Performance and adaptive value of tarsal morphology in rove beetles of the genus Stenus (Coleoptera, Staphylinidae)
Zoologisches Institut der Universität, Ökologie, Olshausenstraße 40, D-24098 Kiel, Germany
* e-mail: obetz{at}zoologie.uni-kiel.de
Accepted 7 February 2002
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Summary |
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Key words: adhesion, climbing, ecomorphology, locomotion, tarsus, tenent seta, water surface, rove beetle, staphylinid, Stenus
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Introduction |
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Rove beetles of the genus Stenus Latreille are well known because
of their protrusible prey-capture apparatus (e.g.
Betz, 1996;
Weinreich, 1968
), which must
be considered a key innovation responsible for the tremendous radiation of
this genus (more than 2100 species worldwide) (see
Herman, 2001
). This radiation
has taken place with significant variation in the structure of the tarsi,
which may be (i) slender with weakly or non-bilobed tarsomeres (subgenera
Stenus s. str., Nestus and Tesnus) or (ii) widened
with distinctly bilobed tarsomeres (subgenera Hypostenus, Metastenus
and Hemistenus) (see fig.
1 in Betz, 2002
)
(Freude et al., 1964
;
Puthz, 1971
). Morphometric
analyses have shown that these two groups are clearly distinct in that wide
bilobed tarsi can accommodate significantly more ventral tarsal setae than can
slender tarsi (Betz, 2002
). On
the basis of outgroup comparisons, slender tarsi in the listed subgenera very
probably represent the ancestral state of these beetles (V. Puthz, personal
communication), whereas wide bilobed tarsi may have evolved as a derived state
in the context of plant climbing, opening a novel adaptive zone for this group
of organisms. This hypothesis is supported by the observation that, at least
in central Europe, Stenus species with slender tarsi are soil
dwellers, whereas species with wide tarsi are predominantly plant-climbers
(Betz,
1998a
,b
).
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However, behavioural observations have revealed that representatives of
ground-dwelling species with slender tarsi are also capable of climbing up the
lower parts of vegetation, e.g. for foraging or finding shelter at night
(Betz, 1994). Moreover, at
least in the temperate zones, most of the species appear to be associated with
wetlands (e.g. Anderson, 1984
;
Horion, 1963
;
Koch, 1989
;
Puthz, 1971
;
Renkonen, 1934
), where they
inhabit waterside environments such as reeds or sparsely vegetated sites on
river or lake margins. In these habitats, the representatives of many species
can regularly be observed voluntarily walking across the surface of water
supported only by their tarsi (Betz,
1999
). Since this support depends on the contact between the
entire tarsus and the water surface (e.g.
Denny, 1993
;
Guthrie, 1989
), it can be
hypothesized that species with wide bilobed tarsi are better adapted to this
mode of locomotion than species with slender tarsi (see
Renkonen, 1934
).
I have therefore tested two alternative hypotheses to explain the primary
biological role of widened bilobed tarsi in this group of organisms: (i) that
widened tarsi allow better adhension to smooth (plant) surfaces and thus more
effective climbing in the (reed) vegetation and (ii) that widened tarsi
provide better support on the water surface as a precondition for successful
colonization of waterside wetland habitats. I have tested these hypotheses
experimentally (i) by analyzing the vertical climbing performance of 18
Stenus and one species of the sister genus Dianous on
differently textured surfaces using a microbalance (similar to the
experimental design used previously) (see
Stork, 1980a), and (ii) by
measuring the contact angles at the interface between the water and the
ventral tarsus surface to calculate the vertical upward component of the
surface tension supporting the body weight of the beetle. Experiments
eliminating either the tarsal claws or the tenent setae have provided
additional insights into the underlying mechanisms of tarsal attachment and
the functional roles played by both these elements on various surface
structures.
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Materials and methods |
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The pulling forces produced by the beetles were calculated by multiplying these masses by gravitational acceleration. As a result, characteristic traces were obtained showing the pulling forces continuously exerted by the individual beetles throughout the experiment (Fig. 1). For further comparative analyses, only the maximal force was selected in each case. Since the microbalance was set to its lowest sensitivity to external vibrations, the recorded maxima accurately reflect the actual maximum pulling performance of the beetles. By direct observation of the beetles during the experiments, it could be confirmed that they actually exhibited their maximum pulling performance, i.e. they attempted to escape from the fixed mount by pulling vertically in the direction of maximum sensitivity of the balance. This could be further ensured by occasionally stroking the beetle with a fine hairbrush to stimulate escape behaviour.
The vertical climbing performance of each beetle was tested on four different surfaces, with approximately 3 h of recovery between tests (Fig. 2): (i) factory-cleaned microscope glass slides (Wetzel, Braunschweig, Germany) (Fig. 2A); (ii) undeveloped fibre-based non-glossy photographic paper (RC DeLuxe Multigrade III, Ilford, UK) (Fig. 2B); (iii) the adaxial surface of young air-dried reed leaves (Phragmites communis Trin.) (Fig. 2E); (iv) thin filter paper (Fig. 2F).
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Experiment 2: measurement of the relative significance of tarsal
claws versus tenent setae for vertical climbing on a variety of surfaces
For these experiments, I compared S. comma (slender tarsi) with
S. pubescens and S. cicindeloides (both with wide bilobed
tarsi). Apart from the above-mentioned surface types i, iii and iv, additional
tests were performed with S. comma and S. pubescens but only
on the adaxial surfaces of young fresh leaves of (v) Glyceria maxima
(C. Hartm.) Holmb. (Fig. 2C)
and (vi) Phragmites communis (Fig.
2D). The surface of Phragmites leaves undulates on
account of the protruding parallel veins, but Glyceria leaves have
much smoother surfaces.
For each species, two groups of 10 beetles (each consisting of five males and five females) were tested. After the pulling forces of all 20 intact beetles had been measured as described above, the beetles were anaesthetized and, in the first group of 10 individuals, the claws of the forelegs were cut at the base, and in the second group, the tarsal tenent setae were `neutralized' by covering them with a thin layer of fast-drying superglue. The latter formed a smooth hard layer covering tarsomeres I-IV; tarsomere V with the claws remained intact. The superglue treatment was not applied to the articulation zones between the single tarsomeres so it did not reduce the mobility of the tarsomeres relative to each other. After 1 day of recovery with food ad libitum, the pulling forces on the various surfaces were measured again in both groups under otherwise identical conditions.
Measurement of the surface roughness of a variety of surfaces
The four test surfaces used in experiment 1 were sputter-coated with gold
to increase their reflection during the following measurements. The surface
roughness (Ra values) was determined with an optical profiler (Veeco
Instruments Inc., type NT3300), which was run in the VSI mode and calibrated
according to an NIST-certified step height standard (10.10 µm step height).
To equalize possible tilts of the test surfaces, one layer was subtracted from
the measured surface images prior to the determination of their roughness.
Measurement of the apparent surface energies of a variety of
surfaces
The apparent surface energies of the majority of test surfaces were
determined by measuring the advancing contact angle with a drop shape analysis
system (DSA 10-G140, Krüss GmbH, Hamburg, Germany) using water and
di-iodomethane as test liquids (volume flux 10 µl min-1, conic
calculation method). On account of the considerable undulation of the dry
surface of Phragmites, the contact angle on this surface was measured
statically, whereas it was measured dynamically (sessile drop method) on all
the other surfaces. The apparent surface energies (made up of their polar and
dispersive components) were automatically calculated by the system according
to Owens and Wendt (1969) and
Rabel (1971
) from the drop
shape data.
Measurement of the surface tension of pond water
Samples of pond water from two different locations near Kiel (Northern
Germany), where Stenus beetles were observed to be active on the
surface of the water, were taken in August. The surface tension was measured
in the laboratory with a processor tensiometer (K12, Krüss, Hamburg,
Germany) as 70.5 mN m-1 at 27.4°C and 66.4 mN m-1 at
26.6°C.
Qualitative demonstration of non-wettable and wettable parts of the
body during locomotion on the surface of water
A test chamber (3 cmx2 cmx1.5 cm) with white-coloured inner
sides was filled with tap water (surface tension 72.0 mN m-1)
according to Baudoin (1976).
The surface of the water was illuminated by fibre optics from an angle of
approximately 70°. Beetles from various subgenera with slender tarsi
(S. comma, S. juno, S. boops) and wide tarsi (S. cicindeloides,
S. solutus, S. pubescens) were observed while walking on the surface of
the water in this container. The shadows on the bottom of the container
produced by those parts of the body that contacted the surface of the water
were recorded by a video system. Depressions caused by the weight of the
beetle pressing down the water surface via hydrophobic body surfaces
gave large roundish shadows at the bottom of the test chamber, whereas
upward-curving menisci caused by hydrophilic surfaces produced luminous points
(Baudoin, 1976
).
Quantification of the wettability of the ventral side of the
tarsi
The wettability of the ventral side of the tarsi by water was determined in
4-5 specimens of each of six equally sized Stenus of various
subgenera with slender or wide tarsi and in one Dianous species with
slender tarsi. The animals were anaesthetized with CO2, decapitated
and fixed on their back on a mechanical stage. The advancing apparent contact
angles between the ventral sides of the middle tarsi and distilled water were
measured dynamically using the drop shape analysis system described above. The
vertical component of the surface tension FS acting on the
ventral tarsal surface and supporting the insect on the surface of water was
calculated according to Baudoin
(1976) and Guthrie
(1989
) as:
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A safety coefficient C, estimating the capacity of the buoyancy
plus surface tension to support the beetle on the surface of the water, was
calculated according to Baudoin
(1976):
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Scanning electron microscopy
To measure the morphometric parameters of the tarsi of the various species
as required for the above calculations, whole legs were stepwise dehydrated in
ethanol/acetone, critical-point-dried (CPD 020, Balzers, Germany), fixed to
stubs with silver paint, coated with gold (Sputter Coater S 150 B), and viewed
in a scanning electron microscope (LEO 420, Leo, Oberkochen, Germany).
Statistical analyses
All the following statistical tests were performed with log-transformed
data. Before this transformation, the square root of the number of tarsal
setae and the cube root of body mass were calculated. Simple linear regression
analysis was used to test for the dependence of the maximal exerted pulling
force on the number of tenent setae. The numbers of tarsal tenent setae were
obtained from a previous publication (see
table 1 in
Betz, 2002), in which the
tenent setae of each species were counted on the hind tarsi of five males and
five females. To correct for body size, two separate linear regression
analyses of both these log-transformed variables against log-transformed body
mass were performed. The non-standardized residuals of these analyses (i.e.
the difference between the data and a linear regression fitted to them) were
then used to test for the final relationship between the two variables. The
Tukey test was used to test for differences between the slopes of the
different regression lines (Zar,
1999
). MannWhitney U-tests were performed with the
non-standardized residuals to test for overall differences in maximum pulling
force between the group of species with slender tarsi (subgenera
Stenus s. str. + Nestus) and the group of species with wide
tarsi (subgenera Hypostenus, Metastenus and Hemistenus) on
each of the various surfaces. To test for differences between the climbing
performance of the same individuals on the different test surfaces, a Friedman
test was performed, followed by the Wilcoxon test for paired comparisons
(Sokal and Rohlf, 1995
). The
Wilcoxon test was also used to perform within-species comparisons of the
climbing performance of individuals before and after manipulation of the claws
and the tenent setae. Interspecific comparisons of pulling forces were
performed with pairwise MannWhitney U-tests. The significance
levels of all the non-parametric pairwise tests that included more than one
comparison were corrected according to the sequential Bonferroni procedure
(Sokal and Rohlf, 1995
). All
statistical analyses were performed with SPSS 6.1. (SPSS Inc., Chicago,
USA).
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Results |
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Surface roughness
Whereas glass represents an evenly smooth surface
(Fig. 2A), the selected type of
photographic paper has a uniformly roughened surface structure at the
micrometre level (Fig. 2B). The
surface of fresh Glyceria plants is smooth (similar to glass), but
regularly interrupted by protruding longitudinal leaf veins
(Fig. 2C). Protruding hairs or
wax blooms are not present. In Phragmites leaves, the surface on and
between the protruding leaf veins bears an array of tiny spines
(Fig. 2D,E) and wax blooms
(Fig. 2G). Dry
Phragmites leaves (Fig.
2E) differ from fresh ones
(Fig. 2D) by their
significantly increased surface topography, indicated by distinct clefts and
ridges. Filter paper represents a coarse network of thick fibres
(Fig. 2F). The quantitative
differences in surface roughness between the four test surfaces used in
experiment 1 are shown in Fig.
3. As indicated by the illustrated surface profiles and
Ra values, the surface roughness of the test surfaces increases in
the order glass, photographic paper, dry Phragmites leaves and filter
paper.
Surface energies and polarities
The apparent surface energies and polarities of the test surfaces, as
calculated from the contact angle measurements, are summarized in
Table 1.
Vertical climbing performance on a variety of surfaces
The absolute maximum pulling forces achieved by the beetles on various
surfaces are summarized in Table
2. The pulling force to body weight ratios for the various
surfaces are illustrated in Fig.
4. In general, the pulling forces in the species investigated
increased in the order photographic paper, glass, dry Phragmites
leaves, filter paper (Fig. 4;
graphs on the left). The overall comparison of the body-weight-corrected
pulling forces revealed that, on all four test surfaces, species with wide
tarsi (subgenera Hypostenus, Metastenus and Hemistenus) on
average exhibited significantly higher forces than species with slender tarsi
(subgenera Stenus s. str., Nestus) (MannWhitney
U-tests; significance levels on photographic paper and glass,
P<0.001; on dry Phragmites leaves and filter paper,
P0.05). However, a clear distinction between the two groups was
observed only on glass and on photographic paper (although, on the latter,
three species with wide tarsi attained only relatively low pulling forces; see
Fig. 4A). On both the other
surfaces, single species with slender tarsi also accomplished high relative
pulling forces and vice versa
(Fig. 4; plots on the left). As
indicated by the values of both the coefficient of determination
(r2) and the slope (b), the importance of the
number of ventral tarsal tenent setae for the attained pulling forces is
greatest on glass and decreases in the order photographic paper, dry
Phragmites leaves and filter paper
(Fig. 4; plots on the right).
The slopes of the regression lines are similar for photographic paper and dry
Phragmites leaves only; they are significantly different for all the
other possible comparisons (pair-wise Tukey tests, P<0.05).
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Relative significance of tarsal claws versus tenent setae for
vertical climbing on a variety of surfaces
The results of the pulling force experiments conducted with manipulated
animals are summarized in Table
3 (original data) and Fig.
5 (corrected for body weight), which compare the performance of
S. comma with slender tarsi with those of S. pubescens and
S. cicindeloides, both of which have wide tarsi.
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Stenus comma
In this species (Fig. 5A),
the pulling forces per body weight on the smooth glass and fresh
Glyceria leaf surfaces were very low even in intact beetles, so
neutralization of the tenent setae had little effect. A considerable decrease
in the pulling forces after the loss of the tenent setae was established on
the dry Phragmites leaves only (on fresh Phragmites leaves,
this decrease was much smaller, although statistically equally significant),
whereas this decrease was trivial and statistically not significant on filter
paper. Claw amputation resulted in a marked decline in the attainable pulling
forces on all test surfaces that showed at least some degree of surface
roughness (i.e. all the tested surfaces except glass).
Stenus pubescens
On the smooth glass and fresh leaf surfaces, intact beetles attained
considerably higher pulling forces per body weight than S. comma
(Fig. 5B). However, on the
rougher surfaces of dry Phragmites and filter paper, the pulling
forces were similar to those of S. comma. Neutralization of tenent
setae reduced the pulling forces most drastically on the smooth glass and
plant surfaces; this decline was more moderate on filter paper, although
statistically equally significant. Removal of the claws had no effect on the
pulling forces on the smooth glass and Glyceria surfaces. However,
with increasing roughness of the test surfaces, the effect of claw removal
became much more significant.
Stenus cicindeloides
This species (Fig. 5C) was
only tested on three different surfaces and showed almost the same results as
S. pubescens.
Significance of the specific structure of tenent setae
The pulling force experiments conducted with animals whose claws had been
removed made it possible to estimate the significance of the size and specific
structure of the single tenent setae by dividing the maximal attained pulling
forces by the number of tenent setae and comparing this variable among species
(Fig. 6). The species-specific
numbers of tarsal tenent setae were obtained from counts performed by Betz
(2002). It can be seen that,
on the majority of test surfaces, the tarsal tenent setae of both the species
with wide tarsi (S. pubescens and S. cicindeloides) allow
significantly higher pulling forces than those of the species with slender
tarsi (S. comma). The only instance when the pulling forces of
individual tenent setae are almost identical between species with wide and
slender tarsi is on dry Phragmites leaves
(Fig. 6).
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Wettability of the tarsi during locomotion on the surface of
water
Videography of beetles of various species walking on the clean surface of
tap water in a test container revealed that both species with wide tarsi and
species with slender tarsi were supported by the surface film. While being
supported, the beetles occasionally bent the apex of the abdomen downwards so
that it came into contact with the water surface and thus supported the body,
in addition to the legs (see Betz,
1999). When contacting the surface film, the bottom surface of the
tarsi produced large roundish shadows surrounded by a bright halo. The centres
of the shadows were sometimes superimposed by one sharp luminous point
(Fig. 7). The measured apparent
contact angles between the bottom surface of the tarsi of the various species
and water together with the other variables necessary to calculate the forces
supporting the beetles on the surface film are given in
Table 4. It can be seen that,
from a theoretical point of view, both tarsal morphologies permit sufficient
support by the surface of water (safety coefficients >1), which is mainly
attributable to the high apparent contact angles between the ventral sides of
the tarsi and the water (Table
4). The effect of buoyancy on supporting the beetles on the
surface of water is negligible in all the species examined
(Table 4).
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Discussion |
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I have evaluated tarsal performance under two different potential selective
regimes: locomotion on a surface film and climbing up vertical structures. The
use of widened tarsi in contexts such as copulation can be ruled out, because
(i) in the vast majority of Stenus species, there are no obvious
sex-specific differences in tarsal morphology, (ii) the tarsi are widened not
only in the forelegs but also in the middle- and hindlegs, and (iii) species
in lineages with widened tarsi copulate in an end-to-end position, which does
not require the male to grasp the female. However, several species with
slender tarsi mate only in a parallel position
(Betz, 1999), a position in
which widened tarsi in the male would indeed be more advantageous. In the
following, I consider the usefulness of widened tarsi versus slender
tarsi in Stenus and in one Dianous species in the context of
locomotion on the surface of water and vertical climbing on various surface
textures. Finally, I discuss the contribution of my results to our
understanding of the mechanism of insect tarsal attachment to differently
textured solids.
The role of tarsal morphology during walking on the surface of
water
Many representatives of the Steninae live in damp environments, where they
can be found running on the ground or climbing on a variety of plants. In
these habitats, the beetles need to be able to cross patches of free surface
water and, indeed, three different modes of locomotion can be distinguished on
a surface film (Betz, 1999):
(i) walking on the surface film as if it were firm ground, following the same
mode of leg coordination as during terrestrial locomotion, (ii) swimming,
while the tarsi, tibiae and the entire undersurface of the body make contact
with the surface of the water and perform typical swimming movements without
becoming immersed, and (iii) expansion skating, i.e. rapidly skimming over the
surface film by releasing an abdominal secretion (see
Jenkins, 1960
;
Linsenmair, 1963
).
The function of the tarsi is most critical during normal walking on the
surface film since, in this case, the bottom surfaces of the tarsi form the
only interfaces with the water, sometimes supported by the ventrally bent apex
of the abdomen. As shown in Fig.
7, the tarsi of the Steninae walking on the surface film in a test
container produce large shadows surrounded by bright halos, indicating their
non-wettability (see Baudoin,
1976). However, a single luminous point can also be seen within
these shadows; this must be produced by some wettable structure on the tarsi.
As in semiaquatic bugs (Baudoin,
1976
; Darnhofer-Demar,
1969
; Møller Andersen,
1976
), these luminous points are probably caused by the smooth,
and thus more-wettable, claws penetrating the surface film.
The contact angle measurements confirm that the investigated Steninae have
especially water-repellent tarsal ventral surfaces with apparent contact
angles with water of up to 150° (Table
4). Contact angles of this size cannot be achieved by the
hydrophobic chemical composition of the cuticle surface alone, but only by
additional surface roughness (e.g. Crisp,
1963; Holdgate,
1955
; Noble-Nesbitt,
1963
). In contrast to smooth tarsal attachment systems such as
euplantulae and arolia, tarsi with an array of obliquely inclined ventral
tenent setae have the potential to form this roughness, causing an increase in
water-repellency as required for safely supporting a walking insect on the
surface film. However, this is achievable only when the tarsal tenent setae
are impregnated with a hydrophobic lipid layer, increasing the true contact
angle beyond 90° (e.g. Holdgate,
1955
).
In several Stenus species, it has been demonstrated that the
beetles release a secretion through their tarsal tenent setae; the lipid
component of this secretion resembles the composition of the superficial lipid
coating of the body surface (Betz,
2002). Since the lipid fraction is mainly a mixture of unsaturated
fatty acid glycerides and aliphatic hydrocarbons, i.e. neutral lipids, it
should have little tendency to spread over a water surface. Hence, it can be
assumed that high contact angles on the bottom of the tarsi can be maintained,
even after prolonged contact with water, by the subsequent delivery of a
hydrophobic secretion, which is distributed over the tarsi and the other parts
of the body surface by intense self-grooming. Grooming has been observed to
take up 5-50% of the time budget in Stenus species
(Betz, 1999
), underlining its
biological significance. When submerged, the tarsi and other parts of the body
have a silvery appearance, provided by air entrapped between the unwettable
hairs. Such air films are well known from other semi-aquatic insects, such as
water striders (e.g. Crisp,
1963
), and are responsible for the considerable increase in the
apparent contact angle beyond the true contact angle
(Adam, 1963
).
The contact angle measurements performed on the bottom surface of the tarsi
demonstrate that, in all the species examined, the tarsi, irrespective of
whether they are wide or slender, provide adequate safety coefficients to
support the beetles on the surface of water
(Table 4). These values are
sufficient to support the body on the surface film, even if the surface energy
of the water is reduced by natural surfactants. As can be deduced from
Table 4, species with slender
tarsi nevertheless attain considerable perimeters of the line of contact
between the tarsus and the water surface because their small tarsal width is
more than compensated for by their increased tarsal length. Consequently,
their safety coefficients are not necessarily lower than those of species with
widened tarsi. According to these results, it can be concluded that there have
been no selective demands in this biological context to drive the widening of
the tarsi within the Steninae. The actual safety coefficients for all the
species examined must be considered to be even higher, because the method used
to calculate buoyancy (equation 5 in Materials and methods) somewhat
underestimates the upward force exerted by the water surface; the calculated
buoyancy only considers that of the tarsus itself, whereas the depression in
the surface film caused by the tarsus has a volume that must be considered as
being larger than that of the tarsus (see
Suter et al., 1997;
Suter and Wildman, 1999
).
According to these authors, such depressions associated with leg/surface-film
contact are employed by many water-walking organisms to produce the horizontal
thrust necessary for propelling themselves over the surface film.
The role of tarsal morphology during climbing on a variety of
surfaces
As in other studies of insect tarsal attachment using straingauge force
transducers (e.g. Dixon et al.,
1990; Lees and Hardie,
1988
; Stork,
1980a
; Walker et al.,
1985
), the present study considers vertical pulling forces exerted
by tethered animals as indicators of tarsal attachment performance. The
advantage of this method is that the animals can be directly observed during
the experiments, ensuring that they actually exert their maximum performance
abilities. While doing so, the Steninae beetles usually attach to the surface
with all six tarsi simultaneously.
In an initial experiment, the attachment performance of beetles of 19 species was tested on four different surfaces (Fig. 4). This experiment revealed that widened tarsi associated with considerably more tenent setae had significantly higher attachment capacities. The presence of a large number of tarsal tenent setae was also of marked significance on smooth plant surfaces such as Glyceria maxima, as used in the second experiment with manipulated beetles (Fig. 5). On surfaces with increased roughness, the importance of the number of tarsal tenent setae decreased, some species with slender tarsi also attaining relatively high pulling forces (Fig. 4). This indicates that, on these surfaces, the claws become functionally more important, since they enable the beetles to cling to protruding elements of the surface and subsequently to use this firm anchoring point to draw up the rest of the body.
To elucidate the relative significance of the tarsal tenent setae versus that of the pretarsal claws, a second series of experiments was undertaken, which allowed a comparison of the attachment performance of beetles before and after neutralization of the tarsal tenent setae and the claws (Fig. 5). This experiment clearly demonstrated the significance of the widened bilobed tarsi on smooth surfaces such as glass or fresh Glyceria leaves: whereas S. comma beetles with slender tarsi did not attain large pulling forces on these surfaces either with intact tarsi or with neutralized tenent setae (Fig. 5A), the neutralization of the tenent setae greatly diminished the attachment performance of both species with wide tarsi (S. pubescens and S. cicindeloides) (Fig. 5B,C). At the same time, claw removal, but with intact tenent setae, did not affect movement on these smooth surfaces. In contrast to the conditions on these smooth surfaces, the tenent setae are of minor importance on very rough surfaces, here exemplified by filter paper. On this surface, claw removal reduced attachment capabilities drastically in all the test species irrespective of their tarsal morphologies, whereas the effect of the neutralization of the tenent setae was much smaller (Fig. 5).
The results attained on both dry and fresh Phragmites surfaces are of special interest since the characteristics of smooth and rough surfaces appear to be united in both these surfaces. This is indicated by the observation that, in all the three test species irrespective of their tarsal morphology, both the neutralization of the tenent setae and the amputation of the claws significantly reduce the attainable pulling forces (Fig. 5). Therefore, pretarsal claws and tarsal tenent setae are probably functionally linked on these surfaces and work synergistically. The possible mechanism behind this is discussed in more detail in the next section.
In Stenus species with especially widened tarsi, not only is the
bottom surface of the tarsus provided with more tenent setae but the quality
of the single tenent setae is also different. In the 19 species investigated,
nine different morphological types of tarsal ventral setae could be identified
(see figs 3 and
4 in
Betz, 2002). Whereas the
majority of these types probably have mechanoreceptive functions, three of
them can be assigned as tenent setae. One of these types occurs in all the
investigated species irrespective of their tarsal morphology. It is terminally
tapered and sub-apically recurved, but otherwise shows no specific terminal
differentiation (see figs 3c,d;
4a,e in
Betz, 2002
). However, species
with widened tarsi have an additional type of tenent seta, which is distally
spatulate (see figs 3h and
4c in
Betz, 2002
). Tenent setae of
this type are also found among many other groups of beetles
(Stork, 1980b
) and can be
assumed to develop higher adhesive forces as a result of their increased area
of contact with the substratum.
The significance of the specific morphology of the tenent setae for attachment performance in Stenus beetles can be roughly assessed by comparing the maximum pulling forces divided by the total number of tenent setae among beetles whose claws were removed (Fig. 6). Indeed, these calculations show that, in addition to the number of tenent setae, their size and morphology are also of vital importance. On the vast majority of the test surfaces, the calculated pulling forces per tenent seta are higher in S. pubescens and S. cicindeloides compared with S. comma with its slender tarsi (Fig. 6). This is probably attributable to the sole-like enlarged apices that are present on the tenent setae in both the first two species and that might also be better supplied with adhesive secretion. These spatulate apices probably provide the setae with a larger area of contact with the substratum. The specific tarsal morphology is of no major importance if the beetles try to move on dry Phragmites leaves. The possible reason for this is discussed in the next section.
As shown above, the especially widened bilobed tarsi in Stenus
beetles are not vital for supporting the beetles on the surface of water.
Indeed, the results of the pulling force experiments suggest that the main
selective demands have come from the attachment to smooth plant surfaces. The
peak pulling forces in species with slender tarsi on smooth surfaces amount to
1-10 times their own body weights (cf.
Table 2 and
Table 3), but this is obviously
not a sufficient safety factor to make possible the more permanent settlement
of the vegetation. First, the listed pulling forces represent the maximum
performance abilities only when the beetles attach themselves to the surface
with all six tarsi. The exerted pulling forces are usually considerably lower
(see Fig. 1), because during
normal walking only three tarsi are simultaneously in contact with the surface
and the attachment to the surface will not be always optimal. Second, wide
tarsi might be not only important for mounting vertical structures but also
for resisting horizontal detachment forces caused by the drag and whiplash of
moving leaves. According to the projections of Stork
(1980a) and assuming that
leaves oscillate in a strong gusty wind in a harmonic motion, insects have to
withstand detachment forces of approximately 16 times their body mass. As can
be seen from Table 2 and
Fig. 4, on smooth surfaces,
such as glass or fresh leaves, only species with wide tarsi (subgenera
Hypostenus, Metastenus and Hemistenus) attain pulling
force/body weight ratios that clearly exceed this value, whereas those of
species with slender tarsi (subgenera Stenus s. str.,
Nestus, and the one Dianous species examined) remain below
this value.
The experimental results of the present study demonstrate the importance of
wide tarsi, accommodating a large number of tarsal setae, for climbing on
vertical plant surfaces. Recalling that, in Stenus beetles, slender
weakly or non-bilobed tarsi most probably represent the phylogenetically
antecedent condition compared with the wide distinctly bilobed tarsi, the
evolution of wide tarsi in the various lineages might represent a key
innovation that has made possible the expansion of the adaptive zone to live
plants, contributing to the tremendous radiation of this genus. Indeed,
approximately 70% of the more than 2100 Stenus species described
belong to three subgenera (Hypostenus, Metastenus and
Hemistenus) whose representatives have wide bilobed tarsi (V. Puthz,
personal communication). Unbalanced clade diversities of this order might
justify consideration of the evolution of wide bilobed tarsi in
Stenus as a key innovation. With reliable phylogenies available, this
hypothesis might be testable in the future using statistical approaches (e.g.
Bond and Opell, 1998).
Mechanisms of tarsal attachment to a variety of surfaces
The present study was conceived mainly to evaluate the adaptive value of
various tarsal morphologies and it also contributes to our understanding of
the general mechanism of tarsal attachment to different substrata. The test
surfaces used in this study differ mainly (i) in their surface roughness (Figs
2,3),
(ii) in their free surface energies and (iii) in their surface polarities
(Table 1), which makes it
possible to infer the relative functional roles of both the pretarsal claws
and the tarsal tenent setae and the possible characteristics of the tarsal
adhesive secretion from the performance data.
Rough surfaces
The mechanism of insect tarsal attachment to differently textured surfaces
has been the subject of various previous studies (e.g.
Dixon et al., 1990;
Gorb et al., 2001
;
Jiao et al., 2000
;
Lees and Hardie, 1988
;
Roth and Willis, 1952
;
Stork, 1980a
;
Walker et al., 1985
), some of
them aiming at illuminating the relative significance of the pretarsal claws
versus the tarsal tenent setae and the smooth attachment pads. Two of
these studies have emphasized the predominant role of the claws on rough
surfaces (Roth and Willis,
1952
; Stork,
1980a
). As discussed in the previous section, this view is in good
agreement with the results of the present study. Hence, on rough surfaces, the
maximally attainable pulling forces should be limited only by (i) the leverage
and maximum power output of the leg muscles and (ii) the yielding strength of
the pretarsus and the surface projections of the substratum. For the latter,
the shape of the claws in relation to the surface topography (structure and
magnitude) may be of special importance. However, even on filter paper, the
tarsal tenent setae have a significant (although weak) supporting effect on
the pulling forces exerted (Figs
4D,
5B), suggesting their
mechanical interlocking with surface irregularities.
Smooth surfaces
The vertical pulling forces measured in this study represent attachment
forces that work parallel (non-normal) to the plane of the surface at the
interface between the tarsal tenent setae and the substratum. In insects, a
tarsal secretion sandwiched between the ventral tarsus surface and the
substratum is considered to be a vital component of attachment in both hairy
and smooth systems (e.g. Jiao et al.,
2000). This is especially true of smooth surfaces which, in the
present study, are represented in various forms by the glass, photographic
paper, fresh Glyceria leaves and, to a lesser degree, fresh
Phragmites leaves. The underlying attachment forces acting parallel
to the substratum are generally considered to be a combination of capillary,
viscous, friction and, at close contacts, molecular forces (e.g.
Gorb and Scherge, 2000
;
Jiao et al., 2000
;
Scherge and Gorb, 2001
;
Wigglesworth, 1987
).
As mentioned above, Stenus species with both slender and wide
tarsi release a tarsal secretion via their tenent setae; this is
visible as footprints on a glass surface. According to its neutral lipid
content (Betz, 2002), this
secretion has two functions: (i) to keep the ventral surface of the tarsi
water-repellent (as discussed above) and (ii) to wet a variety of plant
surfaces to improve attachment forces during locomotion. Despite their high
surface polarities, surfaces such as glass and photographic paper have
sufficiently high free surface energies to allow possible wetting by even
apolar lipid secretions (Table
1) (see McFarlane and Tabor,
1950
). In contrast, surface energies can be extremely low on waxy
plant surfaces (Table 1) and
might thus impede wetting by even non-polar lipid tarsal secretions.
However, the results showing that the pulling forces of clawamputated
beetles on fresh Glyceria and Phragmites leaves are similar
to (or even larger than) those attained on glass
(Table 3;
Fig. 5) suggest that the tarsal
secretion might be capable of spreading on even these extremely hydrophobic
surfaces. This might be understandable under the two-component surface energy
approach (e.g. Wu, 1973),
according to which complete wetting of a low-energy substratum by a low-energy
adhesive might be still possible on condition that the surface polarities of
both the adhesive and the substratum match closely. Hence, insect tarsal
secretions should generally be expected to be mixtures of neutral lipids with
only low (if any) contents of polar components such as fatty acids, esters and
alcohols. The few attempts to analyse the tarsal secretion of insects
chemically support this hypothesis (e.g.
Attygalle et al., 2000
;
Betz, 2002
;
Ishii, 1987
;
Kosaki and Yamaoka, 1996
). The
spread of such adhesives would be further facilitated by maintaining their
viscosity as low as possible (McFarlane
and Tabor, 1950
; Zisman,
1964
). The main contribution of the adhesive to attainable
adhesion is presumably attributable to its viscosity rather than to its
capillarity, because the pulling forces measured in this study are exerted
parallel to the substratum (e.g. Denny,
1993
; Jiao et al.,
2000
).
It has not as yet been possible to determine the thickness of the secretion
sandwiched between the tarsal tenent setae and the substratum; this would be
crucial for a quantitative assessment of the relative contribution of those
forces to the observed pulling forces. However, for the adhesive pads of
Tettigonia viridissima, it has recently been shown that the tarsal
secretion does not completely account for the adhesive forces exerted vertical
to the substratum (Jiao et al.,
2000), so that additional friction and/or intermolecular forces
are probably involved (see Persson, 1998).
Another interesting result of the present study is the low attachment of
the tarsi to the photographic paper. This substratum yielded the lowest
measured pulling forces of all the tested surfaces, especially in species with
wide tarsi (Table 2). Although
its surface is actually uniformly roughened (Figs
2B,
3B), photographic paper is here
treated as a smooth surface because its surface irregularities are obviously
an order of magnitude too small (indicated by its low Ra value of
1.09) to present an opportunity for the claws to cling to it. This can be
deduced from the finding that the pulling forces exerted on photographic paper
are even lower than those attained on glass
(Table 2), on which the claws
have been shown not to contribute to the overall pulling force
(Table 3; Fig. 5). Since the surface
energies and polarities of the photographic paper are very similar to those of
the glass (Table 1), the
reduced attachment forces on photographic paper are not likely to be
attributable to its lower wettability by the secretion. Rather, they must be
considered to be the result of the reduced area of real contact between the
roughened surface of the photographic paper and the tarsal tenent setae; this
would substantially reduce attachment forces caused by both adhesion and
friction (e.g. Persson, 1998; Scherge and
Gorb, 2001). The same effect is probably responsible for the
reduced attachment forces of claw-amputated beetles on dry Phragmites
leaves compared with fresh ones (Table
3; Fig. 5), because
wilting results in an increased surface corrugation (compare
Fig. 2E with
Fig. 2D). This takes place not
only on a large-scale level, resulting in longitudinal semi-cylindrical
ridges, but also at the level of the epidermis cells, forming distinct bulges
(Fogg, 1947
,
1948
).
Surfaces that combine attributes of both rough and smooth
surfaces
Many natural plant surfaces usually combine the surface characteristics of
both rough and smooth substrata, because the smooth plant epidermis might be
regularly disrupted by protuberances, such as cuticular folds, leaf veins,
trichomes or wax crystalloids (e.g.
Juniper and Jeffree, 1983).
The wax crystalloids on glaucous plant surfaces might actually physically
impede the adhesion of insect tarsi (i) by reducing the actual area of
contact, (ii) by contaminating the tenent setae and (iii) by exfoliating as
the insect walks on the surface (e.g.
Brennan and Weinbaum, 2001
;
Eigenbrode, 1996
;
Juniper and Burras, 1962
;
Stork, 1980c
). This effect is
probably responsible for the reduced adhesion, especially of clawamputated
Stenus beetles, to Phragmites surfaces compared with
Glyceria leaves (Table
3; Fig. 5) because
protruding wax blooms have been detected on Phragmites leaves only
(see Fig. 2G). Consequently,
the specific structure of the single tenent setae is of no importance on the
latter (see Fig. 6).
However, as indicated in the previous section, the claws are to some extent capable of making up this shortcoming, suggesting a hitherto overlooked functional synergism between claws and tarsal tenent setae. Since both the removal of the claws (leaving the tarsal tenent setae intact) and the neutralization of the tenent setae (leaving the claws intact) result in a significant decrease in the pulling forces, the claw function probably adds to the adhesion and friction force mediated by the tenent setae. This can be illustrated by a simple mechanical analogue in which the tarsus is considered as a hook-like lever rotating about a projection or crevice of the substratum to which the claws cling (Fig. 8). During vertical upward climbing, this construction results in the tarsus being pressed against the surface, causing spring-like compression of the elastic tenent setae (Fig. 8B). Consequently, according to the counterforce exerted by the tenet setae, the load of the tarsus directed normal to the surface and, thereby, the real area of contact between the tarsus and the substratum are increased.
|
Such conditions would actually enhance the attachment forces according to
both mechanisms of attachment considered, i.e. friction and adhesion,
attributable to the viscosity of the secretion (e.g.
Bowden, 1957; Persson, 1998;
Scherge and Gorb, 2001
).
Whereas the first part of the mechanical analogue (i.e. clinging of the claws
to a surface irregularity and subsequent pressing of the tarsus against the
surface) has been directly observed in tethered beetles, the proposed
spring-like behaviour of single tarsal tenent setae has not as yet been
confirmed by direct observation but can be inferred from the internal setal
flexibility and curved appearance. The increase in attachment forces acting
parallel to the substratum with increasing normal loads has recently been
empirically demonstrated in `smooth' systems, such as the attachment pads of
the locust Tettigonia viridissima
(Jiao et al., 2000
), and in
`hairy' systems as exemplified by gecko feet
(Autumn et al., 2000
). The
latter study has shown that the parallel pulling forces attained by single
gecko foot-hairs depend on the initial preload, which does not have to be
maintained during subsequent pulls. Applied to the mechanical analogue of
Fig. 8, this means that, in
insects that climb upwards on vertical surfaces, an initial grasp of the claws
to some surface irregularity might suffice to increase significantly the
subsequent attachment forces exerted by the tenent setae, even when the claws
no longer cling to the surface. This would be especially important on plant
surfaces with relatively weak and/or small surface projections, which (unlike
filter paper) would easily give way under the subsequent stronger shear forces
exerted by the claws.
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Acknowledgments |
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References |
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