A protein from the salivary glands of the pea aphid,
Acyrthosiphon pisum, is essential in feeding on a
Navdeep S. Mutti*†‡, Joe Louis*, Loretta K. Pappan†§, Kirk Pappan†§, Khurshida Begum*, Ming-Shun Chen¶,
Yoonseong Park*, Neal Dittmer†?, Jeremy Marshall*, John C. Reese*, and Gerald R. Reeck†**
Departments of *Entomology and†Biochemistry, Kansas State University, Manhattan, KS 66506;¶U.S. Department of Agriculture, Department of
Entomology, Kansas State University, Manhattan, KS 66506; and?Cooperative Research Centre for National Plant Biosecurity, Canberra ACT 2617, Australia
Edited by Barry J. Beaty, Colorado State University, Fort Collins, CO, and approved March 26, 2008 (received for review September 20, 2007)
In feeding, aphids inject saliva into plant tissues, gaining access to
phloem sap and eliciting (and sometimes overcoming) plant re-
sponses. We are examining the involvement, in this aphid–plant
interaction, of individual aphid proteins and enzymes, as identified
in a salivary gland cDNA library. Here, we focus on a salivary
protein we have arbitrarily designated Protein C002. We have
shown, by using RNAi-based transcript knockdown, that this pro-
tein is important in the survival of the pea aphid (Acyrthosiphon
protein, its transcript, and its gene, and we study the feeding
process of knockdown aphids. The encoded protein fails to match
any protein outside of the family Aphididae. By using in situ
hybridization and immunohistochemistry, the transcript and the
protein were localized to a subset of secretory cells in principal
salivary glands. Protein C002, whose sequence contains an N-
terminal secretion signal, is injected into the host plant during
c002-knockdown aphids, we find that the knockdown affects
several aspects of foraging and feeding, with the result that the
c002-knockdown aphids spend very little time in contact with
phloem sap in sieve elements. Thus, we infer that Protein C002 is
crucial in the feeding of the pea aphid on fava bean.
aphid–plant interaction ? saliva ? RNAi ? electrical penetration graph ?
the insect and the defense systems of the plant (for recent
reviews, from several perspectives, of aphid–plant interactions,
see refs. 1–5). When, in an ongoing coevolution, the aphid has
(temporarily) established an advantage, it can probe to and enter
its feeding site, the sieve element containing phloem sap, the
aphid’s source of nutrients; can overcome the plant’s defense,
possibly during probing and certainly during its extended feeding
on the phloem sap; and will eventually withdraw from the plant,
having, at least in some cases, not caused great damage. This
sequence of events establishes the host range of the aphid. From
the standpoint of this article, it is important to note that
salivation continues throughout the process, including the ex-
tended feeding on phloem sap (3).
Despite decades of investigation, the steps or phases in aphid
feeding remain poorly understood at a molecular level. Recently,
however, Will et al. (6) have identified a system in which they
have gained considerable insight at the molecular level. In
particular, they demonstrated that calcium-binding proteins of
aphid saliva may undermine a calcium-requiring mechanism of
plant defense. Such aphid proteins, in binding Ca2?in phloem
sap, would cause a conversion of the proteinaceous ‘‘forisomes’’
of sieve elements to a contracted (nonblocking) state, thus
preventing the forisomes from occluding the sieve tubes, which
of phloem sap through the puncture wound from the aphid’s
he ability, or inability, of an aphid to feed on a plant results
from a multifaceted interplay between the feeding systems of
stylets. At this point, the individual proteins involved in this
undermining of this plant defense are unidentified, but the work
of Will et al. (6) illustrates well that the saliva of aphids likely
holds the secret to many aspects of aphid–plant interaction.
Aphid saliva has therefore received a good deal of attention.
Until recently, most studies have been at an enzymatic level. This
large body of work has been comprehensively reviewed by Miles
(7). Summarized briefly, two broad types of enzymes can be
expected in the saliva of any aphid, namely oxidoreductases and
hydrolases. Among the first type, polyphenol oxidase and ‘‘per-
oxidase’’ are of special note because of their occurrence in many
aphid species and their centrality in Miles’ ‘‘redox hypothesis’’ of
detoxification of defensive phytochemicals. The most commonly
and pectinases. The difficulties faced in such work are, however,
considerable, given the minute amounts of saliva produced, and
the biochemical composition of any given aphid’s saliva.
Accordingly, we have opted for a molecular genetics ap-
proach; that is, we aim to access, and then study, individual
proteins of saliva through salivary gland cDNA libraries. From
an initial examination of ?4,700 ESTs from such a library, we
have selected one contig for detailed investigation. It was
arbitrarily designated transcript c002 and was selected on the
basis of its abundance (it was the seventh most populous EST
contig) and the presence of a full open-reading-frame, encoding
peptide for secretion at its N terminus. We have reported that
RNAi-based c002 transcript knockdown dramatically reduces
the life span of pea aphids on fava bean leaves (8).
Here, we report on the transcript c002, its gene, and the
encoded protein, including localization of the transcript and
protein in a subset of secretory cells in the principal salivary
glands and the transfer of Protein C002 to the host plant during
aphid feeding. Using the electrical penetration graph (EPG)
method (3), we have studied the feeding behavior of pea aphids
in which transcript c002 has been knocked down. From these
Author contributions: N.S.M., K.P., M.-S.C., Y.P., J.C.R., and G.R.R. designed research;
N.S.M., J.L., L.K.P., K.P., and K.B. performed research; N.S.M., K.P., M.-S.C., Y.P., N.D., J.M.,
J.C.R., and G.R.R. analyzed data; and N.S.M., Y.P., N.D., J.M., J.C.R., and G.R.R. wrote the
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. CN763138).
‡Present address: School of Life Sciences, Arizona State University, Tempe, AZ 82287.
**To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0708958105 PNAS ?
July 22, 2008 ?
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studies we conclude that Protein C002, a secreted component of
salivary glands, is essential for feeding on fava bean by the pea
Sequences of Transcript c002 and Protein C002. In Fig. 1 we present
the unigene nucleotide sequence of transcript c002 and its
predicted encoded amino acid sequence. The predicted protein
is predicted by SignalP to be a signal peptide for an extracellular
protein (www.cbs.dtu.dk/services/SignalP/), with cleavage pre-
dicted between residues 23 and 24. The mass of the predicted
mature protein is 21.8 kDa. There are no potential O-
glycosylation sites (www.cbs.dtu.dk/services/NetOGlyc/) or N-
glycosylation sites (www.cbs.dtu.dk/services/NetNGlyc/). Pro-
grams for prediction of secondary structure predict a high helix
content interrupted by loops or turns. For instance, the PROF
program of Rost et al. (9) predicts 62% helix in the mature
protein and on this basis tentatively classifies the Protein C002
c002 is an abundant EST in our salivary gland cDNA library.
In ?4500 ESTs from the library, there are 17 occurrences of the
c002 EST, a 14-fold higher frequency than among head and
whole-body ESTs (even though these include a normalized
whole-body library), thus indicating an enrichment of the cDNA
in the salivary gland library.
Blasting c002 against the nonredundant database and the EST
database at National Center for Biotechnology Information
(NCBI) reveals no strong matches to any protein of known
function or indeed to any predicted protein at all outside of the
family Aphididae. Homologs are found among aphids, including
the brown citrus aphid, Toxoptera citricida, the cotton aphid,
Aphis gossypii, the green peach aphid, Myzus persicae, and the
greenbug, Schizaphis graminum (10). An analysis of variation of
the C002 sequence in several aphid species will be presented
elsewhere (J.M., unpublished results).
Transcript Size and Gene Copy Number. Northern blot analysis of
total pea aphid RNA using a full-length c002 probe revealed a
single band of 1,100 bases (Fig. 2 Left). In Southern analysis, we
observed single bands in digests by several enzymes, suggesting
a single gene (locus) encoding Protein C002 (Fig. 2 Right).
Assembly of the c002 gene from the genomic reads available at
the Baylor College of Medicine Human Genome Sequencing
Center (www.hgsc.bcm.tmc.edu/projects/aphid/) indicates a sin-
gle gene, containing a single intron [see supporting information
(SI) Fig. S1].
Localization of Transcript c002 and Protein C002 Within Salivary
Glands. Most of the volume of aphid salivary glands is comprised
of large secretory cells, 4 in each accessory gland and 21 in each
lobe of the bilobed principal gland (11, 12). In situ hybridization
with digoxygenin-labeled c002 DNA revealed the presence of
transcript c002 only in the principal salivary glands and in only
a few of the secretory cells in each lobe (Fig. 3 A and B). The
unstained (differential interference contrast) image shows the
overall morphology expected of an accessory salivary gland and
its associated (bilobed) principal salivary gland (11, 12). (These
authors’ diagrams of the histology of aphid salivary glands can
be seen online, on pages 51 and 53 in ref. 10). Propidium iodide
staining revealed the large nuclei expected within the secretory
cells, and the c002 transcript was largely confined to a handful
of such cells in each lobe of the principal gland.
Immunohistochemical localization of Protein C002, using
rabbit polyclonal antibodies raised against the recombinant
protein, revealed staining in a subset of several cells in the
principal salivary gland (Fig. 3C).
aphids were placed on fresh fava bean plants for 24 h and then
removed. Leaf tissue was then extracted and Western blot
analysis was performed using polyclonal anti-C002 rabbit anti-
bodies. As shown in Fig. 4 (lane 3), a band was detected in plants
fed upon by aphids that matches the position of recombinant
Protein C002 (Fig. 4, lane 1). On the other hand, a Protein C002
band was not detected in plants that had not been exposed to
M G S Y K L Y V A V M A I A I A V V Q E
V R C D W S A A E P Y D E Q E E A S V E
L P M E H R Q C D E Y K S K I W D K A F
S N Q E A M Q L M E L T F N T G K E L G
S H E V C S D T T R A I F N F V D V M A
T N Q N A H Y S L G M M N K M L A F I I
R E V D T T S N K F K E T K E V F E R I
A K T P E I R D Y I K H T T A R T V D L
L K E P V I R G R L F K V V K A F E G L
I K P S E N E E L V K Q R L K R I T N A
P A K M A M G A I N K F G S F L R R F *
sequence of Protein C002. The nucleotide sequence is a unigene sequence
assembled from c002 ESTs from NCBI. It agrees with the sequence obtained
from the c002 gene (see Fig. S1). The arrow indicates the signal peptide
cleavage site as predicted by SignalP.
Nucleotide sequence of transcript c002 and the inferred amino acid
genome DNA digested by: XbaI (first lane), NcoI (second lane), EcoRV (third
lane), and EcoRI (fourth lane).
tein C002 in dissected pea aphid salivary glands. (A) Differential interference
contrast image. PG, principal gland; AG, accessory gland. (B) Differential
interference contrast image overlaid with in situ hybridization for transcript
c002. Green is the positive signal for antisense c002; nuclei are red. (C)
Immunohistochemistry with anti-Protein C002 staining. Nuclei are red; green
is the positive signal for anti-C002 antibody. (Scale bar: 50 ?m.) Negative
controls (sense probe for in situ hybridization and preimmune serum for the
immunohistochemistry) did not show any positive staining.
In situ hybridization and immunohistochemical localization of Pro-
www.pnas.org?cgi?doi?10.1073?pnas.0708958105Mutti et al.
aphids (lane 2). These results indicate that Protein C002 is
transferred from aphid to plant during feeding. Protein C002 was
also detected in extracts from pea aphid heads and salivary
glands (lanes 4 and 5, respectively). No bands were detected in
any of these samples in a Western blot with secondary antibody
only (result not shown).
A band of about 75 kDa detected in the plant extracts, both
before and after aphid feeding (Fig. 4, lanes 2 and 3), was also
detected in plant extracts when a preimmune serum was used
(result not shown). Thus, it appears that the rabbit used for
raising antibodies against Protein C002 had previously devel-
oped antibodies against a plant protein of 75 kDa and that these
antibodies were not fully removed in passing the anti-C002
antiserum over a column of immobilized Protein C002 for
Effects of c002 Transcript Knockdown on Feeding Behavior. EPG
provides a powerful method to study probing behavior by an
aphid during foraging and feeding (3, 13, 14). Three main phases
during stylet penetration have been defined from EPG studies:
the pathway phase, the xylem phase, and the phloem (or sieve
element) phase (3, 14), and details within each of these phases
can be discerned (3, 13–15).
We have used EPG to assess the effect on the foraging and
feeding behavior of pea aphids in which transcript c002 has been
knocked down (8). Representative EPG traces are shown in Fig.
5. These (and EPGs of nine other insects in treatment and
control groups) revealed dramatic differences in feeding (or
attempted feeding) by the two groups. The EPGs as shown in the
figure (and their continuations for 8 h) differ in several ways,
most strikingly in that the c002-knockdown insect never entered
a phloem (or sieve element) phase. In fact, of the 10 knockdown
insects examined, only one exhibited a sieve element phase (and
then only once and for just 30 min). The control insect, however
(Fig. 5), not only entered the sieve element phase, but remained
in that phase for 5 of the 8 h of observation (data not shown in
the figure, where we include only the first 3 h of the trace). There
control insect, for instance, a lower rate of occurrence of cell
puncture signals during the probing phase.
In Table 1, we present a summary of the EPGs recorded from
10 c002-knockdown and 10 control-injected insects (a total of
80 h of EPGs from each group). Several foraging or feeding
parameters can be seen to be affected in a statistically significant
way by the knockdown. First, an aphid’s ability to identify a
suitable location for initiating probing is significantly reduced
because it takes c002-knockdown aphids ?6 times longer than
the control insects to identify such a site and begin probing (P ?
0.0073; Table 1). Second, once probing is initiated, the c002-
knockdown aphids probe individual epidermal and mesophyll
cells at only half the rate of control aphids (P ? 0.031). Third and
most strikingly, c002-knockdown aphids are far less likely to
initiate sieve element phase feeding (P ? 0.0001) than the
control insects. The control aphids fed 52 times longer than the
c002-knockdown aphids (P ? 0.0001), which could be due either
to an inability of the knockdown insects to identify and remain
in a sieve element once a sieve element is probed or to simply not
coming into contact with a sieve element. Finally, the c002-
knockdown aphids spend ?4-fold more time not engaged in
probing behavior than do control insects (P ? 0.0001).
Here and in and a previous article (8), we have identified a
protein, Protein C002, that appears to play an essential role (or
roles) in the foraging and feeding of the pea aphid on fava beans,
a typical host plant for this aphid species.
Protein C002 can, on the basis of our results, be considered a
specialized, salivary gland protein, which does not exclude the
possibility that it is synthesized in other organs in small amounts.
Indeed, we have a preliminary indication that transcript c002
occurs in gut, but at ?100-fold lower amounts than in salivary
C002) is one of the many aspects of this transcript and protein
that will be under continued investigation in our laboratories.
Both Protein C002 and its transcript occur in the principal
salivary glands in the pea aphid but apparently in only some of
those cells (?5 of the 21 cells within each lobe). This restriction
of expression of the c002 gene to a subset of secretory cells is
intriguing. In unpublished work, we have found that the enzyme
laccase is also restricted in its distribution in the principal
salivary gland, but to a different subset of secretory cells than is
Protein C002 (Q. D. Liang and G.R.R., unpublished observa-
tions). Thus, it appears that individual secretory cells in the
principal salivary gland have different ‘‘assignments’’; that is,
of which will be enzymes), and thus different subsets of the
secretory cells could in principle produce salivas of different
Our EPG studies on c002-knockdown insects (and control
insects) demonstrated striking effects of the knockdown. Our
earlier work (8) demonstrated premature deaths of the knock-
down insects feeding on fava bean leaves. The EPG results
Rabbit anti-C002 polyclonal antibodies were used to develop Western blots.
Samples for electrophoresis were as follows: recombinant Protein C002 (lane
1); extract of fava bean tissue, without aphid feeding (lane 2); extract of fava
(lane 4); extract from five dissected pea aphid salivary glands (lane 5). The
experiment was carried out four times with similar results. Further negative
controls, beyond lane 2), were as follows. Omission of primary antibody
resulted in a blank lane (no bands). Use of preimmune serum, rather than the
primary antibody, resulted in a single band in the plant samples of ?75 kDa,
regardless of aphid feeding.
Detection of Protein C002 in fava bean extract after aphid feeding.
SEP, sieve element phase.
Representative EPG waveforms of c002-knockdown insects (a) and
Mutti et al.
July 22, 2008 ?
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no. 29 ?
reported here offer a good deal of insight into that observation.
It is not much of an oversimplification to state that the knock-
down aphids do not feed. They attempt to feed, but are, for all
intents and purposes, unsuccessful in doing so. Thus, of 10
knockdown insects studied by EPG, only one showed a sieve
element phase, and that was quite short (only 30 min). The
knockdown aphids exhibit a probing phase, a typical behavior as
aphids search for a sieve element (3, 13–15), but they do either
not find sieve elements, do not penetrate them, or, if they do, do
not maintain their penetration. In other words, the c002-
knockdown insects essentially do not ingest phloem sap. This
lack of feeding is presumably responsible for the premature
death that we observed in these knockdown insects (8).
Although salivary secretions have long been recognized as
vitally important in the interaction of aphids and plants (3, 7),
this work describes a body of evidence that has been marshaled
to demonstrate the essentiality of an individual, unambiguously
identified salivary protein or enzyme and to provide direct
evidence for that essentiality and for its role in feeding. The
molecular mechanism by which Protein C002 acts in aphid
feeding is not clear from the current results. Because Protein
C002 matches no annotated protein in sequence, it is a total
unknown, functionally, at the molecular level. Thus, we can
exclude little at this stage. For instance, we cannot exclude that
it might correspond to an enzyme activity detected by others in
diluted saliva but that the enzyme occurs in the pea aphid in
unrecognizable form (nonmatching amino acid sequence); we
cannot exclude that it could be a calcium-binding protein.
Indeed, we cannot entirely exclude the possibility that it is a
structural component of the stylet sheath, although this possi-
bility seems unlikely given the rather normal probing of epider-
mal and mesophyll cells exhibited by the knockdown aphids.
Finally, we certainly cannot exclude a role for Protein C002
produced in another organ, especially gut, but this role would
have to be in addition to that of the salivary protein, which
contacts plant tissue. One possibility that intrigues us is that
Protein C002 aids in identifying or maintaining contact with
sieve elements and that its depletion causes a defect analogous
to the inability of Macrosiphum euphorbiae to establish sieve
element phases in transgenic potato plants having half the
normal sucrose content (16). This and other possible mecha-
nisms will be the subject of ongoing investigations in our
Materials and Methods
Aphids, Plants, Standard Procedures, and cDNA Library Construction. The aphid
colony and growth of fava beans are as described (8, 10), as are the standard
methods used in this present work. The PCR-based cDNA library was made
following instructions with the SMART cDNA library construction kit (Clon-
tech), starting with RNA isolated from 250 dissected salivary glands. ESTs are
posted at NCBI, accession numbers DV747494–DV752010.
Expression of Recombinant Protein in Escherichia coli for Antibody Preparation.
A cDNA-encoding Protein C002 was amplified by PCR. We used a forward
and a reverse primer 5?-GTA TGG ACA AGC TTA TTA AAA ACG TCG-3? con-
protein. The PCR product (632 bp) was ligated into a pGEM-T Easy Vector and
used to transform E. coli strain JM109. LB/ampicillin/IPTG/X-Gal (100 ?g/ml
ampicillin, 0.5 mM IPTG, 80 ?g/ml X-Gal) plates were used to grow trans-
formed bacteria. White colonies were selected, and the insert was excised
from the vector by digestion with NcoI and HindIII, purified by low-melting-
point agarose gel electrophoresis, ligated into vector H6pQE60(17), and used
to transform E. coli strain JM109. A single colony from the plate was used to
inoculate 3 ml of 2? YT medium containing 100 ?g/ml ampicillin. The culture
was shaken at 300 rpm, 37°C overnight. The 3-ml overnight culture was then
used to inoculate 200 ml of 2? YT medium with ampicillin and incubated at
by adding IPTG to 1 mM final concentration and the culture incubated for
another 5 h. Bacteria were harvested by centrifuging at 6,800 ? g, 20 min at
4°C, and then resuspended in 4 ml of lysis buffer [8 M urea, 0.1 M NaH2PO4,
0.01 M Tris?Cl (pH 8.0)]. A 5-?l sample was reserved for Western blot analysis.
The bacteria were incubated on ice for 30 min with Triton X-100 at a final
concentration of 2% and lysozyme to a final concentration of 1 mg/ml and
then sonicated on ice. The lysates were centrifuged at 12,000 ? g for 20 min
for Western blot analysis.
Protein C002 expressed in 2 liters of culture medium was purified in 8 M
urea by affinity chromatography with nickel-nitrilotriacetic acid (Ni-NTA)
resin (Qiagen). The protein was concentrated with a YM-10 Centricon mem-
brane (Millipore) to 400 ?l, mixed with 2? SDS loading buffer, separated by
electrophoresis in a 12.5% acrylamide gel (ISC Bioexpress), and stained with
cut out and sliced into pieces for injection into rabbit to generate antiserum
Western Blot Analysis. Polyclonal rabbit antibodies were purified on a column
of immobilized recombinant Protein C002. The purified recombinant protein
was linked to a matrix of cross-linked 4% beaded agarose activated to form
aldehyde functional groups by using AminoLink Plus immobilization kit
then removed after 24 h. Plant tissue (1.5 g) was homogenized in PBS in liquid
nitrogen. Several freeze–thaw cycles were used, and the extract was centri-
fuged at 12,000 ? g for 5 min. Approximately 2 ml of supernatant was
concentrated to 200 ?l by using a YM-3 Microcon centrifugal filter device
(Millipore) and then filtered through a YM-50 centrifugal filter. SDS/PAGE
then subjected to SDS/PAGE on 4–20% gradient gels (ISC Bioexpress) and
transferred onto a PVDF membrane. Nonspecific binding sites were blocked
with purified polyclonal antibody (1:200 dilution) overnight followed by
extensive washing for 3 h with frequent changes of PBS with 0.2% Triton
X-100 (PBST). The antigen–antibody complexes were visualized with horse-
radish peroxidase-conjugated goat anti-rabbit IgG (Pierce) at a dilution of
1:15,000 and detected with a SuperSignal West Femto maximum sensitivity
substrate kit (Pierce) on x-ray film.
Fluorescence in situ Hybridization. A single-stranded DNA probe was synthe-
sized by using a PCR digoxigenin (DIG) probe synthesis kit (Roche). A 397-bp
fragment of clone c002 was amplified by PCR, and single-stranded sense or
Table 1. Analysis of the effects of c002-knockdown on foraging and feeding behavior in the pea aphid
Behavior examined Control group mean (SD) Treatment group mean (SD)
P , Ho: ?1 ? ?2
Time to first pathway phase, min
Rate of PD during initial cell-probing phase, no. per min
Total duration of pathway phase, min
Total time mouth parts not touching the plant, min
Time to first SEP feeding, min
Total duration of SEP feeding in 8 h, min
7 (13) 45 (45)
SEP, sieve element phase, observed during feeding in a sieve element.
*Significant at ? ? 0.05.
www.pnas.org?cgi?doi?10.1073?pnas.0708958105 Mutti et al.
antisense probes were synthesized by asymmetric PCR. Dissected salivary Download full-text
glands were fixed in 4% paraformaldehyde in PBS for 1 h at room tempera-
paraformaldehyde for 1 h at room temperature. Prehybridization was per-
30 min. The fixed glands were exposed to DIG-labeled single-strand sense or
antisense DNA probes (200 ng) in hybridization solution at 48°C for 20–30 h
and washed successively in hybridization solution and in PBST. The glands
were then blocked with 1% BSA by incubating at room temperature for 30
min, washed, and incubated with anti-digoxigenin horseradish peroxidase
Tyramide signal amplification kit (Molecular Probes).
Nuclei were counterstained with 1.5 ?M propidium iodide (Molecular
Probes) for 30 min at room temperature. Finally, the glands were mounted in
glycerol and examined under a fluorescence microscope (Nikon Eclipse E800)
with a triple bandpass filter for FITC (green color) and Cy3 (red color). Photo-
graphs were taken with a digital camera attached to the compound micro-
scope, and pictures were edited with Adobe Photoshop 7.0.
Immunohistochemistry. Pea aphid salivary glands were dissected in PBS,
acid, 24% formaldehyde, 5% glacial acetic acid) for 10 min at room temper-
and incubated with primary antibody (raised in rabbit against recombinant
C002) at 1:100 dilution overnight at 4°C. The glands were then washed three
times at 15-min intervals with PBST and blocked with 5% normal goat serum
in PBST for 1 h, washed three times at 15-min intervals with PBST, and
incubated with secondary antibody Cy3-conjugated goat anti-rabbit (Jackson
ImmunoResearch Laboratories) at 1:500 dilution overnight at 4°C. The glands
were then washed extensively with PBST at 15-min intervals. Nuclei were
stained by using TO-PRO-3 (Molecular Probes, Invitrogen) at 5 ?M for 30 min
in dark at room temperature. Glands were washed extensively with PBST and
mounted on mounting media (Gel/Mount; Biomeda Corporation) on a glass
slide. Photographs were taken with a Nikon Zeiss LSM 5 Pascal (laser scanning
Analysis of Aphid Feeding Behavior. EPG measurements were carried out in an
noise. All experiments were carried out at room temperature (22°C– 24°C). A
gold wire, 2 cm long and 10 ?m in diameter, was glued to the dorsum of the
aphid by using conductive silver paint (Colloidal Silver; Ted Pella, Inc.). The
wiring was done by immobilizing the aphid with a vacuum-operated plate.
The gold wire (insect electrode), which allowed some free movement by the
aphid on the plant’s surface, was connected to the EPG probe. A stiff copper
wire, 10 cm long and 0.2 cm in diameter, from the EPG monitor, was inserted
into soil of the pot in which the plant was rooted (plant electrode). The two
electrodes were connected to an eight-channel GIGA-8 direct current ampli-
fier (Wageningen Agricultural University, Wageningen, The Netherlands),
which has 109? input resistance and an adjustable plant voltage. When the
aphid stylets come into contact with the electrified plant, the circuit is closed,
interpretable signal. The feeding behavior of individual aphids on fava bean
plants was monitored for 8 h with the help of a four-channel amplifier (two
channels for siGFP-RNA-injected aphids and two for siC002-RNA-injected
aphids). Ten replications were completed for each of two groups. The target
group was c002-knockdown insects, injected as described in ref. 8 and main-
tained for 3 days on an artificial diet. These conditions produce ?60% knock-
down of transcript c002 (figure 3 in ref. 8). The control group consisted of
insects injected with siGFP-RNA (8) and otherwise treated the same as the
target group. Waveform recordings were analyzed with the EPG analysis
software PROBE 3.0 installed on a PC.
phase (a measure of how quickly an aphid can identify a suitable location for
probing individual cells); (ii) the rate of occurrence of potential drops during
the initial pathway phase (a measure of how quickly an aphid probes individ-
ual plant cells with its stylet); (iii) the total duration of the pathway phase (a
a nutrient-rich cell for feeding); and (v) total duration of sieve element phase
feeding (a measure of time spent feeding on a nutrient-rich source). If the
sieve element phase is not reached during the entire experiment time, the
time (15), i.e., 8 h for this work. We tested for significant differences between
our two treatments by using a randomization approach with 10,000 itera-
by randomizing the data and determining the probability of getting the
observed ?siC002-RNA. We used the SAS platform (18) and ? ? 0.05.
ACKNOWLEDGMENTS. We thank our colleagues Subbaratnam Muthukrishnan
Department of Agriculture Cooperative State Research, Education, and Exten-
sion Service National Research Initiative G001-35302-09975 by the Cooperative
Research Centre for National Plant Biosecurity, Canberra, Australia, and by the
Kansas Agricultural Experiment Station (publication 08-325-J).
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