Exceptional plant penetration and feeding upon cortical parenchyma cells by the woolly poplar aphid.

Abstract

Forty percent of aphids live wholly or partly on trees, most species being associated with leaves or petioles. Species able to exploit woody parts have either specific adaptations, such as extra long stylets that allow them to reach the phloem, or the ability to induce galls. The woolly poplar aphid, Phloeomyzus passerinii (Signoret) (Hemiptera: Aphididae), colonizes the trunks and base of the lower branches of mature poplars and causes cortical necrosis leading to the death of trees where infestation is heavy. Very little is known about the mode of feeding of P. passerinii. This study looked at the feeding behavior of P. passerinii on stem-cuttings of Populus x canadensis Moench using: (i) histological analyses of the feeding site and stylet pathway and (ii) electrical penetration graphs (EPG, DC) based on parthenogenetic apterous females on woody tissues. The histological and EPG results showed that stylets of P. passerinii penetrated into the plant tissues following a straight unbranched extracellular and intracellular pathway to reach the cortical parenchyma. Compared to EPGs for phloem sap feeding aphids, there were differences in the waveforms A and C whereas a new waveform Icp was described. Based on histological analyses and previous descriptions of EPG waveforms, correlations with the stylet tip position and aphid activities within bark tissues are discussed. A pathway and a sustained intracellular phase were distinguished, both occurring in the cortical parenchyma cells. The bark aphid feeding mode is discussed in relation to the damage caused and in terms of changes in the aphid's diet.

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Exceptional plant penetration and feeding upon cortical parenchyma cells
by the woolly poplar aphid
Sophie Pointeaua, Arnaud Amelineb, Françoise Lauransc, Aurélien Salléa, Yvan Rahbéd,
Stéphanie Bankhead-Dronneta, François Lieutiera,⇑
aUPRES EA 1207, Laboratoire de Biologie des Ligneux et des Grandes Cultures, Université d’Orléans, Rue de Chartres, BP 6759, FR-45067 Orléans Cedex, France
bUnité de Recherche EA 4698, EDYSAN, Ecologie et Dynamique des Systèmes Anthropisés, Laboratoire de Bio-Ecologie des Insectes Phytophages et Entomophages,
Université de Picardie Jules Verne, 33 rue St Leu, FR-80039 Amiens Cedex, France
cEquipe Formation des parois Lignifiées, Unité Amélioration, Génétique et Physiologie Forestières, INRA Orléans, Avenue de la Pomme de Pin, BP 20619 Ardon,
FR-45166 Olivet Cedex, France
dBiologie Fonctionnelle, Insectes et Interactions (BF2I) INRA: UR0203, Institut National des Sciences Appliquées de Lyon INSA Bat. L. Pasteur 20 Avenue Albert Einstein,
FR-69621 Villeurbanne Cedex, France
a r t i c l e i n f o
Article history:
Received 8 December 2011
Received in revised form 5 March 2012
Accepted 14 March 2012
Available online xxxx
Keywords:
Tree–aphid interaction
Phloeomyzus passerinii
Aphididae
Trunk feeder
Bark tissue
Electrical penetration graph
a b s t r a c t
Fortypercent of aphids live wholly or partly on trees,most species being associated withleaves or petioles.
Species able to exploit woody parts have either specific adaptations, such as extra long stylets that allow
them to reach the phloem, or the ability to induce galls. The woolly poplar aphid, Phloeomyzus passerinii
(Signoret) (Hemiptera: Aphididae), colonizes the trunks and base of the lower branches of mature poplars
and causes cortical necrosis leading to the death of trees where infestation is heavy. Very little is known
about the mode of feeding of P. passerinii. This study looked at the feeding behavior of P. passerinii on stem-
cuttings of Populus x canadensis Moench using: (i) histological analyses of the feeding site and stylet path-
way and (ii) electrical penetration graphs (EPG, DC) based on parthenogenetic apterous females on woody
tissues. The histological and EPG results showed that stylets of P. passerinii penetrated into the plant tis-
sues following a straight unbranched extracellular and intracellular pathway to reach the cortical paren-
chyma. Compared to EPGs for phloem sap feeding aphids, there were differences in the waveforms A and C
whereas a new waveform Icp was described. Based on histological analyses and previous descriptions of
EPG waveforms, correlations with the stylet tip position and aphid activities within bark tissues are dis-
cussed. A pathway and a sustained intracellular phase were distinguished, both occurring in the cortical
parenchyma cells. The bark aphid feeding mode is discussed in relation to the damage caused and in terms
of changes in the aphid’s diet.
? 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Aphids living on trees account for approximately 40% of aphid
species throughout the world. They belong to the three families of
the Aphidoidea: Aphididae, Adelgidae and Phylloxeridae (Blackman
and Eastop, 1994). Tree-dwelling aphids generally feed on leaves or
petioles but some are able to exploit the trunks, branches and roots
(Blackman and Eastop, 1994; Pollard, 1973). Some of the aphids
that feed on woody tissues are able to reach the phloem either be-
cause they are large and feed on young stems, branches or large
shrubs (e.g. Tuberolachnus salignus (Gmelin)) or, if they feed on
the trunk (e.g. Stomaphis sp.), because they are large and have long
stylets with a proboscis of approximately twice the length of the
body (Pollard, 1973; Dixon, 1997, 1998). Other aphids that feed
on woody tissues are mainly gall-forming insects living on roots
or stems, such as the woolly apple aphid, Eriosoma lanigerum
(Hausmann) and the grape phylloxera Daktulosphaira vitifoliae
(Fitch) (Pollard, 1973; Miles, 1989; Brown et al., 1991; Kellow
et al., 2004; Kingston et al., 2007). Phloem sap feeding aphids
usually cause limited symptoms on trees whereas more profound
species-specific effects such as galls are produced by parenchyma
feeding aphids (Miles, 1989). Adelgidae induce galls that are visible
ontheoutsideof conifershootsbutsomemorphswithintheAdelges
species feed on the cortical parenchyma cells of the trunk of their
secondary host without causing visible galls (e.g. Adelges piceae
(Ratzeburg) on fir trunks) (Balch, 1952; Balch et al., 1964; Müllick,
1977; Rohfritsch, 1990; Miles, 1989).
Tree-dwelling aphids generally cause little damage to forest
ecosystems. However, severe outbreaks of aphids living on woody
tissues have been reported to cause extensive tree mortality that
0022-1910/$ - see front matter ? 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jinsphys.2012.03.008
⇑Corresponding author. Tel.: +33 2 38 41 72 30; fax: +33 2 38 49 43 26.
E-mail addresses: sophie.pointeau@univ-orleans.fr (S. Pointeau), arnaud.ameli
ne@u-picardie.fr (A. Ameline), Francoise.Laurans@orleans.inra.fr (F. Laurans),
aurelien.salle@univ-orleans.fr(A.Sallé),
stephanie.bankhead@univ-orleans.fr(S.
univ-orleans.fr (F. Lieutier).
Yvan.Rahbe@lyon.inra.fr
Bankhead-Dronnet),
(Y.Rahbé),
francois.lieutier@
Journal of Insect Physiology xxx (2012) xxx–xxx
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Page 2

leads to significant economic and ecological damage, such as the
dieback of North American Fraser firs owing to outbreaks of
A. Piceae (Müllick, 1977; Miles, 1989). The woolly poplar aphid,
Phloeomyzus passerinii (Signoret), the only known species in the
subfamily Phloeomyzinae (Aphididae) (Blackman and Eastop,
1994), is a monoecious aphid that feeds on poplar trunks. Firstly
described in France (Signoret, 1875), this species is native from
the Palaearctic area (Blackman and Eastop, 1994). Its life cycle is
characterized by an autumnal production of sexual alates, occur-
ring while parthenogenetic reproduction through apterous individ-
uals continues (Blackman and Eastop, 1994). Colonies develop in
bark crevices of young or mature trees. During outbreaks in poplar
stands, the aphids can colonize whole trunks, causing extended
necroses of cortical cells resulting in cracks, mortality of lower
branches and inhibition of bud break (Della Beffa, 1936; Arzone
and Vidano, 1984). Aphid outbreaks lead to a reduction in tree
growth and even to massive tree mortality, which causes consider-
able damage to poplar stands in southern Europe, North Africa and
the Near East (Arzone and Vidano, 1984; Lapietra and Allegro,
1990; Sadeghi et al., 2007). Although P. passerinii is a widespread
pest, little is known about its feeding strategy. This lack of knowl-
edge prevents the development of effective control.
The feeding of insects with piercing mouthparts has mainly
been observed indirectly because it occurs inside plant tissues.
Light or transmission electron microscopy can provide such partial
and indirect information about the location of the tissues involved
in feeding and the stylet pathways of these insects (Pollard, 1973;
Tjallingii, 1988). In contrast, since several decades, electrical Pene-
tration Graph (EPG) is used to acquire direct temporal information
on stylet penetration. Probing behavior is generally described by
the characterization of EPG waveforms (or waveform patterns) cor-
related with both stylet tip positions inside plant tissues and insect
activities (Tjallingii, 1978, 1988, 2000). EPGs have been used to
study plant resistance and plant virus transmission and have
mainly been applied to crop pests such as phloem sap feeding
Aphididae (Alvarez et al., 2006; Fereres and Moreno, 2009) and
other piercing-sucking insects such as sharpshooters, thrips,
whiteflies, psyllids and mealybugs (Janssen et al., 1989; Calatayud
et al., 1994; Harrewijn et al., 1996; Miranda et al., 2009; Bonani
et al., 2010). A few EPG studies have investigated the probing
behavior of tree-dwelling aphids feeding on woody stem tissues
(Sandanayaka and Hale, 2003; Cardoso, 2007; Kingston et al.,
2007; Penteado, 2007).
This study investigated the feeding behavior of the woolly
poplar aphid by combining histology and EPG recording. First,
the tissues penetrated and fed upon by apterous parthenogenetic
P. passerinii were identified by light (LM) and transmission electron
microscopy (TEM). Then, EPG waveforms were described and cor-
related with histological observations of salivary sheaths. Together
with comparisons with earlier EPG studies on other plant-sucking
insects the probing behavior of P. passerinii on poplar stems was
characterized.
2. Materials and methods
2.1. Insects and plants
A P. passerinii colony was established from an apterous parthe-
nogenetic aphid collected in October 2008 from a poplar stand in
Reboursin (France). This clonal colony belonged to the most com-
mon haplotype that has so far been identified in France (Pointeau
et al. unpublished data from combined analyses of three mitochon-
drial DNA fragments, Cytochrome oxidase I and II and Cytochrome
b genetic fragments). The colony was maintained under the breed-
ing conditions developed by Arru (1974), in a climate controlled
chamber (20 ± 1 ?C, 70 ± 10% RH and 16L:8D photoperiod), on
Populus x canadensis Moench cv. ‘I214’ stem-cuttings (25 cm long
and 1.5–2 cm diameter), which provide an optimal rearing host
for this aphid (Arru, 1974; Lapietra and Allegro, 1990). Stem-cut-
tings were one-year old poplar stump sprouts cut from mature
trees using a pair of secateurs. They were collected in the experi-
mental nursery of Guéméné-Penfao (France) in late autumn when
winter buds were present. The laboratory colony was transferred
every 15 days onto new stem-cuttings.
Experiments were performed on the poplar genotype ‘I214’ for
both histological and EPG studies. Poplar stem-cuttings (1.5–2 cm
diameter) were collected as described above in late autumn
2008. The stem-cuttings were kept at 2 ?C in dry conditions until
use. Prior to the experiments, the stem-cuttings were trimmed to
20 cm in length and, to simplify their use in experiments, all buds
except the terminal bud were removed. The stem-cuttings were
put in water for one week to rehydrate the plant tissues and initi-
ate bud break and root formation. They were then placed in 0.53 l
pots containing compost (Klasmann substrate 4, recipe no. 267).
Bud break and leaf formation indicated that the potted stem-cut-
tings were in a good physiological condition.
As the aphids prefer the upper part of mature trees (Della Beffa,
1936; Arzone and Vidano, 1984), histological observations were
also performed on one log from a 10 year old tree. The tree was
cut in July 2009 and one 1.50 m long (20 cm diameter) log from
the upper part of the trunk was retained. Infestations for histolog-
ical preparations were performed soon after the log cutting with-
out specific log treatment.
2.2. Histology of stylet pathways
2.2.1. Aphid settling and sample collection
Sets of two apterous parthenogenetic adult females were placed
on the bark of eight stem-cuttings and one 1.5 m log (two sets
10 cm apart per stem-cutting and ten sets 10 cm apart for the
log) and allowed to feed for 48 h. Each set was caged under half
of a transparent gelatin capsule (half a cylinder) (size 000, LGA,
La Seyne-sur-Mer) covered by a band of Parafilm? to hold the cap-
sule on the bark. Plant tissues were pre-fixed for 2 h, with the
aphids in situ, by injecting a glutaraldehyde solution [2.5% in a
0.2 M, pH: 6.8, McIlvain citrate/phosphate buffer (Pépin and Bou-
mendil, 1982)] into the gelatin capsules. The capsules were then
removed and block samples of approximately 1 mm3, including a
zone from the cork layer on the superficial area of xylem, were
carefully cut using a razor blade around the probing site, with
the aphid in situ.
2.2.2. Sample preparation and staining for light microscopy
Block samples were fixed in 2.5% glutaraldehyde and dehy-
drated at room temperature in a graded ethanol series (25–100%)
They were then embedded in historesin Technovit 7100? (Kulzer
& Co, Wehrheim, Germany), allowing 3 lm thick serial transverse
sections (microtome RM 2155, Leica Microsystems, Vienna, Aus-
tria). To view and measure the salivary sheaths, thirteen stem-cut-
ting sections and four log sections containing the salivary sheaths
were stained with 0.2% (wt:vol) acid fuchsin in 1:1 (vol:vol) mix-
ture of 95% ethanol and glacial acetic acid (Backus et al., 1988),
cleared with distilled water and stained with 0.1% malachite green
in distilled water (wt:vol).
All sections were mounted on slides using Canada balsam and
examined using a light microscope (Leica DMR, Leica Microsys-
tems, Vienna, Austria) with a Leica DFC 320 digital camera. The
area round the end of the stylet pathway (cell layer) and salivary
sheath typology (‘‘branched’’ or ‘‘unbranched’’ salivary sheaths
and stylet pathway) were characterized. The mean and maximum
stylet penetration depths were measured on the poplar stem-
cuttings and the mature log using ImageJ 1.40 (National Institutes
2
S. Pointeau et al./Journal of Insect Physiology xxx (2012) xxx–xxx
Please cite this article in press as: Pointeau, S., et al. Exceptional plant penetration and feeding upon cortical parenchyma cells by the woolly poplar aphid.
Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/j.jinsphys.2012.03.008

Page 3

of Health). The thickness of the cork and cortical parenchyma was
also calculated based on three measurements per section. The sty-
lets of five parthenogenetic females were examined using light
microscopy and their total length was measured using ImageJ 1.40.
2.2.3. Sample preparation for transmission electron microscopy
Samples were fixed, dehydrated and embedded in LR-White re-
sin as described above. Series of ultra thin adjacent sections
(150 nm) were cut using an ultramicrotome (Leica Ultracut R, Leica
Microsystems, Vienna, Austria), mounted on 400-nickel wire mesh
grids and counterstained with uranyl acetate (20 min) and lead cit-
rate (30 min). Sections were viewed using a transmission electron
microscope (CM 10 Philips, Eindhoven, The Netherlands). In order
to monitor serial section progression, some thin sections (1.25 lm)
and ultra thin sections were stained using the Richardson azure II
and methylene blue staining method (Richardson et al., 1960),
mounted on slides using Canada balsam and examined using light
microscopy (Leica DMR, Leica Microsystems, Vienna, Austria).
2.3. Electrical penetration graph (EPG) recording
The stylet penetration was EPG recorded using a Giga-4 EPG
system (EPG systems, Wageningen, The Netherlands) (Tjallingii,
1978, 1988), adjusted to about 70? gain. Three to five day old
apterous adult females of P. passerinii were collected from the lab-
oratory colony and immediately immobilized on the tip of an
Eppendorf cone connected to a vacuum device. The waxy wool cov-
ering the apterous parthenogenetic aphid dorsum was gently re-
moved with a fine paintbrush and then a thin gold wire (20 lm
diameter and 2 cm length) was stuck onto the aphid’s dorsum
using water-based conductive silver glue (EPG systems, Wagenin-
gen, The Netherlands). The electrode was then attached to the
amplifier input of the EPG system and the aphid was carefully
placed in contact with the bark at mid-height of the potted
stem-cuttings. Another electrode (10 cm copper wire, 2 mm diam-
eter) was inserted into the soil of the potted stem-cuttings. Twenty
aphids were monitored from 8.00 h. to 16.00 h. The connected
aphid–plant system was placed inside a Faraday cage in a cli-
mate-controlled room at 20 ± 1 ?C under electric fluorescent light.
The EPG data acquisition and analysis were performed using Probe
3.4 software for Windows (EPG systems, Wageningen, The
Netherlands).
Different EPG waveforms were distinguished and described
based on standard characteristics: relative amplitude, frequency,
voltage level, and electrical origin. The relative amplitude of the
waveform was the ratio between the measured amplitude (mini-
mum to maximum deflection) and the highest recorded amplitude
(only waveforms with a main R origin). The frequency (Hz) was
estimated from 40 observations for each waveform (two observa-
tions per aphid). The voltage level of the waveform (positive or
negative) reflected an intracellular or extracellular position of the
stylet tips (Tjallingii, 1985b). The main electrical origin was due
to resistance fluctuation (R), electromotive force (emf) or both
(Tjallingii, 1985a, 2000). To determine the electrical origin of each
waveform, the system voltage was set to positive and negative lev-
els during typical periods in each waveform (Tjallingii, 2000). The
stylet activities and position of the stylet tips in the plant tissues
were inferred from histological observations and similarities to
other aphids and Sternorrhyncha (van Helden and Tjallingii,
2000; Sandanayaka and Hale, 2003).
After waveform characterization, the occurrence and duration
of some EPG parameters were calculated using EPG-Calc 4.9 (Gior-
danengo, 2009). Some non-sequential parameters were calculated:
(1) % of aphids showing a waveform, (2) number of waveform peri-
ods and (3) their mean duration per aphid (Table 2). Some sequen-
tial parameters were also calculated to characterize the probing
behavior of P. passerinii such as: (4) time from the beginning of
experiment to the first probe, (5) time from the start of the first
probe to initiate each specific period (i.e. pathway and sustained
ingestion (>10 min)), (6) total duration of non-probing period from
the start of the first probe to the end of the recording, (7) total
duration of penetration before the first sustained ingestion within
a probe, and (8) mean number of intracellular phases before the
first sustained ingestion.
Typical sequences of waveform events occurring during stylet
penetration were studied to characterize P. passerinii probing
behavior. The likelihood of a specific waveform being followed
by another waveform type was calculated on the basis of 160 h
(8 h per aphid) of EPG recording (Wayadande and Nault, 1996; Al-
meida and Backus, 2004; Miranda et al., 2009).
2.4. Data analysis
Statistical analyses were performed using R 2.7.2 (R Develop-
ment core team, 2007). Comparisons between the depths of the
cortex layers in the stem-cuttings and those in the log were per-
formed using the Wilcoxon rank sum test. The significance level,
a, was set at 5% for all statistical analyses. The mean values in
the text and tables are given with their standard error.
3. Results
3.1. Histology of stylet pathways
The transverse thin sections (1.25 lm thick) of stem tissues
showed that stylets followed a straight route through the bark tis-
sues, not extending deeper than the cortex cell layer, i.e. the
ground tissue region between the vascular system and the epider-
mis (Fig. 1a). Likewise, all salivary sheaths observed, in both the
stem-cuttings and the log, were unbranched and ended in the cor-
tical parenchyma. For both the poplar stem-cuttings and the log,
the thickness of the cork layer averaged 74 ± 2 lm (avg ± SE;
n = 39 and n = 12, respectively). However, the cortical parenchyma
layer was significantly thicker on the log (1685 ± 96 lm; n = 12)
than on the poplar stem-cuttings (692 ± 15 lm; n = 39) (W = 468;
P < 0.001). The mean penetration depth of the salivary sheaths into
the cork and cortical parenchyma was 390 ± 46 lm (n = 13; maxi-
mum 602 lm) on the poplar stem-cuttings and 302 ± 85 lm (n = 4;
maximum 531 lm) on the log. The total length of stylets of P. pass-
erinii was 760 ± 17 lm (n = 5).
Light microscopy of transverse thin sections also showed that
stylets may follow an intercellular pathway, but crossing right
through cell walls may indicate that they probably also follow an
intracellular pathway (Fig. 1b). Intracellular stylet penetration
was confirmed by LM on ultra thin sections (150 nm thick)
(Fig. 1c) and TEM on ultra thin sections that showed punctured cell
with cell remnants (Fig. 1d).
3.2. Electrical penetration graph (EPG) recording
3.2.1. Characterization of EPG waveforms
The woolly poplar aphid produced EPG signals at two clearly
distinct voltage levels, suggesting both intracellular and extracellu-
lar stylet penetration. Three distinct waveforms were distin-
guished and named A, C and Icp (Fig. 2a, Table 1). Despite a few
differences in waveform characteristics when compared to phloem
sap feeding aphids (Tjallingii, 1988; Prado and Tjallingii, 1994; van
Helden and Tjallingii, 2000), waveforms A and C were labeled using
the canonical terminology for EPG waveforms of phloem sap feed-
ing Aphididae, waveform A being the initial electric contact of sty-
lets with plant tissues, and waveform C being the salivary sheath
S. Pointeau et al./Journal of Insect Physiology xxx (2012) xxx–xxx
3
Please cite this article in press as: Pointeau, S., et al. Exceptional plant penetration and feeding upon cortical parenchyma cells by the woolly poplar aphid.
Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/j.jinsphys.2012.03.008

Page 4

secretion and extracellular activities during the stylet penetration
of the cortical parenchyma (Tjallingii, 1988, 2000). A new term
was created for a new intracellular phase, ‘‘waveform Icp’’ (Intra-
cellular cortical parenchyma), because this type of waveform has
not previously been described.
During an 8 h period, P. passerinii performed an average of
2.2 ± 0.5 probes (n = 20) with a mean duration of 115 ± 24 min
per probe (n = 20). Waveform A was always recorded at the start
of probes for the 20 aphids, with a mean duration of 4.5 ±
0.4 min (n = 20) (Fig. 2a, Table 2). This waveform was characterized
by one to three stepwise increases in voltage levels, each starting
with irregular signal at a high maximum frequency (1–11 Hz)
and followed by a low amplitude period. Electrical resistance
appeared its main electrical origin (Table 1).
Waveform C occurred in every recording, always at extracellu-
lar voltage level with a mean duration of 3.2 ± 0.3 min (n = 20)
for each uninterrupted period, and an average occurrence of
18.8 ± 2.0 (n = 20) periods per aphid (Fig. 2a, Table 2). The signal
was irregular, with a pattern of frequently occurring sub-wave-
forms C-I and C-II, and occasionally occurring sub-waveforms
C-III or C-IV, all with a high amplitude, a frequency range of
1–7 Hz, and resistance as the main electrical origin (Fig. 2c, Table
1). Occurrence and duration of the sub-waveforms in waveform
C are not given because of a more or less distinct repetitive pattern
depending on the EPG recordings. Contrary to other aphids, no
clear B waves were shown during waveform C.
Waveform Icp also occurred in all recordings at a distinctly low-
er voltage level than the other waveforms, which suggests that the
stylets had punctured a membrane of a living plant cell (Fig. 2a)
(Tjallingii, 1985b). The Icp waveform showed three sub-wave-
forms, Icp-I, Icp-II and Icp-III, the characteristics of which are given
in Table 1. Two distinct Icp periods occurred (Fig. 3), brief Icp peri-
ods (b-Icp) of about 0.3–2.7 min and sustained Icp (s-Icp) that
lasted longer than about 20 min (Table 2). Brief Icp periods were
found in all recordings embedded in waveform C (Fig. 2a). They oc-
curred 16.2 ± 1.6 times on average per individual with a mean
duration of 1.0 ± 0.1 min (n = 20) per period (Fig. 3a, Table 2). Dur-
ing a b-Icp period, Icp-I was followed by Icp-II (only one period of
each). The sustained Icp periods were observed in 65% of the 8 h
recordings (Table 2) and occurred 1.3 ± 0.2 times per individual
Fig. 1. Sections of stylet pathways of Phloeomyzus passerinii adult parthenogenetic females feeding on the poplar bark of Populus x canadensis cv. ‘I214’. (a) Light microscopy of
transversal section (1.25 lm thick) of poplar stem-cutting showing a straight stylet pathway ending in the cortical parenchyma (b) details of break in cell wall and (c) light
microscopy of ultra thin transversal section (150 nm thick) of stylet tips in cortical parenchyma. (d) Transmission electron microscopy (TEM) of transversal sections (150 nm
thick) of stylet in a cortical parenchyma cell showing presence of cell remnants. The neighboring cell without stylets didn’t show such remnants. c, cork; cc, cork cambium; cp,
cortical parenchyma; sc, sclerenchyma; st, stylets; ssh, salivary sheath; cw, cell wall; es, extracellular space.
4
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Please cite this article in press as: Pointeau, S., et al. Exceptional plant penetration and feeding upon cortical parenchyma cells by the woolly poplar aphid.
Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/j.jinsphys.2012.03.008

Page 5

with a mean duration of 106.4 ± 28.8 min (n = 13) (Table 2). The s-
Icp periods showed all three sub-waveforms, Icp-I, Icp-II and Icp-III
(Fig. 3a). High -resolution graphs (Fig. 3) showed that Icp-I has two
types of peaks, positive and negative, which seem independent
from each other (Fig. 3b). The positive peaks showed somewhat
smaller amplitude than the negative ones, those later being very
similar to those in Icp-II. This suggests that Icp-I and -II have these
negative peaks in common, although in Icp-II they are occurring at
a higher frequency than in Icp-I (3–8 Hz vs. 1–7 Hz; Fig. 3a, insets).
Icp-I occurred 34.5 ± 21.7 times per individual with a mean dura-
tion of 0.67 ± 0.02 min, whereas Icp-II occurred 256.5 ± 23.4 times
with a mean duration of 0.31 ± 0.04 min (n = 20). The last sub-
waveform Icp-III showed a nearly flat signal (low amplitude and
no distinguishable frequency) and occurred 216.3 ± 30.0 times on
average per individual, usually between two Icp-II sub-waveforms,
with a mean duration of 0.16 ± 0.02 min (n = 13). The main electri-
cal origin of each Icp sub-waveform was emf (Table 1).
3.2.2. Time partitioning and sequence of waveforms
During 8 h of EPG recording, P. passerinii spent on average 26.5%
of the time non-probing (Np) from the start of the first probe
(n = 20). During probing activities in the cortical parenchyma, it
spent more time in intracellular than in extracellular activities
(62% vs. 38%, n = 20). Several EPG parameters were selected to
describe the probing behavior of P. passerinii (Table 3). On average,
the first probe occurred after 3 h of non-probing activity
(192.5 ± 29.0 min). The first C + b-Icp period occurred soon after
the onset of the first probe, while the first s-Icp after the onset of
the first probe was 2 h (123.0 ± 34.5 min). Within a probe, the total
time spent in waveforms A + C + b-Icp before the first s-Icp was
49.2 ± 3.8 min, including on average 14.1 ± 1.8 b-Icp periods.
Sequence analysis of waveforms during all 160 h of EPG record-
ing (20 recordings of 8 h each) showed that all individuals began
probing with waveform A which was followed by waveform
C (Fig. 4). The general scheme that came after the A and the first
C periods consisted in alternate b-Icp patterns and waveform C.
86.9% of waveforms C were followed by b-Icp patterns that all re-
turned to waveform C. Only 4.5% of all waveforms C initiated were
followed by a sustained Icp pattern (s-Icp). More than half of the
initiated s-Icp (58.8%) was maintained until the end of the record-
ing, while the others returned to waveform C. Finally, 8.5% of
waveforms C were followed by non-probing activity, which was
maintained until the end of recording in 22.6% of cases and re-
turned to waveform A in the other cases.
4. Discussion
Histological analysis of salivary sheaths and EPG waveforms re-
vealed that P. passerinii likely feeds from cortical parenchyma tis-
sue. This strategy is in agreement with both the little honeydew
secretion of P. passerinii when compared to phloem sap feeding
aphids (personal observation) and the stylet length of P. passerinii.
This tiny aphid (1.6–1.7 mm in length; Arzone and Vidano, 1984)
does not have a very long stylet as do aphids of the Stomaphis spe-
cies (Blackman and Eastop, 1994; Dixon, 1998) which feed on the
trunks of mature trees, and is not a large species like phloem sap
Fig. 2. Electrical penetration graph (EPG) signals of the woolly poplar aphid, Phloeomyzus passerinii probing on the bark of Populus x canadensis cv. ‘I214’ stem-cuttings. (a)
One hour general scheme of a typical EPG recording. (b) Waveform A, representative trace of the starting point of an EPG recording showing three main voltage levels (shown
by arrows). (c) Waveform C, typical succession of several sub-waveforms C: C-I, C-II, C-III (d) and C-IV (e). Sub-waveforms C-III and C-IV, occasionally occurring.
S. Pointeau et al./Journal of Insect Physiology xxx (2012) xxx–xxx
5
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Page 6

feeding aphids that probe on tree bark such as Lachninae. Even if P.
passerinii inserted the whole of its stylet (i.e. approximately
760 ± 17 lm) into the mature poplar bark – its preferred feeding
site – the stylet are not long enough to penetrate the distance of
1685 ± 96 lm into the inner cortical parenchyma tissue to reach
the phloem.
The stylet pathways of P. passerinii within the cortical paren-
chyma appeared to differ from those of phloem sap feeding aphids.
Phloem sap feeding aphids leave many empty branches of salivary
sheaths (Tjallingii and Hogen Esch, 1993), whereas P. passerinii
leave only straight, unbranched salivary sheaths. This observation
suggests that P. passerinii does not need to locate phloem vessels,
which is not surprising if it feeds exclusively in the cortical paren-
chyma. Changes in the stylet pathway of P. passerinii were only ob-
served when stylets encountered blocks of sclerenchyma (Deprost
E., unpubl. data), which has also been observed for other aphids
feeding on woody tissues such as E. lanigerum (Staniland, 1924).
Previous transmission electron microscopy studies showed that
aphid stylet pathways were extracellular (sometimes intramural)
with brief intracellular punctures (potential drop [pd] waveforms)
of cells bordering the stylet pathway (Kimmins, 1986, 1988; Tjall-
ingii and Hogen Esch, 1993). The stylet pathway of P. passerinii was
also extracellular. However, some cells encountered along the
pathway seemed to have been crossed and punctured (as sug-
gested also by the presence of cell remnants in damaged cells),
which seems to match the straight route of P. passerinii’s salivary
sheath.
One waveform, Icp, is new for P. passerinii. Given the non-vascu-
lar feeding strategy of P. passerinii, all waveforms except for wave-
form A appeared to be correlated with stylet tip positions within
the cortical parenchyma. Although not new, waveform A differed
from the typical brief, high amplitude signals reported from aphids
feeding on soft plant tissues (Tjallingii, 1988) but was similar to
woolly apple aphids, E. lanigerum, which penetrate their stylets
into woody apple shoots (Sandanayaka and Hale, 2003). Initially,
this aphid also had long waveform A periods with one to three sud-
den increases in voltage level, indicating an extracellular stylet
activity generating a continual decrease in resistance component,
probably as a result of cork penetration. The bark probably be-
comes more electrically conductive closer to the cortical paren-
chyma, explaining the increasing voltage levels. Waveform A in
EPGs of phloem sap feeding aphids is soon followed by waveform
C correlated to both salivary sheath secretion and extracellular sty-
let penetration within the parenchyma tissue (Tjallingii, 1978,
1988; Tjallingii and Hogen Esch, 1993). On the basis of similarities
in waveform characteristics and tissue location it is most likely
Table 1
Characteristics of electrical penetration graph (EPG) waveforms of the woolly poplar aphid, Phloeomyzus passerinii during an 8 h access period to the bark of Populus x canadensis
cv. ‘I214’ stem-cuttings. Correlations with stylet activities in P. passerinii were made using light microscopy of P. passerinii’ salivary sheaths and feeding sites, comparisons with
stylet activities from phloem sap feeding aphids (van Helden and Tjallingii, 2000) and from Eriosoma lanigerum, a tree-dwelling aphid that feeds on lignified plant tissue
(Sandanayaka and Hale, 2003).
EPG waveformWaveform characteristicsCorrelations
Amplitude
(%)
Frequency
(Hz)
Voltage
level
Electrical
origin
Stylet tips in
plant tissue
Stylet activitiesSub-
waveform
Np
A
C
0
0–50
–
1–11
–
Extrac.
–
R
Not in plant
Epidermis/cork
Cortical
parenchyma
Non-probing
Initial electric contact of stylet-plant
Salivary sheath secretion and extracellular activities during stylet penetration
C-I
C-II
C-III
C-IV
100
20–300
20–150
150–500
1–7
1–7
1–7
1–7
Extrac.
Extrac.
Extrac.
Extrac.
R
R
R
R
IcpCortical
parenchyma
Ingestion? [Correlation work is needed to verify that waveform Icp (or a specific
Icp sub-waveform(s) actually represents ingestion]
Icp-I (+)
Icp-I (-)
Icp-II
Icp-III
– 1–7Intrac.emf
emf
emf
emf
–
–
3–8
0.1–0.5
Intrac.
Intrac.
Note: Relative amplitude (minimum to maximum deflection) only for R components: compared to sub-waveform I of waveform C (C-I); voltage level extracellular (extrac.) or
intracellular (intrac.); main electrical origin (R) resistance fluctuation or (emf) electromotive force; Np, Non probing; Icp-I (+), positive peak of sub-waveform Icp-I; Icp-I (-),
negative peak of sub-pattern Icp-I.
Table 2
Occurrence (% and numbers) and duration (min) of EPG waveforms in the woolly poplar aphid, Phloeomyzus passerinii during 8 h recordings on the bark of Populus x canadensis cv.
‘I214’ stem-cuttings. Np, non-probing.
EPG waveforms & sub-waveforms % Aphids showingPeriods/aphid Period duration/aphid
Mean ± SE (Min–Max) Mean ± SE(Min–Max)
Np
A
C
b-Icpa
s-Icpb
Icp-I
Icp-II
Icp-III
100
100
100
100
65
100
100
65
2.6 ± 0.5
2.2 ± 0.5
18.8 ± 2.0
16.2 ± 1.6
1.3 ± 0.2
34.5 ± 1.7
256.5 ± 23.4
216.3 ± 30.4
(1–11)
(1–10)
(8–39)
(7–29)
(1–3)
(26–44)
(128–396)
(91–369)
176.5 ± 24.7
4.5 ± 0.4
3.2 ± 0.3
1.0 ± 0.1
106.4 ± 28.8
0.67 ± 0.02
0.31 ± 0.04
0.16 ± 0.02
(32.1–431.4)
(1.6–8.4)
(1.7–6.6)
(0.3–2.7)
(20.1–377.0)
(0.59–0.84)
(0.09–0.48)
(0.12–0.28)
Note: Occurrence and duration of waveform C sub-waveforms are not given because of a more or less distinct repetitive pattern depending on recordings.
ab-Icp are the brief Icp events (Icp < 10 min) that occurred during stylet pathway.
bs-Icp are the sustained Icp events (Icp > 10 min).
6
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Please cite this article in press as: Pointeau, S., et al. Exceptional plant penetration and feeding upon cortical parenchyma cells by the woolly poplar aphid.
Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/j.jinsphys.2012.03.008

Page 7

that waveform C of P. passerinii is correlated to the same activities.
However, the sub-waveforms of waveform C showed more or less
distinct repetitive patterns, as described in Janssen et al., (1989) on
whitefly. These authors suggested that they reflected an alterna-
tion of intercellular secretion of sheath saliva and mechanical pro-
gress of the stylets. Repetitive patterns in P. passerinii might be
related to such alternation of activities during penetration, but fur-
ther analyses are required to link sub-waveforms to the aphid
activities. Waveform B features from other aphids were less clear
or missing (Tjallingii, 1988).
The main difference between P. passerinii and other aphids
appeared in waveform Icp, the intracellular phase in cortical
parenchyma cells. The EPGs showed two distinct waveform Icp
periods: (1) the brief b-Icp, usually lasting about one minute, oc-
curred 16 times on average, and (2) a sustained s-Icp, lasting more
than 20 min, that often occurred subsequently. The return to wave-
form C at the end of a pd waveform in phloem sap feeding aphids
(lasting 5–15 s, typically) seems to be due to the withdrawal of the
stylet tip from the living cell (Tjallingii, 1985b). Alternatively, the
return could be prompted by the stylet piercing the membrane
at the opposite side of the cell or by the death of the punctured cell.
In the case of P. passerinii, the two latter explanations seem more
appropriate as shown by intracellular passages in the straight sty-
let pathways. The relatively small number of b-Icp waveforms and
their much longer duration than the pd waveforms support the
hypothesis that they might be related to crossing the cells, mean-
while possibly ingesting their contents. The biological significance
of the s-Icp waveform is much more difficult to explain. The sus-
tained intracellular phase in non vascular cells is rather different
from committed phloem ingestion in phloem feeders (Pettersson
et al., 2007). The way in which P. passerinii ingests nutrients from
the cortical parenchyma cells during these periods of s-Icp, is a
question open to speculation. Kunkel (1967) suggested that paren-
chyma feeding Homoptera might stimulate a symplastic nutrient
flow from surrounding cells and allowing subsequent sucking of
the fluid contents of several cells by puncturing only one cell.
Although rather speculative, this hypothesis corresponds to the
clear indication of an intracellular position of stylet tips during s-
Icp. The difference between b-Icp and s-Icp, in terms of waveform,
is that Icp-III only occurred in the latter. However, this sub-wave-
form represents a ‘silent period’, suggesting no apparent activities,
in contrast to Icp-I and Icp-II that showed regular repetitions of
peaks (Fig. 3). The Icp-II peaks may suggest similarity to the E2
phloem ingestion waveform in phloem sap feeding aphids. How-
ever, the E2 peaks in other aphid EPGs have mixed emf/resistance
Fig. 3. (a) Waveform Icp found in a 1 h Phloeomyzus passerinii EPG recording on the bark of Populus x canadensis cv. ‘I214’ stem-cuttings: (1) b-Icp: brief Icp events
(Icp < 10 min) observed during stylet pathway and composed of sub-waveform Icp-I followed by sub-waveform Icp-II, (2) s-Icp: sustained Icp events (Icp > 10 min) composed
of a typical succession of sub-waveforms Icp-I, Icp-II and Icp-III. Higher resolution views of representative sub-waveforms; (a) Icp-I, (b) Icp-II and (c) Icp-III.
Table 3
Electrical penetration graph (EPG) parameters used to study the probing behavior of
the woolly poplar aphid, Phloeomyzus passerinii during an 8 h access period to the
bark of Populus x canadensis cv. ‘I214’ stem-cuttings. Np, non-probing.
EPG parameters Mean ± SE (Min–Max)
Time parameters (min)
Time from the beginning of
experiment to first probea
Time from start of first probe to first C + b-Icpb
Time from start of first probe to first s-Icpc
Total duration of Np from start of
first probe to end of recording
Total duration of A + C + b-Icpbbefore
the s-Icpcwithin a probe
192.5 ± 29.0 (11.4–437.3)
4.5 ± 0.5
123.0 ± 34.4 (17.9–375.1)
127.2 ± 25.9 (0–316.37)
(1.6–8.1)
49.2 ± 3.8 (17.9–67.2)
Occurrence parameter
Number of b-Icpbbefore the first s-Icpc
14.1 ± 1.8(5–29)
aStarting point of probe is waveform A.
bb-Icp are brief Icp events (Icp < 10 min) that occurred during stylet pathway.
cs-Icp are sustained Icp events (Icp > 10 min).
S. Pointeau et al./Journal of Insect Physiology xxx (2012) xxx–xxx
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Page 8

origin and are not related to ingestion but to salivation, concur-
rently with ingestion. Ingestion is reflected by a regular sine-wave
like signal of about 5 Hz, similar to but with a much lower ampli-
tude than the waveform during xylem ingestion (Prado and Tjallin-
gii, 1994). The peaks shown in our Icp-I and -II may, therefore, be
related to salivation, the Icp-I peaks resembling the E1 salivation
peaks and the Icp-II downward peaks resembling the E2 salivation
peaks. Furthermore, Icp-I always occurred before Icp-II, similar to
E1 always preceding E2. However, similarities in waveform shape
are generally not reliable and further experimental studies are re-
quired to explain them. Sub-pattern Icp-III was only observed dur-
ing s-Icp events between two sub-patterns Icp-II. This sub-pattern
was associated with intracellular position of stylets in parenchyma
cells and showed a flat trace usually lasting less than 10 s. Under
the hypothesis that a symplastic nutrient flow occurs during s-
Icp period, Icp-III might correspond to the period during which
the punctured cell has been emptied of its content and is filled
from the contents of surrounding cells. Alternatively, an intracellu-
lar osmotic pump hypothesis could be possibly applied to Icp-III, a
‘pressure release’ (into the food canal) period between Icp-II peri-
ods, during which salivation may have increased the osmotic pres-
sure in the punctured cell. Further studies involving correlation
work by running EPG recordings in parallel with histological stud-
ies, quantifications of honeydew secretion or stylectomy will be
necessary to confirm these hypotheses.
The average time needed to reach a sustained intracellular
phase in a cortical parenchyma cell after plant access in P. passerinii
(5 h) is similar to that reported in phloem sap feeding aphids: 5–
7 h in some aphid–plant combinations (Tjallingii and Mayoral,
1992), although this includes a longer non-probing time before
the first probe (3 h). It would be interesting to perform further as-
says to test the effects of tethering on this first long non-probing
period. In phloem sap feeding aphids, 1 h of complex probing activ-
ity distributed over several separate probes, frequently including
some short sieve element punctures has been observed regularly
(Tjallingii and Mayoral, 1992). In P. passerinii, the sustained intra-
cellular phase was preceded by 2 h of a relative more simple
sequence of events with an average of two probes per recording.
This simplicity is probably due to the fact that mainly one type
of plant tissue is penetrated. Only waveform A and early waveform
C represent penetrating a different tissue: cork and cortical
cambium, respectively. Then, alternations of waveform C and b-
Icp reflected extracellular stylet penetration and crossing of corti-
cal parenchyma cells.
Pollard (1973) underlined that Aphidoidea penetrate plant tis-
sues targeting phloem sieve tubes, with exceptions for some
tree-dwelling aphids species for which the destination of stylets
is the cortical parenchyma such as in Adelgidae. In studying the
probing behavior of Adelgidae (i.e. Pineus boerneri Annand on Pinus
taeda L.), Cardoso (2007) described two main waveforms (one
extracellular and one intracellular), which could be comparable
to the waveforms C and Icp of P. passerinii’s EPGs. The most striking
difference is the absence of short intracellular events (comparable
to b-Icp) occurring during the pathway phase. In P. boerneri’s EPGs,
aphids generally stay on average 1.7 h in the extracellular phase
before shifting to a sustained intracellular phase (Cardoso, 2007).
However, penetration of cortical parenchyma tissue of conifer
stems by Adelgidae is intercellular and only occasionally intracel-
lular (Balch, 1952; Pollard, 1973; Müllick, 1977). This could explain
the absence of short intracellular events related to crossing of cor-
tical parenchyma cells and could confirm the biological signifi-
cance of b-Icp pattern. Some Adelgid species have a 2-year life
cycle consisting of two different feeding strategies according to
the years (Havill and Foottit, 2007). For instance, A. piceae feeds
on galls initiated on its primary host (i.e. spruce) the first year,
but feeds by inserting its stylets inside cortical parenchyma cells
of the trunk of its secondary host (i.e. firs) the second year (Balch,
1952; Balch et al., 1964; Müllick, 1977). When A. piceae penetrate
cortical parenchyma cells of fir trunks, they trigger a hyperplasy
and a hypertrophy of cortical parenchyma cells that might increase
the sugar content and nutritional quality of these cells (Balch,
1952; Balch et al., 1964; Müllick, 1977). The prolonged intracellu-
lar phase in P. passerinii’s EPGs might be related to feeding in such
nutritious tissue. However, such cellular modifications are gener-
ally visible after a few days (Müllick, 1977). P. passerinii initiated
a sustained intracellular event after a time lag of 5 h from the
beginning of the experiment.
Tree-dwelling aphids have been considered to cause less dam-
age than other sucking insects because they are rarely responsible
for virus infection and their damage is mainly linked to reduction
in tree growth (Blackman and Eastop, 1994). However, it seems
that we are here confronted with an exception where bark damage
Fig. 4. Sequence analysis of waveforms. Typical sequence probability of waveforms as occurred during probing behavior of the woolly poplar aphid, Phloeomyzus passerinii in
the bark of poplar stem-cuttings of the genotype Populus x canadensis cv. ‘I214’. The values near the arrow correspond to the likelihood that one waveform is followed by any
other type. Probabilities <1% are not shown.
8
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Please cite this article in press as: Pointeau, S., et al. Exceptional plant penetration and feeding upon cortical parenchyma cells by the woolly poplar aphid.
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Page 9

may result in extensive tree mortality. Two main hypotheses might
explain such a phenomenon. Firstly, damage might be the conse-
quence of tissue modifications caused by normal feeding. For in-
stance, morphs of A. piceae in their secondary host are, with P.
passerinii, the only known trunk-dwelling aphids that do not cause
apparent galls but induce extensive tree mortality. A. piceae trig-
gers a non specific response consisting in the formation of a
‘‘wound periderm’’ within cortical parenchyma cells and a histo-
logical modification of tracheids similar to ‘‘compression wood’’
which disturbs sapwood translocation and causes tree mortality
(Müllick, 1977; Miles, 1989). Secondly, herbivores having intimate
relationships with their host trees, such as sessile Hemipteran, may
induce a hypersensitive reaction (HR), i.e. a localized host tissues
death reducing the negative impact of the insect (Fernandes,
1990). Species inducing strong HR may be particularly damaging
to their host tree by causing widespread tissue necrosis and tree
mortality (Radville et al., 2011). The presence of an extended
necrosis of cortical cells and the rapid and important poplar mor-
tality following P. passerinii infestation suggest that its feeding
mode induces such pronounced HR. Moreover, a plant that has
no co-evolutionary history with a specific herbivore is more likely
to exhibit a HR resulting in widespread tissue necrosis (Ryan et al.,
1990). Poplar grown in poplar stands are recent man selected hy-
brids. Thus, the lack of co-evolutionary history between P. passeri-
nii and theses hybrids might also contribute to the development of
a strong HR. However, the consequences of the feeding mode on
poplars are still unknown and it is not known whether symptoms
of damage are linked to a comparable tree responses.
In a broad ecological perspective, this study extends knowledge
of the bark-feeding nutritional ecology of Aphididae. P. passerinii is
the only Aphididae species known to feed on cortical parenchyma
cells without causing apparent galls, a diet more usually encoun-
tered in Adelgidae (Pollard, 1973; Müllick, 1977; Miles, 1989).
Parenchyma feeding is a characteristic of the two primitive fami-
lies, i.e. Adelgidae (all associated with conifers) and Phylloxeridae
(mainly associated with oaks and pecans) (Pollard, 1973; Blackman
and Eastop, 1994; von Dohlen and Moran, 2000). Heie, (1987) sug-
gested that the success of the Aphididae lineage was partly due to
the emergence of phloem sap feeding as inferred from the evolu-
tion of a large cauda in recent families of Aphidoidea. Because a
large cauda gives a morphological advantage by allowing the
excretion of the large quantity of honeydew produced by phloem
feeders, it may have developed during the shift from parenchyma
feeding to phloem sap feeding (Heie, 1987). P. passerinii is the only
species of the sub-family Phloeomyzinae (Blackman and Eastop,
1994), a very peculiar family characterized by a rounded rudimen-
tary cauda (Heie, 1987). This morphological characteristic is not
surprising given the mode of feeding of P. passerinii and might be
associated with a primitive parenchyma feeding lifestyle. The phy-
logenetic position of Phloeomyzinae has not yet been elucidated.
However, the mode of feeding of P. passerinii suggests that it might
be a primitive sub-family in the Aphididae lineage that evolved be-
fore the emergence of the phloem sap feeding lifestyle in Aphidi-
dae. Its exact position however, still needs to be clarified.
In conclusion, despite the feeding preference of P. passerinii for
the bark of mature trees, both histological and EPG analysis per-
formed on poplar stem-cuttings were appropriate ways of studying
the mode of feeding of this trunk feeder. They gave significant new
insight into the feeding ecology of Aphididae and showed a simpler
probing behavior than phloem sap feeding aphids, probably owing
to the small number of tissues crossed. The distinction between the
short and sustained intracellular events of waveform Icp made it
possible to separate P. passerinii EPG waveforms into two main
phases: a pathway phase (gathering waveforms A, C and b-Icp)
and a sustained intracellular phase (s-Icp) in cortical parenchyma
tissue. The information obtained on the feeding site and stylet
penetration of P. passerinii should serve as a basis for advanced
studies on the mechanisms that lead to tree mortality and the pop-
lar’s resistance mechanism.
Acknowledgments
This work is part of the Ph.D. thesis of S. Pointeau carried out
under the directorship and supervision of F. Lieutier with the par-
ticipation of S. Bankhead-Dronnet as a co-supervisor. It was sup-
ported by grants from the French Ministry of Agriculture, Food
and Fisheries (MAAP) and the French Ministry of Research and
Education. We thank Freddy Tjallingii for his suggestions and cor-
rections, which greatly improved the quality of this manuscript.
We are grateful to Alain Moreau (INRA Orléans, France) and Berna-
dette Delaleu (INRA Nouzilly, France) for their technical support in
histology.
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Journal of Insect Physiology (2012), http://dx.doi.org/10.1016/j.jinsphys.2012.03.008