Écoscience

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Effects of sublethal attack by a sucking insect,

Hyalymenus tarsatus, on Sesbania drummondii
seeds: Impact on some seed traits related to
fitness

Lilian Ceballos, Claude Andary, Maxime Delescluse, Marc Gibernau, Doyle

Mckey & Martine Hossaert-Mckey

To cite this article: Lilian Ceballos, Claude Andary, Maxime Delescluse, Marc Gibernau,
Doyle Mckey & Martine Hossaert-Mckey (2002) Effects of sublethal attack by a sucking insect,
Hyalymenus tarsatus, on Sesbania drummondii seeds: Impact on some seed traits related to
fitness, Écoscience, 9:1, 28-36, DOI: 10.1080/11956860.2002.11682687

To link to this article: http://dx.doi.org/10.1080/11956860.2002.11682687

Published online: 23 Mar 2016.

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 ECoSCIENCE 9 (1) : 28-36 (2002)

Effects of sublethal attack by a sucking insect,

Hyalymenus tarsatus, on Sesbania drummondii
seeds: Impact on some seed traits
related to fitness1
Lilian CEBALLOS2, Centre d’Ecologie Fonctionnelle et Evolutive (CNRS, UPR9056), 1919 Route de Mende, 34293
Montpellier Cedex 5, France.
Claude ANDARY, Laboratoire de Botanique, Phytochimie et Mycologie (CNRS, UPR9056), Faculté de Pharmacie,
15 Avenue C. Flahault, 34060 Montpellier Cedex 2, France.
Maxime DELESCLUSE3, Marc GIBERNAU4, Doyle MCKEY, Martine HOSSAERT-MCKEY5,
Centre d’Ecologie Fonctionnelle et Evolutive (CNRS, UPR9056), 1919 Route de Mende, 34293 Montpellier
Cedex 5, France, e-mail: Martine.Hossaert@cefe.cnrs-mop.fr
Downloaded by [Universite Laval] at 02:59 06 August 2017

Abstract: Developing seeds of Sesbania drummondii are attacked by nymphs and adults of the bug Hyalymenus tarsatus
(Heteroptera: Alydidae), which kill some seeds and weaken others. Parasitism by this piercing-sucking insect reduced the
resources for the future seedling and affected seed physiology, including dormancy and exudation of allelochemicals of
imbibing seeds. Seeds attacked by H. tarsatus had reduced mass (20-80% reduction, depending on intensity of attack).
Heavy attack led to irregular shape, changes in seed coat color, and disruption of dormancy. While intact seeds did not
imbibe during a 48-hour test in water, a high proportion of bug-attacked seeds germinated, from 51 to 94%, depending on
intensity of attack. Attack by H. tarsatus also affected accumulation of allelochemicals and their exudation by imbibing
seeds. There were no quantitative differences in proanthocyanidin content between exudates of attacked and unattacked
seeds. In contrast, concentrations of total condensed tannins were higher in exudates of attacked seeds on the third day of
imbibition. This change may reflect induction of chemical defenses by herbivore attack and/or a mechanism to restore seed
coat impermeability. Although difficult to quantify, effects of sublethal attack by this sucking insect on the seed bank are
likely to have important consequences for the demography of S. drummondii, a short-lived perennial in habitats where
conditions for recruitment are variable and unpredictable among years.
Keywords: seed predation, seed size, physical dormancy, seed bank, seed exudation, seedling defenses, condensed tannins,
induced plant defenses, Sesbania drummondii, legume, Hyalymenus tarsatus, Alydidae.

Résumé : Les graines en développement de Sesbania drummondii sont attaquées par les nymphes et les adultes de
Hyalymenus tarsatus (Hétéroptère : Alydidae), qui peut soit les détruire soit les altérer partiellement. Le parasitisme des
graines par cet insecte réduit la quantité de ressources disponibles aux plantules mais influe aussi sur la physiologie des
graines, comme la dormance et la production de substances allélochimiques. L’attaque des graines par H. tarsatus entraîne
une réduction de leur masse (20-80 % de réduction selon le degré d’attaque). Les attaques répétées entraînent des changements
dans la forme et la couleur des graines et provoquent la rupture de dormance. Alors que les graines intactes restent
imperméables durant des tests d’imbibition de 48h, une grande proportion des graines attaquées germent rapidement (entre
51 et 94 % des graines, selon le degré d’attaque). Les attaques par H. tarsatus peuvent aussi affecter l’accumulation de
substances allélochimiques et leur exsudation au cours de l’imbibition des graines. Il n’y a cependant pas de différence dans
le contenu en proanthocyanidines entre les exsudats des graines attaquées et des graines saines. Par contre, la concentration
en tannin condensés totaux est plus élevée chez les graines attaquées lors du troisième jour de l’imbibition. Ce changement
dans la teneur en tanins condensés totaux peut refléter l’induction de défenses chimiques suite à l’attaque par un phytophage
et / ou un mécanisme pour restaurer l’imperméabilité du tégument. Bien que difficiles à quantifier, les effets de ces
attaques sublétales par des insectes suceurs sur le stock de graines au sol doivent être importants sur la démographie de
S. drummondii, espèce pérenne à cycle de vie court occupant des habitats où les conditions pour le recrutement fluctuent
entre années.
Mots-clés : prédation des graines, taille des graines, dormance, banque de graines, protection des plantules, tannins conden-
sés, défense induites, Sesbania drummondii, légume, Hyalymenus tarsatus, Alydidae.

1Rec. 2001-03-19; acc. 2001-12-29.

2Alternate address: Laboratoire de Botanique, Phytochimie et Mycologie (CNRS,
UPR9056), Faculté de Pharmacie, 15 Avenue C. Flahault, 34060 Montpellier Cedex,
France.
3Present address: Laboratoire de Génétique et de Biologie des Populations Végétales,

Bat. SN2, Université de Lille I, 59855 Villeneuve d’Ascq Cedex, France.

4Present address: Laboratoire d’Ecologie Terrestre (UMR 5552), Université Paul

Sabatier, 118 Route de Narbonne, Bât 4R3, 31062 Toulouse Cedex 4, France.
5Author for correspondence.

Introduction
In plants, various critical life history functions are inte-
grated within seeds, and several seed traits interact to reduce
the effects of environmental variability. Dormancy and dis-
persal of seeds allow them to escape unfavorable conditions
in time and space (Venable & Brown, 1988). These risk-
reducing traits are substitutable under certain conditions,
and trade-offs between dispersal and dormancy have been
found (Templeton & Levin, 1979). Seed banks associated
with dormancy buffer the negative effects of bad years,
limit fluctuations of population size, decrease the risk of
extinction, and potentially increase the genetic diversity of
populations (Venable, 1989; Levin, 1990; Baskin & Baskin,
1998).
For many plants, most seeds do not germinate immedi-
ately when mature, but remain dormant for long periods.
Dormancy may be imposed by physiological inhibition of
germination of the embryo and/or by physical mechanisms
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(such as impermeable seed coats). Physical dormancy is
found in many families and has evolved many times inde-
pendently (Baskin & Baskin, 1998). Persistence of seeds in
soil seed banks makes them an attractive and predictable
resource for various predators. Accordingly, selection has
favored the evolution of various resistance mechanisms in
seeds capable of long periods of dormancy. In legume
seeds, for example, dormancy is caused by progressive
dehydration of seed tissues and impermeabilization of the
seed coat, preventing water uptake by dormant seeds. Seed
coats of many legumes contain high concentrations of tan-
nins, which deter granivores and contribute to making seed
coats impermeable to water and oxygen (Egley et al., 1985).
They thus physically impede the entry of pathogens (Kulik
& Yaklich, 1991; Helsper et al., 1994) and also protect seed
lipid and protein reserves from oxidative degradation. These
protective mechanisms have been studied extensively in
seeds of legumes because of the economic interest of this
family. Some legumes have seeds capable of prolonged
periods of dormancy (e.g., 158 years for Cassia multijuga;
Baker, 1989).
Among the threats to seed coat integrity are attacks by
insects that pierce and suck developing or mature seeds.
Attacks by such insects, if sufficiently severe, can kill
seeds. Those that survive will have reduced nutrient
reserves. Piercing-sucking insects may also introduce
pathogens that kill or weaken dormant seeds or germinat-
ing seedlings (Kremer & Spencer, 1989). To the best of our
knowledge, few studies have examined the effects of
attacks by such insects on permeability of the seed coat and
thus on the seed’s capacity for dormancy (Karban and
Lowenberg, 1992).
This study examines the effects of attack of seeds of
Sesbania drummondii (Rydb.) Cory (Fabaceae: Papil-
ionoideae) by a piercing-sucking insect, Hyalymenus tarsat-
us (F.) (Heteroptera: Alydidae, subfamily Alydinae). Most
members of this subfamily feed on seeds, principally of
legumes, but their hosts also include other plant families
(Schaefer, 1980). Many species are polyphagous on
legumes, and some are important pests on pulse crops
(Yonke & Medler, 1968; Aina, 1975). As in other alydines,
adults and nymphs of H. tarsatus pierce immature pods
ÉCOSCIENCE, VOL. 9 (1), 2002
with the proboscis and suck the reserves of developing
seeds. The fate of the attacked seed depends on the intensity
of attack and on the rapidity of seed response. The bugs can
kill seeds when repeated attacks deplete seed nutrients or
kill the embryo (predation). When intensity of attack is
lower, seeds can often mature. However, these non-lethal
attacks might have negative effects on seeds (parasitism).
Because seed size can influence seedling establishment
(Leishman & Westoby, 1994), a reduction in the reserves
stored in the seed should reduce fitness. Little is known
about how developing seeds tolerate attacks by potential
seed predators (Rosenthal & Kotanen, 1994; Mack, 1998).
Not only can reserves of S. drummondii seeds be reduced by
bug attack, but damage to the seed coat could facilitate
transmission of pathogens (Harman, 1983; Mills, 1983).
Alydids have been implicated in transmission of seed-borne
diseases (Yonke & Medler, 1968). Furthermore, damage by
bugs could increase seed coat permeability and disrupt seed
dormancy (Van Staden, Manning & Kelly, 1989).
In this study, we characterize alterations of physiological
mechanisms in attacked seeds and attempt to understand
how seed parasitism affects the physiology of young
seedlings. First, we determine seed mass as a function of the
class of attack described by a semi-quantitative scale.
Secondly, we compare seed coat morphology and rates of
spontaneous germination in attacked and unattacked seeds.
This comparison suggests that parasitism affects the devel-
opmental and physiological processes involved in imperme-
abilization of the seed coat. Thirdly, we describe plant
response to bug attack, focusing on profiles of the polyphe-
nolic compounds that may explain the observed differences
between attacked and intact seeds in both seed coat color
and permeability. Finally, we discuss the potential impact of
sublethal attacks on seed fitness.
Methods
SPECIES STUDIED
Sesbania drummondii is a perennial shrub that grows in
humid areas (lakeshores, riversides, rice fields, and similar
habitats) along the Gulf coastal plain, from Florida to Texas
and south to the state of Veracruz in Mexico. It is often con-
sidered a weed. It can reach 4 m in height and survive for
10 years. Seeds of S. drummondii are spherical, about
110 mg in mass (Marshall, 1982), and can stay dormant for
10 years or more in the soil, where they form a seed bank.
Seeds are protected by an indehiscent pod with the consis-
tency of tough paper, which contains 3 to 8 seeds (mean 6).
The pods possess a winged pericarp. They float and are dis-
persed by water, blown by wind like sailboats. Fruits and
seeds used in this study were collected in December 1995
on parent plants from natural populations in the coastal
plain of Texas. Dry fruits were stored in paper bags in dark-
ness at 10°C until their use in this study, conducted in
April-May 1998.
Three populations were studied: LBJ (for Lyndon
Baines Johnson) State Park (population 1: 33° 18’ N ,
100° 25’ W); Gerhardt ranch (population 2), near Vienna
(29° 45’ N, 96° 94’ W); and Lake Texana (population 3),
near Edna (28° 58’ N, 90° 38’ W). To analyze inter-individ-
ual variation in the intensity and effects of bug attack, we
29
 CEBALLOS ET AL.: ATTACK OF SESBANIA SEEDS BY HYALYMENUS BUGS
sampled 10 plants in each population. For each plant, 10
pods, each containing 6 seeds, were sampled (1,800 seeds in
total) to take into account within-individual variation. We
chose only pods containing 6 seeds in order to control for a
possible effect of seed number per pod in our observations
and experiments.
Hyalymenus tarsatus is a wide-ranging species and has
been recorded on Sesbania spp. and on undetermined
mimosoid legumes in Texas and Mexico (Schaefer, 1980)
but feeds principally in infructescences of Asteraceae in
Brazil (Oliveira, 1985). We observed H. tarsatus only on S.
drummondii in this study, but other local hosts are not
excluded. It does not, however, feed on S. vesicaria (Jacq.),
which occurs abundantly in the study area. Adults are rapid
fliers. Life history, duration of development of immature
stages, and movements of adults appear to be unknown.
Both adults and the ant-mimetic nymphs (Oliveira, 1985)
feed on developing seeds of S. drummondii. Seeds in mature
fruits, with lignified seed coats and pod walls, are not fed
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upon. We never observed H. tarsatus feeding on seeds or
pods on the ground beneath the plant. Field observations
show that bugs arrive in numbers at the time when young
pods first begin to appear in large numbers and that bug
density varies among individual host plants. However, noth-
ing is known about host orientation and the cues determin-
ing host recognition and host preference.
DEGREE OF ATTACK OF SEEDS
This analysis, conducted in April-July 1998, aimed at
characterizing the impact of parasitism of H. tarsatus on
easily defined seed morphological traits. Pods with six
seeds were carefully opened, and the position of each seed
within the pod was noted from 1 (closest to the peduncle) to
6 (farthest from the peduncle). We defined different classes
of attack as follows: 0 (aborted seeds), 1 (unattacked seeds),
2 (slightly attacked seeds; bearing usually 1-2 puncture
marks, visible as tiny brown spots), 3 (moderately attacked
seeds; usually with 3-5 punctures), 4 (heavily attacked
seeds; usually ≥ 6 punctures), and 5 (seeds completely
decomposed). For each seed, we noted the position, class of
attack, color of the seed coat (cream or yellow-green,
brown, red) and the shape (normal or misshapen). Seeds
were weighed individually to the nearest mg.
Variation in the intensity of seed attack was analyzed
using a type III ANOVA with multinomial error (GEN-
MOD procedure of SAS [SAS Institute, 1999]). The full
model included as explanatory variables the effects of
source population, of individual (nested within population),
of pod (nested within individual), and of position of seeds
(1 to 6). Backwards selection was used to identify the fac-
tors with the most significant effects in the full model.
The same type of ANOVA was performed on the color
and shape of seeds as a function of intensity of attack. This
analysis was restricted to population 2, the only population
in which seeds of all attack classes were present in suffi-
cient number to carry out the analysis. The full model
included the effects of individual, of pod (nested within
individual), and of seed position, as well as the intensity of
attack of each seed. As in the previous ANOVA, backwards
selection was used to identify the factors with the most sig-
nificant effects in the full model.
30
Data on seed mass were analyzed using type III
ANOVA (GLM procedure of SAS [SAS Institute, 1999]) in
a backwards selection model using the same explanatory
variables as in the analyses of seed color and shape.
Comparisons of mean seed mass among populations and
among different degrees of attack were performed using a
Tukey-Kramer test (P < 0.05).
PERMEABILITY OF SEEDS FOLLOWING ATTACK
Three lots of 100 seeds from population 2 (10 replicates
of 10 seeds for each damage class) were set to imbibe at
room temperature (20°C) in petri dishes (100% humidity):
unattacked seeds (class 1), attacked seeds (classes 2 and 3),
and heavily attacked seeds (class 4). After 48 hours, the
number of imbibing seeds, indicated by protrusion of the
radicle, was counted. Dormancy in seeds of S. drummondii
is physical, and once the radicle emerges, the seed is com-
mitted to germination. The effect of attack level on perme-
ability of seeds was analyzed using a type III ANOVA with
multinomial error (GENMOD procedure of SAS [SAS
Institute, 1999]) and comparisons of mean number of seeds
germinated were performed using the Tukey-Kramer test (P
< 0.05).
RELEASE OF ALLELOCHEMICALS DURING IMBIBITION OF SEEDS
For this experiment, from each of six different plants
we chose a lot of 120 intact seeds and another lot of 120
attacked seeds, giving a total of 12 lots. For attacked seeds,
moderately attacked seeds (classes 2 and 3) were used
because seeds from classes 4 and 5 were heavily infested by
fungi. Fungal infection could have had confounding effects
on exudation of allelochemicals.
Each of the 12 lots was arbitrarily divided into four 30-
seed subsamples, and each of these subsamples was further
divided into three replicates of 10 seeds each. Seeds were
mechanically scarified with a razor blade next to the lens,
the part where papilionoid seeds usually become permeable
(Baskin & Baskin, 1998). The exact mass of each seed and
the total mass of each 10-seed replicate were noted.
Scarified seeds were then soaked in water (10 seeds per 100
ml distilled water). To examine exudation over the time
course of imbibition and to avoid any effect of removal of
exudate on the exudation process, exudate from each of the
four arbitrary subsamples was collected only once, on day
1, 2, 3, or 4 of imbibition. Extracts from each replicate were
freeze-dried and weighed separately (results expressed in
mg exudates g dormant seeds-1).
The rate of exudation was analyzed using a type III
ANOVA (GLM procedure of SAS [SAS Institute, 1999]) in
a backwards selection model with the time (day of imbibi-
tion), the degree of attack, and the individual plant as
explanatory variables. Comparisons of the dry mass of the
exudates released by seed at different time of imbibition
were performed using a Tukey-Kramer test (P < 0.05).
ANALYSIS OF IMBIBITION EXTRACTS
Imbibition extracts were dissolved in 70% acetone to
the final concentration of 1% (Weight/Volume), then cen-
trifuged at 10,000 rpm. Extracts were analyzed using a
spectrophotometer (PYE/UNICAM/Vis®, Angleton, Texas)
to quantify contents of both total condensed tannin and

proanthocyanidins. Condensed tannins, as measured by our
analyses, are constituted of both monomers (catechins) and
polymers. Proanthocyanidins correspond to the polymerised
portion of the condensed tannins. These oligopolymers
account for much of the activity of condensed tannins. It is
useful, however, to take into account all condensed tannins,
including monomers, because the latter are sometimes pre-
dominant and have particular biological effects.
QUANTIFICATION OF TOTAL CONDENSED TANNINS (CT)
A 0.1-ml aliquot of acetonic extract was mixed with
0.5 ml of a solution of 0.1% DMCA (dimethylaminocin-
namaldehyde) in a methanol and 12N HCl (3:1) mixture.
DMCA reacts specifically with catechin monomers and oli-
gopolymers of catechins to yield a complex, blue-green in
colour, whose maximum absorbance is measured at 640 nm,
after 10 minutes of contact (Treutter, Freucht & Santos-
Buelga et al., 1994). The control was an acetone blank.
Concentrations were expressed as mg of catechin equiva-
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lents per g dormant seeds.
QUANTIFICATION OF PROANTHOCYANIDINS (PA)
A 0.15-ml aliquot of acetonic extract was mixed with
0.9 ml of butanol-HCl (90:10) with 50 mg of Fe3+ added as
a catalyst, in sealed tubes. After being shaken vigorously
(Vortex), tubes were heated for 50 seconds in a microwave
oven (power output 900W). After 10 minutes of cooling,
PAs (in acid solution and in the presence of Fe as a catalyst)
hydrolyze to give molecules of anthocyanins, absorbance of
which is measured at 545 nm and compared to an acetone
blank (Treutter, Freucht & Santos-Buelga et al., 1994).
Concentrations were measured as mg cyanidin equivalents
per g dormant seeds.
POLYMERIZATION DEGREE (PD)
Solubility in water and biological activity of exuded
polyphenols depend partly on their molecular weight, i.e.,
their degree of polymerization. PD was obtained by divid-
ing PA concentrations (BuOH/HCl assay) by CT
concentrations (DMCA assay). The higher the PD, the
larger the size of PAs in the extract. For each sample (N = 6
replicates sample-1), concentrations of CT and PA, and
polymerization degree, were compared and differences
within samples were tested for statistical significance, using
a two-factor (degree of attack, individual from which seeds
were collected) ANOVA (GLIM, 1985) and LSD tests
(P < 0.05, STATISTIX, 1994). Data on polymerization
degree were analyzed after normalizing them by arcsin
square root transformation.
Results
Usually, seeds were attacked by H. tarsatus during
intermediate stages of seed development, i.e., when seed
coats were not yet lignified but seeds were large enough to
contain substantial amounts of nutritive reserves. The
immediate response of individual seeds to attack involved
secretion of a whitish latex that formed a plug. In the fol-
lowing minutes, this plug turned tan and hardened, sealing
the opening. The greater the intensity of attack on develop-
ing seeds, the more pronounced the alteration of their mor-
phology was. Seeds attacked were small, misshapen, and
ÉCOSCIENCE, VOL. 9 (1), 2002
fragile. Nevertheless, many of them gave positive reac-
tions in tetrazolium tests for viability (Ceballos, unpubl.
data), and a proportion of such seeds are capable of giving
healthy seedlings. When a developing seed was submitted
to numerous and/or prolonged attacks, its seed coat turned
red (possibly due to production of quinones) and lignified
precociously, at a stage when seed filling was not yet com-
plete. Parasitism thus affected at least some biochemical
processes in developing seeds, those responsible for pig-
mentation and hardening of the seed coat.
DEGREE OF ATTACK OF SEEDS
Intensity of seed attack by H. tarsatus varied signifi-
cantly among populations studied (Genmod, ANOVA:
c22 = 1,182.16, P < 10-4; Table I). Population 1 had very lit-
tle attack, while population 2 had a very high level, and
population 3 a moderate level of attack (Figure 1). Also,
within each population, there was great variation in intensi-
ty of attack among individuals (c218 = 258.71, P < 10-4;
Table I) and among pods within individual plants
(c290 = 427.40, P < 10-4; Table I). Position of seeds within
the pod showed no variation in attack. None of the interac-
tions between factors tested were significant.
The ANOVA performed on data for population 2
showed that intensity of attack significantly influenced the
color (c24 = 726.30, P < 10-4) and shape (c24 = 383.49,
P < 10-4). Unattacked seeds (class 1) were creamy-white or
yellow-green in color and never misshapen; slightly
attacked seeds (class 2) were mostly (about 95%) brown in
color and 7% were misshapen; moderately attacked seeds
(class 3) were brown (66%) or red (34%), with 39% mis-
shapen; heavily attacked seeds (class 4) were almost all red
(97%) and misshapen (84%); seeds that had been heavily
attacked and obviously fungus-infected (class 5) were with-
out exception red in color, and almost all (97%) were mis-
shapen.
EFFECT OF ATTACK BY H. TARSATUS ON SEED MASS
The effect of attack on seed mass was highly signifi-
cant (F4,1670 = 32.58, P < 10-3; Table II), with mean mass
of seeds decreasing as intensity of attack increased. Mean
mass of unattacked seeds (class 1) was 104.7 ± 28.3 mg.
Mean mass of seeds of attack class 2 (102.7 ± 52.0 mg) was
not significantly different from that of class 1, but was more
variable. Mean mass progressively decreased in seeds of
classes 3 (80.7 ± 23.1 mg) and 4 (37.9 ± 23.1 mg). Finally,
mean mass of heavily attacked and fungus-infected seeds
(class 5) was 22.2 ± 16.1 mg. The effects of population and
of individual (within population) were also significant, par-
tially due to significant interactions of these variables with
the degree of attack (Table II). Mass of seeds from popula-
tion 2 (mean = 47.0 ± 47.6 mg) was much lower than in the
TABLE I. Results of the ANOVA of degree of attack of seeds of
S. drummondii by H. tarsatus (multinomial error, GENMOD
procedure of SAS [SAS Institute, 1999]).
Source df c2 P
Population 2 1,182.96 < 10-4
Individual (population) 18 258.71 < 10-4
Pod (individual) 90 427.40 < 10-4
31
 CEBALLOS ET AL.: ATTACK OF SESBANIA SEEDS BY HYALYMENUS BUGS

Population 1 Population 2 Population 3

5 5 5

4 4 4
Class of attack

3 3 3

2 2 2

1 1 1

0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100

Percentage of seeds
FIGURE 1. Frequency of different classes of attack of seeds by Hyalymenus tarsatus in the three studied populations of Sesbania drummondii. Class
1 = unattacked; 2 = slightly attacked; 3 = moderately attacked; 4 = heavily attacked; 5 = seeds heavily attacked and obviously fungus-invested.
Downloaded by [Universite Laval] at 02:59 06 August 2017

other populations (population 1: mean = 102.4 ± 17.0 mg; T ABLE II. Results of the ANOVA (Type III) of seed mass of
population 3: mean = 103.9 ± 42.8 mg). This difference was Sesbania drummondii (GLM procedure of SAS [SAS Institute,
due to the fact that level of attack was higher in population 1999]).
2. When only unattacked seeds (class 1) were compared
among these three populations, mass was not significantly Effect F P
different (F2,713 = 1.41, P = 0.245). Attack F4, 1670 = 32.58 < 10-4
Population F2, 1670 = 15.50 < 10-4
Individual (population) F27, 1670 = 1.66 0.018
PERMEABILITY OF SEEDS FOLLOWING ATTACK Population•attack F8, 1670 = 2.29 0.020
The effect of parasitism on permeability of seeds was Individual•attack F72, 1670 = 1.55 0.003
highly significant (c22 = 65.92, P < 10-4). None of the intact
seeds imbibed water during the 48 hours of the test, whereas
an average of 51.0 ± 7.4% of slightly attacked seeds (class 2 TABLE III. Effect of attack by Hyalymenus tarsatus on permeabili-
and 3) and 94.0 ± 8.4% of heavily attacked seeds (class 4) ty of seeds. Values given are mean (± SD) percentages of seeds of
three attack classes (see text for definition of classes) that imbibed
imbibed spontaneously, triggering their germination. Table
and germinated when placed in water.
III shows that differences were significant in each pairwise
comparison of attack classes. Seeds Mean (%) Tukey-Kramer test*
Unattacked 0 a
ANALYSIS OF IMBIBITION EXTRACTS Moderately attacked 51.00 (± 7.38) b
DRY MASS OF EXUDATES Attacked 94.00 (± 8.43) c
Mass of exuded matter did not differ significantly *Different letters in the rightmost column indicate significant differences
(comparison of means, Tukey-Kramer test, P < 0.05).
among seeds from the six maternal parents (F5,44 = 0.54,
P = 0.75) or between intact and attacked seeds
(F1,44 = 2.61, P = 0.11). Thus, attack caused no quantitative singly, only the difference on day 3 was significant. Seeds
change in the mass of exudates. In contrast, there was a produced by different maternal parents did not differ in con-
strong time effect on exudation of seeds (F3,47 = 12.81, centrations of CT. None of the interaction terms (between
P < 10-5) (Figure 2). The difference between successive his- individual, time and degree of attack) were significant.
tograms indicates the mean additional mass exuded during As for condensed tannins, the effect of time on exuda-
each day. Comparison of means (LSD test, P < 0.05) tion of proanthocyanidins (PA) was highly significant
showed significant differences in mass of matter exuded (Figure 3b; F3,15 = 60.21; P < 10-5, Table IV). PA concen-
between the different days. Rate of exudation was about tration continued to increase in the exudates over the 4
equal on days 1 and 2, but a smaller mass of material was days, but only the difference between days 1 and 4 (the
exuded between days 2 and 3, and an even smaller amount beginning and end of the experiment) was significant. In
between days 3 and 4. contrast to the results for CT, seeds produced by different
individual plants differed significantly (F 5,15 = 5.34;
QUANTIFICATION OF TANNINS P = 0.01) in proanthocyanidin (PA) concentrations, con-
Condensed tannin concentrations in the exudates varied tributing to the much greater standard deviations for PA
significantly over time (F3,15 = 19.74, P = 2. 10-5), increas- (Figure 3b) than for CT (Figure 3a). While degree of attack
ing from day 1 to day 3 of imbibition, then decreasing on did not significantly affect concentrations of proanthocyani-
day 4 (Figure 3a). Overall, attacked seeds exuded signifi- dins exuded, two interaction terms (time•individual and
cantly more tannins than did unattacked seeds (F1,15 = 5.2, attack•individual) were marginally significant (Table IV),
P = 0.04). This difference was consistently observed over showing that individual plants varied in the time course of
the entire time course of imbibition, but for each day taken PA exudation and in their response to attack.
32
 ÉCOSCIENCE, VOL. 9 (1), 2002

40 a) Total condensed tannins Unattacked seeds

bc c
Attacked seeds
Dry mass of exudates (mg)

0.1 b
30 b
a 0.09

Mean concentration
0.08 a
0.07 ab ab ab
20
0.06
a a
0.05
10 0.04 a
0.03
0.02
0 0.01
1 2 3 4 0
Time (days)

F IGURE 2. Cumulative dry mass of exudates released by seeds of b) Proanthocyanidins

Sesbania drummondii after 1, 2, 3, and 4 days of imbibition. Means and
SD of 12 lots of 120 seeds each. Different letters above histograms indi- 1.8
cate significant differences (comparison of means, Tukey-Kramer tests, b
1.6 b
Downloaded by [Universite Laval] at 02:59 06 August 2017

P < 0.05).

Mean concentration
1.4 ab
ab ab
TABLE IV. Results of the ANOVA of concentration of proantho- 1.2 ab
cyanidins (PA) in seed exudates (GLM procedure of SAS [SAS 1
Institute, 1999]).
0.8
Effect F P 0.6
Time F3, 15 = 60.21 < 10-5 0.4 a a
Individual F5, 15 = 5.34 < 0.01
Attack F1, 15 = 0.078 ns 0.2
Individual•attack F5, 15 = 2.95 0.047 0
Individual•time F15, 15 = 2.58 0.038
c) Polymerization degree
45 c
Polymerization degree (PD) is the ratio PA/CT, and c
Degree of polymerization

40
patterns for this variable thus reflected those of its two com- 35
ponents (Figure 3c). There were significant differences
30
among individuals (F 5,15 = 3.7, P = 0.02) and the time b
25
effect was very strong (F3,15 = 70.99, P < 10-5). PD changed b
b
20
little during the first three days, but increased abruptly on ab
the fourth day (Figure 3c). Tannins were apparently subject- 15 a a
ed to marked polymerization on the fourth day of imbibi- 10
tion. Effect of attack on PD of exudates was not significant, 5
and none of the interaction terms were significant. 0
1 2 3 4
Time (days)
Discussion
FIGURE 3. Total concentrations of phenolic compounds exuded by
A large proportion of developing seeds of S. drum- attacked and unattacked imbibing seeds of Sesbania drummondii after 1,
mondii that were fed on by adults and nymphs of H. 2, 3, and 4 days of imbibition: (a) total condensed tannins (CT), concen-
tarsatus survived these attacks. However, feeding by this tration expressed as catechin equivalents, in mg g of seeds-1; (b) proantho-
cyanidins (PA), concentration expressed as cyanidin equivalents, in
bug affected several fitness-relevant traits of seeds. Bug mg g of seeds-1; (c) degree of polymerization (PD). For each day, means
attack was associated with reduced mass of mature seeds, and standard deviations for 6 individuals are given (30 seeds per individual
and the strength of the effect depended on the intensity of per day for each of the two attack classes). Different letters above his-
attack. Severe bug attack on developing seeds also led to tograms indicate significant differences (comparison of means, LSD
loss of seed dormancy. Finally, seed attack was associated tests, P < 0.05).
with changes in the metabolism of polyphenolic compounds
of seed coats, resulting in changes in seed coat color and in of polyphenolics suggest that the plant may have a limited
increased concentration of condensed tannins exuded by capacity for adaptive response to bug attack.
seeds during imbibition. Reduced seed mass is likely to
have strong negative effects on fitness. While loss of dor- EFFECTS OF ATTACK ON SEED MASS
mancy could conceivably benefit some seeds, its conse- Parasitism of developing seeds of S. drummondii by H.
quences overall are also likely to be strongly negative. The tarsatus resulted in substantial reduction in the amount of
effect of sublethal attack on seeds by this insect can thus be stored reserves, which should reduce the probability of
described as parasitism. The observed effects on metabolism establishment of seedlings upon emergence. The mecha-
33
 CEBALLOS ET AL.: ATTACK OF SESBANIA SEEDS BY HYALYMENUS BUGS
nisms behind this effect may be complex. In addition to
removing reserves already stored in the seed, seed-feeding
bugs may disrupt development and reduce further storage of
reserves (Bates et al., 2001). Parasitism by H. tarsatus also
results in misshapen seeds. Attack appears to lead to preco-
cious lignification of the seed coat, which may physically
limit the volume available for stored reserves. Whatever the
mechanisms behind it, reduced seed mass leads to decreased
fitness under competition (Winn, 1988). Reduced seed mass
is also likely to have a strong negative effect when a fluctu-
ating and unpredictable abiotic environment leaves only a
narrow window of time for establishment. Seedlings of S.
drummondii establish in habitats characterized by strongly
fluctuating water level and must grow quickly to a size
that enables them to survive flooding. This rapid initial
growth is supported by protein and energy reserves stored
within the seeds (and by the photosynthetic cotyledons of
seedlings). Substantial losses of seed reserves could thus
greatly reduce seedling growth and survival.
Downloaded by [Universite Laval] at 02:59 06 August 2017
EFFECTS OF ATTACK ON SEED DORMANCY
Parasitism of seeds of S. drummondii by H. tarsatus
also had a striking effect on seed coat permeability. In
legumes, dormancy results from development of imperme-
ability of the seed coat. Seeds whose coats were pierced by
this bug became permeable to water. The extent of this
effect, in terms of rupture of dormancy, depended on inten-
sity of attack. Even at moderate intensity of parasitism,
however, physical dormancy was disrupted, resulting in
spontaneous imbibition by 51% of seeds. In the most heavi-
ly attacked classes, this proportion exceeded 90%, meaning
a near absence of dormancy for these seeds. Our findings
are similar to those of Karban and Lowenberg (1992), who
found that attack by lygaeid and scutellerid seed bugs
removed the physical dormancy of seeds of Gossypium spp.
(Malvaceae). The ecological and evolutionary consequences
of such enhancement of germination are difficult to pre-
dict and may not necessarily be negative (Karban and
Lowenberg, 1992). More rapid germination may sometimes
confer an advantage. However, some effects are likely to be
decidedly negative. Attacks on the integrity of the seed
coat may facilitate pathogen infection or initiate biochemi-
cal changes that reduce seed fitness. Impermeability of
dormant seeds to oxygen protects lipid and protein reserves
from oxidative degradation (peroxidation, conformational
changes of proteins, etc.). Oxygen entering pierced seed
coats leads to deterioration, characterized by reduction in
levels of proteins, lipids, ATP pools, RNA, and polysaccha-
rides (Anderson & Baker, 1983). As a consequence, the
metabolism of seedlings is seriously disrupted.
Loss of seed dormancy is also likely to reduce fitness in
this plant. Conditions for seedling establishment vary great-
ly among seasons and years as a consequence of unpre-
dictable fluctuations of water level in the temporarily flood-
ed habitats where this plant grows. Dormancy allows seeds
to survive the large proportion (in most sites) of years that
are either too dry or too wet for seedling establishment.
EFFECTS OF ATTACK ON METABOLISM AND BIOCHEMISTRY OF
SEEDS
While our study did not address effects of bug attack on
biochemical processes affecting seed contents, it showed
34
that attack affected metabolic processes in the developing
seed coat. Seed coat color at maturity was conspicuously
affected. The higher the intensity of attack, the redder the
seed coat was. This pattern suggests that attack on develop-
ing seeds led to oxidation of phenols to quinones. Werker,
Marbach and Mayer (1979) reported the presence of quinones
in the palisade layer of mature legume seeds and suggest-
ed that reticulation of tannins to polysaccharides of
macrosclereid cell walls involved the oxidation of phenols
to quinones. The response of developing seeds to intense
bug attacks suggests that attack may trigger the early
expression of a normal developmental program that renders
seeds impermeable. Such an effect could be interpreted in
two ways. It might simply reflect damage-caused disruption
of development. Alternatively, it could be an adaptive repair
response to restore impermeability. If the second alternative
holds, then the mechanism appears to be of limited effec-
tiveness when levels of attack are high.
Parasitism of seeds by H. tarsatus influenced biochemi-
cal profiles of seed exudates, being associated with an
increase in the amount of condensed tannins exuded during
imbibition, an increase that became significant the third day
of imbibition (Figure 3). Seed coat condensed tannins are
known to be involved not only in defense (Graham, 1991;
Kulik & Yaklich, 1991; Helsper et al., 1994), but also in
processes of impermeabilization (Marbach & Mayer, 1975;
Egley et al., 1985). They also affect seed coat color (Powell,
Oliveira & Matthews, 1986; Cabrera & Martin, 1989), and
may have contributed to the color effect noted above.
Our data support the hypothesis that parasitism of
developing seeds results in the induction of increased pro-
duction of condensed tannins in the seed coat. Because
seed-coat tannins are regarded as constitutive components
of these organs, induction of their synthesis in response to
environmental stresses or signals has been little examined.
Our data suggest that it would be interesting to examine this
question in other plant species.
Proanthocyanidin concentration in the exudates, in con-
trast, was not significantly correlated with level of seed par-
asitism, although there was a non-significant trend in this
direction. Polymerization of catechins began on the third
day and experiments ran only four days; a longer duration
of imbibition might have revealed an effect. PA concentra-
tion appeared to respond differently to attack in different
individuals, with two of the six individuals tested containing
lower concentrations of PA in attacked lots.
Finally, parasitism did not affect PD, which reflects the
size of oligopolymeric PAs. PD (and thus the size of tan-
nins) increased over the time course of imbibition (Figure 3c).
Mirroring variation in one of its components (PA), PD was
variable among individuals. Exudation of tannins by imbib-
ing seeds is a dynamic process, with continual release and
oxidative condensation of tannins over the course of
imbibition (L. Ceballos, unpubl. data). This polymerization
of exuded tannins should enhance their overall biological
activity against pathogens. Antifungal activity of S. drum-
mondii exudates remains significant over 9 days (Ceballos
et al., 1998). The fact that polymerization during imbibition
was not affected by parasitism of seeds suggests that the
mechanisms behind it were neither negatively affected by
damage to developing seeds nor enhanced by induction.

Conclusion
In contrast to seed predation, the impact that seed-
attacking insects have on fitness when they do not kill seeds
outright has been relatively little studied. Compared to the
effects of chewing insects, the effects of sucking insects on
plants are in general difficult to quantify, and the effects of
piercing-sucking insects such as H. tarsatus on seeds that
survive attack will be more difficult to quantify than those
of chewing insects such as bruchids and weevils (Janzen,
1976). For unknown reasons, intensity of bug attack var-
ied greatly among the three populations of S. drummondii
we studied and among individuals within populations.
Observations also suggested variation in attack among fruits
of the same individual plant. This variation is likely to
affect the relative importance of seed predation and seed
parasitism as outcomes of the interaction, with parasitism as
the predominant effect under lower attack intensities. The
effects of seed parasitism are likely to be cryptic unless
studies are specifically designed to incorporate effects of
Downloaded by [Universite Laval] at 02:59 06 August 2017
seed mass and dormancy on seedling survival.
Acknowledgements
We thank J., H., and L. McKey for help in collecting pods, J.
and H. McKey and P. and F. Gerhard for allowing access to S.
drummondii populations on their property, A. Caizergues for
help in statistical analysis, J. Schaffner (Department of
Entomology, Texas A&M University, emeritus) for identifying
H. tarsatus; and the French Government (Ministère de l’Éducation
Nationale, de la Recherche, et de la Technologie) for a doctoral
grant to L. Ceballos.
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