Structure and properties of the glandular surface in the digestive zone of the pitcher in the carnivorous plant Nepenthes ventrata and its role in insect trapping and retention
1 Evolutionary Biomaterials Group, Max Planck Institute for Metals Research,
Heisenbergstr. 3, D-70569 Stuttgart, Germany
2 Botanique et Bioinformatique de l'Architecture des Plantes, UMR CNRS 5120,
Boulevard de la Lironde - TA40/PS2, F-34398 Montpellier, Cedex 5,
France
* Author for correspondence (e-mail: o.gorb{at}mf.mpg.de)
Accepted 3 June 2004
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Summary |
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Key words: trapping function, insect, insect attachment, smooth/hairy attachment, fly, Calliphora vicina, bug, Pyrrhocoris apterus, profile, elasticity, Young's modulus, adhesion, tenacity, surface free energy, claw interlocking, adhesive pad, friction force
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Introduction |
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In most Nepenthes species, the pitcher is composed of a lid, a
peristome (ribbed upper rim of the pitcher), a waxy zone and a digestive zone
with a pool of digestive fluid (Adams and
Smith, 1977; Owen and Lennon,
1999
). These zones differ greatly in their geometry, structure,
surface architecture and functions. The lid and the peristome of pitchers are
supposed to be involved in animal attracting and trapping
(Jebb and Cheek, 1997
;
Owen and Lennon, 1999
;
Gaume et al., 2002
), and the
waxy zone has been widely studied for its implications in trapping and
preventing the escape of prey (Lloyd,
1942
; Juniper and Burras,
1962
; Juniper et al.,
1989
; Gaume et al.,
2002
,
2004
). By contrast, the
digestive zone is believed to contribute solely to prey utilisation. For this
reason, the digestive zone has been studied mostly for its chemical function
in digestion, absorption and transport of the insect-derived nitrogen
compounds (Juniper et al.,
1989
; Schulze et al.,
1999
; Owen et al.,
1999
; An et al.,
2001
). However, this zone may also influence the trapping
efficiency of the pitcher, since animals are usually captured and drawn into
the digestive fluid (Juniper et al.,
1989
; Adams and Smith,
1977
; Owen and Lennon,
1999
). Recent experiments with flies (Drosophila
melanogaster) and ants (Iridomyrmex humilis) on epidermal
surfaces of the Nepenthes alata pitchers showed that the glandular
secretion probably acts mechanically, like a glue, and impedes insect
locomotion (Gaume et al.,
2002
).
The principal aim of the present study is to investigate whether the glandular surface of the digestive zone in pitcher plants is also involved in the trapping and retention mechanism. This surface may constitute an area for retaining insects. We hypothesise that (1) the roughness of the glandular surface does not accommodate claw interlocking; (2) stiffness of digestive zone material precludes both claw and pad attachment; (3) adhesive properties of the surface prevent insect attachment and locomotion and (4) gland secretion lessens insect adhesive pad attachment. To test this, structural and biomechanical studies of the glandular surface of Nepenthes ventrata were combined with insect behavioural experiments. Laboratory experiments were carried out on fresh and air-dried glandular surfaces with flies (Calliphora vicina) and bugs (Pyrrhocoris apterus). The two species have adhesive pads of two different types: hairy and smooth, respectively. The following questions were asked: (1) which morphological features of the glandular surface may contribute to trapping insects, (2) do mechanical properties of the digestive zone, such as plant material elasticity, adhesive properties and surface free energy of the glandular surface, promote insect retention and (3) how does the presence of gland secretion influence insect attachment?
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Materials and methods |
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The two insect species used in friction tests were selected for similarity
in size (values are means ± S.D.; fly body
length=9.33±0.44 mm, n=15; bug body length=9.30±0.60
mm, n=15) and weight (fly mass=44.58±10.67 mg, n=15;
bug mass=42.67±8.59 mg, n=15). They bear adhesive pads
belonging to two alternative types of locomotory attachment devices in insects
(Gorb and Beutel, 2001;
Beutel and Gorb, 2001
;
Gorb, 2001
). The fly
Calliphora vicina Robineau-Desvoidy (Diptera, Brachycera,
Calliphoridae) possesses hairy pulvilli, whereas the bug Pyrrhocoris
apterus L. (Hemiptera, Heteroptera, Pyrrhocoridae) has smooth ones. Both
types of attachment pads have been reported to produce secretory fluid, which
is delivered onto the contact area during contact formation with the substrate
(Hasenfuss, 1977
,
1978
;
Bauchhenss, 1979
;
Walker et al., 1985
; Gorb,
1998
,
2001
). Representatives of both
insect orders were reported to be captured by the traps of Nepenthes
plants (Juniper et al., 1989
;
Kato et al., 1993
;
Moran, 1996
;
Moran et al., 1999
). Flies
were taken from a laboratory colony at the University of Würzburg
(Germany) and kept in a small terrarium at 20-24°C, 65-70% humidity.
Insects were fed with crystalline sugar and tap water. Bugs were collected in
a lime-tree garden in Tübingen (Germany) and used on the same day.
Structural studies
The general organisation of the glandular surface in the digestive zone and
gland arrangement were studied on a fresh, untreated pitcher surface with a
binocular microscope (Leica MZ 12.5) with a built-in video chip (Leica IC A)
and a scanning electron microscope (SEM). For SEM, small pieces (1
cm2) of the pitcher wall were cut out with a razor blade from the
upper and middle parts of the digestive zone, mounted on holders and examined,
without sputter-coating, in a Hitachi S-800 scanning electron microscope at 5
kV. Gland dimensions as well as the density of the glands per mm2
were quantified from digital images using SigmaScan software (SPSS Inc.,
Chicago, IL, USA).
The surface profile was studied on fresh, untreated samples (1
cm2) cut out, using a razor blade, of the upper and middle parts of
the digestive zone and glued with double-sided tape to a glass slide. The
samples were examined in a scanning white-light interferometer (Zygo NewView
5000; Zygo Corporation, Middlefield, CT, USA) using objectives 20 and 50 at
magnifications of 20x0.4, 20x1.3, 20x2.0, 50x0.4,
50x1.3 and 50x2.0. The device included optics for imaging an
object surface and a reference surface together onto a solid-state imaging
array, resulting in an interference intensity pattern that was read
electronically into a computer. A series of interferograms were generated as
the objective was scanned perpendicular to the illuminated surface. They were
then individually processed, and finally a complete 3-D image, constructed
from the height data and corresponding image plane coordinates, was created.
From 3-D images, profiliograms of sections with and without glands as well as
height parameters were obtained.
For anatomical studies of digestive glands, tissue specimens (1
cm2) were cut out, using a razor blade, of the middle part of the
digestive zone, fixed overnight in a 5% sucrose solution in a desiccator under
vacuum, infiltrated in 15% and 30% sucrose solutions, embedded in
Tissue-Tek® (O.C.T. Compound Sakura Finetek USA, Inc., Elkhart, IL, USA),
frozen and sectioned in longitudinal and transverse planes perpendicular to
the surface using a cryotome Leica CM 3050. Sections (7, 10 and 14 µm
thick) were collected on pre-coated glass slides, both unstained and stained
for 0.5 min with a warm (60°C) ethanol solution of Sudan Black, rinsed for
1 min in 50% ethanol, mounted in mounting medium (Depex; Serva, Heidelberg,
Germany) and analysed in a light microscope (Zeiss Axioplan) under
bright-field conditions and under phase contrast. In addition, unstained
sections and fresh non-sectioned tissue specimens were mounted on a glass
slide and checked for autofluorescence using fluorescence microscopy with an
HBO 100 W/2 UV filter pack (Carl Zeiss, Germany) in one of three bands of
wavelength: green (excitation 512-546 nm; emission 600-640 nm), red
(excitation 710-775 nm; emission 810-890 nm) and UV bands (excitation 340-380
nm; emission 425 nm). Digital images were obtained using a Zeiss AxioCam MRc
video camera mounted on the light microscope.
To obtain detailed information about the attachment devices in 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 gold-palladium (10 nm) and examined in the SEM at 20 kV. After friction experiments (see below), insects were killed by freezing individually at -20°C in small plastic jars. The insects were returned to room temperature, air-dried, mounted on holders, sputter-coated with gold-palladium and studied in the SEM.
Estimation of material properties
The material properties of the digestive zone were measured with a
micro-force measurement device (Basalt-BT01; Tetra GmbH, Ilmenau, Germany;
Gorb et al., 2000;
Jiao et al., 2000
). A small
piece (
1 cm2) of the pitcher wall, cut out of the middle part
of the glandular digestive zone with a razor blade and glued with double-sided
tape to a platform, was used as a lower sample
(Fig. 1). A small sapphire
sphere (0.75 mm in radius), connected to a glass spring with a spring constant
of 290 N m-1, was an upper sample. The sphere was brought into
contact with the plant sample to set a normal force of 300-1800 µN, which
corresponded to the weight of middle-sized and large syrphid flies
(Gorb et al., 2001
), and then
retracted from the surface. After each measurement, the sapphire sphere was
cleaned with acetone. The recorded force-distance curves were used to
calculate the elasticity of the plant sample (loading process) and to estimate
the adhesion force (retracting process)
(Fig. 2). Experiments were
carried out at room temperature (20-25°C) at a relative humidity of
58-76%. Three pitchers from three different plants were tested. Elasticity was
estimated from five randomly chosen points (1-2 tests per point; in total, 7
tests); adhesion was measured at 23 points (1-10 tests per point; in total, 85
tests).
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The plant surface deformed when the sapphire sphere pressed against the
sample. The indentation of the plant material was obtained by comparing the
spring deflections induced by the plant sample and a hard sample (a glass
plate). By subtracting the displacement of the plant sample from that of the
hard sample at the same spring deflection under the same applied force, the
indentation of the plant material was calculated
(Gorb et al., 2000;
Jiao et al., 2000
).
To estimate the elasticity of the digestive zone tissue, the Hertz theory
(Hertz, 1881), describing the
deformation of two smooth elastic bodies in contact under applied force, was
used. The relationship between the indentation (
) and the applied force
(Fn) is given by:
![]() | (1) |
where K is the reduced elasticity modulus of materials and
a is a radius of the circular contact area. K is related to
the Young's modulus as:
![]() | (2) |
where E and are the Young's modulus and Poisson ratio of the
plant sample, respectively, and Eb and
b
are the same parameters for the sapphire sphere. Since the material of the
sapphire sphere is much stiffer than that of the plant sample tested
(Eb>>E):
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Contact radius, a, depends on the indentation depth and the
sapphire sphere radius (R) as:
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Equations 1, 3 and 4 imply that the indentation can be expressed with
applied force as:
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From equation 5, the Young's modulus of the plant material was calculated.
To compare data on adhesion at different points of the glandular surface,
obtained with different normal forces applied, tenacity T (the
adhesive force, Fa, per unit of contact area) was used:
![]() | (6) |
where A is contact area. From the Hertz theory, for two elastic
bodies in contact:
![]() | (7) |
By substituting equations 4, 5 and 7 into equation 6, we obtain:
![]() | (8) |
Contact angle measurements and evaluation of surface free energy
Estimates of surface free energy and its components on the glandular
surface of the digestive zone were carried out with a high-speed optical
contact angle measuring device (OCAH 200; DataPhysics Instruments GmbH,
Filderstadt, Germany). The device was equipped with a high-speed CCD video
system, four manual and/or electronic (motor-driven and software-controlled)
dosing units with a manual or electronic multiple-dosing system,
micro-controller module for control of the electronic syringe units and
motor-driven sample stages. The software offered static contact angle
measurements according to the sessile drop method, estimation of the surface
free energy of solids and their components according to standard evaluation
methods, and provided statistics and measurement error analysis
(Maier, 2002).
A series of well-characterised liquids (water, density=1.000 kg
m-3; diiodomethane, density=3.325 kg m-3; ethylene
glycol, density=1.113 kg m-3) were used to calculate the surface
free energy of the glandular surface. Using the sessile drop method (drop
volume 2 µl) with circle or ellipse fitting, static contact angles of
liquids to the surface were evaluated on fresh, untreated glandular tissue.
Samples (3-6 cm2) were cut out using a razor blade from the upper
and middle parts of the digestive zone and then attached with double-sided
tape to a glass slide. The surface free energy and dispersion and polar
contributions of the surface energy were calculated according to two universal
methods: Owens-Wendt-Kaelble (Owens and
Wendt, 1969) and Wu (Wu et
al., 1995
). The surface free energy of three pitchers belonging to
three plants was calculated. For each pitcher, 3-5 measurements of the contact
angle of each liquid to the surface were conducted with a total of 39
measurements.
Friction experiments with insects
Friction experiments with two insect species were designed to study the
influence of glandular surface on the functional efficiency of the insect
attachment systems composed of claws and adhesive pads of two alternative
designs: smooth and hairy. Two questions were asked: (1) does the glandular
surface covered with gland secretion lessen insect attachment and (2) how does
the glandular surface affect attachment systems with different pad
designs?
Force measurements were carried out with a load cell force transducer (10 g
capacity; Biopac Systems Ltd, Santa Barbara, CA, USA)
(Gorb and Popov, 2002).
Experimental insects were made incapable of flying, prior to experiments, by
either gluing the forewings together (bugs) with a small droplet of molten wax
or cutting off the wings (flies). The insect was attached to the force sensor
by means of a hair (10-15 cm long) glued to the dorsal surface of the insect
thorax with a drop of molten wax (Fig.
3A). Plant surface samples (6-15 cm2) were cut out of
the digestive zone using a razor blade and then, either fresh or air-dried,
connected with double-sided tape to a horizontal glass plate so that insects
had to move from the bottom to the upper part of the zone, simulating the
situation of escaping from the bottom of the pitcher. Three types of
substrates were used: (1) fresh glandular surface, (2) glandular surface that
had been air-dried for 3-5 days at 20-24°C, 60-75% humidity and (3) a
glass plate as a control. The force generated by the insect walking
horizontally on the test substrates was measured. Force-time curves were used
to estimate the maximal friction force produced by insects
(Fig. 3B). Experiments were
carried out at 23°C and 76% humidity. For each substrate type, experiments
with five individual flies and five individual bugs (4-5 repetitions per
insect) were conducted. Five pitchers from five different plants were tested.
In all, 30 insects were used and 149 individual tests were carried out.
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Results |
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Both the shape and size of the glands vary depending on the level of the digestive zone. Glands of the upper part are smaller (area=12563.31±5420.47 µm2, mean ± S.D., n=30 samples, N=3 pitchers), almost round or slightly elongated in the longitudinal direction. In the centre of the zone, glands are larger (area=49101.90±10843.28 µm2, n=30, N=3), round or slightly elongated in the transverse direction. Glands situated at the bottom of the pitcher are much larger (area=92242.52±14139.50 µm2, n=15, N=3), round or noticeably elongated in the transverse direction. Density of the glands is considerably higher in the distal part of the digestive zone (8.87 glands mm-2, n=15, N=3) than at the bottom of the pitcher (3.72 glands mm-2, n=15, N=3).
The surface of the gland is not even but corrugated and bears small scale-like irregularities (Fig. 4D,E). On the surface of some glands, beam-shaped crystals are found (Fig. 4E).
Surface profile
The surface of the glandular zone is regularly covered with glands. The
surface between glands is relatively smooth and shows small height differences
at low magnification in the white-light profiliometer (objective 50,
magnification 50x0.4). At a higher magnification (objective 50,
magnification 50x2.0), the surface appears to be uniformly structured
with almost similar irregularities (height, 0.1-0.4 µm; length, 3.0-5.0
µm) (Fig. 5A,B).
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Glands show different profiles in the upper (Fig. 5C,D) and middle (Fig. 5E,F) parts of the digestive zone. In both cases, they are located in deep (22-28 µm) depressions with downward-projecting hoods distally and a slope slanting in the proximal direction of the pitcher. However, the height of the actual gland and hood can vary over the digestive zone: at the top, glands are lower (11-14 µm) and hoods higher (25-28 µm) than those of the middle part (both glands and hoods: 14-16 µm). In upper glands, both the upper slope surface and at least part of the central region are covered by the hoods, whereas in glands situated at lower levels of the digestive zone only the upper slope surface is hooded.
Generally, downward-pointing hoods and the slanting slopes of depressions result in the anisotropy of the glandular surface profile. This anisotropy is less developed in the middle part of the digestive zone and significantly reduced at the bottom of the pitcher.
Anatomy of the digestive zone
In both longitudinal and transverse sections of the digestive zone, the
pitcher wall appears as a layered structure (thickness=212.28±31.50
µm, n=12, N=4) with an outer epidermis facing the
environment, a layer of inner epidermis facing the trap cavity, and a
multilayered spongy mesophyll in between
(Fig. 6A). The epidermal cells
are covered with cuticle, are thick-walled (especially outer walls) and vary
greatly in shape (from almost round to oval, laterally elongated) and size
(outer epidermis, length=15.71±3.22 µm, width=25.90±10.55
µm, n=23; inner epidermis, length=14.81±5.02 µm,
width=28.16±10.18 µm, n=30). The optical properties of the
outer wall of the inner epidermis differ from those of the internal wall and
the walls of mesophyll cells. The outer wall shows weak autofluorescence in UV
light (Fig. 7A,B). Mesophyll
cells are thin-walled, relatively large (length=33.35±11.25 µm,
width=42.63±12.79 µm, n=37) and irregularly shaped.
Vascular bundles are present in the mesophyll tissue.
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In the inner surface of the pitcher wall, glands are distributed equally, and each is situated at the bottom of a small epidermal depression and either does not extend beyond it or protrudes from the epidermal surface to a maximum of no more than half the thickness of the gland (Fig. 6B,C). Sections indicate that the hood, called the epidermal ridge, is comprised of a modified epidermal cell extending over at least part of the depression, as described above (Fig. 6B). The hood may also appear as paired structures surrounding the gland on each side (transverse sections across the upper part of the gland; Fig. 6C). The entire hood shows weak autofluorescence in UV light in whole-mount surface preparations (Fig. 7C).
The gland is a multicellular structure consisting of three cell layers.
Although an epidermis is absent from the gland, a thin (up to 1 µm thick)
cuticle covers its surface (Fig.
6D). The outermost cell layer, known as a glandular head
(terminology from Juniper et al.,
1989), consists of columnar cells
(Fig. 6D-F) that do not vary
much in shape but do vary in size (longitudinal sections,
length=22.59±5.11 µm, width=14.42±2.65 µm, n=52;
transverse sections, length=21.82±4.30 µm, width=12.51±1.15
µm, n=39). The cells bear thicker lateral walls (longitudinal
sections, thickness=2.27±0.44 µm, n=19; transverse
sections, thickness=2.55±0.97 µm, n=7) and an extremely
thick external wall (longitudinal sections, thickness=3.47±0.53 µm,
n=18; transverse sections, thickness=4.08±0.73 µm,
n=11) having different optical properties than the internal walls
(Fig. 6D) and walls in other
gland cells and mesophyll cells. The external wall differs in its material
structure at the inner and outer margins
(Fig. 6D). Inner margins are
half as thick (longitudinal sections, thickness=1.22±0.29 µm,
n=18; transverse sections, thickness=1.26±0.33 µm,
n=11) as outer ones (longitudinal sections,
thickness=2.33±0.44 µm, n=19; transverse sections,
thickness=2.75±0.81 µm, n=11). In these cells, as well as
in adjoining epidermal cells and in the ridge, the outer margin of the
external wall weakly reflects UV light
(Fig. 7A,B). Only a few cells
of the first layer are found to have cytoplasm. Most cells contain secretion
inclusions that may fill up to three-quarters of the cell volume
(Fig. 6F).
The cells of the second layer usually vary more in shape (from almost columnar, radially elongated to round or even oval, laterally elongated) and size (longitudinal sections, length=14.91±3.60 µm, n=35, width=19.43±4.70 µm, n=33; transverse sections, length=15.64±4.17 µm, width=19.30±3.91 µm, n=30) than those of the first layer (Fig. 6E-G). These cells have relatively thin walls and have cytoplasm.
The third layer could be clearly observed, mainly in longitudinal sections. It appears as a chain of elements and is composed of large (longitudinal sections, length=12.00±3.19 µm, width=29.93±7.39 µm, n=11; transverse sections, length=10.41±2.77 µm, width=27.20±5.85 µm, n=15), in most cases laterally elongated, cells (Fig. 6E,G). As in the cells of the previous layer, these cells have thin walls and cytoplasm. In a very few sections, not a layer but just several separate cells are found beneath the third layer.
A glandular head and cells of the second and third layers comprise the
so-called glandular component of the gland. The gland is separated from a
sunken epidermal layer and other underlying tissue by a band of laterally
elongated and radially flattened cells (longitudinal sections,
length=4.96±1.71 µm, width=29.50±7.09 µm, n=11;
transverse sections, length=4.35±0.86 µm, width=21.27±3.10
µm, n=12) with thickened walls (longitudinal sections,
thickness=1.44±0.43 µm, n=15; transverse sections,
thickness=0.90±0.07 µm, n=4) (Figs
6G,
7A). In these cells, lateral
and partly external and internal walls, as well as lateral walls in cells of
the adjoining third gland layer and sunken epidermis, strongly reflect UV
light (Fig. 7B). This indicates
large deposits of cutin or suberin and is characteristic for an endodermoid
component of the gland (Juniper et al.,
1989; Owen and Lennon,
1999
; Schulze et al.,
1999
). On whole-mount surface preparations, these walls appear as
two nets that differ in mesh dimension
(Fig. 7C).
Tracheid elements of vascular bundles are seen close to digestive glands (Fig. 6G). Usually they are located one or two layers beneath the base of the gland.
Properties of the glandular surface
Elasticity of the material
The force-time curves indicated that the plant material has visco-elastic
properties (Fig. 8A). During
the loading process, a gradual deformation of the sample was observed. The
deformation fitted well with the Hertz model (nonlinear regression according
to equation 5, P<0.001, one-way ANOVA) in the entire range (up to
1800 µN) of the applied force (Fig.
8B). The indentation at the applied forces of 450-1800 µN was
8-16 µm. Since the Poisson ratio known for plant materials ranges from 0.2
to 0.7 (Mohsenin, 1986;
Niklas, 1992
), we used an
average ratio for calculations. By applying the Hertz theory for load forces
up to 1800 µN and the Poisson ratio of 0.5, we obtained the Young's modulus
of 637.19±213.44 kPa (n=7 points, N=3 pitchers).
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Adhesive properties
Measurements of the adhesion revealed that the glandular surface was not
homogeneous: points with adhesion (n=14) and without adhesion
(n=9) were found. At points with adhesion, the adhesion force varied
from 24.74 to 647.32 µN. This variation depended significantly, in
decreasing order of importance, on the plant, the applied force and the point
of application (Table 1). On
the whole, the adhesion increased linearly with the applied force
(Fn, significant), but the slope of the relation varied
according to the point studied [Fn x point (plant),
significant], while it did not vary significantly according to the plant
(Fn x plant, not significant)
(Fig. 9A). Measured at the same
point, the adhesion force usually increased slightly with an increasing
applied force ranging from approximately 300 to 1000 µN
(R2=0.72, F1,5=12.78, P=0.01;
Fig. 9B; point chosen as an
example among six others) or remained constant (R2=0.006,
F1,8=0.05, P=0.84;
Fig. 9C; point chosen as an
example among two others). Points with adhesion differed in tenacity from 1.39
to 28.24 kPa. Data on adhesion force and tenacity were not normally
distributed and skewed to the left (skewness for adhesion was 1.74 and for
tenacity was 1.76), because of the numerous points without adhesion
(Fig. 10). There were
considerably more points with weak adhesion (adhesion force up to 400 µN,
tenacity up to 15 kPa) than with strong adhesion (adhesion force 500-650
µN; tenacity 22-28 kPa) (Fig.
10). No points with intermediate adhesive properties were found.
Hence, points with strong adhesion were probably randomly situated directly on
the digestive glands, while points with poor adhesion were located in the
vicinity of glands.
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Surface free energy
In all three pitchers studied, the glandular surface was readily wetted
with all three liquids (Table
2). Contact angles of both polar liquids - water (surface
tension=72.1 mN m-1, dispersion component=19.9 mN m-1,
polar component=52.2 mN m-1;
Busscher et al., 1984) and
ethylene glycol (surface tension=48.0 mN m-1, dispersion
component=29.0 mN m-1, polar component=19.0 mN m-1;
Erbil, 1997
) - were similar
and significantly lower than that of disperse diiodomethane (surface
tension=50.0 mN m-1, dispersion component=47.4 mN m-1,
polar component=2.6 mN m-1;
Busscher et al., 1984
) (one-way
ANOVA, F2,37=8.934, P<0.001;
Table 3). On the external
pitcher surface, the difference between contact angles of all three liquids
was highly significant (one-way ANOVA, F2,38=48.069,
P<0.001; Table 3).
All liquids wetted this surface; however, the contact angle of water was
relatively high, especially compared with that on the glandular surface
(Tables 2,
3).
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|
The glandular surface has a higher surface energy than the external surface of the pitcher, the difference being highly significant and independent of the calculation method used (paired t-test, d.f.=5, t=11.284, P<0.001). The surface energy calculated for the glandular surface according to two methods had similar total values (one-way ANOVA, F1,4=0.113, P=0.753) but different values of its polar and dispersion components. According to the Owens-Wendt-Kaelble method, the polar component was very high, exceeding the dispersion component by a factor of 3-6 (Table 2). According to the Wu method, both components contributed almost equally to the surface energy: the polar component exceeded the dispersion component only by one-third (Table 2). For the external surface, values of the total surface energy were different depending on the method of calculation (one-way ANOVA, F1,4=11.435, P=0.028). The dispersion component, with one exception, was either slightly or up to twice as high as the polar component (Table 2).
Morphology of the attachment systems of the bug Pyrrhocoris apterus and the fly Calliphora vicina
The tarsus of Pyrrhocoris apterus has three segments, which are
ventrally covered with hooked setae, curved proximally
(Fig. 11A). The pretarsus
bears two pulvilli connected to the pretarsus in the region between the
unguitractor plate and claws. Pulvilli appear rather smooth in SEM images
(Fig. 11B); however, slight
corrugation of the surface may be observed at a high magnification
(Fig. 11C). Pulvilli material
seems to be very compliant and it collapses in air-dried samples.
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The tarsus of Calliphora vicina has five segments, ventrally covered with setae, pointed proximally. The pretarsus bears two pulvilli connected to claws through mobile sclerites called auxilia. The pulvilli are densely covered with tenent setae (Fig. 11D). Each seta is composed of a shaft (15-20 µm long) and a terminal plate, or spatula (1.5-2.0 µm wide) (Fig. 11E).
Friction force generated by insects on the glandular surface
Values of the maximal friction force generated by insects differed
significantly according to the substrate studied, the insect species and the
individual for a given species and a given substrate
(Table 4). Moreover, the
substrate influenced the performance of insects differently according to
whether they were bugs or flies (interaction species x substrate,
significant). For example, on a fresh glandular surface, the friction force
generated by bugs was reduced compared with glass, while it was enhanced for
flies.
|
Effect of substrate
The effect of substrate on insect performance was the most significant
effect (Table 4). For the bug
Pyrrhocoris apterus, the highest force was measured on a dry
glandular surface, and the lowest one was measured on a fresh glandular
surface (Fig. 12A). The fly
Calliphora vicina showed the highest friction force on both glandular
substrates and performed much worse on glass
(Fig. 12A). There was no
significant difference in the flies' performances on the fresh or dry states
of the glandular surface, whereas the force on glass differed significantly
from both glandular surfaces (Table
4; Fig. 12A). SEM
observation of insects immediately killed after the experiments clearly
demonstrated that pitcher secretion did not contaminate the pads of either
insect studied.
|
Differences between insects
Insect species studied showed significant difference in maximal friction
force on all three substrates tested (Fig.
12B). On both glass and dry glandular surfaces, bugs produced a
higher friction force than flies, while on fresh glandular surfaces flies
performed better (Fig.
12B).
![]() |
Discussion |
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Once an animal is captured in the digestive fluid, it may struggle in the
liquid and succeed in leaving the pool and try to escape. It was previously
indicated that, as well as all other functional pitcher surfaces, the
glandular surface displays properties responsible for the retention of insects
within the pitcher (Gaume et al.,
2002). However, the possible role of the glandular surface, the
glands or the gland secretion in the trapping and retaining system of the
pitcher has not been fully elucidated.
Our data on the morphology, material and surface properties of the glandular surface and the experimental results obtained in friction tests with two insect species provide a good basis for discussing the possible anti-attachment function of the glandular zone and the mechanism of trapping in general.
Possible role of surface morphology in preventing insect attachment
Our results on the morphology and surface profile showed that the glandular
surface is not uniform and smooth but bears structures causing significant
roughness of the surface. Surface roughness results from (1) epidermal
depressions (up to 30 µm deep) with glands placed at the bottom, (2)
prominent hoods (15-30 µm high) overhanging the upper margins of
depressions and (3) prominent sessile glands (up to 15 µm high) that do not
extend beyond depressions (in the upper and middle part of the digestive zone)
or protrude out of the epidermal surface to a depth of no more than one half
of the height of the gland (in the lower part of the zone). The presence of
depressions, containing digestive glands, and hoods has been previously
described in other Nepenthes species
(Lloyd, 1942;
Adams and Smith, 1977
;
Pant and Bhatnagar, 1977
;
Juniper et al., 1989
;
Owen and Lennon, 1999
;
Owen et al., 1999
;
Gaume et al., 2002
). Towards
the bottom of the pitcher, the dimensions and prominence of hoods decrease,
whereas those of glands increase, so that the surface becomes increasingly
uneven.
Prevention of claw interlocking
It is known that insects attach well to a macroscopically rough surface
(Dai et al., 2002;
Betz, 2002
). In this case,
insects usually use surface irregularities as anchorage sites for their claws
(Nachtigall, 1974
;
Cartmill, 1985
). It has been
previously shown that insects having tarsi equipped only with claws are able
to attach to vertical substrates only if the diameter of surface asperities is
much larger than the diameter of the claw tips
(Dai et al., 2002
). In N.
ventrata, the size of depressions, hoods and glands greatly exceeds claw
tip diameter (1-5 µm depending on insect size), therefore the
rough-structured glandular pitcher surface can guarantee a sufficient number
of sites for successful claw interlocking for insects.
However, the geometrical features of some structures covering the glandular
surface may prohibit the use of large surface asperities for clinging.
Firstly, pendant hoods covering either both the upper slope and the central
region in upper glands or only the upper slope of glands (in the lower part of
the zone) not only protect gland cells from being scraped by insect feet but
may also prevent insects from clinging to both hoods and glands
(Lloyd, 1942;
Juniper et al., 1989
).
Moreover, anisotropic hoods may induce insects to fall and impede their escape
in a similar way as the downward-directed lunate cells of the waxy zone in
N. alata (Lloyd,
1942
; Gaume et al.,
2002
). Secondly, depressions with the angle of slope in a proximal
direction of the pitcher may preclude insects from using them as foot-holds.
However, in the lower part of the digestive zone and particularly at the
bottom of the pitcher, the surface roughness increases and the anisotropy of
the surface profile almost disappears. Interestingly, this probably does not
affect the retaining function of the glandular surface because, as in mature
pitchers, the lower regions of the glandular surface are usually submerged in
the pool of digestive fluid (Juniper et
al., 1989
; Owen and Lennon,
1999
; Owen et al.,
1999
).
Attachment by means of adhesive pads
In order to attach to smooth substrates, many insects have evolved
specialised adhesive pads based on two different mechanisms: smooth flexible
pads and hairy structures (Gorb,
2001). Compliant pad material in the first case and fibrillar
surface structures in the second guarantee to maximise the contact area and
ensure a high attachment force on smooth substrates
(Gorb, 2001
;
Beutel and Gorb, 2001
). It has
been previously reported that many insects showed excellent attachment ability
on a variety of smooth artificial and natural substrates
(Way and Murdie, 1965
;
Stork, 1980
;
Edwards, 1982
;
Eigenbrode, 1996
;
Eigenbrode et al., 1999
;
Federle et al., 2000
). An
experimental study of the attachment ability of the beetle Chrysolina
fastuosa (Chrysomelideae), possessing claws and hairy adhesive pads, on
various plant substrates revealed successful attachment to 46 smooth surfaces
of 41 plant species (Gorb and Gorb,
2002
). The presence of cellular sculpturing, sinuous fissures and
additional subcellular irregularities on these surfaces did not lessen the
attachment. It was suggested that on smooth substrates, even if they are not
perfectly smooth, the area of real contact between the substrate and the
terminal elements of the beetle is large enough to provide a high attachment
force. In the digestive zone of the N. ventrata pitcher, the surface
between glands has a smooth appearance and is slightly uneven due to low
convexities. The effect of various smooth plant substrates on insect
attachment (Gorb and Gorb,
2002
) may also be extended to the glandular surface of
Nepenthes. This surface could provide appropriate substrate for
insect attachment in the case of pad-bearing insects. The surface of the
glands, although slightly corrugated and covered with tiny scale-like
subcellular asperities, may also present a proper support for adhesive
pads.
Mechanical strength of the surface
Anatomical study of the epidermal surface and glands of the digestive zone
revealed no specialised structures that could influence insect attachment.
Nevertheless, some features of the inner morphology may contribute to the
enhancement of the pitcher wall rigidity resisting external forces from
insects attempting to cling to the glandular surface. The surface between the
glands is covered by epidermal cells with thick lateral and greatly thickened
outer walls. The glandular head cells also have thicker lateral walls and an
extremely thick external wall. It has been previously hypothesised for the
carnivorous plant Sarracenia purpurea that the thick lateral walls in
the epidermal cells of the absorption zone may reinforce the pitcher against
struggling prey (Barckhaus and Weinert,
1974). The same function may also be suggested for the glandular
surface of Nepenthes plants. Moreover, cutin impregnations in these
thick external cell walls in the epidermis, epidermal ridges and glandular
head cells, indicated from UV fluorescence, contribute not only to the
formation of a barrier to apoplastic transport
(Juniper et al., 1989
;
Owen and Lennon, 1999
) but
also may contribute to an increase in the mechanical strength of the glandular
surface. Besides the cutinised wall layer, the surface between glands and the
gland surface itself are covered with a cuticle layer, relatively thick in the
first case and very thin on the glands. The presence of the thick cuticle that
covers the epidermis and extends over the top of the digestive glands was also
found in N. alata pitchers (Owen
and Lennon, 1999
). Using our microscopy methods, cuticular gaps,
reported as characteristic cuticular discontinuity in glands of Nepenthes,
Drosophyllum, Sarracenia and Utricularia (reviewed by
Juniper et al., 1989
), were
not detected in N. ventrata.
Mechanical properties of the digestive zone and insect retention
It was previously suggested that the pitcher wall in Nepenthes is
extremely strong owing to the thick-walled epidermis of the outer and inner
surfaces, as well as mechanical reinforcement by heavily sclerenchymatised
veins and nonvascular idioblasts (Lloyd,
1942; Juniper et al.,
1989
). This strengthening was believed to prohibit escape through
the sides of the pitcher by leaf-cutting insects, that was sometimes observed
in representatives of several genera of carpenter wasps. However, the material
properties of the pitcher wall and the surface properties of its inner surface
have not been previously estimated, although these parameters may be important
for trapping and retaining.
The results of microindentation experiments on the glandular surface allow us to conclude that the material of the inner surface in the digestive zone has visco-elastic properties. However, taking into account the relatively low load forces, which insects may apply to this substrate, the system of the plant pitcher wall may be considered to function mainly in the elastic regime. For this reason, in our estimations, we neglected, as less important for insect-retention, the viscous component of the material properties of the plant tissue studied.
Effect of surface material stiffness on insect attachment
Previous authors have shown that substrate stiffness as well as the
diameter of surface asperities may influence the friction force of the tarsal
claw system (Dai et al.,
2002). In order to allow insect attachment and locomotion,
elements of the surface roughness should be greater in dimension than the claw
tip diameter, and the substrates should be sufficiently stiff to prevent its
penetration by claws, otherwise the claws will slide over the surface. As was
shown above, despite appropriate roughness, the glandular surface does not
appear to allow effective claw interlocking because of the surface anisotropy
such as the direction and orientation of structures such as the hooded
epidermal cells and the proximally sloped surfaces of gland depressions.
In terms of surface stiffness, claw clinging might be possible due to indentations formed by `punching' the surface, via forces applied by the claws. In this case, the stiffness of the substrate may play a crucial role in providing a sufficient attachment force.
We estimated whether the glandular surface could have accommodated claw
attachment via a `punching' process. On the basis of the
microindentation experiments, the Young's modulus of the material in the
digestive zone was found to be 637.19 kPa. It is known that insect claws are
made of a hard cuticle that was reported to have a Young's modulus of 9
GPa (Neville, 1975
). We
assumed that insects can attach to the surface and move on it if the claws
make deep dents, which noticeably exceed the diameter of the claw tip. To each
claw, the insect applies a force equal to its weight divided by 12, when all
six legs are used in attachment; during locomotion, this weight is divided by
six, when only three legs are in contact with the substrate. According to the
Hertz model (Hertz, 1881
),
light insects (3 mg) possessing claws with a tip diameter of 1 µm indent
the surface to a depth of 2.14 µm (using six legs) or 3.39 µm (using
three legs). For heavier insects (300 mg) with a claw tip diameter of 5 µm,
the dents are 26.91 µm (for six legs) and 42.72 µm (for three legs)
deep. Thus, in cases of small and relatively larger insects relevant to the
plant species studied, the depth of dents made by claws is much larger than
the claw tip diameter. In summary, this indicates that the glandular surface
can guarantee claw interlocking if the required surface roughness is
lacking.
To obtain strong attachment, using adhesive pads, a large contact area
between the surface and the pads is required in order to achieve a strong
attracting force. For this purpose, materials of either or both contacting
bodies should be very compliant. In insects having a smooth type of attachment
device, a very low stiffness of pad material (Young's modulus 27.2±11.6
kPa) coupled with a specific ultrastructural architecture of the cuticle have
been previously found (Gorb et al.,
2000). This type of cuticle architecture allows surface
replication and thus an increase of the area of real contact between a
substrate and the pad. Insects with hairy adhesive pads were reported to be
able to replicate the substrate profile not only due to the division of one
large contact into many small ones
(Scherge and Gorb, 2001
;
Arzt et al., 2003
) but also
because of the high flexibility of terminal elements called tenent setae
(spring constant 1.31 N m-1;
Niederregger et al., 2002
).
Our microindentation experiments showed that the material of the digestive
zone is relatively stiff. This means that it is not the plant tissue, but the
adhesive pads, that contribute mostly to an increased contact area between
these surfaces.
Influence of surface adhesive properties on insect attachment and locomotion
Another mechanism contributing to the attachment strength is the adhesive
energy of the contacting surfaces. It is known that pad-bearing insects
deliver secretory fluid onto the contact zone to increase the attachment force
between the pad and the substrate (Ishii,
1987; Kosaki and Yamaoka,
1996
; Gorb, 1998
;
Eisner and Aneshansley, 2000
;
Voetsch et al., 2002
;
Federle et al., 2002
). Insects
are thus able to increase the capillary and viscous forces contributing to
overall adhesion. For insects without adhesive pads, attachment to smooth
surfaces may only be possible because of specific properties of the substrate.
In our further estimations, we discuss the possible effect of the glandular
surface on the adhesion of pad-less insects.
Adhesive properties of the glandular surface are very inhomogeneous. There
are many non-adhesive areas that cannot accommodate insect attachment. The
adhesive areas also vary greatly in values of measured adhesion force. Most
adhesive points showed a weak adhesion force and low tenacity. To estimate
whether this adhesion is sufficient to guarantee the insects attachment, we
performed the following calculation. In order for small and middle-sized
insects (up to 40 mg) to adhere to a substrate with all six legs having an
approximate contact area of 8000 µm2 per leg (estimated for
insects used in this study), a surface tenacity of at least 5 kPa is required.
In order for the plant surface to accommodate locomotion, when only three legs
are in contact with the substrate, a tenacity of at least 10 kPa would be
required. To fulfil this, all the attachment structures should contact the
substrate areas with these adhesive properties. However, a tenacity of more
than 10 kPa was actually found to be rather rare on the glandular surface.
Therefore, if insects do not use specialised adhesive organs, this surface
cannot provide sufficient adhesion for insect attachment. For heavier insects
with the same contact area, a higher substrate tenacity is required for
adherence to the glandular surface (Fig.
9A). Although very few points with strong adhesion and high
tenacity (22-28 kPa) were also found, they were not believed to be significant
for the effective attachment and locomotion for large insects but may function
as solitary adhesive spots that glue small insects and impede their locomotion
(Gaume et al., 2002). This may
limit their escape potential from the trap.
We found that the adhesion force increased at a lower rate than the load (Fig. 9A,B, slope <1). Thus, lighter insects should adhere relatively more strongly than heavier insects. Moreover, in nature, the surface is vertical and heavier insects will have a greater disadvantage due to their weight, which tends to pull them inside the pitcher. This may support the idea that light insects can attach to the glandular surface without using specialised adhesive organs if the surface adhesion exceeds the insect weight (Fig. 9A; points located on the left side of the line Fa=Fn).
Physico-chemical properties of the surface and pad adherence
Surface free energy of the substrate is another important parameter
affecting adhesion between two contacting bodies. It is known that, in solids,
surface energy plays a crucial role in contact, since it leads to an increase
of the real contact area, which results in a high attracting force
(Israelachvili, 1992). In
contrast to other pitcher surfaces, the glandular surface has a rather high
surface free energy. This must ensure a high adhesion force based on the
capillary interaction. Moreover, the polar component of the surface energy
greatly exceeded the dispersal component. As the polar component demonstrates
the presence of far-ranging forces, this surface will additionally contribute
to attractive interaction between contacting bodies
(Israelachvili, 1992
). The
glandular surface was found to be hydrophilic (strong attractive interaction
between the surface and water). The pad secretion produced by insects has been
reported to contain oily substances
(Ishii, 1987
;
Kosaki and Yamaoka, 1996
;
Eisner and Aneshansley, 2000
).
Recent work has further demonstrated the presence of water and water-soluble
substances in pad secretion (Gorb,
2001
; Voetsch et al.,
2002
; Federle et al.,
2002
). Therefore, it may be assumed that on the hydrophilic
glandular surface, the polar water component of the pad secretion contributes
substantially to adhesion, in addition to the disperse oily component. Thus,
both components of the pad secretion can probably promote an increase of
capillary forces that provides successful insect attachment on the glandular
surface of the pitcher.
Attachment of insects with different pad design to the glandular pitcher surface
Our results showed that the bug Pyrrhocoris apterus, having a
smooth attachment system, generated a significantly lower friction force on
the fresh glandular surface than on a smooth dry glass plate used as a
reference substrate. However, on the air-dried glandular surface, the force
was higher than on both glass and fresh glandular surfaces. This indicates
that the gland secretion on a fresh plant surface reduces the attachment
force. We assume that the gland secretion does not function as a glue, as was
previously hypothesised (Gaume et al.,
2002). Rather, the gland secretion may increase the thickness of
the fluid layer between the pad surface and the substrate. This causes a
lubrication effect in the contact between surfaces and decreases friction
force. In other words, the substrate becomes slippery. On the fresh glandular
surface, bugs were able to use their claws to interlock to surface
irregularities, since the surface anisotropy was probably not as effective
with the plant sample placed horizontally. The highest friction force obtained
on the dry glandular surface may be explained by the use of both adhesive pads
to attach to smooth areas between glands and claws to cling to
macro-asperities of the substrate.
The fly Calliphora vicina, having a hairy attachment system, performed equally well on both fresh and air-dried glandular surfaces and much worse on the glass plate. This indicates that on the glandular surface, insects presumably used both claws and adhesive pads, whereas on glass, only pads were used for attachment. In contrast to the smooth pads of bugs, the hairy pads of flies were not disabled by the gland secretion. Superfluous fluid, occurring on the glandular surface, probably fills the spaces between tenent setae and therefore did not affect the friction force through lubrication.
A comparison of the performance of the two insect species on glass showed that bugs generated a significantly higher force than flies. The same trend was also found on the dried glandular substrate. This means that on dry substrates, using either adhesive pads or both pads and claws in the attachment, both smooth and hairy structures succeed. However, on the fresh plant surface, flies showed a significantly higher friction force. In this case, not only claws but also pads seemed to contribute to the generation of a friction force. Thus, we assume that the presence of the additional layer of fluid in a contact zone may disable smooth adhesive pads, whereas it does not worsen the effectiveness of hairy systems. This might be explained by the fact that in the hairy system, superfluous fluid may be drawn off into intersetal spaces. The secretion does not appear to contaminate either type of adhesive pads.
Concluding remarks
This study represents a complex investigation of plant-insect surface
interactions, including general morphology, internal structure, material and
adhesive properties, and surface energy in the highly specialised glandular
tissue of Nepenthes. The study is also combined with measurements of
attachment forces generated by insects on this plant surface. On the basis of
structural, physico-chemical and mechanical characterisation of the plant
surface, we tried to estimate the possible functional importance of the
glandular surface in the trapping and retaining mechanism of N.
ventrata. We assume that the influence of the plant surface on insect
attachment differs depending on insect weight and design of their attachment
systems. In general, the glandular surface is probably not responsible for
prey capture and retention.
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Acknowledgments |
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