With a flick of the lid: a novel trapping mechanism in Nepenthes gracilis pitcher plants.
ABSTRACT Carnivorous pitcher plants capture prey with modified leaves (pitchers), using diverse mechanisms such as 'insect aquaplaning' on the wet pitcher rim, slippery wax crystals on the inner pitcher wall, and viscoelastic retentive fluids. Here we describe a new trapping mechanism for Nepenthes gracilis which has evolved a unique, semi-slippery wax crystal surface on the underside of the pitcher lid and utilises the impact of rain drops to 'flick' insects into the trap. Depending on the experimental conditions (simulated 'rain', wet after 'rain', or dry), insects were captured mainly by the lid, the peristome, or the inner pitcher wall, respectively. The application of an anti-slip coating to the lower lid surface reduced prey capture in the field. Compared to sympatric N. rafflesiana, N. gracilis pitchers secreted more nectar under the lid and less on the peristome, thereby directing prey mainly towards the lid. The direct contribution to prey capture represents a novel function of the pitcher lid.
- SourceAvailable from: Walter Federle[Show abstract] [Hide abstract]
ABSTRACT: Plant-insect interactions are determined by both chemical and physical mechanisms. Biomechanical factors play an important role across many ecological situations, including pollination, herbivory and plant carnivory, and have led to complex adaptations in both plants and insects. However, while mechanical factors involved in some highly specific interactions have been elucidated, more generalised effects may be widespread but are more difficult to isolate, due to the multifunctional properties of the plant surfaces and tissues where interactions occur. Novel methodologies are being developed to investigate the mechanisms of biomechanical interactions and discover to what extent adaptive structures could be exploited via biomimetics.Current opinion in plant biology 12/2012; · 10.33 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Many plants combat herbivore and pathogen attack indirectly by attracting predators of their herbivores. Here we describe a novel type of insect-plant interaction where a carnivorous plant uses such an indirect defence to prevent nutrient loss to kleptoparasites. The ant Camponotus schmitzi is an obligate inhabitant of the carnivorous pitcher plant Nepenthes bicalcarata in Borneo. It has recently been suggested that this ant-plant interaction is a nutritional mutualism, but the detailed mechanisms and the origin of the ant-derived nutrient supply have remained unexplained. We confirm that N. bicalcarata host plant leaves naturally have an elevated (15)N/(14)N stable isotope abundance ratio (δ(15)N) when colonised by C. schmitzi. This indicates that a higher proportion of the plants' nitrogen is insect-derived when C. schmitzi ants are present (ca. 100%, vs. 77% in uncolonised plants) and that more nitrogen is available to them. We demonstrated direct flux of nutrients from the ants to the host plant in a (15)N pulse-chase experiment. As C. schmitzi ants only feed on nectar and pitcher contents of their host, the elevated foliar δ(15)N cannot be explained by classic ant-feeding (myrmecotrophy) but must originate from a higher efficiency of the pitcher traps. We discovered that C. schmitzi ants not only increase the pitchers' capture efficiency by keeping the pitchers' trapping surfaces clean, but they also reduce nutrient loss from the pitchers by predating dipteran pitcher inhabitants (infauna). Consequently, nutrients the pitchers would have otherwise lost via emerging flies become available as ant colony waste. The plants' prey is therefore conserved by the ants. The interaction between C. schmitzi, N. bicalcarata and dipteran pitcher infauna represents a new type of mutualism where animals mitigate the damage by nutrient thieves to a plant.PLoS ONE 01/2013; 8(5):e63556. · 3.73 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The digestion of prey by carnivorous plants is determined in part by suites of enzymes that are associated with morphologically and anatomically diverse trapping mechanisms. Chitinases represent a group of enzymes known to be integral to effective plant carnivory. In non-carnivorous plants, chitinases commonly act as pathogenesis-related proteins, which are either induced in response to insect herbivory and fungal elicitors, or constitutively expressed in tissues vulnerable to attack. In the Caryophyllales carnivorous plant lineage, multiple classes of chitinases are likely involved in both pathogenic response and digestion of prey items. We review what is currently known about trap morphologies, provide an examination of the diversity, roles, and evolution of chitinases, and examine how herbivore and pathogen defense mechanisms may have been coopted for plant carnivory in the Caryophyllales.Current opinion in plant biology 07/2013; · 10.33 Impact Factor
With a Flick of the Lid: A Novel Trapping Mechanism in
Nepenthes gracilis Pitcher Plants
Ulrike Bauer1,2*, Bruno Di Giusto3, Jeremy Skepper4, T. Ulmar Grafe2, Walter Federle5
1Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom, 2Department of Biology, University Brunei Darussalam, Gadong, Brunei
Darussalam, 3English Language Center, Ming Chuan University, Taipei, Taiwan, 4Department of Physiology, Development and Neuroscience, University of Cambridge,
Cambridge, United Kingdom, 5Department of Zoology, University of Cambridge, Cambridge, United Kingdom
Carnivorous pitcher plants capture prey with modified leaves (pitchers), using diverse mechanisms such as ‘insect
aquaplaning’ on the wet pitcher rim, slippery wax crystals on the inner pitcher wall, and viscoelastic retentive fluids. Here we
describe a new trapping mechanism for Nepenthes gracilis which has evolved a unique, semi-slippery wax crystal surface on
the underside of the pitcher lid and utilises the impact of rain drops to ‘flick’ insects into the trap. Depending on the
experimental conditions (simulated ‘rain’, wet after ‘rain’, or dry), insects were captured mainly by the lid, the peristome, or
the inner pitcher wall, respectively. The application of an anti-slip coating to the lower lid surface reduced prey capture in
the field. Compared to sympatric N. rafflesiana, N. gracilis pitchers secreted more nectar under the lid and less on the
peristome, thereby directing prey mainly towards the lid. The direct contribution to prey capture represents a novel
function of the pitcher lid.
Citation: Bauer U, Di Giusto B, Skepper J, Grafe TU, Federle W (2012) With a Flick of the Lid: A Novel Trapping Mechanism in Nepenthes gracilis Pitcher Plants. PLoS
ONE 7(6): e38951. doi:10.1371/journal.pone.0038951
Editor: Jeff Ollerton, University of Northampton, United Kingdom
Received February 7, 2012; Accepted May 16, 2012; Published June 13, 2012
Copyright: ? 2012 Bauer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by a Henslow Research Fellowship of the Cambridge Philosophical Society and a field work grant by the Charles Slater Fund,
Cambridge, to UB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Carnivorous pitcher plants have recently emerged as a model
system for studying the evolution of functional traits in plant
morphology in an ecological context [1–7]. Members of the
paleotropical genus Nepenthes capture prey with specialised, highly
modified leaves (pitchers) acting as passive pitfall traps . Most
species produce two morphologically distinct pitcher types: ‘lower’
pitchers that usually rest on the ground and develop on immature
rosette plants, and hanging ‘upper’ pitchers on climbing vines.
Each pitcher consists of the main pitcher body, partly filled with
digestive fluid, a collar-like upper rim (peristome), and the pitcher
lid which in most species forms a ‘roof’ above the pitcher opening,
protecting the pitcher from being flooded by rain.
Pitchers of all Nepenthes species secrete nectar to attract insect
prey . Extrafloral nectaries are scattered across the outside of
the pitcher and both the upper and lower lid surface, and are
densely packed around the inner margin of the peristome. The
quantity of nectar secreted on different parts of the pitcher (and
other parts of the plant) varies with pitcher development, and
between species [9,10]. In fully developed, open pitchers (i.e.
functional traps) the largest quantities of nectar are secreted on the
peristome and under the pitcher lid .
A number of distinct trapping mechanisms have been described,
such as specialised slippery surfaces on the peristome  and the
inner pitcher wall [12,13], as well as viscoelastic pitcher fluids .
The peristome is highly wettable and under humid conditions, thin
stable water films form on the surface, rendering it extremely
slippery . Due to its wetness-dependence, the peristome only
activates the trap intermittently, and visiting insects can safely
harvest nectar during inactive (i.e. dry) times . By this means,
the plant may promote the survival of ‘scout’ ants that ultimately
recruit larger numbers of worker ants to the trap.
The slipperiness of the inner wall is based on a dense layer of
platelet-shaped wax crystals that are orientated perpendicularly to
the surface. These crystals drastically reduce the available contact
area for insect adhesive pads . In addition, the platelets have
been reported to break off easily and contaminate the insects’
adhesive pads [13,17]. The wax crystal layer is a common feature
of many Nepenthes although there are several species in which it is
reduced or absent [5,6]. N. gracilis is unusual in that it has wax
crystals not only on the inner pitcher wall but also on the
underside of the pitcher lid (Fig. 1A). This characteristic prompted
us to investigate whether the lid is involved in prey capture in this
We observed that ants harvesting nectar from the lower lid
surface of N. gracilis in the field (in Brunei, Northern Borneo) were
able to walk upside down on the wax crystal surface without
difficulty, while the same ants would slip and fall from the waxy
inner pitcher wall. Nevertheless, the presence of such a unique
structure strongly suggested a trapping function. A casual
observation of a Coccinellid beetle being flicked into a N. gracilis
pitcher by a raindrop after seeking shelter under the pitcher lid
prompted us to hypothesise that the wax crystal layer, while
providing a secure foothold under normal conditions, causes
insects to detach more easily under sudden impacts. The
horizontal orientation of the lid directly above the pitcher opening
(Fig. 1B) and its comparatively high stiffness could further aid this
trapping function. Field observations of increased prey numbers in
PLoS ONE | www.plosone.org1 June 2012 | Volume 7 | Issue 6 | e38951
N. gracilis pitchers after rainy days (C. Clarke, personal commu-
nication) support this idea; however, they might as well be due to
the increased trapping efficiency of the peristome under wet
conditions. We therefore investigated the role of the N. gracilis lid
for prey capture in the laboratory and in the field, and
characterised the detailed structure of the wax crystal surface
using scanning electron microscopy (SEM).
The impact of heavy ‘rain’ drops causes ants to fall from
the lower lid surface of N. gracilis
In the laboratory, we allowed Crematogaster sp. ants (a common
prey species of N. gracilis at our study site) to forage on freshly
harvested pitchers. Rain was simulated using an infusion drip
system (Fig. 2A; see Methods). The effect was striking:
40.6169.62% of all ants visiting the lower lid surface were
knocked off by the impact of ‘rain’ drops and fell into the pitcher
(Video S1). In contrast, not a single ant fell from the lid before or
after the simulation of rain, confirming that the slipperiness of the
lower lid surface was not altered by the increase of humidity after
the ‘rain’. This result was not changed when an isolated pitcher lid
(mounted horizontally using a paper clip) was tested: in this case
44% (11 of 25) ants were knocked off by ‘rain’ drops (Video S2).
Ants were observed to be relatively ‘safe’ when holding onto the
thicker mid-rib and get knocked off more frequently when they
were positioned further out towards the (thinner) sides of the lid.
Whether this was due to the mid-rib providing additional grip or
to the dampened impact of the rain drops in this thicker section of
the lid is not clear.
N. gracilis pitchers rely on different trapping surfaces
under different weather conditions
We investigated the contribution of each surface (inner wall,
lower lid surface, peristome) under different experimental condi-
tions (before/during/after simulated rain). We found a highly
significant dependence of the surfaces’ trapping efficiency on the
experimental conditions (ANOVA comparing two separate
Generalised Linear Mixed Models, for details see Methods,
df=4, x2=185.97, P,0.001; Fig. 2B). Ants fell from the lower
lid surface only under the impact of simulated ‘rain’ drops, in
which case up to 57% of the visitors were captured. The (weather-
independent) wax crystal layer on the inner pitcher wall provided
a low but more or less constant baseline trapping efficiency
(c. 7%). The peristome was not slippery when dry but reached
high efficiency (up to 80%) under wet conditions. In our
experiment, the peristome became slippery approximately 2–
3 min after the start of the simulated rain, and stayed slippery for
7–10 min after we stopped the dripping.
The lid of N. gracilis pitchers contributes to natural prey
capture in the field
We tested the biological relevance of the lid capture mechanism
by comparing prey numbers between an untreated control and
pitchers with experimentally modified lids (underside coated with
a thin layer of a non-toxic, transparent silicon polymer). Ants are
able to walk on this polymer surface under both wet and dry
conditions. The ‘anti-slip coating’ of the lower lid surface caused
a significant reduction of captured prey in the field (Mann-
Whitney U test, n1/2=15, Z=1.97, P,0.05; Fig. 3). Prey numbers
over the course of the experiment (19 days) were highly variable
both between pitchers and between sampling intervals (3 days).
Remarkably, the lid manipulation did not render pitchers
completely ineffective: all pitchers did capture some prey over
the course of the experiment. This indicates that the pitchers were
still able to trap prey with the peristome and the inner wall.
N. gracilis has evolved two structurally and functionally
different wax crystal surfaces
Scanning electron micrographs of the inner pitcher wall and
underside of the pitcher lid revealed that both wax crystal surfaces
are radically different in structure. The inner wall surface (Fig. 4A–
B) was similar in morphology to wax crystal surfaces studied in
other Nepenthes species, with a continuous, 3.0560.36 mm (mean 6
Figure 1. Morphology of N. gracilis pitchers. (A) N. gracilis pitcher with visiting Polyrhachis pruinosa ant, showing the epicuticular wax crystal
surfaces on the inner pitcher wall and on the underside of the pitcher lid. (B) The horizontal orientation directly above the pitcher opening puts the
lower lid surface in an ideal position for prey capture.
New Trapping Mechanism in Nepenthes Pitcher Plants
PLoS ONE | www.plosone.org2June 2012 | Volume 7 | Issue 6 | e38951
s.d., n=21) thick layer of leaf-like wax platelets connected to an
underlying matrix of shorter wax crystals [13,17]. In contrast, the
lower lid surface (Fig. 4C–D) was covered with discrete, pillar-like
wax structures, 1.7860.36 mm (mean 6 s.d., n=18) in height and
1.57 mm (median, range=3.45 mm, n=37) in diameter. The
individual micropillars were unevenly distributed across the
surface and sometimes densely clustered so that they appeared
merged into solid blocks. The cuticular surface in between the
micropillars was perfectly smooth and free of any crystal structures
(Fig. 4C). The largest gaps between (clusters of) micropillars were
typically 2.3460.62 mm (mean 6 s.d., n=17) wide.
Investment in prey attraction by N. gracilis is
concentrated on the pitcher lid
We compared the nectar production of N. gracilis and N.
rafflesiana (without wax crystals under the lid) in the same field site,
sampling every second day from both peristome and lower lid
surface over a period of two weeks. The median area-specific daily
amount of sugar secreted on the lower lid surface was 3.4 times
higher in N. gracilis than in N. rafflesiana (Mann-Whitney U test,
n1=9, n2=10, Z=3.184, P,0.01; Fig. 5). In contrast, N. rafflesiana
pitchers secreted slightly higher amounts of sugar onto the
peristome; however, this difference was not statistically significant
(Mann-Whitney U test, n1/2=10, Z=1.285, P=0.2).
The trapping function of the lower lid surface in N. gracilis
constitutes a new trapping mechanism that has not been described
previously. So far, the lid was only thought to play a role in prey
attraction and as a protection against rain that would otherwise
dilute the pitcher fluid . The precarious position directly above
the pitcher opening (Fig. 1B), however, makes the lower lid surface
highly suitable as a trapping device. Our experiments have
demonstrated the effectiveness of the lid trapping mechanism for
ants, but we have observed it to work for beetles (Coccinellidae)
and flies (Musca domestica) as well (Video S3).
The microroughness created by the wax pillars is likely to
reduce the contact area between the ants’ smooth adhesive pads
and the lid surface . However, some of the gaps between the
micropillars may be large enough ($3 mm) for ant claws to
Figure 2. Contribution of the individual N. gracilis pitcher surfaces to prey capture under different environmental conditions. (A)
Experimental setup to test how rain drops falling onto the pitcher lid affect ant capture. (B) Proportion of ant visitors to each pitcher surface that fell
into the pitcher, under ‘dry’, ‘raining’, and ‘wet’ treatment condition, respectively. The interaction of pitcher surface and experimental condition was
highly significant (P,0.001).
Figure 3. Biological relevance of the lid capture mechanism.
The natural prey capture rate of pitchers with a non-slippery PDMS
coating applied to the lower lid surface is reduced in comparison to the
untreated control group (*: P,0.05).
New Trapping Mechanism in Nepenthes Pitcher Plants
PLoS ONE | www.plosone.org3June 2012 | Volume 7 | Issue 6 | e38951
interlock [16,19]. Furthermore, the compact, clustered arrange-
ment of the micropillars should render them less likely to break
under the claw-induced stress. Effective claw use may explain the
observed good walking performance of ants in the absence of rain
Intermittent slipperiness of the peristome has been suggested as
a strategy to ensure the survival of ‘scout’ ants and promote
subsequent recruitment or worker ants to the pitcher. The lid
capture mechanism may have a similar effect on the recruitment of
social insects to the nectaries under the lid. Rainfalls in the
distributional range of N. gracilis (Borneo, Sumatra, Malay
Peninsula and central Sulawesi ) are typically brief and heavy
with intensities of up to over 90 mm h21and most rain falling
within less than one hour [20,21]. The lower lid surface is
therefore a safe place to forage for most of the time. The
marginally significant reduction of trapping success by the ‘anti-
slip coating’ on the lower lid surface, despite the presence of other
effective trapping mechanisms, indicates an important contribu-
tion of the lid towards natural prey capture in N. gracilis. It is
currently not clear whether this mechanism is a unique feature of
N. gracilis or a more widespread phenomenon in Nepenthes. A
similar manipulation of the lid in N. rafflesiana pitchers in a recent
study did not show an effect on prey capture , and wax crystals
are absent from the lower lid surface of this species. Thus, it is
likely that the wax micropillars are crucial for the trapping
function of the lid in N. gracilis.
Our results suggest that N. gracilis has not only evolved special
morphological adaptations to capture prey with the pitcher lid, but
has also adjusted its nectar secretion patterns to increase prey
attraction to the lower lid surface. The peristome of N. gracilis
pitchers, although fully functional (Fig. 2B), is very narrow, and
larger insects can easily use their claws to hold on to its outer edge
while harvesting nectar (Fig. 1A). It has recently been demon-
strated that many Nepenthes species have specialised to prioritise
either the peristome or the inner wall for trapping, manifested in
two extreme pitcher morphologies: strongly enlarged peristomes
and smooth inner walls on one hand, and narrow peristomes and
well-developed wax crystal layers on the other . N. gracilis has
further specialised by evolving a new type of wax crystal surface
under the pitcher lid. These wax crystals appear to provide the
right level of slipperiness to cause insects to fall into the pitcher
when the lid vibrates while allowing them to approach the
nectaries safely at other times. Further experiments and field
studies should be conducted to elucidate the detailed biomechan-
ical underpinnings of this new trapping mechanism, and to
investigate what implications it has for the prey spectrum of N.
Figure 4. Microscopic structure of the wax crystal surfaces. (A–B) Microstructure of the crystalline wax layer on the inner pitcher wall. (A) Top
view, showing a dense network of thin, upright wax platelets (scale bar: 5 mm). The freeze-fracture side view (B) reveals the internal organisation of
the surface (scale bar: 2 mm). (C–D) Microstructure of the lower lid surface. (C) Top view: the wax crystals form solid, pillar-like structures, unevenly
distributed across the surface and surrounded by smooth cuticle (scale bar: 5 mm). (D) Side view of the wax pillars (scale bar: 2 mm).
New Trapping Mechanism in Nepenthes Pitcher Plants
PLoS ONE | www.plosone.org4 June 2012 | Volume 7 | Issue 6 | e38951
Materials and Methods
Permission to conduct field research in Brunei Darussalam was
granted through the appointment of UB as a research associate
with University Brunei Darussalam. No further permits were
required as the study was conducted on publicly owned, not
protected land. N. gracilis and N. rafflesiana are not protected under
Brunei law. CITES export (No. BA/MAP/02/1003) and import
(No. 358814/05) permits were obtained to export plant material
for SEM analysis.
Plant material and field site
The study was conducted in the Tutong district of Brunei,
Northern Borneo, in July 2011. Our field site was a heavily
degraded roadside habitat with open, shrub-dominated vegetation
over white silica sands. Both N. gracilis and N. rafflesiana are highly
abundant in this site while the hybrid N. gracilis 6N. rafflesiana is
rare. All experiments were performed on upper pitchers of N.
gracilis, using either live pitchers in the field, or freshly collected
pitchers in the laboratory. Upper pitchers of N. rafflesiana were
used for comparison in the measurements of nectar production.
Plant material for SEM analysis was obtained from the field (two
pitchers from different plants) and from the Royal Botanic
Gardens of Kew (three pitchers from two different clones/three
Effect of simulated rain on the capture efficiency of N.
Five upper pitchers were tested under three different conditions:
dry, during and directly after simulated rain. Each pitcher was
collected from the field immediately (,30 min) before the start of
the experiment by cutting the leaf at the base. In the laboratory,
the leaf was fixed to a tripod stand to simulate the natural
orientation of both leaf and pitcher. A colony of Crematogaster sp.
ants was collected from the same field site three days in advance
and kept in a plastic container. The ants were given access to the
experimental pitcher via a wooden skewer and immediately started
to recruit workers to forage on the secreted nectar.
Rain was simulated experimentally using an ExadropTMdrip
infusion system with a precision flow control (B. Braun,
Melsungen) attached to a 1.5-litre plastic bottle with distilled
water. The outlet of the drip tube was fixed to a standard
photographic tripod and positioned in 50 cm distance directly
above the pitcher lid (Fig. 2A). The drip frequency was adjusted to
0.22–0.32 s21. The simulated rain drops had a mass of 38–44 mg
(range of n=60 droplets), corresponding to a spherical drop
diameter of 4.2–4.4 mm, and reached a velocity of 3.060.3 m s21
(mean 6 s.d. from n=10 droplets measured using an A-602f
Basler camera at 304 frames per second) which is roughly one
third of the terminal velocity for that drop size . For
comparison, most rain drops in tropical rains are typically between
1.5 and 3 mm in diameter, and frequently reach .4 mm at the
leading edge of storms or after interception by vegetation [23,24].
The impact momentum of our simulated droplets (0.125 g m s21)
is within the range of natural rain (0.028–0.60 g m s21) .
A digital video camera (Sony DCR-SR35E) was positioned in
front of the pitcher so that a full size view of the peristome and the
underside of the pitcher lid could be recorded. The foraging ants
were observed and videotaped for a total of 30 min on each
pitcher, 10 min each before, during and directly after simulated
rain. Videos were analysed by counting the number of falls from
each surface (underside of the lid, peristome, and inner pitcher
wall) in relation to the number of visitors on the respective surface.
Since the inner pitcher wall bears no nectaries it is not normally
visited by foraging ants, but ants foraging on the peristome
nectaries occasionally stray out onto the inner wall and get
trapped. We therefore counted the number of falls from both
peristome and inner pitcher wall in relation to the number of
visitors on the peristome.
An additional experiment was performed on an isolated N.
gracilis lid that was fixed in natural horizontal orientation on
a tripod using a paper clip. Crematogaster sp. ants were given access
to the lid as described above for the whole pitcher. We videotaped
the performance of the ants on the lower lid surface while
simulating rain with the above described drip method.
Anti-slip coating of the lower lid surface of naturally
Thirty N. gracilis pitchers (each on a different plant) were
labelled in the field and randomly assigned to an experimental or
a control group. Using a fine paint brush, a thin layer of a non-
toxic, transparent and odourless PDMS polymer (SylgardTM184,
Dow Corning, Midland) was applied to the lower lid surface of the
pitchers in the experimental group. SylgardTM184 has been
shown to have no measurable effect on insect attraction but
provide a hydrophobic, non-slippery surface for insects .
All prey was removed from the pitchers and the fluid was
filtered through a NucleporeTMtrack edge membrane filter
(25 mm diameter, 12 mm pore size, Dow Corning). A small
polyurethane cone (cut from a commercial ear plug) was inserted
into the tapered bottom end of the pitcher to prevent the loss of
prey. Prey was sampled every third day for a total of 19 days by
sucking out the pitcher fluid using a 10 mL syringe with an
attached silicone tube, transferring the fluid to a petri dish, and
removing all prey manually with a pair of fine spring steel
Figure 5. Area-specific nectar secretion onto the peristome and
the lower lid surface. N. gracilis pitchers secrete significantly larger
amounts of nectar under the lid than those of sympatric N. rafflesiana
New Trapping Mechanism in Nepenthes Pitcher Plants
PLoS ONE | www.plosone.org5 June 2012 | Volume 7 | Issue 6 | e38951
Comparison of the wax crystal structure on the lid and
the inner pitcher wall
Five N gracilis upper pitchers were collected in airtight plastic
bags and transported in a cool box to the laboratory where they
were quench-frozen (3–4 hours after collection) in liquid propane
cooled in liquid nitrogen. Approximately 1 cm2pieces of the
pitcher lid and inner pitcher wall were cut with a razor blade,
freeze-dried, mounted on SEM stubs and sputter-coated with
a 20 nm layer of gold. Alternatively, samples were freeze-fractured
before freeze-drying. The microstructure of the wax crystal layers
on the lower lid surface and inner wall surface was examined using
a Philips FEI XL30-FEG SEM with an accelerating voltage of
Measurement of nectar production in the field
In a plot of approximately 15650 m, 10 pitchers each of N.
gracilis and N. rafflesiana, each on a different plant, were labelled
and roofed with transparent sheets of stiff plastic foil to protect the
nectar from being washed off by rain. To prevent insects from
collecting the nectar, we applied sticky TangletrapTMresin to the
base of the leaf and enclosed each pitcher in a fine-mesh gauze
bag. At the start of the experiment, we removed all nectar from the
peristome and lower lid surface by repeatedly wiping the surface
with approximately 1 cm2sized squares of wet laboratory wipe
(KimwipeTM, Kimberley-Clarke, Reigate) held with a pair of self-
closing blunt forceps.
Nectar from both the peristome and the lower lid surface was
sampled every second day over a period of two weeks. Samples
were obtained by moistening the surface with a wet KimwipeTM
square and then wiping it with a dry piece of a highly absorbent
medical swap (SugiTM, Kettenbach Medical, Eschenburg) made
from cotton and cellulose. Individual SugiTMswaps were cut into
3–4 small pieces to minimise the use of absorbent material, and
were handled using forceps and latex gloves to avoid contamina-
tion. Samples from the peristome and from the lid of both Nepenthes
species were collected separately in Eppendorff tubes and dried
over silica gel for 5–7 days. The completely dried samples were re-
diluted in the smallest possible amount of distilled water (between
0.1 and 0.6 mL) depending on the amount of absorbent material
used, and the sugar content was measured with a handheld
refractometer (ATAGO, L. Ku ¨bler, Karlsruhe).
The measured sugar secretion was corrected for the varying
area of the sampled surfaces to allow for a direct comparison
between the two Nepenthes species regardless of their different
pitcher size and geometry. Surface areas were measured after the
final nectar sampling by mounting the surfaces flat (cut in smaller
pieces where necessary) on graph paper, pressing them down with
a glass plate, and taking a photograph from above. The areas were
then measured digitally using Scion Image (release Alpha 18.104.22.168,
Scion Corporation, Frederick) software.
Statistical analysis of data
Statistical tests were conducted using the software packages
BiAS. for Windows and R. Data were tested for normality using
Shapiro-Wilks tests, and non-parametric tests were used were
appropriate. Throughout the paper, descriptive statistics denote
mean 6 s.d. for normally distributed data and median and range
in all other cases. Effects were considered significant when
To analyse the results of the rain simulation experiment,
General Linear Mixed Models (GLMMs, appropriate for pro-
portional count data) were fitted to the data. The experimental
conditions (before/during/after simulated rain) and the individual
surfaces (peristome, lower lid surface, inner pitcher wall) were
considered fixed factors. To improve the accuracy of the model,
‘surface’ (nested in the random factor ‘pitcher’) was also included
as a nested random factor. We calculated two separate GLMMs,
with and without interaction of the fixed factors. In a second step,
a conventional one-way ANOVA was performed to compare both
models: significant differences between the models indicate
a significant fixed factor interaction.
the underside of the pitcher lid of N. gracilis.
Effect of simulated rain on ants foraging on
the underside of an isolated N. gracilis pitcher lid.
Effect of simulated rain on ants foraging on
rate: 428 s21, playback frame rate: 10 s21) of a house fly
(Musca domestica) being knocked off the underside of
an N. gracilis lid by a simulated rain drop and captured.
High-speed video recording (recording frame
The authors would like to thank the Royal Botanical Gardens at Kew for
kindly providing plant material, and the B. Braun Melsungen AG for
providing the drip systems used to simulate rain. We are grateful to
University Brunei Darussalam, Mr Harith Tinggal and family, and Dr Dan
Thornham for logistic support during the field work. Dr Charles Clarke
contributed thoughts and observations in fruitful discussions. Dr Francisco
Rodriguez-Sanchez gave valuable advice on the use of GLMM statistics to
analyse our data.
Conceived and designed the experiments: UB BDG WF. Performed the
experiments: UB. Analyzed the data: UB. Contributed reagents/materials/
analysis tools: JS TUG. Wrote the paper: UB WF. Prepared samples for
1. Ellison AM, Gotelli NJ (2009) Energetics and the evolution of carnivorous plants
– Darwin’s ‘most wonderful plants in the world’. J Exp Bot 60: 19–42.
2. Chin L, Moran JA, Clarke C (2010) Trap geometry in three giant montane
pitcher plant species from Borneo is a function of tree shrew body size. New
Phytol 186: 461–470.
3. Moran JA, Hawkins BJ, Gowen BE, Robbins SL (2010) Ion fluxes across the
pitcher walls of three Bornean Nepenthes pitcher plant species: flux rates and gland
distribution patterns reflect nitrogen sequestration strategies. J Exp Bot 61:
4. Bauer U, Grafe TU, Federle W (2011) Evidence for alternative trapping
strategies in two forms of the pitcher plant, Nepenthes rafflesiana. J Exp Bot 62:
5. Bauer U, Clemente CJ, Renner T, Federle W (2012) Form follows function:
morphological diversification and alternative trapping strategies in carnivorous
Nepenthes pitcher plants. J Evol Biol 25: 90–102.
6. Bonhomme V, Pelloux-Prayer H, Jousselin E, Forterre Y, Labat J-J, et al. (2011)
Slippery or sticky? Functional diversity in the trapping strategy of Nepenthes
carnivorous plants. New Phytol 191: 545–554.
7. Grafe TU, Scho ¨ner CR, Kerth G, Junaidi A, Scho ¨ner MG (2011) A novel
resource-service mutualism between bats and pitcher plants. Biol Lett 7: 436–
8. Juniper BE, Robins RJ, Joel DM (1989) The carnivorous plants. London:
9. Merbach MA, Zizka G, Fiala B, Maschwitz U, Booth WE (2001) Patterns of
nectar secretion in five Nepenthes species from Brunei Darussalam, Northwest
Borneo, and implications for ant-plant relationships. Flora 196: 153–160.
New Trapping Mechanism in Nepenthes Pitcher Plants
PLoS ONE | www.plosone.org6 June 2012 | Volume 7 | Issue 6 | e38951
10. Bauer U, Willmes C, Federle W (2009) Effect of pitcher age on trapping
efficiency and natural prey capture in carnivorous Nepenthes rafflesiana plants. Ann
Bot 103: 1219–1226.
11. Bohn HF, Federle W (2004) Insect aquaplaning: Nepenthes pitcher plants capture
prey with the peristome, a fully wettable water-lubricated anisotropic surface.
PNAS 101: 14138–14143.
12. Knoll F (1914) U¨ber die Ursache des Ausgleitens der Insektenbeine an
wachsbedeckten Pflanzenteilen. Jahrb Wiss Bot 54: 448–497.
13. Juniper BE, Burras JK (1962) How pitcher plants trap insects. New Scientist 13:
14. Gaume L, Forterre Y (2007) A viscoelastic deadly fluid in carnivorous pitcher
plants. PLoS ONE 2: e1185.
15. Bauer U, Bohn HF, Federle W (2008) Harmless nectar source or deadly trap:
Nepenthes pitchers are activated by rain, condensation and nectar. Proc R Soc B
16. Scholz I, Bu ¨ckins M, Dolge L, Erlinghagen T, Weth A, et al. (2010) Slippery
surfaces of pitcher plants: Nepenthes wax crystals minimize insect attachment via
microscopic surface roughness. JEB 213: 1115–1125.
17. Gorb E, Haas K, Henrich A, Enders S, Barbakadze N, et al. (2005) Composite
structure of the crystalline epicuticular wax layer of the slippery zone in the
pitchers of the carnivorous plant Nepenthes alata and its effect on insect
attachment. JEB 208: 4651–4662.
18. Clarke C (1997) Nepenthes of Borneo. Kota Kinabalu: Natural History
19. Dai Z, Gorb SN, Schwarz U (2002) Roughness-dependent friction force of the
tarsal claw system in the beetle Pachnoda marginata (Coleoptera, Scarabaeidae).
JEB 205: 2479–2488.
20. Bidin K, Chappell NA (2006) Characteristics of rain events at an inland locality
in northeastern Borneo, Malaysia. Hydrol Process 20: 3835–3850.
21. Cranbrook G G-H Earl of, Edwards DS (1994) Belalong: a tropical rainforest.
London: Royal Geographical Society; Singapore: Sun Tree Publishing.
22. Gunn R, Kinzer GD (1949) The terminal velocity of fall for water droplets in
stagnant air. J Meteor 6: 243–248.
23. Ulbrich CW, Atlas D (2007) Microphysics of raindrop size spectra: tropical
continental and maritime storms. J Appl Meteor Climatol 46: 1777–1791.
24. Brandt CJ (1989) The size distribution of throughfall drops under vegetation
canopies. Catena 16: 507–524.
25. Kimble P (1996) Measuring the momentum of throughfall drops and raindrops.
Masters thesis (Western Kentucky University). Available: http://
New Trapping Mechanism in Nepenthes Pitcher Plants
PLoS ONE | www.plosone.org7June 2012 | Volume 7 | Issue 6 | e38951