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First published online August 8, 2008
Journal of Experimental Biology 211, 2647-2657 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.019273
An insect trap as habitat: cohesion-failure mechanism prevents adhesion of Pameridea roridulae bugs to the sticky surface of the plant Roridula gorgonias
Evolutionary Biomaterials Group, Department of Thin-Films and Biological Systems, Max-Planck Institute for Metals Research, Heisenbergstraße 03, D-70569 Stuttgart, Germany
* Author for correspondence (e-mail: voigt{at}mf.mpg.de)
Accepted 28 May 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: adhesion, biomechanics, cuticle, insect–plant interaction, plant resin
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Several
carnivorous and protocarnivorous plants use `fly-paper' traps with tentacles
and/or capitate trichomes to capture their prey
(Lloyd, 1942;
Juniper, 1986). The released
glandular fluids make the surface shiny, and, by means of these reflective
surface properties, a large number of insects are attracted and can be
suddenly entangled when coming in contact with the sticky surface of the
plant.
Viscid secretions at trichome tips can be, in general, of three kinds: oily
(often aromatic), mucilaginous and resinous
(Lloyd, 1942). In the genera
Byblis, Drosera, Drosophyllum and Pinguicula, a hydrous
mucilage is found, whereas, in the South African perennial shrubs Roridula
gorgonias Planch. and R. dentata L. (Roridulaceae), the
transparent exudates of their glands are very different from those of
Drosera (Lloyd,
1934). The water-insoluble pellucid droplets of Roridula
plants have been observed to persist indefinitely on dead and dried-up leaves
(Bruce, 1907) or leaves
preserved in formalin (Lloyd,
1934). Marloth (Marloth,
1925) washed dried leaves with chloroform and found a very viscid
resinous residue containing 
10% of caoutchouc. These results were
confirmed by Lloyd (Lloyd,
1934), who extracted fresh leaves with acetone, yielding a resin
or a mixture of resins. Following this by extraction with petroleum ether
yielded an acetone-insoluble material having properties of caoutchouc.
However, no distinct chemical analyses on the composition of the
Roridula secretion have been performed.
The presumably resinous nature led to the conclusion that proteolytic
enzymes can be neither transported nor dissolved in the adhesive secretion
(Marloth, 1925). That is why
the `carnivorous syndrome' of Roridula plants is considered
controversial. Primarily, these plants were supposed to be carnivorous because
of the similarity of the tentacle-shaped trichomes to those in representatives
of the genera Drosera and Drosophyllum
(Darwin, 1875;
Marloth, 1903;
Fenner, 1904;
Bruce, 1907). As no digestive
organs in the trichomes and leaves were found, Roridula was placed in
the group of protocarnivorous plants – those that trap insects without
the ability to digest them (Marloth,
1910; Marloth,
1925; Lloyd, 1934;
Lloyd, 1942;
Juniper et al., 1989).
However, Midgley and Stock (Midgley and
Stock, 1998) found higher levels of nitrogen and stronger
ultraviolet reflectivity in the mature leaves of plants that captured insects,
compared with control plants. These data confirm the carnivorous nature of
R. gorgonias, despite the apparent lack of proteolytic enzymes.
However, Plachno and colleagues (Plachno
et al., 2006) observed phosphatase activity in the leaf epidermis
using enzyme-labeled fluorescence microscopy.
The leaf surface of Roridula is covered with numerous
tentacle-shaped and capitate trichomes of different size but similar
structure, concentrated along leaf margins and the main vein
(Fenner, 1904;
Bruce, 1907;
Uphof, 1962). The adhesive
secretion of the trichomes is extremely effective, as shown by the conspicuous
number of trapped insects of considerable size and mass, particularly flying
ones (Marloth, 1910;
Barthlott et al., 2004).
Hartmeyer (Hartmeyer, 1998)
suggested that the secretion of Roridula is the strongest glue of all
insect-trapping plants. Stuck insect parts and entire corpses of occasionally
considerable size and body mass have been found on the plant surface
(Marloth, 1903;
Marloth, 1910). Mean prey
length averaged 3.55±0.57 mm (±s.d.; N=109)
(Ellis and Midgley, 1996).
However, no representative systematic study of the insect species trapped by
Roridula plants has been performed to date. From a small sample of
R. dentata, representatives of Hymenoptera (25 individuals from
subfamilies Sphecinae and Apinae), Diptera (20 individuals from the family
Muscidae), Coleoptera (a few individuals from the families Coccinellidae and
Scarabaeidae), Hemiptera (single individuals from the families Lygaeidae,
Reduviidae and Membracidae) and Lepidoptera have been reported previously,
whereas bees, wasps and flies were the most abundant specimens found
(Marloth, 1903;
Marloth, 1910). Samplings of
individual leaf rosettes of 15 R. gorgonias plants after an
eight-week period resulted in a total of 109 trapped individuals belonging to
32 macro-invertebrate species (>2mm in length) consisting mainly of
dipterans and coleopterans (Ellis and
Midgley, 1996). Additionally, 122 micro-invertebrates (<2mm in
length) mainly from the orders Thysanoptera and Diptera have been counted.
Illustrating their potency, South African farmers call the plant `Vliegebos'
and suspend the plants in their houses as flytraps
(Marloth, 1925).
However, mirid bugs of the genus Pameridea (Heteroptera, Miridae,
Bryocorinae, Dicyphini) are obligately associated with Roridula
plants (Reuter, 1907;
Dolling and Palmer, 1991;
Picker et al., 2004) in a form
of digestive and pollinating mutualism
(Ellis and Midgley, 1996;
Reiner, 2003;
Anderson, 2005;
Anderson, 2006;
Anderson and Midgley, 2002;
Anderson and Midgley, 2003;
Anderson and Midgley, 2007;
Anderson et al., 2003). They
live omnivorously and walk confidently and quickly on the sticky plant surface
without becoming entangled and without hindrance. The bugs feed on the glued
insects and defecate on the leaves
(Marloth, 1903;
Lloyd, 1934). The nitrogen in
the faeces of the bugs is absorbed through the thin leaf cuticle of the plant,
thus providing up to 70% of the total plant nitrogen uptake
(Ellis and Midgley, 1996;
Anderson and Midgley, 2002;
Anderson and Midgley, 2003).
Similar relationships between mirid bugs and carnivorous plants are known from
representatives of the genera Byblis and Drosera in
Australia (Schuh, 1995).
Moreover, mirid bugs from the subfamilies Orthotylinae and Bryocorinae seem to
be specialized for living on glandular hairy plants
(Reuter, 1913;
Dolling and Palmer, 1991;
Falkingham, 1995;
Schuh, 1995;
Wheeler, 2001;
Sugiura and Yamazaki, 2006).
For example, the mirid species Dicyphus errans Wolff (Bryocorinae,
Dicyphini) avoids contact with sticky glandular secretions by means of
morphological (slim body, long and slender legs, elongated curved claws) and
behavioral (mode of locomotion, grooming) adaptations to hairy plant
substrates (Southwood, 1986;
Voigt et al., 2006a;
Voigt et al., 2007). D.
errans is often observed grooming various body parts
(Voigt, 2005). By contrast,
Pameridea bugs frequently touch the released viscid plant secretion,
which is spread evenly over ovoid glands and often over the trichome
multiseriate stalks and the plant stem
(Marloth, 1910).
Previous authors (Lloyd,
1934; Hartmeyer,
1996; Hartmeyer,
1998) have suggested that bugs must have some kind of defense and
resistance mechanisms to adhesive substances such as a sophisticated
arrangement of short bristles capable of protecting the body against
adhesives, specialized body cleaning adaptations and/or a locomotion mode in
which the body is kept elevated above sticky trichomes. Intensive body
grooming has been reported previously to be an essential adaptation of mirid
bugs to living permanently, and walking, on hairy and glandular hairy plant
substrates (Kullenberg, 1946;
Voigt et al., 2006b). Our
preliminary observations showed that Pameridea roridulae Reuter,
after being wrapped in a sticky leaf of R. gorgonias, continued
normal walking without body grooming.
Why does P. roridulae not stick to the adhesive secretion, whereas
numerous other insect species do? Is such an anti-adhesive property of the
mirid bug surface related to some specialized microstructure
(Fig. 1C), similar to that
previously described on unwettable surfaces of aquatic bugs (Anderson, 1976;
Anderson, 1977; Anderson, 1982; Perez
Goodwyn, in press; Perez
Goodwyn et al., 2008)? Does the solid epicuticle have a strong
repelling ability to nonpolar fluids (Fig.
1B)? Are there some fluids or easy-to-break solid layers on the
epicuticle that function to prevent direct contact between plant secretions
and the insect surface (Fig.
1D,E), as suggested by Lloyd
(Lloyd, 1934)?
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Adult C. vicina blow-flies were reared from commercially offered larvae (Angelmarkt Stephan, Stuttgart, Germany).
Light microscopy
A stereomicroscope Olympus SZX 12 with a DF PLAPO 1xPF objective
(Olympus Corporation, Tokyo, Japan) was used to observe P. roridulae
mirid bugs and captured insects on R. gorgonias. Images were taken
using a Nikon Coolpix E995 digital camera adapted to the stereomicroscope with
a C-Mount adapter and a MDC 2 relay lens MXA 29005 (Nikon Corporation, Tokyo,
Japan).
Cuticle prints of living and dead P. roridulae and C. vicina, pressed against a glass slide, were visualized using an upright AXIOPLAN microscope with an AxioCam MRc digital camera (Carl Zeiss MicroImaging GmbH, Jena, Germany) and AxioVision 3.1 software (AxioVision GmbH, München-Hallbergmoos, Germany).
Cryo-SEM
To visualize the details of interactions between plant and insect surfaces
in the freshly frozen condition, microscopy studies were carried out using a
Hitachi S-4800 cryo-SEM (Hitachi High-Technologies Corp., Tokyo, Japan)
equipped with a Gatan ALTO 2500 cryo-preparation system (Gatan, Inc.,
Abingdon, UK). Samples were mounted on metal holders, frozen in the
preparation chamber at –140°C, sputter-coated with
gold–palladium (3nm) and examined in a frozen state in the cryo-SEM at 3
kV accelerating voltage while at –120°C.
Plant trichomes, droplets and insect surface
Fresh samples of R. gorgonias, P. roridulae and C. vicina
were prepared and studied as described above.
Plant adhesive secretion on different surfaces
Cryo-SEM has been reported previously to be a successful method for
visualizing droplets of glycerine, water, lipids and other biological fluids
and their mixtures (Gorb et al.,
2007). Using tweezers, droplets of single tentacle-shaped and
capitate trichomes were manually brought into contact with surfaces of freshly
killed insects (C. vicina, P. roridulae), and small hemispherical
pieces of smooth Au–Pa-metallized epoxy resin Spurr
(Spurr, 1969;
Gorb, 2006), and examined
according to the method described above. The solid metallized Spurr surface is
known to increase the material contrast between the substrate and fluid
droplets as well as, owing to the substrate profile, allowing the observation
of droplets at an appreciable angle (Gorb
2006, Gorb et al.,
2007).
Plant adhesive drops treated with different solvents
Using tweezers, single tentacle-shaped trichomes with glandular droplets
were removed from the leaf margin, washed for 5 min in ethanol (Rotipuran®
99.8%, p.a., Carl Roth GmbH & Co. KG, Karlsruhe, Germany), acetone
(Rotipuran® 99.8%, p.a., ACS, ISO, Carl Roth) or cold chloroform
(99.8%, p.a., Merck KgaA, Darmstadt, Germany), placed on sample holders
and observed as described above. The results were compared with data on
untreated trichomes and those washed with aqua millipore.
Insect cuticle
Samples of P. roridulae and C. vicina – living,
dead, and dead washed with cold chloroform (5 min) – were mounted on
holders. Using a cold scalpel, fractures of frozen legs were performed in the
preparation chamber of the cryo-SEM at –140°C. Next, samples were
sputter-coated in the frozen condition (3 nm thickness of Au–Pa) and
examined in the cryo-SEM at –120°C and an accelerating voltage of 3
kV.
From digital images of 10 randomly selected points, the thickness of the epicuticular grease layer was estimated, using Sigma Scan Pro 5 (SPSS, Inc., Chicago, IL, USA) software. The data obtained were statistically processed using Kruskal–Wallis one-way ANOVA on ranks and an all pairwise multiple comparison Tukey test (SigmaStat 3.1.1® software, Systat Software, Inc., Richmond, CA, USA).
Measurements of adhesion forces
The measurements were carried out using a force transducer (10g capacity,
Biopac Systems, Santa Barbara, CA, USA), attached to a motorized DC3314R
micromanipulator with MS314 controller (World Precision Instruments, Sarasota,
FL, USA). A piece of double-sided carbon tape (5x5mm) was firmly
attached to the force transducer. Using tweezers, a tentacle-like glandular
trichome was removed from the leaf margin and attached by its base to the
tape, perpendicular to the force transducer
(Fig. 2). The trichome could be
moved up and down with a velocity of 100µm s–1. The
adhesive droplet on the trichome tip was brought into contact with the insect
cuticle of the ventral side of the abdomen (living and dead bugs and flies, as
well as chloroform-washed bugs) or with the glass surface. The droplet was
preloaded to a maximum of 50µN force and then withdrawn. Force–time
curves were used to estimate the maximum pull-off (adhesion) force. For each
test surface, 20 measurements on different sites (N=5 insects,
n=4 measurements per insect), and 120 single measurements in total
were carried out (N=6 surfaces, n=20 measurements per
surface). Kruskal–Wallis one-way ANOVA on ranks followed by an all
pairwise multiple comparison procedure (Tukey test) was used to evaluate
differences in the adhesion force values between the substrates (SigmaStat
3.1.1® software, Systat Software, Inc.). Laboratory conditions were
similar to those mentioned previously.
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Using a few representative force–time curves, measured on both living P. roridulae (N=3) and C. vicina (N=3), force–distance curves were calculated to estimate the work necessary to retract the adhering plant trichome from an insect cuticle at a distance of 1.5 mm.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Properties of the plant adhesive secretion
Shapes of trichome tips
The capitate and tentacle-shaped trichomes of R. gorgonias consist
of multicellular stalks and multicellular glandular heads releasing the
adhesive secretion (Fig. 4).
The width in the middle of secretion droplets ranged from 129 to 405 µm
(226.4±54.8 µm, mean ± s.d., N=120). The consistency
of the secretion seems to vary slightly between the very long, tentacle-shaped
and shorter capitate trichomes. The latter possess round, apparently more
viscous, stable droplets (Fig.
4B,C) that appear rough in the cryo-SEM preparations
(Fig. 4D). The tips of the
longer tentacle-shaped trichomes are equipped with ovoid, apparently less
viscous, secretion droplets, having a smooth surface at high magnification
(x1000) in the cryo-SEM (Fig.
4E). The adhesive fluid of different trichomes can spread over the
trichome stalk (Fig. 4E,F) and
even the leaf lamina (Fig. 4B).
Furthermore, we observed fluid filaments of up to 5 cm in length into which
the secretion, in particular those produced by the tentacle-shaped trichomes,
can be pulled after contacting a surface
(Fig. 3F,H,
Fig. 4A).
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Solubility of the adhesive secretion in different solvents
Treatment of the secretion droplets of the tentacle-shaped trichomes with
different fluids demonstrated differences in droplet solubility. The droplets
kept their volume and ovoid shape after treatment with aqua millipore
(Fig. 5A). After trichome
treatment with absolute ethanol, they were deformed, and the secretion was
partially removed such that some intact, convex glandular cells could be seen
(Fig. 5B,C).
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Contact behavior of the adhesive secretion on different substrates
Intact droplets were manually attached to insect cuticles or metallized
epoxy-resin surfaces. Contact formation between the glandular tip of
tentacle-shaped trichomes and P. roridulae was hardly detectable. The
secretion spreads on the surface of the mirid bug
(Fig. 6A,B). However, on single
bug setae, residual round droplets of the secretion were observed
(Fig. 6C). In contrast to the
mirid bug, the surface of the fly C. vicina induced formation of
round, droplet-shaped patches with a distinct boundary
(Fig. 6D–F). Similar
droplets formed on the surface of epoxy resin coated with Au–Pa
(Fig. 6G,H). Furthermore,
numerous flat micro-droplets were observed when the tip of a tentacle-shaped
trichome briefly contacted the metallized surface
(Fig. 6I).
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The adhesive force of the plant secretion on different surfaces
The measured adhesive force depended on the surface to which the
tentacle-shaped trichomes were brought into contact
(Fig. 7). The lowest values
were obtained on living mirid bug cuticle, and the highest ones on the cuticle
of living and dry flies, as well as on the cuticle of chloroform-washed mirid
bugs. These differences were statistically highly significant. During the
experiment, secretion frequently formed filaments with lengths ranging from 2
to 51 mm in 48% of the individual tests (for details, see Materials and
methods). The length of filaments (l) did not influence the measured
pull-off force (FA)
(FA=158.20–0.68l, r2=0.01,
F1,58=0.41, P=0.52, linear-regression analysis).
The work required to retract the trichome from the insect cuticle was
estimated from force–distance curves
(Fig. 8). It was much lower
(0.07±0.026 J) in living P. roridulae than in living C.
vicina (0.18±0.093 J) (for each species, N=3).
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Insect cuticle
Both insect species studied have an integument that bears large setae
(Fig. 6A,D) and a layer of much
smaller, procumbent microtrichia. When light pressure was applied by a clean
glass slide to the insect cuticle, prints of epicuticle grease became visible
in the phase-contrast mode of a light microscope
(Fig. 9). The cuticle of living
and dried P. roridulae mirid bugs left a distinct amount of fluid
residues (Fig. 9A–D), in
most cases forming a continuous layer on the glass surface. In contrast to the
mirid bug cuticle, cuticle prints of living C. vicina flies were seen
as discrete micro-droplets on the glass slide
(Fig. 9E,F).
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Cryo-SEM fractures clearly revealed an amorphous epicuticular grease layer
on the surface of the cuticle in living and dry P. roridulae. This
layer covers the entire insect surface, including procumbent microtrichia
(Fig. 10A,B,D,E). In living
and dry C. vicina, such a clear grease layer was not observed. The
thickness values of the layer were significantly higher (30-fold) in
living and dry mirid bugs compared with those of living and dry flies
(Table 1). In both insects
treated with chloroform, the grease layers seemed to be nearly removed
(significantly lower thickness values were measured; see
Table 1). Furthermore, in
chloroform-rinsed mirid bugs, a clean cuticle with clear uncovered
microtrichia was found (Fig.
10F, compare with Fig.
10D,E).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Barthlott et al., 2004). We
showed here that not only the tips of tentacle-shaped trichomes, but also
their stalks and the leaf lamina, are mostly coated with an adhesive
secretion. This coverage is responsible for the adhesive property of almost
the entire plant. The adhesive secretion is released in the form of shiny
droplets on the ovoid glandular heads of numerous capitate trichomes of
varying lengths. Very long tentacle-shaped trichomes, shorter trichomes and
very short capitate trichomes together comprise a three-dimensional trap. Once
stuck to a fluid glandular droplet of a long, flexible, tentacle-shaped
trichome, an insect will struggle and move, pulling the droplet into a long
fluid filament. A moving insect usually remains trapped and contacts further
droplets from the long tentacle-shaped trichomes. The arrangement of trichomes
in combination with the insect behavior results in an entanglement of the
insect prey between trichomes and pulled filaments. Finally, the insects,
caught by the presumably more viscous secretion of the stiffer short capitate
trichomes, adhere firmly to the plant surface. Our results show that the work
required to break contacts between the fluid filaments of the plant and the
insect surface is higher in flies than in mirid bugs. This result suggests
that struggling flies, compared with P. roridulae mirid bugs, must
apply considerably more energy to free themselves from the plant.
Captured insects were usually observed sticking ventrally or laterally to the plant surface. Insects initially contact the plant surface with their feet but finally become trapped at their ventral and lateral surfaces, which seem to be more sensitive to the sticky traps.
The presence of long, thin, extensible fluid filaments, caused by the
pulling of adhesive droplets, and their ability to recover their shape
afterwards, supports previous assumptions about the existence of a
caoutchouc-like fluid in this plant
(Marloth, 1925;
Lloyd, 1934). This behavior
indicates that the secretion possesses a viscoelastic property. The trichomes
seem to release substances of varying viscosity. The fibrous residues we found
on trichome tips after washing them with cold chloroform might indicate a
composite nature of the secretion consisting of a fibrous network embedded in
a fluid matrix. Such a composition of the secretion could well explain its
behavior as being similar to that of an elastomere with fluid-like behavior.
Our efforts in visualizing solvent-treated droplets indicate the presence of a
multicomponent substance, partly soluble in ethanol and chloroform and
entirely soluble in acetone. These solubility characteristics suggest that the
adhesive fluid might comprise various lipids and terpenes (polar resins).
Thus, our results support previous observations that the adhesive secretion
has a resinous character (Marloth,
1925; Lloyd,
1934). However, we did not observe an acetone-insoluble material,
as reported by Marloth (Marloth,
1925). Terpenes in plant secretions have been reported to be
released in great diversity and, normally, as a mixture of several terpenes
(Michie and Reid, 1968;
Schnepf, 1969;
Dell and McComb, 1978). Fatty
or oily substances have been found as concomitant fluids of essential oils. In
the sticky sage Salvia glutinosa L. (Lamiaceae), for example, sticky
secretions of glandular trichomes contain a large amount of lipids
(Schnepf, 1969). In addition
to the solvent-treatment experiment, the probable presence of lipid-like
components was supported by the particular pattern of droplet prints spreading
into numerous micro-droplets on a metallized epoxy-resin surface. The
formation of such extremely small fluid droplets is hardly possible for a
highly viscous material. That is why we assume that the margins of the large
droplets contain oily substances. Also, the affinity of the adhesive secretion
to the grease-covered, hydrophobic insect cuticle is an additional indicator
of the possible presence of oily substances in the plant adhesive fluid.
The adhesive secretion of R. gorgonias has been reported
previously to be very viscous and to be the strongest glue of all plant
flypapers (Hartmeyer, 1998).
In our adhesion measurements, we obtained pull-off forces of the secretion in
tentacle-shaped trichomes at the micro-Newton scale. In the protocarnivorous
tar flower Befaria racemosa Venten (Ericaceae), the stickiness of the
viscid secretion corresponded to that of commercial flypapers and ranged
between 40 and 50 kPa (Eisner and
Aneshansley, 1983). If we assume the width of secretion droplets
(100 µm; see Fig. 6I)
to be the contact area, we can estimate the adhesive strength in R.
gorgonias as being 13 kPa. Such a discrepancy between our results
and those in the literature might be explained by the fact that our
experimental design differed drastically from that used in experiments on
B. racemosa. Eisner and Aneshansley
(Eisner and Aneshansley, 1983)
measured the pressure-dependent adhesive strength on the fluid trapped between
two flat surfaces, whereas we estimated the adhesion strength of a single
ovoid droplet slightly brought into contact with the insect or glass surface
and then retracted from it. Direct comparison of these two approaches is not
possible because of the strong differences in the fluid thickness (a thinner
adhesive layer attaches two bodies much more strongly), which was not measured
in both studies. Our experiment was designed to simulate the natural situation
in which insects contact the surface of R. gorgonias (usually through
long tentacle-shaped trichomes) and then pull the adhesive secretion into the
form of filaments. This is also the reason why we used long tentacle-shaped
trichomes in our experiments.
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;
Voigt et al., 2006a;
Voigt et al., 2007), whereas
P. roridulae bugs seem not to avoid particular plant parts, even
regions between the trichomes, and frequently touch the glandular fluid. We
observed that they also seldom groom their body, contrary to previous
suggestions (Lloyd, 1934;
Hartmeyer, 1996;
Hartmeyer, 1998). These
authors discussed the arrangement of short setae as being the mechanism
protecting the mirid bug against adhesives. Our comparative SEM study has
revealed that the surfaces of both P. roridulae bugs and C.
vicina flies are covered with cuticle outgrowths of different origins.
Adhesive droplets adhere to cuticle protuberances of both mirid bugs and
flies. However, flies are caught by R. gorgonias, whereas mirid bugs
are not caught. Thus, we presume that the hairy covering of the surface is not
the main reason for the resistance of the bug to the sticky secretion. The
present study shows that P. roridulae bugs employ a different
mechanism to resist plant adhesion.
Our experiments show that the adhesion force was significantly lower on
surfaces of live and dead P. roridulae bugs compared with that of
live and dead C. vicina as well as compared with that of
chloroform-washed mirid bugs (the latter still bear a hairy covering). That is
why our results support the assumption of Lloyd
(Lloyd, 1934) that mirid bugs
release some substances repelling the adhesive secretion of the plant. Even if
the greasy layer does not really repel the adhesive secretion but rather seems
to have a strong affinity for it (the adhesive wets the surface of the bug),
this layer sloughs off easily (cohesion failure), preventing trapping of the
bug. According to both light-microscopic data on cuticle prints of P.
roridulae and to the cryo-SEM results showing the distinct amorphous
layer on the surface of the epicuticle of P. roridulae, we
hypothesize that an epicuticular, greasy substance, removable with chloroform,
is responsible for the anti-adhesive property of the mirid bug surface.
Lipid-containing epicuticular grease has been reported previously from other
arthropods: Calliphora erythrocephala Meigen (Diptera, Calliphoridae)
(Wolfe, 1954), Rhodnius
prolixus Stål (Wigglesworth,
1933) (Heteroptera, Reduviidae), Periplaneta sp.
(Blattaria, Blattidae) (Beament,
1945; Beament,
1958; Gilby and Cox,
1963), Acheta domesticus L. (Orthoptera, Gryllidae)
(Hendricks and Hadley, 1983),
Locusta migratoria L. (Orthoptera, Acrididae)
(Vötsch et al., 2002),
Leptinotarsa decemlineata Say (Coleoptera, Chrysomelidae)
(Tower, 1906), Cicindelinae
(Carabidae, Coleoptera) (Stegemann,
1929), Boophilus microplus Canestrini (Acarina, Ixodidae)
(Gilby, 1957) and
Cupiennius salei Keys (Araneae, Ctenidae)
(McConney et al., 2007). Such
a covering has been extracted from the fresh cuticle of the cockroach either
as hard wax, mobile grease (Beament,
1945; Beament,
1958) or as a relatively fluid lipid layer hardening through
chemical reactions (Gilby,
1962; Gilby and Cox,
1963). In Periplaneta sp., the polar, reducing-agent
material spreads over the entire surface
(Lees and Beament, 1948).
Epicuticular grease has been considered to play a fundamental role in limiting
water loss (e.g. Ramsay, 1935;
Neville, 1975) and serving as
a behavioral cue for insects (Dubis et
al., 1987; Espelie et al.,
1991). Its thickness previously has been suggested to be at a
molecular scale (Beament,
1955), varying from less than 1 nm to several microns
(Hendricks and Hadley, 1983),
or ranging from 0.1 µm to 1.0 µm
(Locke, 1964). In the hunting
spider Cupiennius salei, the thickness of the surface viscous layer
has been quantitatively estimated to be 20–40 nm
(McConney et al., 2007).
Atomic force microscopy of elytra of the Colorado potato beetle L.
decemlineata revealed a grease layer with a thickness of 8 nm
(Gorb et al., 2008;
Voigt et al., in press). Our
measurements of grease thickness on leg cuticle in flies and mirid bugs
confirm the previously suggested nanoscale dimensions. In C. vicina,
a thin layer ranging from 19.8 to 29.7 nm was found. Remarkably, the grease
thickness in P. roridulae is much more prominent, ranging from 516 nm
to 713 nm. This layer covers the surface of the mirid bugs, embedding also
their microtrichia.
Assuming that the epicuticular grease appears lipid like, we suggest that it functions as a layer causing cohesion failure when the plant adhesive contacts the surface of the mirid bug (Fig. 11). This means that plant adhesive secretions can adhere to the layer of grease, but, under pulling force, the grease layer breaks apart, preventing adherence to the underlying solid cuticle. On the surface of the fly, the very thin, greasy coverage presumably consists of fragmentary patches where the sticky plant secretion can get into contact with the solid epicuticle by filling the gaps of the discontinuous greasy layer (Fig. 11A). The 30-fold thicker epicuticular layer of the surface of the mirid bug prevents contact formation between the plant adhesive and the bug cuticle (Fig. 11B). The microtrichia, submersed in the grease layer, might be interpreted as a microstructure maintaining the thickness of the layer. The sticky droplets cannot penetrate the epicuticular grease of the mirid bug. As a result, these mutualistic mirid bugs cannot be trapped by the plant.
Conclusion and outlook
An adhesive secretion enables the R. gorgonias plant to capture
effectively a large number of prey insects. The secretion exhibits adhesive
properties, presumably differing depending on the type of trichome. Further
mechanical characterization of the adhesive secretion combined with the
analysis of the chemical composition of secretion from different trichomes
will aid in further understanding the trapping strategy of protocarnivorous
R. gorgonias.
The mutualistic mirid bug P. roridulae bears a thick, anti-adhesive greasy layer, which is a `smart' surface adaptation for living on the strongly adhesive surfaces of the plant. The anti-adhesion mechanism of the mirid bug is based on the cohesion failure caused by grease. The non-continuous, patch-like pattern of grease found in potential dipteran prey does not provide sufficient protection against the plant adhesive. Additionally, the presence of specific substances in the bug grease targeted to prevent adhesion of Roridula secretion cannot be entirely excluded. In future studies, this mechanism should be compared with that of the cuticle structures of various Heteroptera. Possibly, similar adaptations might be found in other insect–plant associations. Finally, the cohesion failure anti-adhesive mechanism might be potentially interesting for technical developments of novel biologically inspired surfaces.
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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0.001
and Tukey test, P<0.05).
