Entomologia Experimentalis et Applicata 97: 203–210, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

203

Difficulties in location and acceptance of phloem sap combined with

reduced concentration of phloem amino acids explain lowered
performance of the aphid Rhopalosiphum padi on nitrogen deficient barley
(Hordeum vulgare) seedlings

K. L. Ponder, J. Pritchard, R. Harrington1 & J. S. Bale

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK; 1 Department of
Entomology and Nematology, IACR-Rothamsted, Harpenden, Herts. AL5 2JQ, UK

Accepted: June 8, 2000

Key words: EPG, stylectomy, phloem amino acids, nitrogen, aphid-plant interaction

Abstract
Effects of nitrogen deficiency in hydroponically grown barley seedlings (Hordeum vulgare L.) on the development
and reproduction of the aphid Rhopalosiphum padi (L.) (Hemiptera: Aphididae) were investigated.
Plant growth was significantly reduced in seedlings grown without nitrogen. Aphid intrinsic rate of increase
(rm ) was also significantly lower on these plants compared with that on plants grown with 8 mol m−3 nitrogen.
Phloem sap was collected from seedling stems by aphid stylectomy and amino acids quantified by HPLC. There
was a significant reduction in the concentration of non-essential amino acids as a group, but not of essential amino
acids. Electrical penetration graphs (EPG) indicated that aphids reached the phloem more quickly and fed for
longer on plants grown with nitrogen. This is the first reported study in which this combination of techniques has
been used to understand the interactions of an aphid and plant under different environmental conditions.

Introduction 1987; Kazemi & van Emden, 1992; Sandström &

The phloem from nitrogen deficient plants has been
shown to have an altered spectrum, and in most cases a
reduced concentration of amino acids (Weibull, 1987;
Peuke et al., 1994; Tillard et al., 1998), although this is
not always the case (e.g., Shelp, 1987). Unfortunately,
due to technical difficulties in sampling phloem, most
work has used phloem bleeding methods to collect
phloem sap (e.g., Peuke et al., 1994; Tillard et al.,
1998). Phloem sap collected by stylectomy is believed
to provide a better representation of its composition in
vivo (Weibull et al., 1990; Girousse et al., 1991).
Studies of aphids feeding on artificial diets (e.g.,
Mittler, 1967; Srivastava & Auclair, 1975; Prosser
et al., 1992) have shown that the concentration and
composition of dietary amino acids influence their per-
formance. On plants, although many studies have cor-
related the concentration and / or spectrum of phloem
amino acids with aphid performance (e.g., Weibull,
Pettersson, 1994), a relationship is not always ap-
parent (Girousse & Bournoville, 1994). The lack of
consistent relationships between aphid performance
and plant nitrogen status highlights the complexity
and inherent variability of the interaction in vivo and
points to the need for a well-controlled experimental
approach.
In this study, the interrelationships between the
performance of the bird cherry-oat aphid R. padi
feeding on barley (H. vulgare) seedlings grown with
different nitrogen inputs were investigated under con-
trolled environmental conditions. Phloem amino acids
(sampled by stylectomy) were quantified as an indi-
cator of the nutritional quality of the plant and the
probing behaviour of the aphid (measured by electri-
cal penetration graphs) analysed in parallel with this.
These parameters were considered likely to be critical
components of the interaction and have not been quan-
204
Table 1. Chemical composition of hydroponic nutrient so-
lutions A (nitrogen deficient) and B (8 mol m−3 nitrogen)
barley grown in well-watered compost at the Univer-
used in experiments sity of Birmingham. All aphids were maintained in a
L18:D6 regime at 18–20 ◦ C.

Concentration in solution (mol m−3 ) Phloem collection and amino acid analysis. Speci-
Solution A Solution B men cages (Agar Scientific Ltd.), 1 cm in diameter,
CaCl2 2 2 were secured to the stems of barley seedlings by foam
MgSO4 1 1 bungs forming a close but non-restricting seal. A max-
KH2 PO4 4 4 imum of ten aphids (R. padi) were caged overnight
NaFeEDTA 0.015 0.015 onto the plant. Stylectomy was performed using high-
HBO3 0.25 0.25 frequency microcautery (Unwin, 1978) between 3.5
MnSO4 0.002 0.002 and 7.5 h after the start of the photoperiod to reduce
CuSO4 0.002 0.002 effects of diurnal variation. When a successful cut had
NaMoO4 0.00025 0.00025 been made, the cage was flooded with paraffin oil to
NH4 NO3 – 4 eliminate evaporation and the exuding phloem sap col-
Total nitrogen 0 8 lected into an oil-filled micropipette (Pritchard, 1996).
Sap was collected for a maximum of 4 h and samples
stored at –70 ◦ C until analysis.
The osmotic pressure of all samples was measured
by picolitre osmometry (Tomos et al., 1994) using
tified using this combination of techniques before, in
NaCl standards, to give a measure of total solutes in
a single experimental system.
the sample. Sample volume was calculated by measur-
ing the diameter of a droplet suspended in oil. Samples
Materials and methods were then dissolved in 10 µl of 0.1 M HCl/95%
ethanol (1:1) to limit enzymatic degradation (Weibull
Plant material. Barley seeds (H. vulgare L. cv Igri) et al., 1986). Amino acids were quantified by HPLC,
were germinated for 48 h on damp paper in the dark. following the methodology of Jones et al. (1981). A
Germinated seeds were transferred to a mesh over an Beckman 157 Fluorescence detector at 490 nm was
aerated hydroponic nutrient solution (solution A in Ta- used to detect the amino acids. Standards were AA-S-
ble 1) so that their roots were suspended in the solution 18 (Sigma) supplemented with asparagine, glutamine,
but the seed itself remained above it. These were left γ -aminobutaric acid and tryptophan and diluted with
for a further 24 h in the dark and then exposed to a double distilled water to 25 µM. Due to small sample
L16:D8 regime (18–20 ◦ C). sizes (8 nitrogen-deficient and 14 nitrogen-sufficient
Approximately 24 h later (when the shoots were plants) amino acids were divided into ‘non-essential’
1–2 cm long) half the seedlings were transferred to and ‘essential’ groups and each group analysed by
hydroponic nutrient solution B (Table 1) which was manova. Histidine and serine could not be reliably
identical to solution A but also contained 8 mol m−3 separated; these were included together in the ‘non-
nitrogen (as NH4 NO3 ). This concentration of nitrogen essential’ group as serine (non-essential) was likely to
is similar to that used in other formulations of nutrient contribute much more to their combined concentration
solutions (Salisbury & Ross, 1991). At this stage all than histidine (essential).
seedlings were thinned to 8 per pot (capacity 900 ml)
and held in the solution by foam bungs. Experiments Aphid performance. Aphids were caged singly on
were carried out using seedlings 13–18 days after ger- the stems of barley. They were maintained on their ex-
mination (2–3 leaf stage). Leaf lengths were measured perimental treatments for a complete generation prior
every few days to monitor the growth of seedlings in to the experiment to reduce maternal effects on subse-
the different solutions and dry weights and water con- quent performance and to produce nymphs of a known
tents were calculated from plants harvested at 5 day age. Aphids were checked daily until the start of repro-
intervals. duction and then every other day, when offspring were
counted and removed. Performance was assessed by
Aphid culture. A clone of R. padi was started with a calculation of the intrinsic rate of increase (rm ) (Wyatt
single aphid from a long-term culture maintained on & White, 1977).

Aphid population growth. To determine if the rm
values calculated from aphids caged individually on
stems could be extrapolated to uncaged populations,
the increase in numbers of aphids allowed to roam
freely over whole plants was monitored. Three ten-
eral alates were introduced into a muslin-covered cage
containing three plant pots, each with five seedlings
grown in vermiculite. A total of 10 cages were used.
Plants were watered 2–3 times a week, 5 of the cages
with nitrogen-sufficient and 5 with nitrogen-deficient
nutrient solutions (Table 1). After 14 days, the num-
bers of aphids in each cage were recorded. Five repli-
cates of each nitrogen treatment were made and the
results analysed with a Mann–Whitney U-test.
Aphid feeding behaviour. Electrical penetration
graphs (EPG) were obtained over 5 h periods us-
ing a 4-channel DC system (Tjallingii, 1988). Plant
and aphid treatments were as described for previous
experiments. Apterous adults, taken from the stock
culture, were starved for an hour before being attached
to a 2 cm length of 25 µm gold wire (Goodfellow)
with silver conductive paint (RS Components). The
paint had previously been tested for adverse effects
on aphid reproduction and longevity–none was found.
Aphids were gently lowered onto the stems of plants
and recording started. Stylet 2.5 software (supplied by
W. F. Tjallingii, Wageningen, the Netherlands) was
used for acquisition and subsequent analysis.
Duration and frequencies of non-penetration and
penetration activities were calculated. Penetration ac-
tivities were divided into pathway phases (waveforms
A, B, C), xylem (waveform G), phloem salivation
(waveform E1) and phloem ingestion (waveform E2).
Detailed descriptions of waveforms are given by
Tjallingii (1990). The time taken to reach sieve ele-
ments and the time to sustained ingestion (>10 min)
were recorded from the start of the first penetration
and from the start of the probe that contained them.
Ingestion from a sieve element for more than 10 min
is thought to be indicative of sieve element acceptance
(Tjallingii & Mayoral, 1992). Aphids that failed to
locate the sieve elements during the 5 h were said to
take 300 min to start phloem feeding. To overcome the
possibility of making Type I errors, only parameters
directly related to phloem feeding were statistically
analysed (see Table 3) using Mann–Whitney U tests
with the Bonferroni correction, resulting in the critical
value of α being 0.01. Lengths of individual E1 and
E2 periods were analysed with t-tests.
205
To investigate whether there were any effects of
tethering on the subsequent probing behaviour of the
aphids, ‘free’ aphids were placed on stems of plants
and their behaviour checked every 5 min for 30 min,
and every 15 min thereafter up to 3 h. Behaviours were
classified as walking, surface testing (aphid station-
ary, antennae waving), and probing (aphid stationary,
antennae flat against back). Results were compared
with those obtained from EPG recordings where walk-
ing and surface testing were taken to be equivalent to
non-penetration, and probing equivalent to all other
waveforms.
Results
Intrinsic rate of increase (rm ). rm was significantly
reduced on plants grown without nitrogen (means ±
standard errors 0.405 ± 0.007 on nitrogen deficient
seedlings compared with 0.456 ± 0.015 on seedlings
grown with nitrogen; t(42) = 3.37; P=0.002). Num-
bers of aphids that were unrestricted on plants also
showed these trends: after 14 days there were signifi-
cantly fewer aphids on barley watered with nitrogen-
free nutrient solution compared with barley watered
with solutions containing 8 mol m−3 nitrogen (medi-
ans 190 and 325, respectively; P=0.02).
Plant growth. Barley grown in the absence of nitro-
gen showed classic deficiency symptoms. Root length
was increased relative to shoot length and leaves were
small, yellow and started to senesce earlier. Difference
in leaf lengths between the two nitrogen treatments
was highly significant at 8 days after germination (just
after the second leaf had emerged) (t(38) = 6.196;
P<0.0001) and increased as plants got older. Dry
weights showed the same trend (P=0.05 at 8 days after
germination and P<0.001 at 13 days after germina-
tion). The water content of plants was also reduced on
nitrogen deficient plants at 13 days after germination
compared with those grown with nitrogen (89.9% and
91.8% of the wet weights respectively); these differ-
ences were significant (t(17) = 10.9; P=0.000; on
arcsine transformed figures). All experimental work
described in this paper was carried out using seedlings
between 13 and 18 days after germination, when
nitrogen deficiency symptoms were clearly visible.
Phloem composition. Osmotic pressure of phloem
sap was 1.25 ±0.15 and 1.5 ±0.2 MPa (means ± stan-
dard errors) from seedlings grown without and with ni-
206
Table 2. Mean concentrations (mol m−3 (s.e.)) and percentages of amino acids (grouped
into essential and nonessential amino acids) in phloem sap of barley seedlings grown with or
without nitrogen in the hydroponic nutrient solution. ‘Conc N-O’ refers to the difference in
concentration of each amino acid between the two treatments

Animo acid No nitrogen 8 mol m−3 nitrogen Conc. N–O

Concentration % Concentration %

Non-essentials
Asp 3.6 (1.1) 3.1 3.3 (1.0) 1.8 -0.3
Glu 17.7 (4.3) 15.4 18.0 (6.0) 9.6 0.3
Asn 9.5 (5.3) 8.3 7.0 (1.4) 3.8 -2.5
Gln 38.0 (17.1) 33.1 89.6 (16.8) 48.05 1.6
Gly 0.1 (0.1) 0.1 0.5 (0.2) 0.3 0.4
His+ser 5.2 (0.8) 4.6 10.3 (3.5) 5.5 5.1
Ala + Tyr 5.7 (0.8) 4.9 15.9 (1.8) 8.5 10.2
GABA 0.6 (0.3) 0.5 1.3 (0.7) 0.7 0.7

Total 80.4 70.0 145.9 67.2 65.5

Essentials
Arg 14.6 (3.6) 12.7 15.4 (5.2) 8.2 0.8
Thr 2.8 (0.9) 2.4 3.1 (0.9) 1.7 0.3
Trp 0.4 (0.1) 0.3 1.0 (0.3) 0.6 0.6
Met 2.6 (0.7) 2.3 2.7 (0.6) 1.5 0.1
Val 1.9 (0.4) 1.7 3.6 (1.0) 1.9 1.7
Phe 2.2 (0.7) 1.9 2.0 (0.4) 1.1 -0.2
Ile 2.7 (1.0) 2.4 1.3 (1.6) 0.7 -1.3
Leu 1.7 (0.9) 1.5 3.0 (0.9) 1.6 1.3
Lys 1.3 (0.5) 1.1 1.5 (0.4) 0.8 0.3
Total 30.1 30. 33.6 32.8 3.5

Total 110.5 (13.9) 100 179.5 (23.0) 100 79.0

trogen respectively. These differences were not signif-
icant (t(18) = 1.73; P=0.33). Table 2 shows the com-
position of phloem amino acids from each treatment.
Total amino acid concentration was 110.52 mol m−3 in
nitrogen deficient plants and 179.47 mol m−3 in plants
grown with nitrogen, which was a significant differ-
ence (t(20) = 2.12; P=0.047). No differences were
found in the concentrations of essential amino acids
(Pillai’s criterion=0.596; F(10,11) = 1.62; P=0.22).
There was a significant reduction in the concentration
of non-essential amino acids as a group from nitrogen
deficient plants (Pillai’s criterion=0.688; F(8,13) =
3.59; P=0.02). This difference could not be ascribed
to any particular amino acids though greatest reduc-
tions were in glutamine and alanine + tyrosine.
Aphid feeding behaviour. Table 3 summarises the re-
sults from EPG analysis. Duration of phloem ingestion
(pattern E2) was decreased on plants grown without
nitrogen, compared with plants grown with 8 mol m−3
nitrogen (P=0.01); on the former, there was a quali-
tative increase in time spent not probing. Time taken
to reach the phloem (first E1 pattern) from the start
of the recording was greater on plants grown with-
out nitrogen compared with those grown with nitrogen
(P=0.01). The time taken to first phloem ingestion
pattern (E2) and to sustained ingestion (E2>10 min)
was also increased (P=0.02 and P=0.01, respectively)
though after the Bonferroni correction P<0.01 was
required for rigorous statistical significance. These
trends in sieve element location and acceptance be-
tween each nitrogen treatment were also observed
when measured from the start of the probe that con-
 207
Table 3. Summary of EPG results. Values are means (s.e.). Rows in bold are
those that were statistically analysed. Letters (a,b) indicate significant differences
(Mann–Whitney with Bonferroni correction P<0.01). Numbers in superscript
brackets indicate n values that differ from n = 21

Parameter No nitrogen 8 mol m−3

n=21 nitrogen
n=21

Duration of (min)
Non-penetration 99.7 (16.1) 49.6 (11.4)
Pathway (A, B, C) 122.4 (12.5) 123.7 (14.4)
Xylem ingestion (G) 4.1 (3.1) 4.3 (3.3)
Salivation into sieve element (E1) 13.6 (5.1) 6.2 (1.6)
Ingestion from sieve element (E2) 50.9 (12.8)a 103.1 (18.6)b
Longest E2 50.8 (14.3) 85.7 (17.8)
Average E1 2.66(83) (0.74)a 0.91(72) (0.1)b
Average E2 40.7(37) (11.0) 30.8(42) (8.1)

Time to (min)
from start of recording
First E1 160.7 (23.1)a 85.4 (18.4)b
First E2 175.5 (22.6)a 105.4 (20.0)b
First sustained E2 211.1 (19.2)a 135.6 (19.8)b
from start of probe
First E1 100.3 (28.4) 38.4 (14.4)
First E2 121.4 (28.6) 62.6 (19.0)
First sustained E2 157.5 (28.5) 73.2 (21.7)

Time between (min)

E1 & sustained E2 70.6(15) (21.8) 52.7(20) (19.0)

Number of
Periods of non-penetration 9.9 (1.6) 7.2 (0.85)
E2 probes 0.95 (0.17) 1.45 (0.18)
E1 events 4.5 (0.88) 5.05 (0.92)
E2 events 1.86 (0.41) 2.48 (0.49)
E1 periods before sustained E2 1.43(13) (0.78) 1.79(17) (0.89)
E1 events per E2 event 2.6(15) (0.26) 1.9(20) (0.31)

Proportion of
probes containing E2 0.19 (0.05) 0.34 (0.07)

tained them, but were not statistically analysed. There
were no qualitative differences in the numbers of
probes made, or in number of phloem feeding peri-
ods. On nitrogen deficient plants a higher proportion
of aphids reached a sieve element (showed pattern E1)
but did not achieve phloem ingestion from it (P=0.02)
and the average length of each E1 period was signif-
icantly longer (P=0.01). Periods of xylem ingestion
were infrequent in both treatments.
A smaller proportion of tethered aphids was
recorded probing the plant at any one time, but there
were no apparent effects of host plant nitrogen treat-
ment on the discrepancies between tethered and free
aphids during the 3 h of recording.
208
Discussion
The intrinsic rate of increase (rm ) of R. padi feeding
on barley was reduced on seedlings grown without ni-
trogen. These seedlings showed clear differences in
leaf lengths, dry weights and water content, all of
which were reduced under nitrogen deficiency. Such
a relationship between an aphid and its host plant has
been documented before (Weibull, 1987; Petitt et al.,
1994) and is generally attributed to the fact that plants
grown under nitrogen deficiency exhibit a reduction
in the amino acid ‘quality’ available to aphids which
are already limited by the nitrogen component of their
diets.
Phloem amino acids. The concentration of non-
essential amino acids was significantly lower in the
nitrogen deficient plants. Although artificial diet work
has shown that lack of certain essential rather than
non-essential amino acids limit aphid performance, a
reduction in the concentration of non-essential amino
acids may limit the amount of excess dietary nitro-
gen for use in symbiotic conversion to essential amino
acids. Without detailed chemical analyses of aphids
and honeydew, such a hypothesis remains speculative.
Altered nitrogen nutrition could affect the plant in
a number of other ways (Klingauf, 1987). Experiments
in which the sucrose and amino acid composition of
phloem was mimicked in artificial diets show that
aphid performance cannot be attributed to these simple
nutritional factors alone (Rahbé et al., 1988; Febvay
et al., 1988). For example, other chemicals (deterrents
or attractants) in the phloem or in other tissues could
be affected by altered nitrogen nutrition and may make
it harder for the aphid to locate and/or feed from the
sieve elements.
Aphid feeding behaviour. Aphids used in experi-
ments were taken from the stock culture, which were
reared on plants that were not nitrogen-limited. There-
fore the probing behaviour of aphids recorded on
nitrogen-deficient plants may have been affected by
introduction to a ‘new’ plant treatment. On nitrogen
deficient plants, aphids showed a greater proportion
of time failing to penetrate tissues (increased non-
penetration, np) and spent less time in phloem in-
gestion (E2). The time taken to first penetration was
similar on each treatment as was the number of short
(< 2 min) probes, suggesting that epidermal factors
were not involved. Time taken to reach the phloem
was increased on nitrogen deficient plants indicat-
ing that the aphids experienced problems in locating
sieve elements. However, EPG analysis cannot dis-
tinguish a brief (10s) penetration into a sieve element
from penetration of other cells, which occur frequently
(Tjallingii & Hogen Esch, 1993). Consequently, lo-
cation and subsequent rejection of sieve elements is
likely to have been underestimated. The frequency
of potential drops (pds) has been shown to increase
shortly before a sieve element puncture occurs. Pds
were not analysed in this study but could have pro-
vided some additional information on sieve element
location and acceptance, and consideration of this be-
haviour should be made in future studies on phloem
feeding behaviour. Compared with aphids on plants
grown with nitrogen, on nitrogen deficient plants more
aphids that reached the sieve elements (showed pattern
E1) failed to follow this with phloem ingestion (pat-
tern E2), and it took longer within a probe to show
sustained (>10 min) ingestion. These observations in-
dicate that as well as possibly being harder to locate,
once reached and sampled, the sieve element was more
frequently rejected on nitrogen deficient plants. The
average length of each E1 period was significantly
longer on nitrogen deficient plants which implies that
some factor in the sieve elements prevents or delays
the aphids from ingesting phloem on nitrogen defi-
cient plants. Caillaud et al. (1995) and Prado (1997)
working on resistant and susceptible wheat lines also
showed these trends in occurrence and duration of E1
and E2 periods. There was no evidence for deterrent
chemicals causing the difficulties in phloem location
and acceptance. Duration of pathway activities (pat-
tern A, B and C) was similar in both treatments and
there were no occurrences of a sudden cessation of
phloem feeding, suggesting that there was not a toxic
or deterrent factor in the phloem. However, lack of an
appropriate feeding stimulant in phloem of nitrogen
deficient plants cannot be ruled out.
Sampling by stylectomy may introduce a bias and
only phloem that an aphid has ‘accepted’ can be quan-
tified. Therefore, there could have been a greater dif-
ference in the average sieve element composition be-
tween the two treatments, which would not have been
detected. No significant differences were recorded in
the average length of each E2 period during the 5 h
recordings. During this time span at least, once ‘ac-
cepted’ the phloem may remain equally suitable on
both treatments. The phloem rejected by the aphid
could be of insufficient nutritional quality, and that ac-
tually accepted above a certain nutritional ‘threshold’.
Alternatively, the aphid could reach a point when it can

no longer ‘afford’ to search for more suitable phloem
and must ingest what is available. In this case in par-
ticular, the reduction in concentration of amino acids
from the sampled phloem sap may be responsible for
the lowered aphid performance on nitrogen deficient
plants. Complementary experiments with artificial di-
ets to test the phagostimulatory and nutritional value
of changes in concentrations of certain amino acids
may help determine the relative suitability of phloem
from the two plant treatments.
EPG recordings over longer time periods are re-
quired to determine if there are any longer term effects
on feeding behaviours, such as a reduction in time
spent in each E2 period or compensatory feeding re-
sponses, such as increased ingestion rate. In addition,
examination of the route of the stylets in the plant
tissue would determine how frequently the sieve el-
ements are being reached and rejected without any
phloem related waveforms detected by EPG. Chemi-
cal analyses of honeydew and aphid carcasses would
provide a more complete picture of the nutritional in-
teractions involved and of any differences in aphid
metabolic efficiencies.
Acknowledgements
Thanks to Mark Baird for making and maintaining the
EPG equipment and to Glen Powell, Freddy Tjallingii,
Caroline Awmack and Ken Jakeman for technical ad-
vice. This work was supported by a BBSRC CASE
studentship with IACR-Rothamsted. IACR receives
grant-aided support from the Biotechnology and Bi-
ological Sciences Research Council of the United
Kingdom.
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