context dependency of nectar reward-guided oviposition
TRANSCRIPT
14TH INTERNAT IONAL SYMPOS IUM ON INSECT-PLANT INTERACT IONS
Context dependency of nectar reward-guided ovipositionDanny Kessler*Department ofMolecular Ecology, Max Planck Institute for Chemical Ecology Hans-Knoll-Straße 8, 07745, Jena, Germany
Accepted: 30March 2012
Key words: Manduca quinquemaculata, Datura wrightii, Nicotiana attenuata, sugar concentration,
flower, herbivory, pollination, nectar volume, Solanaceae, Sphingidae, Lepidoptera
Abstract Nectar is the most common floral reward used to recruit pollination services. Changes in nectar vol-
umemay affect not only pollination services, but also the attraction of antagonists such as herbivores,
especially if the same insect species acts as herbivore and pollinator. Plants compete with each other
for the best pollination services, at the same time employing various strategies to avoid herbivory.
Datura wrightii Regel and Nicotiana attenuata Torr. ex Watson, two sympatric solanaceous species,
compete for the same hawkmoth pollinator, Manduca quinquemaculata (Haworth) (Lepidoptera:
Sphingidae), although standing nectar volume per flower differs 50-fold (70 vs. 1.3 ll, respectively)between these species. This large difference may also result in differences in oviposition rates. I con-
ducted a detailed analysis of diurnal changes in nectar volume and sugar concentration in field- and
glasshouse-grown D. wrightii and N. attenuata plants, and tested how well nectar production is buf-
fered against the loss of large amounts of foliar tissue that frequently occurs due toM. quinquemacu-
lata larval herbivory. I examined the influence of nectar volume on herbivore damage in the field and
compared the results with previously published data fromN. attenuatawhich were collected simulta-
neously. Oviposition by M. quinquemaculata moths increased significantly in D. wrightii plants
whose nectar volume had been experimentally increased five-fold compared to untreated control
plants, and correlated with the numbers of flowers per plant in native populations. The results sug-
gest that a hawkmothmother may use standing nectar volume of a potential host plant to estimate its
size, and possibly health, to make the optimal decision for her progeny. This mode of assessment,
however, is apparently not used with another plant species, as other more herbivory-related cues,
such as olfaction or vision, are more influential in determining oviposition rates on other plant spe-
cies. Yet within a plant species, regulating nectar volume strongly influences future herbivory.
Introduction
Animal-pollinated plants must attract pollen-transferring
floral visitors to ensure outcrossing. They do so by using a
broad spectrum of floral shapes, colors, volatiles, and other
characteristics, and by producing a variety of rewards as
payments for pollination services. Floral nectar is the most
common reward (Simpson & Neff, 1983): with its main
constituents, sugars and amino acids, it is a primary food
source for nectar-seeking pollinators (Luttge, 1977). Plants
compete with each other for the best pollination services
by advertising themselves via flowers, which makes them
visible not only for mutualistic pollinators, but also for
antagonists such as herbivores, or nectar robbers whomay
use the same cues as pollinators to find host plants (Galen
& Cuba, 2001; Andrews et al., 2007). In solving the prob-
lem of attracting pollinators for pollen transfer while try-
ing to remain inconspicuous to herbivores, plants have
evolved sophisticated strategies to achieve their goal of
maximized fitness in a complex interactive environment
(Schiestl, 2010).
Recent research focusing on this trade-off between
attracting pollinators and repelling antagonists has
revealed that floral scents, as well as nectar chemical com-
position, are major factors affecting flower visitation by
friends and foes. Toxins or antimicrobial compounds in
nectar, for example, can reduce the volume of nectar
ingested per visit even by adapted pollinators (Kessler &
Baldwin, 2007), while deterring nectar thieves and flori-
vores (Stephenson, 1981; Kessler et al., 2008). At the same
*Correspondence: Danny Kessler, Department ofMolecular Ecology,
Max Planck Institute for Chemical Ecology, Hans-Knoll-Straße 8,
07745 Jena, Germany. E-mail: [email protected]
© 2012 The Author Entomologia Experimentalis et Applicata 144: 112–122, 2012
112 Entomologia Experimentalis et Applicata© 2012 The Netherlands Entomological Society
DOI: 10.1111/j.1570-7458.2012.01270.x
time, repellent or toxic chemicals in nectar may increase
visitation frequency by decreasing the reward per visitation
(Kessler et al., 2008), which can benefit the plant by
increasing the likelihood of successful outcrossing (Kessler
et al. in press). Not only nectar quality, but also nectar
quantity can influence a pollinator’s decision to visit a
flower (Gass & Sutherland, 1985; Pyke et al., 1988; Thom-
son, 1988), and volume and composition of nectar may
therefore affect both pollen removal from anthers and pol-
len deposition on stigmas (Mitchell, 1993; Cresswell, 1999;
Irwin & Adler, 2008; Kessler et al., 2008).
The sacred Datura, Datura wrightii Regel, and the wild
tobacco, Nicotiana attenuata Torr. ex Watson (both Sola-
naceae), are sympatric species which share pollinators as
well as herbivores. Both plants bloom at night, and flower
morphology and floral scent are well suited for hawkmoth
pollination (Adler & Bronstein, 2004; Kessler et al., 2008).
Hawkmoths are known to pollinate brightly colored, large,
tubular night-blooming flowers with powerful fragrances
(Grant, 1983; Nilsson et al., 1987; Willmott & Burquez,
1996). Although both plant species compete for the same
group of pollinators they vary greatly in flower size and
floral nectar accumulation.
As for many other plant species, the same insect species
that function as pollinators forN. attenuata andD. wrightii
may also be devastating herbivores. The adults ofManduca
sexta (L.) andManducaquinquemaculata (Haworth) (Lepi-
doptera: Sphingidae) are important pollinators (unpubl.
data), whereas the larvae are the plants’ most damaging
herbivores. Under such circumstances, a plant faces the
problem of maximizing outcrossing services from a polli-
nator species, while preventing its oviposition. More spe-
cifically, how does a ‘pollinating herbivore’ choose when
being simultaneously exposed to host plants with different
nectar rewards as in D. wrightii and N. attenuata? Could
standing nectar volume of a plant alone determine the fre-
quency of oviposition? Hawkmoths need to fuel the energy
demands of their metabolically expensive hovering (Casey,
1976; Raguso et al., 2003) and long-distance flights (Pow-
ell & Brown, 1990) by imbibing large quantities of nectar.
This high energy requirement could tempt moths to
remain in the vicinity of plants producing higher nectar
volumes than others, which possibly also increases the
chance of oviposition. Adler & Bronstein (2004) showed
in an elegant glasshouse study that increased nectar vol-
ume can increase the oviposition by M. sexta on Datura
stramonium L. The same effect was found if sugar water
was artificially added to flowers of N. attenuata, which led
to increased oviposition by M. quinquemaculata in the
field (Kessler et al., 2010). These data suggest that nectar
volume plays an important role in a moth’s decision to
oviposit. Female moths may gain important information
from the standing nectar volume about a plant’s size or
even the presence of other herbivores, including conspeci-
fics. Within one species, smaller plants (Brys et al., 2011)
as well as plants facing herbivory (Bronstein et al., 2007)
are known to produce fewer flowers per plant, which also
presumably decreases the total amount of nectar available
per plant.
Little is known about the phenology of nectar accumu-
lation or the factors that control it. Flowers tend to be
more developmentally determinant than leaves (Brad-
shaw, 1965) in their development, showing plasticity only
in response to pollination and herbivory (Strauss et al.,
1996; Kessler & Halitschke, 2009). Hence, one might
expect that nectar volume is tightly regulated. Nectar vol-
ume, however, has been reported to vary in response to
environmental factors, such as water or light availability
(Boose, 1997), elevated CO2 levels (Lake & Hughes, 1999),
or evaporation potential (Corbet & Delfosse, 1984),
which are factors that are inherently variable and
impact the entire plant population. Less is known about
the timing of nectar secretion or the influence of can-
opy area on nectar secretion. Loss of a substantial part
of a plant’s photosynthetic area, as is frequently caused
by herbivore damage (McFadden, 1968), could have a
large impact on nectar volume in its flowers and hence
on pollination efficiency or secondary herbivory. Foliar
herbivore damage frequently decreases flower number,
floral display, and therefore a plant’s total floral volatile
emission, all of which may lead to reduced visitation
rates by pollinators, resulting in less outcrossing and
lower seed set (Bronstein et al., 2007) and possibly also
to reduced oviposition.
Here, I measured the diurnal patterns of nectar produc-
tion in glasshouse- and field-grown D. wrightii and N. at-
tenuata plants – species which differ 50-fold in nectar
accumulation per flower – and investigated the influence
of loss of photosynthetic tissue on standing nectar volume
and nectar sugar concentration by removing rosette, stem,
or all leaves of a plant. Finally, I examined the conse-
quences of experimentally increasing nectar volume on
herbivory in natural populations of D. wrightii, and com-
pared oviposition rates in D. wrightii with those found in
N. attenuata.
Materials and methods
Plant material
Datura wrightii plants used in glasshouse experiments
were germinated from seeds bought from B & T World
Seeds (http://b-and-t-world-seeds.com) and further
inbred for two generations. Seeds were incubated in 0.1 M
gibberellic acid (www.carl-roth.de) for 6 h and transferred
High nectar volumes increase oviposition 113
directly into soil for germination. Two-week-old seedlings
were transferred into 2-l pots.
Nicotiana attenuata grown in 1-l pots, from field-
collected seeds (Baldwin, 1998) and 17 generations of
inbreeding were used for all glasshouse experiments. Seeds
of N. attenuata were sterilized and incubated in 0.1 M gib-
berellic acid and 1:50 diluted liquid smoke (vol/vol; House
of Herbs, Passaic, NJ, USA) for 1 h before being germi-
nated on Gamborg’s B5medium (Duchefa, St. Louis, MO,
USA) as described previously (Kruegel et al., 2002).
Both D. wrightii and N. attenuata are entirely self-com-
patible, and thus inbreed if other pollen sources are
excluded. Inbreed lines were obtained by isolating single
plants in the glasshouse and collecting viable seeds.
For glasshouse experiments seeds of N. attenuata were
germinated on Gamborg’s B5 under a day/night cycle of
16/8 h, day light intensity 155 lm s�1 m�2 at tempera-
tures of 26 °C (day) and 24 °C (night) (Percival; http://
www.percival-scientific.com/). Plants of both species were
grown under a day/night cycle of 16/8 h at 26–28 °C(day)/22–24 °C (night), during daytime under supple-
mental light from Master Son-T PIA Agro 400 lights
(400 W; 200–230 lm s�1 m�2; Philips; http://www.
unielektro.de/).
NativeD. wrightii plants used for field experiments grew
close to the Lytle ranch road, Santa Clara, UT, USA (37°07′49.02′N, 114°00′53.91′W) in a population of about 130
plants.
Measuring nectar volume and sugar concentration
Corollas ofN. attenuata remain open for 2–3 days in both
the glasshouse and the field. To be able to distinguish
flower stages, all flowers were removed with a razor blade
1 day before experiments and newly opened flowers
were labeled. Datura wrightii flowers remain open for
only 1 day. For field-collected samples, inflorescences
(N. attenuata) or flowers (D. wrightii) were covered over-
night with mesh bags (80 9 20 9 24 cm Breather plant
bags; www.kleentest.com) to exclude pollinators and allow
for evapotranspiration., or were covered with translucent
plastic bags (Plastibrand, www.merckeurolab.be/app/cata-
log/Product?article_number = 129–0131) to reduce
evapotranspiration. In the glasshouse, inflorescences were
covered with translucent plastic bags, similar to those used
in the field, to reduce evapotranspiration or remained
uncovered. Nectar was collected between 04:00 and
07:00 hours by inserting a clean, calibrated 25 ll glass cap-illary into the corolla tube until it reached the base of the
nectaries. The sugar concentration was measured using
a portable refractometer (Optech; http://www.reichert-
labtec.de/p6.html) with a range of 0–32% and a resolution
of 0.2% (Kessler & Baldwin, 2007). To obtain data on the
diurnal pattern of nectar secretion, nectar of one flower of
each of 20 plants was collected every hour in N. attenuata,
or one flower of three plants in D. wrightii. In D. wrightii,
measurements had to be conducted over several days, as
flower number wasmuch lower than inN. attenuata.
To investigate the role of photosynthetic tissue in
maintaining nectar volume and sugar concentration, I
examined changes in nectar production and sugar concen-
tration after removing either all leaves, all stem leaves, all
rosette leaves, or no leaves with a razorblade from both
plant species. Leaves ofN. attenuata can be clearly divided
into rosette and stem leaves, whereas D. wrightii is a bushy
plant with only stem leaves. However, for simplification I
use the same nomenclature inD. wrightii as inN. attenuata
and call the upper half of leaves stem leaves, and the lower
half, rosette leaves (Figure 2A and B). All re-growing leaves
were cut every 2nd day. For N. attenuata (Figure 2C and
D), two flowers of 20 replicate plants in each defoliation
treatment-group were measured 1 week after defoliation
began andmeans of all flowers from single plants were used
for statistical analysis. In an earlier experiment with older
plants, 3–7 flowers of 6–7 replicate plants (N. attenuata)
were measured. For D. wrightii, one flower of eight repli-
cate plants was measured 9–11 days after the first treat-
ment, as plants did not produce flowers every day.
Oviposition in relation to plant size or flower number
To evaluate how plant size as well as flower number
may influence oviposition of M. quinquemaculata in a
D. wrightii population, I counted newly oviposited eggs on
D. wrightii plants of various sizes or plants carrying differ-
ent numbers of flowers. The experimental plants were
monitored daily for eggs of M. quinquemaculata. All eggs
were removed during the day before the night that an
experiment occurred. To check for plant size effects, the
size of all plants was recorded, and all flowers were
removed between 19:00 and 21:00 hours, just before flow-
ers started to open. Plant size was estimated by measuring
the plant height and the diameter from which the volume
of a circular cylinder was calculated as a proxy of plant size.
The diameter of plants included in this experiment ranged
between 42 and 120 cm. To test whether or not flower
number can influence oviposition, the flower number on
all plants was reduced to 1–4 per plant with a pair of scis-
sors in the dusk. Freshly laid eggs were counted the follow-
ingmorning.
Nectar supplementation experiment
To examine the influence of nectar volume on oviposition
rates in a natural population of D. wrightii, five pairs
of similarly sized D. wrightii plants were chosen from a
population in two consecutive nights and trimmed to one
114 Kessler
open flower per plant. The nectar volume of the flower
was experimentally increased for one plant in each pair by
adding 400 ll of a 25% (wt/vol) sucrose solution at dusk
(20:00–21:00 hours), thereby increasing standing nectar
volume five-fold. In control plants, flowers were left
untreated. All eggs were removed from plants at the time
of supplementation and plants did not differ in the num-
ber of eggs found before supplementation. Newly oviposit-
ed M. quinquemaculata eggs were counted the morning
after nectar supplementation. Oviposition data on N. at-
tenuata have been published in Kessler et al. (2010), but
are shown again to enable direct comparisons with data
fromD. wrightii. Experiments withN. attenuata proceeded
in a similar manner and were conducted simultaneously in
a population which was located � 10 km away from the
D. wrightii population and thus shared hawkmoth popula-
tions, inMay and June 2004. Treatment and control mem-
bers of a plant pair were matched by plant size and flower
number was reduced to five flowers. Nicotiana attenuata
flowers were treated by adding 5 ll of a 12.5% (wt/vol)
sucrose solution, to increase the standing nectar volume
on average 20-fold.
With D. wrightii, additional nectar supplementation
experiments were conducted to disentangle the response
of Manduca to floral volatile emission and floral display
from its response to the presence of nectar (sugar solution)
in the field. To compare oviposition among plants con-
taining the same total volume of nectar either in one or in
four flowers, plants of the same size were chosen, control
plants had four untreated flowers and treatment plants
were reduced to one flower which was filled with 400 ll25% (wt/vol) sucrose solution. To measure the impact of
nectar accessibility, pairs of plants having the same size
were chosen and reduced to the same number of flowers
within a pair (1–3 flowers per plant). Carpels of treatment
flowers were closed with a teaspoon of wheat flour tomake
nectar collection impossible, while flowers of control
plants remained untreated.
Sugar concentrations used for nectar supplementa-
tion experiments – 25% (D. wrightii) and 12.5% (N. atte-
nuata) – were chosen according to the nectar sugar
concentrations found for both species in newly opening
flowers.
Statistical analysis
Correlations between plant size and flower number, flower
number and laid eggs, or plant size and flower number,
were analyzed using Microsoft Excel (version 2007). Cor-
relation of analysis was done by residual analysis followed
by calculating the coefficient of determination (R2) by sub-
tracting the residual sum of squares divided by the total
sum of squares, from one.
Data on the influence of leaf ablation on nectar volume
as well as oviposition data met the assumption of homo-
scedasticity and had not to be transformed. Fisher’s
protected least significant difference (PLSD) tests follow-
ing ANOVA, as well as paired Student’s t-tests, were done
using Statview 5.0 (SAS Institute; www.statview.com).
Results
Phenology of nectar accumulation
In both species the nectar volume was much smaller in
field- than in glasshouse-grown plants (Figure 1 A andD).
In D. wrightii, nectar accumulated for 1 day and was
produced over the entire night (Figure 1A). Flowers on
glasshouse-grown plants accumulated on average (± SD)
159.6 ± 11.7 ll, or 29 the nectar volume of field-grown
plants (69.9 ± 27.1 ll) if allowing for evapotranspiration.If flowers were covered with plastic bags, they produced
196.0 ± 5.3 ll of nectar in the glasshouse, or
100.5 ± 17.3 ll in the field. Nectar sugar concentration
ranged between 17.5 and 27.4% (Figure 1B), and was rela-
tively constant over the entire period in which flowers
offered nectar, although it increased slightly in the first
half of the night. Total sugar per flower averaged
46.1 ± 3.0 mg in the glasshouse and 20.5 ± 8.9 mg in the
field (Figure 1C). The nectar of N. attenuata accumulated
for 2 days and was produced over the entire night on both
days (Figure 1D). Flowers on glasshouse-grown plants
accumulated on average 3.7 ± 0.9 ll, or 39 the nectar
volume of field-grown plants (1.1 ± 0.7 ll) if allowing forevapotranspiration. If flowers were covered with plastic
bags, they produced 3.8 ± 1.3 ll of nectar in the
glasshouse, or 1.5 ± 0.8 ll in the field, in the 1st night
(Figure 1D). Nectar volume during the 2nd night
increased 8.5% under glasshouse conditions, or about
49.3% if inflorescences were covered with plastic bags,
compared to the 1st night. This difference likely resulted
from daytime evaporation, because sugar concentration
increased in direct proportion to the decrease in nectar
volume, and the total sugar content of each flower
remained relatively stable after 04:00 hours (Figure 1E).
Sugar concentration increased with the age of the flower in
disclosed plants from 11 to 36% in the glasshouse and
from 19 to 52% in the field. Sugar concentrations after the
1st night increased only 10.2% in flowers enclosed in
plastic, while disclosed flowers increased sugar concentra-
tions 31.4% relative to the 1st night. The production of
sugar per flower was the same for flowers enclosed in
plastic (0.291 mg) and disclosed (0.296 mg) flowers
from the first to the 2nd night. The amount of sugars in
flowers not exposed to pollinators increased significantly
between the 1st and 2nd nights (ANOVA: F2,34 = 6.86,
High nectar volumes increase oviposition 115
P = 0.003), but not between the 2nd and the 3rd nights
(Fisher’s PLSD test: P>0.05) despite an 87% loss of nectar
volume. Total sugar per flower averaged 0.80 ± 0.05 mg
in the glasshouse and 0.39 ± 0.04 mg in the field
(Figure 1F).
Dependence of nectar volume on photosynthetic tissues
To determine the influence of photosynthetic tissue on
nectar volume and nectar sugar concentration of D.
wrightii and N. attenuata plants, I compared nectar from
untreated plants (1), with nectar from plants which had
the following leaves removed: rosette leaves (2); stem
leaves (3); or all leaves (4). In D. wrightii the removal of
rosette, as well as stem leaves significantly reduced nectar
produced per flower [29.2% (2), 39.2% (3); ANOVA:
F2,21 = 43.50, P<0.0001; Fisher’s PLSD test: P<0.0001].The removal of all leaves led to abortion of all buds
produced before the treatment and led to a stop of flower
production. Sugar concentration did not change due to
leaf ablation (ANOVA: F2,21 = 2.33, P = 0.12; Fisher’s
PLSD test: P>0.05; Figure 2B).
In N. attenuata the removal of rosette leaves, stem
leaves, and all leaves significantly decreased nectar pro-
duction [42.2% (2), 30.8% (3), 67.8% (4); ANOVA:
F3,56 = 58.37, P<0.0001; Fisher’s PLSD test: P<0.0001](Figure 2C). In a replicate experiment with older N. atte-
nuata plants, only the removal of stem leaves (ANOVA:
F3,22 = 26.24, P<0.0001; Fisher’s PLSD test: P = 0.018) or
all leaves (P<0.0001) significantly decreased nectar
production [23.2% (3), 45.6% (4)], and removing rosette
leaves did not reduce nectar volume (Fisher’s PLSD
test: P = 0.39). Cutting photosynthetic tissue did not
dramatically alter nectar sugar concentration, although
sugar concentration significantly increased if rosette leaves
or all leaves were removed (ANOVA: F3,56 = 9.19,
P<0.0001; Fisher’s PLSD test: P<0.05) (Figure 2D).
D. wrigh i N. a enuata
○●
□■
gh-baggedghfield-baggedfield
A
B
C
D
E
F
Figure 1 Diurnal changes in nectar volume and sugar concentration of flowers from a single genotype ofDatura wrightii orNicotiana
attenuata plants grown in the glasshouse (gh) or a native population in SWUtah (field). (A)Mean (± SE) volume and (B) sugar
concentration of nectar from threeD. wrightii flowers per harvest time or (D)mean volume and (E) sugar concentration of nectar from
20 N. attenuata flowers per harvest time through 20 h, spanning an 8-h dark period (gray bar). The analysis lasted from 21:00 to
16:00 hours the next day and 20 min were required tomeasure all replicates, which were collected from different individuals, at each given
harvest time. To compare glasshouse- and field-grown plants over the 3-day flower lifespan ofN. attenuata, nectar volume and sugar
concentrationmeasurements were conducted between 04:00 and 06:00 hours on flowers of different ages, but from the same plant, for
three consecutive days. Experiments were repeated for one time point (05:00 hours) with flowers enclosed in plastic bags, to reduce nectar
evaporation in the glasshouse, or with flowers enclosed in plastic bags or textile bags to exclude evaporation or flower visitors in the field.
Total sugar per (C)D. wrightii or (F)N. attenuata flower was calculated fromflower-specific volume, sugar concentrations, and the density
at a certain sugar concentration.
116 Kessler
Oviposition in relation to plant size and flower number
When all flowers were removed from D. wrightii plants of
different sizes, oviposition rate was independent from
plant size (y = 0.00001x + 3.3191; R2 = 0.09; n = 112),
even if plant diameter differed several-fold. In contrast, the
oviposition rate of M. quinquemaculata positively corre-
lated with the number of flowers per plant (y = 19.043x +9.1956; R2 = 0.72; n = 59; 0–4 flowers per plant). The
mean ± SD number of eggs laid per plant almost doubled
if a plant contained two flowers (37 ± 25) instead of one
flower (19 ± 12; Student’s t-test: t = 2.52, d.f. = 27,
P = 0.018), or four (66 ± 44) instead of two flowers
(t = 1.33, d.f. = 8, P = 0.22).
Nectar supplementation increases oviposition in nature
Adding 400 ll of a 25% sucrose solution per flower to
field-grown D. wrightii plants – and thereby increasing theaverage flower nectar volume five-fold – significantly
increased the frequency of M. quinquemaculata oviposi-
tion (paired Student’s t-test: t = 3.91, d.f. = 9, P = 0.004).
Treated plants received twice as many eggs (mean ± SD =47 ± 20) as did control plants (22 ± 9) (Figure 3A).
In pairs of plants for which the control plant contained
four untreated flowers and the treatment plant one flower
filled with 400 ll (the equivalent of four untreated
flowers) of 25% sucrose solution, I found no oviposition
preference of M. quinquemaculata (t = 1.49, d.f. = 7,
P = 0.18). Treatment plants received a mean of 25 ± 8
eggs, and control plants 21 ± 6 eggs. If nectar access in
flowers was restricted for moths by closing carpels using
flour in one plant of plant pairs containing the same
number of flowers, moths significantly preferred to
oviposit on untreated plants containing normal amounts
of nectar (t = 7.49, d.f. = 9, P<0.0001).
Discussion
Research on the ecological function of floral nectar has
primarily focused on its function as a reward for pollina-
tion. Recently, however, researchers have started to dis-
cuss potential costs of nectar production. Herein, I
describe the nectar accumulation patterns of two sym-
patric solanaceous species which share pollinators as well
as herbivores, show how losses of photosynthetic tissue
may alter nectar accumulation, and finally examine the
relationship between floral nectar and simulated leaf her-
bivory in the field. Nectar is an important mutualism-
mediating currency that the plant keeps homeostatic,
because selective pressures exerted by pollinators as well
as herbivores may act simultaneously in optimizing nec-
tar accumulation. Adler & Bronstein (2004) showed that
even nectar volume differences that fall within the range
of natural production change the frequency of oviposi-
tion in the glasshouse, which confirms that even
D. wrigh i N. a enuataA
B
C
D
Figure 2 Effect of defoliation onmean (+SE) nectar volume and sugar concentration in (A and B)Datura wrightii and (C andD)Nicotiana
attenuata. Either no (1), rosette (2), stem (3), or all (4) leaves were removed fromeight replicateD.wrightii, or 15 replicateN. attenuata
plants with a razor blade. Nectar sugar concentration and volumeweremeasured fromone flower (D.wrightii) or two flowers (N. attenuata)
per plant, 1 week after defoliation treatments were imposed.Datura wrightiiplantswhich had all leaves removedwere unable to produce
flowers. Asterisks represent significant differences from control (ANOVA followed by Fisher’s PLSD test: *P<0.05, ***P<0.0001).
High nectar volumes increase oviposition 117
relatively small variation in nectar volume may nega-
tively affect a plant.
The two solanaceous species I investigated in this study
face the same ‘dilemma’, namely attracting moths for pol-
lination services, but at the same time preventing their ovi-
position. In terms of nectar production, the species deal
with this problem very differently. In the field, nectar accu-
mulation per flower differs 50-fold between D. wrightii
and N. attenuata. With its relatively low nectar reward,
N. attenuata seems to have adopted the strategy described
by Baker (1975): sugar is most efficiently used when it is
present in large enough quantities to attract and hold the
attention of a pollinator, but in small enough quantities to
force the pollinator to visit the maximum number of flow-
ers. Datura wrightii, in contrast, produces huge amounts
of nectar, thereby most likely increasing the chance of pol-
lination services in competition with other species like, for
example, N. attenuata. Pollinators have been shown in
many studies to visit more flowers on a plant if nectar is
presented in greater quantities (Pyke, 1978; Hodges,
1995). How important higher nectar rewards can be
becomes apparent when an invasive plant species provides
more nectar than does the native floral community (Chitt-
ka & Schurkens, 2001). Local plants in the vicinity of the
invasive plant Impatiens glanduliferaRoyle had fewer polli-
nator visits as well as lower seed set. This model, however,
builds on a simple relationship between plant and pollina-
tor. Most systems are more complex, as is the case for
N. attenuata and D. wrightii, in which the pollination ser-
vice of the hawkmothsM. sexta andM. quinquemaculata is
to be paid for by receiving herbivory: hawkmoth eggs laid
on the plants hatch into voracious caterpillars. In cases like
these, plants with a higher nectar reward than surrounding
plants or plant species are vulnerable to receiving more
eggs in comparison to plants which accumulate less nectar.
Secretion of floral nectar ofD. wrightii andN. attenuata
plants is not completed at the time when flowers open in
the dusk. Rather, nectar is secreted throughout the entire
night (Figure 1). Although the plants differ greatly in per
flower nectar accumulation, there seems to be a benefit of
secreting nectar overnight instead of presenting the maxi-
mum nectar volume already at dusk. It may be that it is
beneficial to keep producing nectar over night to keep
pollinators searching for nectar, as nectar could have been
removed by previous floral visitors, including opportunis-
tic pollinators or nectar robbers, by the time new pollina-
tors arrive. Several pollinators are thought to sense the
presence of nectar (Wetherwax, 1986; Goulson et al.,
2001), which could lead to a complete loss of pollination
services if nectar were collected by nectar robbers or other
floral visitors already in the dusk. On the other hand,
plants may also try to avoid attracting too much attention
from antagonistic floral visitors by offering nectar contin-
uously rather than in one large pool, especially if a moth
decides based on the nectar volume whether she is going
to oviposit or not. Female moths could use nectar accu-
mulation of a plant as cue to gain information about a
plant’s size. Indeed, I found that the plant size is correlated
with the number of flowers produced per day in plants of a
D. wrightii population (y = 110290x + 156244; R2 = 0.32;
n = 131). If a moth oviposits on larger plants, it would be
beneficial for her progeny not only in terms of food avail-
ability, but also in terms of avoiding predators which may
not be able to locate eggs and caterpillars as easy as on a
smaller plant.
I found a strong positive correlation between the num-
ber of eggs laid and the number of flowers per plant. The
Control Supplement0.0
0.1
0.2
0.3
0.4
M. q
uinq
uem
acul
ata
eggs
/pla
nt/n
ight
D. wrightii
(from Kessler et al., 2010)
0
10
20
30
40
50
B
*N. attenuata
*A
Figure 3 Influence of artificially increased (A)Datura wrightii
and (B)Nicotiana attenuata nectar volume on the oviposition
rate ofManduca quinquemaculata adults on plants in a native
population in Utah, USA. Pairs ofD. wrightii plants of equal size
were chosen and flower number was reduced to one and
supplemented with 400 ll of a 25% sucrose solution – thusincreasing nectar volume five-fold – at dusk or left untreated. ForN. attenuata, the recently opened flowers of size-matched plant
pairs were reduced to five and supplemented with 20 ll of a12.5% sucrose solution – thus increasing nectar volume 20-fold –at dusk or left untreated (Kessler et al., 2010). Control and
supplementedmembers of a pair were at least 5 m apart and all
eggs were removed at the time of supplementation. Newly
ovipositedM. quinquemaculata eggs were counted the following
morning and are expressed asmean (+ SE) eggs per plant [paired
Student’s t-test (D. wrightii), or Fisher’s exact test (N. attenuata):
*P<0.05].
118 Kessler
increased attraction of female moths to plants with more
flowers could be caused by the total amount of nectar per
plant, greater floral volatile emission, or a larger floral dis-
play. The nectar supplementation experiments showed
that nectar volume plays a significant role in the oviposi-
tion decision of a M. quinquemaculata moth, because a
D. wrightii plant with four untreated flowers (total nectar
production approximately 400 ll) received just as many
eggs as a plant with only one flower filled with 400 llsucrose solution. Equally, preventing moths from access-
ing nectar by blocking carpel entrances with flour reduced
oviposition compared to plants which produced normal
amounts of accessible nectar. These results underline the
importance of the nectar itself as cue forM. quinquemacu-
lata oviposition, independent of the number of flowers,
and associated attractive floral components such as floral
scent or floral display. Whereas the strong scent of the
flower is probably required to lure moths for pollination
from far away, nectar may play a role as short-distance cue
helping moths to make an oviposition decision. It is not
known for this study system how many moths lay eggs per
night on D. wrightii. In a scenario where we expect only
one or a few moths to visit a plant per night, it is easy to
imagine that the nectar volume could be used to estimate a
plant’s size. If, however, a huge number of moths visit the
same flowers within one night, then the nectar removal by
the first moth will impact the oviposition decision of the
next. By taking only the total nectar volume of a plant into
account the system yet could be perfectly regulated in both
cases. If many moths visit, increasingly fewer would chose
to oviposit, with the net result of having amaximum num-
ber of eggs per plant, no matter how many moths are in
the pool. The results from the nectar supplementation
experiments suggest that only a few moths visit the same
plant per night, because otherwise the increase in oviposi-
tion events in response to artificially added nectar to one
flower would not be so clear. More field observations of
Manduca behavior, however, are required to be able to
draw definitive conclusions.
In a simultaneous nectar supplementation experiment
with N. attenuata, I filled five flowers per plant each with
20 ll of sucrose solution to reach a total of 100 ll nectarper plant (Figure 3B; Kessler et al., 2010), which is equiva-
lent to one flower of D. wrightii. Is N. attenuata able to
reduce the oviposition rates found in D. wrightii by offer-
ing smaller nectar rewards? A range of nectar accumula-
tion typical for a certain plant species could, together with
nectar composition, provide important information for a
potential herbivore. In addition, the likelihood of an ovi-
position could increase if a moth can fill her energetic
demand by visiting only a few plants. OneM. sextamoth is
able to collect nectar from over 1300 N. attenuata flowers
or approximately 250 plants per night (Kessler & Baldwin,
2007), which may decrease the likelihood of oviposition
on a single plant.Datura wrightii, in contrast, seems to fol-
low another strategy as a floral visit by a Manduca female
is much more likely to include an oviposition event,
because the nectar produced by two or three plants would
be sufficient for one moth (Raguso et al., 2003; Kessler &
Baldwin, 2007). Even after increasing the total nectar vol-
ume of an N. attenuata plant to a volume comparable to
one D. wrightii flower, maximally one egg per plant was
laid in the field (Kessler et al., 2010). In D. wrightii, 16–57eggs were laid per night on plants which had one flower.
This difference implies that other, perhaps more, herbiv-
ory-related selective pressures play a bigger role in an
insect’s choice to oviposit on a plant. The role that the
plant species plays for a moth in making an oviposition
decision is much bigger than the role of nectar, but within
a plant species it is of great importance not to accumulate
more nectar than neighboring plants to prevent oviposi-
tion (Adler & Bronstein, 2004). Increasing five N. attenu-
ata flowers to a total volume of one D. wrightii flower
increased the chance of an oviposition eight-fold in com-
parison to a plant with five untreated flowers (Kessler
et al., 2010).
An alternative hypothesis to explain the more frequent
oviposition in treated plants could also be the better qual-
ity of the nectar. Nectar constituents like the alkaloid
nicotine, which is known to decrease nectar removal in
N. attenuata (Kessler & Baldwin, 2007; Kessler et al.,
2008), were diluted by supplementation with a pure
sucrose solution. Experiments were targeted only on the
nectar volume itself and neglected other nectar constitu-
ents. Adler & Bronstein (2004) showed nicely that also by
adding real nectar to the flowers, oviposition increased in
treated plants, which supports the conclusions that can
be drawn from my data. Furthermore, I expect some of
the compounds also to accumulate in the added sugar
solutions, as secondary volatile components of the nectar
are known to be a hydrophilic subset of the compounds
emitted by surrounding floral tissues, and thus could be
found also in the artificial nectar after a while (Raguso,
2004). However, I cannot exclude effects from altered
nectar chemistry.
Just as plant nectar accumulation can impact herbivory,
herbivory could impact nectar volume if feeding reduces
leaf area by a large amount such as caused byM. quinque-
maculata larvae. InN. attenuata, the loss of more than half
of all leaves to an established Manduca larva is the rule
rather than the exception. Eggs are usually laid on the
lower stem leaves of N. attenuata (Kessler & Baldwin,
2002) and the hatchingManduca larva defoliates the com-
plete stem if it is not predated. InD. wrightii, oviposition is
High nectar volumes increase oviposition 119
distributed over the whole plant. Although D. wrightii
plants are in general bigger than N. attenuata plants, a
Manduca larva will also cause severe damage and is able to
reduce the leaf area by a large amount (McFadden, 1968)
comparable to my rosette and stem-leaf removal treat-
ments. I testedD. wrightii andN. attenuata for their ability
to maintain nectar accumulation as well as nectar sugar
concentration when lacking large portions of their photo-
synthetically active tissue. In both species, nectar accumu-
lation per flower was found to be dramatically reduced
even if half of the leaf area was missing. The ablation of all
leaves inD. wrightii led to abortion of all floral buds and so
to a cessation of any reproductive activity. Reduced nectar
accumulation as a consequence of previous herbivory and
the resulting loss of leaf area could, just as induced volatile
emission (Kessler & Baldwin, 2001), cause a moth to leave
a plant without laying an egg (N. attenuata) or at least lay
fewer eggs (D. wrightii). The caterpillars hatching from
eggs would have a great benefit if they started feeding on a
larger, healthier plant, not only because of food availability
and quality, but also to avoid competition with conspecif-
ics or other herbivores.
Nectar sugar concentration is, in comparison to nectar
volume, not subject to change. Even removal of the entire
canopy did not influence sugar concentration. Sugar con-
centrationmay, through the secretion process, not be plas-
tic, or it may play too important a role in attracting
pollinators. The concentration of sugar in nectar seems to
be most important for determining the pollinator guild at
an earlier evolutionary step: bees, especially, are known to
prefer more concentrated nectars. Low sugar concentra-
tions such as those found in D. wrightii orN. attenuata on
the 1st night the corolla opens are typical for humming-
bird- and moth-pollinated plants (Baker & Baker, 1983)
and thus may be necessary to keep hawkmoths visiting a
plant.
Nevertheless, I found higher nectar sugar concentra-
tions with increasing flower age in N. attenuata. This
increase in sugar concentration is very likely due to evapo-
ration, as nectar volume is decreasing over the day, while
the total amount of sugar produced per flower remains
stable at day time (Figure 1D–F). Because flowers await
visits in the 1st night, the increasing sugar concentrations
during the following daysmay be an artifact of initially low
pollinator densities. Higher sugar concentrations in the
later flower stages may be able to compensate for the lack
of specialist pollinators by attracting generalist pollinators
from guilds such as bees or bumblebees. No cost is associ-
ated with nocturnally visited flowers remaining open
through the following day and being visited by diurnal
pollinators (Pettersson, 1991). Natural selection may
favor adding new pollinators over old ones (Aigner, 2001).
Secondary pollinator recruitment has been shown in
Oenothera elata Kunth and Desmodium setigerum (E.
Mey.) Benth. ex Harv. (Barthell & Knops, 1997; Willmer
et al., 2009).
In summary, this study connects nectar volume to leaf
herbivory in the field and suggests a role for nectar volume
in the host choice ofM. quinquemaculatamoths. I suggest
that herbivory-related selective pressures rather than nec-
tar secretion determine the extent to which oviposition
occurs in different plant species. Within one plant species,
however, overall nectar accumulation by a plant could be
used by moths to estimate the size or even the health of a
plant, as reduction of leaf area led to significant decreases
in nectar secretion.
Acknowledgements
I thank Ian T. Baldwin for sharing ideas, inspiring discus-
sions, and for offering support, Celia Diezel for help in the
field, Andre Kessler and Meredith C. Schuman for edito-
rial assistance, Brigham Young University for use of its
awesome field station, the Lytle Preserve, and the Max
Planck Society for financial support.
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