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
1 Evolutionary Biomaterials Group, Department Arzt, Max Planck Institute for
Metals Research, Heisenbergstrasse 3, 70569 Stuttgart, Germany
2 Institute of Botany, University of Hohenheim, Garbenstrasse 30, 70593
Stuttgart, Germany
* Author for correspondence (e-mail: o.gorb{at}mf.mpg.de)
Accepted 18 October 2005
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Summary |
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Key words: trapping function, wax layer, microstructure, platelet, compound class, chain length distribution, hardness, elasticity modulus, friction force, pad contamination, real contact area, Nepenthes alata
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Introduction |
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To trap insects, pitcher-like leaves or analogous leaf arrangements have
been evolved in carnivorous plants. Traps of this type developed repeatedly in
the evolution of angiosperms and occur in rather unrelated taxa
(Juniper, 1986;
Barthlott et al., 2004
). The
pitchers are passive `pitfall' traps, but may also contain devices for insect
restraint and impedance. In representatives of Sarraceniaceae, for example,
long hairs directed downward on the inner surface of pitchers enable such
traps to act like lobster pots (or weir baskets), preventing insects from
moving back once they have entered (Adams
and Smith, 1977
; Slack,
1983
). Anti-adhesive surfaces within the pitchers covered by
epicuticular wax (EW) crystals are another special adaptation. Such wax layers
occur in Brocchinia reducta Baker, Darlingtonia californica
Torr. (combined with downward-pointed hairs) and other carnivorous plant
species, and are probably most developed in Nepenthes spp.
(Juniper and Burras, 1962
;
Juniper, 1995
; Gaume et al.,
2002
,
2004
).
Within Nepenthes pitchers, surfaces having different functions are
arranged in distinct zones serving for insect attraction, retention, digestion
and uptake of released nutrients (Juniper
et al., 1989; Gaume et al.,
2002
). The slippery zone, located above the digestive one, is
obviously crucial for successful trapping
(Lloyd, 1942
;
Juniper and Burras, 1962
;
Juniper et al., 1989
; Gaume at
al., 2002
,
2004
). It is covered by a layer
of crystalline EW. Insects are not able to get a foothold on this layer, they
glide in the digestive fluid and are restrained from further escape. This
function of the wax covering was first observed by McFarlane
(1893
) and then confirmed by
others (e.g. Knoll, 1914
).
The micromorphology of EW from the slippery (waxy) zone in N.
rafflesiana was studied by Juniper and Burras
(1962), using the carbon
replica technique and inspection by transmission electron microscopy (TEM).
The wax covering was shown to consist of two superimposed layers of EW
crystals, with the upper layer composed of wax platelets arranged parallel to
the surface and overlapping each other. The layer beneath was a complex
network of partially fused crystals. Since micrographs of detached upper
platelets often showed thin stalk-like structures, it was concluded that the
platelets are arranged like roof tiles and fastened to the lower layer only by
thin `stalks' (Martin and Juniper, 1980). Insects moving on the upper layer
would therefore readily cause detachment of the platelets and, because of
this, would lose their footing.
Recent studies on N. alata pitchers using scanning electron
microscopy (SEM) show the upper platelets oriented perpendicular to the
surface (Gaume et al., 2002;
Riedel et al., 2003
). In wax
isolation sequences employing both mechanical and dissolution steps, gradually
changing wax compositions and polymeric aldehydes were found in the
arrangement of EW crystals (Riedel et al.,
2003
). It is known that polymeric aldehydes are responsible for
partial resistance of crystals to solvent attack
(Haas et al., 2001
). They
probably also enhance the mechanical stability of EW microstructures. These
components may therefore play an important role in the interaction of insect
feet with the underlying wax.
The results presented so far are focused either on wax structure or on insect behaviour, and a convincing explanation of the anti-adhesive mechanism in the waxy zone is still lacking. The goal of this study was to combine investigations on wax micromorphology, chemical composition and mechanical properties with behavioural experiments using insects in order to explain the function of the EW layers in insect capture.
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Materials and methods |
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The two-spotted ladybird beetle Adalia bipunctata L. (Coleoptera,
Coccinellidae) was chosen as a model insect species for friction tests because
of its availability and appropriate size (body length=4.8±0.3 mm, mean
± S.D., N=20). Capture of beetles by the
pitchers of several Nepenthes species has previously been reported
(Kato et al., 1993;
Moran, 1996
;
Moran et al., 1999
).
The beetles bear adhesive pads, belonging to the hairy type of locomotory
attachment devices in insects (Gorb and
Beutel, 2001; Beutel and Gorb,
2001
; Gorb, 2001
).
Adult insects were obtained from a commercial supplier (Katz Biotech Services,
Welzheim, Germany) and kept in a small terrarium at 2224°C,
4060% humidity. Beetles were fed with a weak solution of honey in
tapwater.
Micromorphological studies
Epicuticular wax layers
The micromorphology of the waxy zone was studied using SEM and TEM.
For SEM, small pieces (ca. 1 cm2) of the pitcher wall were cut out with a razor blade from the middle part of the waxy zone, mounted on holders by means of conductive carbon double-sided adhesive tape, air-dried for several days, and sputter-coated with gold (10 nm). Pitcher samples were observed either intact or after treatment with chloroform for 20 s at room temperature, or at 60°C. Solvent-free removal of the upper EW layer was achieved by application of a two-component dental wax Coltène President light body (Coltène Whaledent Dentalvertriebs GmbH., Konstanz, Germany) for 5 min, peeled off after polymerisation. Detachment of the upper EW layer was similarly carried out using collodion films (see below). Intact and treated samples were examined in a Scanning Electron Microscope LEO 1530 VP at 37 kV. Crystal dimensions were quantified from digital images using SigmaScan software (SPSS).
Isolated wax crystals of the upper wax layer
Mechanically detached crystals were also examined by the
carbonplatinum coating technique
(Schwarz and Gorb, 2003). Wax
crystals were collected by wiping off the inner surface of the pitcher using a
soft paintbrush. The crystals were transferred onto the copper TEM slit, and
covered with thin transparent pioloform film, by shaking the brush over the
film. The crystals were coated with carbonplatinum at a shallow angle
(about 15°). This method resulted in an increase of image contrast and
better appearance of surface structures. The preparations were observed and
photographed in a Philips CM10 TEM. This method enabled observations of wax
crystals from the upper layer only.
Attachment organs of insects
To obtain detailed information about the attachment organs of the insect
species used in the experiments, tarsi were cut from legs, fixed in 70%
ethanol, dehydrated in an increasing series of ethanol, critical-point dried,
mounted on holders, sputter-coated with goldpalladium (10 nm) and
examined in a Hitachi S-800 SEM at 20 kV. The density of hairs per 1
mm2 of the pad area was quantified from digital images using
SigmaScan software (SPSS).
To study the effect of wax surfaces on attachment pads, insects were allowed to walk for 1 min on each surface used in the friction experiments (see below). The beetle was transferred to a clean glass plate, and its legs were immediately cut off with a sharp razor blade and mounted on SEM-holders using conductive carbon double-sided adhesive tape. On each plant surface, at least three beetles were tested. Insects demonstrating cleaning behaviour of the tarsi were excluded from the further microscopic procedure. Preparations were air-dried, sputter-coated with goldpalladium, and examined in the SEM.
Epicuticular wax analysis
Prior to extraction of EW from the waxy zone, intact pitchers were immersed
in hot chloroform (60°C, 1 min) with the solvent level kept carefully
below the peristome in order to remove wax material from the upper surface of
pitchers only. Afterwards, cylinders containing only the waxy zone were
excised out of pitchers. To obtain EW from this zone, a two-step extraction
procedure was employed by immersing cylinders first in cold (room temperature,
20 s) and then in hot (60°C, 20 s) chloroform. In other samples, the upper
EW layer was obtained by application of highly viscous solution of collodion
in acetone/n-amyl acetate (2:1, v:v). After evaporation of the
solvent, resulting films were peeled off and wax attached to the films was
extracted with cold chloroform (Haas and
Rentschler, 1984). Areas from which the EW was extracted, were
determined on cylinders dissected in longitudinal strips. The strips were
taken by a CCD camera, and the areas were quantified from digital images using
WinDIAS software (Delta-T Devices Ltd., Cambridge, UK).
Wax extracts were analysed by thin-layer chromatography (TLC) and gas
chromatography (GC) as already described
(Haas et al., 2003). The
individual extracts were fractionated into compound classes by preparative TLC
on silica gel G (Riedel-deHaen) with the two solvents: (1) n-hexane
and (2) chloroform/n-hexane (75:25, v:v). Spray reagent for detection
(UV 360 nm) was 0.005% primuline in acetone/water (80:20, v:v). Individual
fractions were analyzed by GC using a Shimadzu GC-17A gas chromatograph
(Kyoto, Japan) equipped with a CP-Sil 8 CB capillary column (25 mx0.32
mm, Varian-Chrompack, Middelburg, The Netherlands), on-column injector and
FID. Operating conditions of the chromatograph were as follows: detector
temperature, 360°C, linear velocity of helium carrier gas, 30 cm
s1. The column temperature was initially set to 160°C
for 2 min and then increased by 8°C min1 up to 340°C
(or 360°C for esters). The fractions of primary alcohols were analysed as
trimethylsilyl ethers after derivatization with
N,O-bis-(trimethylsilyl)-acetamide/pyridine (1:1, v:v). Free fatty acids were
converted to methyl esters using BF3/methanol (14%, w:w).
Appropriate internal standards were employed for each fraction.
Data shown in Table 1 and Fig. 3 represent mean values of at least three independent samples, each analysed in duplicate. A fraction presumably containing triterpenoids was further analysed by gas chromatography and mass spectrometry (GC/MS).
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|
Nanoindentation experiments with wax samples
Preparation of wax samples
For the first set of nanoindentation experiments, the samples of the waxy
zone of the pitcher were prepared in the same way as for SEM study but without
coating. Two types of samples were studied: (1) intact pitchers and (2)
pitchers after solvent-free removal of the upper EW layer by application of
the two-component dental wax (see above).
In the second set of experiments, waxes transferred onto a glass substrate were tested. (1) Wax of the upper EW layer was extracted with cold chloroform as described above. The solvent was evaporated and the residue applied onto glass coverslips (10 mm in diameter). (2) Wax from the lower layer was extracted with hot chloroform and transferred to the glass substrate as described above. Coverslips with waxes were attached to holders by means of double-sided adhesive tabs.
Nanoindentation experiments
Nanoindentation is widely used to determine material properties of a
variety of materials and material systems. The layers under investigation were
expected to be highly structured and compliant. In addition, the exact layer
thickness and the properties of the substrate (for the measurements on the
fresh plant) were unknown. Therefore, it was necessary to perform very
sensitive tests to determine the first point of contact of the indenter tip
with the surface and to select a method which provides information about the
contact behaviour according to the indentation depth.
All tests were carried out using the Nano Indenter SA2 (MTS Systems
Corporation, Oak Ridge, TN, USA). As an indenter tip, a three-sided diamond
pyramid (Berkovich-shape) was used. During the indentation process, the tip
was pushed into the surface at a constant strain rate of 0.05
s1 until the programmed depth limit of 1 µm was reached.
Time, load and displacement were continuously recorded using the CSM
(Continuous Stiffness Measurement) method, which allowed further determination
of mechanical properties as a function of indentation depth in each single
indentation test (Enders at al.,
2004). With this extension, the system applies a load to the
indenter tip in order to push the tip into the surface. Simultaneously, an
oscillating force was superimposed with force amplitude several orders of
magnitude smaller than the nominal load. Hardness and elasticity modulus were
calculated after Oliver and Pharr
(1992
). For each sample type,
10 different samples were used, and on each sample an array of 20 indents was
performed. In all, 800 indentation experiments were carried out.
Friction experiments with insects
To study the influence of waxy surfaces on the attachment ability of the
insects, friction experiments were carried out using tethered walking beetles.
Force measurements were performed using a load cell force transducer (10 g
capacity, Biopac Systems Ltd., Santa Barbara, CA, USA;
Gorb et al., 2004). Both males
and females were used for tests. Prior to the experiments, the elytra were
glued together with a small droplet of molten wax to make the insects
incapable of flying. Each beetle was attached to a force sensor by means of a
human hair (1015 cm long) glued to the dorsal side of the insect thorax
with a drop of molten wax. Plant samples (ca. 15 cm2) were cut out
of the waxy zone of fresh pitchers using a razor blade and then intact or
treated samples were glued with double-sided tape to a glass plate. In the
experiments, insects walked from the lower to the upper part of the zone,
simulating the situation of escaping from the pitcher. The force produced by
the insect moving on a horizontal test substrate was recorded.
Forcetime curves were used to estimate the maximal friction force
generated by beetles. The behaviour of insect individuals during force
experiments was observed and registered.
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Results |
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On TEM preparations of isolated wax crystals from the upper layer, platelets are often stuck together (Fig. 2A). Crystals consist of many layers orientated parallel to each other and to the crystal plane (Fig. 2C). Each platelet bears a `stalk' situated in the same plane as the crystal plate (Fig. 2A,C). In many cases, tiny pieces had broken off the platelets (Fig. 2B,C). Some crystals are partly exfoliated or even broken into several parts. This damage was the result of the preparation process, since a certain force was applied to brush the crystals mechanically from the plant surface.
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The platelets of the upper layer were readily removed either by dissolving in cold chloroform or by application of the dental wax, which was stripped off with the platelets adhered to it. This was also achieved by use of collodion films. The lower layer resembles foam composed interconnected membraneous platelets (Fig. 1C,D). Cavities between platelets forming the cells of the foam are usually irregular in shape and vary considerably in size: length=0.65±0.17 µm, mean ± S.D., N=20; width=0.42±0.11 µm, N=20 (measured in the plane parallel to the pitcher wall surface). Crystals protrude from the surface at acute angles and show no clear orientation. They are mostly irregular in shape with pointed extensions, although almost oval or rectangular crystals also occur. These platelets are smaller that those of the upper layer: length=0.82±0.11 µm, N=20; width=0.53 ±0.11 µm, N=20; thickness=36.54±7.87 nm, N=20; all means ± S.D.). Complete removal of this layer may be achieved by the use of hot chloroform, resulting in a wax-free cuticle surface (Fig. 1E).
Chemical composition of epicuticular waxes
Treatment with cold chloroform dissolved the upper platelets; this extract
amounted to 7.8 µg cm2.
The second extraction step using hot chloroform removed the remaining lower wax layer, yielding an amount of wax=31.6 µg cm2.
The compound classes of alkanes, aldehydes, primary alcohols, free fatty
acids, esters and triterpenoids occur in both wax extracts, but in different
percentages (Table 1). Wax
detached by collodion films (i.e. from the upper layer) showed relative
amounts of fractions rather similar to those of the first extract. Aldehydes
are the predominating compound class, especially in the second extract with
hot solvent, where they represent about 60% of total wax. Primary alcohols
contribute about 30% of the wax mass. Free fatty acids occur in relative
amounts of less than 10% with some deviation in the second extract. The other
fractions are minor constituents. Triterpenoids consist of triterpenones
(- and ß-amyrenone, taraxerone,
-taraxasterone) and probably
methoxylated triterpenols.
Chain length distributions of the aliphatics do not differ from those usually found in plant waxes, i.e. odd-numbered homologues predominate in alkanes, with even-numbered ones in the other fractions (Fig. 3). Alkane profiles from the two subsequent extracts are relatively similar, having C29 as main constituent (Fig. 3B). The homologue distributions of both aldehydes and primary alcohols from the first extract are bimodal with maxima at C24 and C30 (Fig. 3C,D). In their profiles from the second extract, however, only the peak at C30 is pronounced. For the aldehydes, the relative amount of C30 represents about 60% of all homologues, being twice as high as that from the first extract (Fig. 3C). For the primary alcohols, the increase of C30 in the second extract is less marked (Fig. 3D). Free fatty acids also show bimodal profiles with the first maximum at C24 or C26 (Fig. 3E). In the second extract, a slight tendency towards higher chain lengths is seen. The same tendency was also found with esters (Fig. 3, compare A with E).
Taking into account the different proportions of compound classes and diverging profiles of the corresponding fractions, the two layers of EW from consecutive extraction steps are clearly distinguishable by their chemical composition.
Mechanical properties of the waxes
The nanoindentation data revealed significant differences between the
deformation behaviour of the fresh intact plant surface and samples with the
upper wax layer removed (Fig.
4A,B; Table 2). The
data on hardness (H) and elasticity modulus (E) differed in these samples by
more than one order of magnitude. The intact surface appeared much softer and
more compliant than when the upper wax layer was removed. The hardness data
scatter more widely for the intact surface, implying several `breaking'
processes during the deformation (Fig.
4A). After removal of the upper layer, such deformation behaviour
was no longer seen. The curves shown in
Fig. 4A,B represent the mean
values of 200 indentation experiments performed on each sample type. However,
for the intact surface, only the first 35 experiments on each sample
could be used (3050 indents), since after that the data showed an
unusual deformation behaviour. We believe that this was due to a gradual
contamination of the diamond tip with broken wax crystals. Reference
measurements on fused silica (SiO2), used as a calibration material
in nanoindentation tests, showed that for the very first indent
(Fig. 4C, Indent 1) on the
silica after the series of tests on the wax surface, an atypical
loaddisplacement curve was obtained due to the contaminated tip. Only
after a deep imprint in silica did data represent typical values for the
reference material (Fig. 4C, Indent 2). This effect might be explained by the tip cleaning during the first
indentation on the reference material.
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Comparison of the deformation behaviour in all measured samples showed significant differences between the extracted waxes applied on the glass substrate, and the samples from fresh plant surfaces (Fig. 5; Table 3). However, no difference was observed between the extracts from the upper and the lower layers (Table 2). Since the H and E values of wax samples were lower than those of the glass substrate (Hglass=4.3 GPa), the influence of the underlying material on indentation results can be excluded.
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Morphology of the attachment devices of the beetle Adalia bipunctata
The tarsus of A. bipunctata females consists of three segments
(Fig. 6A). The pretarsus bears
two claws curved ventrally. Attachment pads are of the hairy type. The ventral
side of the two first proximal tarsomeres (12) is densely covered by
tiny setae (1st tarsomere: ca. 11.5x103 setae
mm2; 2nd tarsomere: ca. 22.5x103 setae
mm2; Fig.
6B). The setae are more or less uniform, with curved flattened and
widened tips (width=2.08±0.36 µm, mean ±
S.D., N=35) called spatulae
(Fig. 6C,D).
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Since the friction force generated by different insect individuals varied greatly, even on glass (mean=2.635±1.314 mN, N=44), we used data, normalised to values obtained on glass, for comparison of different surfaces. For each individual, the force, obtained on a test plant surface, was compared to that on glass (considered as 100%).
The maximal friction force was significantly different for the substrates tested (KruskalWallis one-way ANOVA on Ranks, H3,56=47.105, P<0.001). The highest force was measured on the plant surface lacking wax (Fig. 7B). On the plant substrates covered with wax, insects generated the lowest force. There was no significant difference in the beetles' performances on waxy plant surfaces either with only one lower wax layer, or with both lower and upper wax layers. In comparison with glass, the force on the plant surface with wax completely removed was significantly higher, whereas on waxy surfaces, insects performed much worse.
Behaviour of insects on tested surfaces
Experimental insects performed well and showed normal locomotion on the
glass plate and on the plant surface with wax removed. On both types of waxy
surfaces, beetles usually were not able to get a grip and slid over the
surfaces, refused to walk and came to a standstill or even turned over on
their backs.
Adhesive pads of insects walked on different surfaces
SEM observation of adhesive pads in insects revealed the difference between
insects walking on different surfaces. After experiments on both treated waxy
substrates (when only the upper wax layer or both wax layers were removed),
the pads remained clean (Fig.
8A,B). In insects used in tests on the intact waxy surface,
contamination of the tenent setae with wax crystals was observed
(Fig. 8CE).
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Discussion |
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Micromorphology of the two superimposed wax layers
The upper EW layer comprises densely arranged platelets oriented more or
less perpendicular to the inner pitcher surface. These platelets show, after
mechanical detachment and examination in the TEM, particular characteristics
of crystallinity. They are very fragile and break easily into tiny pieces.
This fragility is rather uncommon among plant waxes, since EW crystals exposed
to mechanical stress are usually rendered to a smear with almost completely
disintegrated microstructure rather than to fragments
(Neinhuis and Barthlott,
1997). The brittleness of the platelets, in contrast, seemingly
reflects their high crystallinity. Further signs of crystallinity are provided
by the microstructure. The platelets are built up by many layers oriented
parallel to each other, which could indicate strata of crystal growth. Such
crystallization of wax constituents as subsequent layers has recently been
demonstrated in wax crystals of e.g. Galanthus nivalis
(Koch et al., 2004
).
The connection of the upper platelets with the subjacent EW layer remains
uncertain. Due to their spatial orientation, points of contact are not visible
in SEM images. `Stalks' were never found projecting from the upper (i.e.
visible) rims of the platelets. This implies that the `stalks' should be
directed downward and could well provide the connection to the lower layer.
Our results from N. alata do not agree with earlier interpretations
of the arrangement of these platelets
(Martin and Juniper, 1970;
Juniper et al., 1989
). The
upper platelets are not situated parallel to the surface like overlapping roof
tiles. Additionally, earlier interpretations imply bending of the `stalks' at
right angles to provide contact with the lower layer. Signs of such bending,
however, were never observed in the TEM images of isolated platelets.
On the other hand, the length of the `stalks' (up to 0.5 µm), as found in some examples, would not be sufficient to extend downward to the very cuticle surface. However, since the `stalks' probably represent the most fragile part of the platelets, they may act like preformed sites of breakage and would usually not be preserved in full length after mechanical detachment. Therefore, the upper platelets often show only parts of the `stalks', broken at different lengths.
The lower EW layer is composed of interconnected platelets, which do not have a preferred orientation and project from the surface at different angles. These crystals resisted low applied forces, e.g. brushing well. Complete removal of this layer may be achieved mechanically by applying higher shear forces or by the use of hot chloroform.
These morphological distinctions in the two wax layers correlated with differences in chemical composition of waxes.
Comparison of the chemical composition of waxes
The lower EW layer is not soluble in cold chloroform. Chloroform at this
temperature would be sufficient for complete dissolution of wax crystals if
only monomeric constituents are present
(Holloway, 1984;
Walton, 1990
). Our results
show that this treatment offers the opportunity to expose the lower layer,
since its micromorphology is indistinguishable from that disclosed by removal
of the upper platelets, peeled off adhering to the dental wax or collodion.
Hence, wax of the two layers can be isolated selectively by subsequent
dissolution steps using chloroform at different temperatures. The upper
platelets are dissolved in cold chloroform and the lower layer in hot
chloroform. Furthermore, the composition of the first extract closely
resembles that of the upper layer adhered to the collodion film. The reduced
solubility of the lower layer coincides with the presence of polymeric
aldehydes (Haas et al., 2001
).
The polymeric aldehydes are suspected to be responsible for dissolution
resistance against cold chloroform and the structural integrity of crystals
exposed to this treatment. The depolymerization of polymeric aldehydes using
hot chloroform usually leads to increased aldehyde contents in the extracts
obtained. This holds true for the subsequent extraction of wax from N.
alata pitchers, since the aldehyde content of the second extract is
clearly increased. As indicated by different solubility and aldehyde content,
the lower layer is obviously reinforced by polymeric aldehydes whilst crystals
of the upper layer are not. The fourfold increase in wax yield from the lower
layer is probably due to the higher density of interconnected wax crystals
composing this layer.
Primary alcohols are also present in appreciable amounts in the extracts
and could well influence crystal habits, since waxes rich in primary alcohols
often tend to crystallize as platelets
(Barthlott et al., 1998). High
percentages of aldehydes and primary alcohols in the Nepenthes wax
have also been reported by Riedel et al.
(2003
), and co-crystallization
of alcohols and aldehydes was deduced from this fact. The two EW layers are
also discernible by diverging homologue profiles of the most prominent
compound classes. The bimodal profiles of the upper layer show two separate
main constituents (C24, C30). This profile
characteristic is not present in the aldehydes from the lower layer and
primary alcohols are also rather inconspicuous. Aldehydes are markedly
restricted in the lower layer to one single compound (C30), as are
primary alcohols to a lesser extent.
The different chemical compositions of the two layers, concerning both
percentages of compound classes and homologue distributions, indicate their
independent formation. Recent findings on the transport of wax constituents to
the cuticle surface and subsequent crystallization would hardly allow a
differentiation of wax compounds and crystallization in two modifications at
the same time (Neinhuis et al.,
2001), implying that the formation of the two layers occurs at
somewhat different stages of pitcher development. However, detailed studies on
EW in developing pitchers are lacking, and it is not known whether the two
layers develop independently at the same time, or in sequence.
Thus, the two EW layers clearly differ in micromorphology and chemical composition, and this may influence their mechanical properties.
Differences in mechanical properties of waxes
The mechanical properties of plant waxes were measured using
nanoindentation experiments for the first time. Our nanoindentation tests with
intact and treated pitcher surfaces and wax extracts solidified on a hard
substrate showed that waxes of the upper and lower layers exhibit different
mechanical properties: wax of the lower layer is harder and stiffer than that
of the upper layer. The results of the mechanical tests on the fresh plant
surfaces lead to the conclusion that the wax layers studied may affect insect
attachment in different ways.
The hardness values obtained on the pitcher surfaces give information about
the mean pressure the crystals can withstand. For the whole span of the
penetration depth, this pressure is in the MPa range. A rough calculation of
the pressure that one seta of an insect applies onto the surface leads to
values of several kPa (Enders et al.,
2004). Thus, both wax layers appear to insects as mechanically
stable surfaces.
Regarding the upper wax layer, an indentation test performed using a pyramidal indenter tip gave additional information. Because of the angled sides of the tip, the crystals experienced a lateral force during further displacement, which led to a shear motion. Starting at the displacement of ca. 100 nm, hardness values began to scatter, and a tendency to lower values was observed. As described above, this implied several `breaking' processes during loading. It can be assumed that the crystals are mechanically relatively stable under loading in the normal direction, but more unstable when a lateral force is applied. However, to verify this assumption, more quantitative nanotribological tests are needed.
Moreover, the fast contamination of the indenter tip in the experiments with the upper wax layer (Fig. 4C) indicates that crystals, once broken or otherwise detached from surface tended to stick strongly to the tip. This result explains why wax crystals of the upper layer adhere well to the attachment organs of insects, and thus impede proper attachment. Such contamination was not observed in all other the samples studied here. During the indentation tests on the lower layer, as well as in the samples with extracted waxes, no adherence of wax material on the tip was revealed in reference tests on fused silica even after a lengthy series of measurements. These effects may be explained by the weak detaching ability of the crystals of the lower layer. In the case of extracts, it may be assumed that the extraction and solidifying processes caused the changes in the structure and chemical composition of waxes, and these changes led to differences in mechanical properties, both the hardness and elasticity moduli, of both wax layers, and in the behaviour of wax samples under load.
Effect of different wax layers on insect attachment
Laboratory experiments with Adalia bipunctata beetles showed that
waxes significantly reduce the attachment force of insects compared with
reference surfaces, such as glass and wax-free pitcher surface. Interestingly,
there was no difference in the behaviour and forces generated by beetles on
the substrates covered with wax from either the upper or lower layers. Thus,
both wax layers contribute to the trapping and retention functions of the
pitcher by means of a reduction in insect attachment. However, the
morphological study of pads in insects, walked on different surfaces, revealed
the essential difference between effects of each wax layer on insect pads. The
upper layer caused pad contamination with wax material, whereas the lower
layer did not contaminate the pad surface.
To explain the anti-adhesive properties of plant waxes, four hypotheses
have recently been proposed: (1) roughness hypothesis, (2) contamination
hypothesis, (3) wax-dissolving hypothesis and (4) fluid-absorption hypothesis
(Gorb and Gorb, 2002).
According to the first hypothesis, the crystalline wax results in
micro-roughness, leading to a considerable decrease in the real contact area
between the surface and setal tips of insect adhesive pads. The contamination
hypothesis states that wax crystals are easily detachable structures, which
may contaminate pads. The third and the fourth ones deal with the insect pad
secretion, which either dissolve the wax or is absorbed from the setal surface
by the wax layer, respectively. Previous studies provided data confirming the
first (Stork, 1980
;
Gorb, 2001
) and the second
hypotheses (Juniper and Burras,
1962
; Martin and Juniper,
1970
; Dixon et al.,
1990
; Gaume et al.,
2004
; Gorb and Gorb, in
press
). It was found that surface roughness in the range from 0.3
to 3 µm reduces strongly the attachment force
(Gorb, 2001
;
Peressadko and Gorb, 2004
),
and the increased size and aspect ratio of wax crystals causes stronger pad
contamination (Gorb and Gorb, in
press
).
|
On the basis of our data on the micromorphology of wax layers and wax crystals, the chemical composition of the waxes, results of nanoindentation experiments on different wax samples and friction tests with insects, a scheme of the functioning of the waxy zone in the N. alata pitcher and the role of the two waxy layers in prevention of insect attachment may be proposed.
Although the crystals of the upper layer are densely arranged, they remain separate and form no network. Their planes are oriented perpendicular to the pitcher surface, and they are attached to the adjacent substrates through small `stalks'. Therefore, the contact area between a platelet and a substrate is rather small. This causes the ease breakage of `stalks' and thus the facile removal of crystals of the upper layer, especially if a lateral force is applied. Moreover, these platelets are very brittle and may be easily exfoliated or broken to tiny pieces. Both entire crystals and small fragments may adhere well to insect feet and contaminate attachment organs (Fig. 9C). This prevents proper contact formation between adhesive pads and the surface, and leads to an essential reduction in the attachment force.
Platelets of the lower layer are highly interconnected and form a relatively stable network of crystals, oriented at acute angles to a substrate. The crystals do not detach easily and may remain intact even if the upper layer is removed. This network would be able to resist lateral forces applied by climbing insects, but due to its high micro-roughness, causes a considerable decrease in the real contact area with insects' adhesive organs (Fig. 9E). This provides the further reduction of insect attachment. Hence, the lower layer ensures the effectiveness of the whole system of the waxy zone of the pitcher for its trapping function.
Thus, our results on the waxy surface of the N. alata pitcher lead to the conclusion that a decrease in the attachment force of insects is provided by the two different mechanisms: (1) the contamination of insects' attachment organs (the upper wax layer) and (2) the reduction of the real contact area (the lower layer). However, effects of wax dissolving by the insect pad secretion and the secretion adsorption by wax layer cannot be entirely excluded. These hypotheses need to be additionally proven for the case of N. alata.
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List of abbreviations |
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Acknowledgments |
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References |
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