finland
* Author for correspondence (e-mail: hilker{at}zedat.fu-berlin.de)
Accepted 2 March 2005
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Summary |
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Key words: elicitor, egg deposition, egg parasitoid, induction, oviduct secretion, sawfly, Diprion pini, volatiles
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Introduction |
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Egg deposition by the phytophagous sawfly Diprion pini has been
shown to induce the plant to release volatiles attracting Chrysonotomyia
ruforum, an egg parasitoid of D. pini
(Hilker et al., 2002b). This
induction of volatiles is not restricted to the oviposition site, but also
occurs in surrounding tissue that is not damaged (systemic effect). During egg
deposition, D. pini females make a tangential slit in the pine
needles with their sclerotized ovipositor valves, and insert the eggs into the
wounds in the needles. Finally, eggs are covered on the top by a mixture of a
frothy secretion and needle tissue that hardens within a few hours
(Eliescu, 1932
). The elicitor
inducing the pine's response was found to be located in the oviduct secretion
coating the eggs, since application of the oviduct secretion into artificially
wounded pine needles also resulted in the induction of volatiles attractive to
the egg parasitoid, whereas artificial wounding alone did not
(Hilker et al., 2002b
). For
application of the oviduct secretion to artificially wounded pine needles,
oviducts were dissected from sawfly females and secretion was obtained by
washing the oviducts in distilled water. The freshly isolated secretion was
directly transferred into the wound of a pine needle
(Hilker et al., 2002b
).
Prior to the study presented here, nothing was known about the chemistry
and stability of the elicitor located in the oviduct secretion. Thus, we
tested whether the elicitor could be isolated by distilled water or other
solvents and we examined how to store it. Furthermore, we did not know whether
wounding of the pine needle is necessary for the eliciting process or whether
the elicitor is also active when being applied onto an intact, non-wounded
pine needle. Additionally, since our method of obtaining oviduct secretion
from sawfly females did not avoid contamination with hemolymph, the role of
hemolymph in the eliciting activity was unclear. Since oviducts and accessory
reproductive structures are known to be rich in proteins (Gillot, 2002;
Hinton, 1981), we examined
whether a proteinase destroys the eliciting activity of the oviduct
secretion.
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Materials and methods |
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Olfactometer bioassay - general procedures and data collection
All bioassays were conducted with a four-arm olfactometer as described in
detail by Hilker et al.
(2002b). The airflow was
adjusted to 155 ml min-1. We recorded how long the parasitoid was
present within each of the four odor fields over a period of 600 s using the
software program The Observer 3.0 (Noldus, Wageningen, The Netherlands). Only
data obtained from active parasitoids walking for at least 300 s of the
observation period were used for statistical analyses (see below). Parasitoids
preferentially walking in the olfactometer field provided with the test odor
were defined as being `attracted' since significantly longer walking periods
in the odor field is usually interpreted as a response of the parasitoid to an
attractive odor (Hilker et al.,
2002b
). The number of parasitoids used for each bioassay was 22 to
36 (see Table 1). The odor
source was changed after five to nine parasitoids had been tested. One to two
odor sources were tested per day.
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Plant treatments
Small pine twigs of about 20 cm length with approximately 90-120 needle
pairs were cut, placed into water, and treated for a period of 72 h at
25°C and 18 h:6 h L:D. In all but one bioassay (see
Table 1) artificially wounded
pine twigs were used. For artificial wounding, pine needles were slit
tangentially with a scalpel prior to treatment, as described by Hilker et al.
(2002b). If not mentioned
otherwise, the differently treated oviduct secretion samples or respective
control samples were applied into artificially wounded pine needles (see
below). Eight needles of each twig were treated. All pine twigs were tested
systemically as described in detail by Mumm et al.
(2003
), i.e. the lower half of
a pine twig was treated, while the upper part was left untreated but was
wrapped in a polyethylene terephthalate (PET) foil to avoid adsorption of
volatiles from other parts of the twig. This ensured that any volatiles
emitted by the lower, treated half of the twig could be adsorbed by the upper,
untreated half. After 72 h, the upper half of the twig was cut off, the
PET-foil was removed, the cut end of the twig was tightly wrapped with
Parafilm®, and the twig was transferred into the olfactometer to test for
systemic induction. If the twig had been systemically induced, the parasitoids
were expected to be attracted to its volatiles
(Hilker et al., 2002b
).
Bioassay I: solution and storage of the oviduct secretion
(a) In a first experiment, the oviduct secretion was obtained by dissecting
the oviducts (oviductus lateralis and o. communis) of four D. pini
females. Oviducts were transferred to 8 µl of ice-cold Aqua dest
(=distilled water). To remove oviduct cell fragments, samples were centrifuged
(10 min at 12,700 g) and the supernatant containing the
oviduct secretion was immediately applied, in 1 µl portions, to wounded
pine needles (8 needles per twig). After 72 h, the untreated upper part of the
pine twig was tested in the olfactometer.
(b) We next examined whether oviduct secretion remains active after 3 h of storage at room temperature (ca. 20°C). This storage period was chosen because proteinase K treatment (see below) needs an incubation time of 3 h at room temperature. Oviduct secretion was obtained, diluted in Aqua dest. and treated as described for bioassay a. However, prior to application to the wounded pine needles, the oviduct secretion was left in the dark at room temperature for a period of 3 h.
(c) To test whether oviduct secretion diluted in Aqua dest. keeps its activity when frozen directly after dissection, it was stored in a freezer for at least 2 days at -80°C. Samples defrosted at room temperature within a few minutes and were then directly applied to wounded pine needles. Other conditions were the same as described for bioassay (a). The retention of activity of the oviduct secretion after freezing would be very useful to yield larger amounts that are necessary for further analyses.
(d) As a control, slit pine needles were treated with Aqua dest. Eight needles were treated and 1 µl was applied per wounded needle.
(e) Oviduct secretion was obtained as described above (bioassay a), but transferred into Ringer solution (pH 7.2, Merck, Darmstadt, Germany) and then stored in the dark for 3 h at room temperature (ca. 20°C). Treatment of pine needles was the same as described for bioassay a.
(f) To test whether oviduct secretion diluted in Ringer solution can be stored frozen without loosing activity, we kept it frozen as described for bioassay c. For the bioassay, defrosted oviduct secretion in Ringer solution was directly applied to wounded pine needles.
(g) As a control, eight wounded pine needles were treated with Ringer solution (1 µl per needle) only.
Bioassay II: determination of whether damage of needles is necessary for the plant to respond to oviduct secretion
(h) Oviduct secretion was obtained as described for bioassay a and
transferred into Ringer solution (pH 7.2, Merck, Darmstadt, Germany). However,
pine needles were not wounded prior to application of the secretion. Instead,
1 µl secretion was slowly applied to an intact needle.
Bioassay III: the effect of hemolymph in eliciting activity
(i) Legs of sawfly females were cut and 1 µl of the emerging hemolymph
was obtained with a glass capillary and transferred into Ringer solution.
After 3 h storage in the dark at room temperature, slit pine needles were
treated with these samples (compare bioassay e).
Bioassay IV: the effect of proteinase K on the activity of the elicitor
(j) Oviduct secretion was obtained and stored in Ringer solution as
described for bioassay e. 10 vol.% proteinase K (pH 7.2; Merck, Darmstadt,
Germany) was added to the sample. During an incubation period of 3 h, samples
were stored in the dark at room temperature (ca. 20°C). The proteinase K
was covalently bound to small latex bead so that it could be separated from
the secretion after incubation, by centrifuging the sample for 10 min at 8800
g. The supernatant was then applied to artificially wounded
needles as described above.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
In order to examine the digestion of proteins in the oviduct secretion by
proteinase K treatment, and to check that all proteinase K had been removed
prior to the treatment of needles, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) of digested and undigested oviduct secretion was
performed. Samples were prepared as described for bioassays e and j. Digested
samples were analyzed after removal of proteinase K by centrifugation. For
comparison, hemolymph samples were also analyzed by SDS-PAGE. Electrophoresis
was performed on a gradient gel (T=5-17.5%; C=4%) according to the procedure
of Laemmli (1970). Protein
molecular mass markers (Precision Plus Protein, BioRad, München, Germany)
were used. The starting voltage for electrophoresis was 100 V until a uniform
front occurred. Then, the voltage was raised to 200 V. The gels were stained
with Coomassie Brilliant Blue R-250 (Roth, Germany). To obtain a better
visualization of the bands, the gels were analyzed using the software program
Scion Image (Scion Corp., Frederick, MY, USA).
Statistics
Bioassay data were statistically analyzed using the Friedman analysis of
variance (ANOVA) for comparing residence time within each of the four
olfactometer fields using the software program SPSS 11.0. (SPSS Inc., USA).
The Wilcoxon-Wilcox test was used for post-hoc comparisons
(Köhler et al.,
1995).
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Results |
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Wounding of pine needles was found to be essential for the induction process. Volatiles from undamaged pine twigs treated only topically with oviduct secretion did not attract C. ruforum (Table 1, bioassay h). Therefore, the elicitor is only effective when transferred into damaged needle tissue.
Hemolymph of D. pini females did not induce the emission of pine volatiles attractive for the egg parasitoids (Table 1, bioassay i). Since the oviduct secretion does not normally come into contact with hemolymph during oviposition, free hemolymph in the female's abdomen does obviously not play a role per se in the induction of the volatile compounds as a result of egg deposition.
The activity of the elicitor in the oviduct secretion was lost after treatment with proteinase K, because volatiles of pine twigs treated with proteinase K-digested oviduct secretion were not attractive to the parasitoids (Table 1, bioassay j).
SDS-PAGE
Electrophoresis of undigested oviduct secretion showed seven bands with
molecular masses from ca. 10-250 kDa, visualized by seven peaks in the Scion
image (Fig. 1). The band
pattern of the hemolymph was similar, but one band (no. 1) was missing
compared to the oviduct secretion. Band 2 was regularly visible in the
hemolymph samples, although only a small peak is shown in
Fig. 1. SDS gels of digested
oviduct secretion showed no bands, confirming that all proteins were destroyed
by proteinase K treatment. Furthermore, no proteinase K residues were
detected, thus confirming that the enzyme was completely removed from the
sample prior to its application to pine needles
(Fig. 1).
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Discussion |
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To our knowledge, the biochemistry of only two elicitors inducing plant
responses to egg depositions have been intensively investigated
(Hilker et al., 2002a). (1)
`Bruchins' isolated from bruchid beetles, have been found to elicit a response
in peas (Pisum sativum) that directly affects the herbivore. Egg
deposition by the beetles induce growth of neoplasms at the site of egg
attachment. The plant's response to egg deposition may protect the pea pod
from larval feeding damage, because the egg on the neoplasm may easily be
detached from the pod (Doss et al.,
1995
,
2000
). When isolated bruchins
are applied onto responsive pea pods, they also induce neoplastic growth. The
bruchins are long-chain
,
-monounsaturated C22-diols
and
,
-mono- and diunsaturated C24-diols (Oliver et
al., 2000
,
2002
).
(2) In the tenthredinid sawfly Pontania proxima, an
elicitor of a plant reaction is known to be located in the secretion of
accessory glands. When P. proxima lays an egg onto a Salix
fragilis leaf, the egg deposition induces mitogenesis of plant tissue and
growth of a gall is initiated. Chemical analyses revealed that the secretion
of the accessory glands contains nucleic acids, protein
(Hovanitz, 1959), uric acid,
adenine derivatives, glutamic acid and possibly uridine
(McCalla et al., 1962
). Leitch
(1994
) suggested that
precursors of cytokinins may be present in the ovipositional fluid and act as
gall-initiators, however, Higton and Mabberley
(1994
) doubt that cytokinins
induce the galls.
Several elicitors of plant defensive responses to feeding herbivores have
been isolated from regurgitate of herbivorous larvae
(Felton and Eichenseer, 1999).
The components are known to be proteinous or to contain a peptide bond.
ß-Glucosidase has been isolated from the regurgitate of Pieris
brassicae larvae (Mattiacci et al.,
1995
). Volicitin (N-[17-hydroxylinolenoyl]-L-glutamine)
and other fatty acid-amino acid conjugates have been isolated from the
regurgitate of several lepidopteran species (Alborn et al.,
1997
,
2000
;
Halitschke et al., 2001
;
Mori et al., 2001
;
Pohnert et al., 1999
;
Turlings et al., 2000
). Musser
et al. (2002
) isolated a
glucose oxidase from the saliva of Heliothis zea. Their results
indicate that release of this glucose oxidase suppresses the plant's defensive
response, and thus, has been interpreted as counteradaptation by the
herbivore. Little is known about the mode of action of herbivore elicitors
(Dicke and van Poecke, 2002
).
In contrast to elicitors isolated from plant pathogens, no herbivore
defense-related plant perception mechanisms have been identified so far
(Dicke and van Poecke, 2002
;
Felton and Eichenseer, 1999
;
Ham and Bent, 2002
;
Martin et al., 2003
).
Our results revealed that the elicitor present in the oviduct secretion of
D. pini needs to come into contact with wounded plant tissue. Similar
results were found by Meiners and Hilker
(2000) in another tritrophic
system consisting of elm (Ulmus minor), the leaf beetle
Xanthogaleruca luteola, and the egg parasitoid Oomyzus
gallerucae. The elicitor present in the oviduct secretion of the elm leaf
beetle also needs to contact wounded elm tissue to induce production of
volatiles which attract the egg parasitoid of the elm leaf beetle. Elicitors
inducing plant responses to feeding herbivores have also been shown to need
disrupted plant tissue to become active
(Mattiacci et al., 1995
;
Turlings et al., 1990
). In
contrast, Colazza et al.
(2004a
) could show that the
pentatomid Nezara viridula lays its eggs on bean leaves without
wounding the plant tissue. These egg depositions induce the bean leaves to
release volatiles that attract the egg parasitoid. However, bean leaves
carrying eggs of the bug only released volatiles attractive to the parasitoid,
when the leaves had also been damaged by feeding. Leaves with eggs, but
without feeding damage did not emit the attractive volatiles. Thus, in this
tritrophic system also, leaf damage seems to be necessary to induce the
volatile blend attractive for the parasitoid. However, the elicitor associated
with the egg deposition seems to be able to become active also when being
applied to intact leaf surface.
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Acknowledgments |
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Footnotes |
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Present address: Laboratory of Entomology, Wageningen University,
Binnenhaven 7, NL-6709 PD Wageningen, The Netherlands
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