‘Insect aquaplaning’ on a superhydrophilic hairy surface: how Heliamphora nutans Benth. pitcher plants capture prey

Ulrike Bauer, Mathias Scharmann, Jeremy Skepper, Walter Federle

Abstract

Trichomes are a common feature of plants and perform important and diverse functions. Here, we show that the inward-pointing hairs on the inner wall of insect-trapping Heliamphora nutans pitchers are highly wettable, causing water droplets to spread rapidly across the surface. Wetting strongly enhanced the slipperiness and increased the capture rate for ants from 29 to 88 per cent. Force measurements and tarsal ablation experiments revealed that wetting affected the insects' adhesive pads but not the claws, similar to the ‘aquaplaning’ mechanism of (unrelated) Asian Nepenthes pitcher plants. The inward-pointing trichomes provided much higher traction when insects were pulled outwards. The wetness-dependent capture mechanisms of H. nutans and Nepenthes pitchers present a striking case of functional convergence, whereas the use of wettable trichomes constitutes a previously unknown mechanism to make plant surfaces slippery.

1. Introduction

Plant hairs (trichomes) occur in all major groups of terrestrial plants and perform key functions such as thermoregulation, reduction of transpirational water loss and defence against radiation, herbivory and pathogen attack [13]. Trichomes usually render leaf surfaces water-repellent [48], although some are wettable and absorb water [9,10]. In carnivorous plants, they often have special functions, such as secretion (e.g. Drosera, Pinguicula), absorption of nutrients (e.g. Drosera, Brocchinia, Catopsis), sensing visitors and triggering trap movements (e.g. Dionaea, Utricularia), adhesion (sticky hairs in Drosera and Drosophyllum) and prey retention (e.g. Genlisea, Sarracenia) [1113].

The genus Heliamphora (Sarraceniaceae) comprises 23 species of pitcher plants found predominantly in the highlands of southern Venezuela [14]. The pitchers are partly fluid-filled and function as passive pitfall traps for insects, mainly ants [15]. While the morphology has been studied [12,13,1517], the detailed trapping mechanisms are still unknown. Heliamphora nutans (figure 1) grows in altitudes between 2000 and 2700 m where it forms rosettes of funnel-shaped leaves, each up to 18 cm tall and 7 cm wide [14,18]. Prey is attracted by nectar secretion on the inner wall and under an appendage at the rear of the pitcher mouth [12]. The upper (exposed) part of the inner pitcher wall is covered with a dense carpet of inward-pointing hairs that are believed to play a role in prey capture [12] and retention [14]; however, experimental evidence is missing. This pubescent zone extends downwards to a constriction below which the pitcher widens in a bulbous shape and the inner wall is smooth (figure 1b). Further trichomes are found in the narrow tubular section near the bottom of the pitcher; however, these differ in size, density and structure from those on the upper wall surface [12].

Figure 1.

Habitus and inner wall morphology of H. nutans. (a) Dense clusters of H. nutans pitchers growing in the natural habitat on Mt. Roraima, Venezuela (photograph: C. C. Lee). (b) H. nutans pitcher (lengthwise cut in half) illustrating the different morphological zones found on the inner pitcher wall. The fluid level is regulated by a drainage hole (arrow) at the lower end of the pubescent zone (photograph: R. Severitt).

Jaffe et al. [15] observed that the pubescent inner wall of H. nutans pitchers is readily wettable and water rises by capillarity up to the pitcher rim (see the electronic supplementary material, video S1). This was interpreted as a mechanism to drain excess water and maintain a constant fluid level. However, complete wettability leads to the formation of thin water films that may cause insects to slip. Water lubrication, in combination with a directional (anisotropic) surface topography, is critical for the trap function of (unrelated) Asian pitcher plants (Nepenthes) [1921]. Here, we investigate whether the observed wettability plays a similar role for H. nutans, and how surface wetness and trichome anisotropy affect the friction forces of ant tarsi.

2. Material and methods

All experiments were performed on three H. nutans plants grown at Kew Gardens, UK. The plants were kept under natural light in a greenhouse (8–21°C, min. 75% relative humidity), misted daily with reverse osmosis water and not fertilized for several months prior to the experiments.

(a) Pitcher micromorphology

We used scanning electron microscopy (SEM) to characterize the pubescent inner wall surface of H. nutans pitchers. Approximately 15 × 15 mm sections were cut with a razor blade, transported in an airtight container with moist tissue paper and quench-frozen in liquid propane within 4 h of sampling. After freeze-drying, the samples were sputter-coated with a 20 nm thick gold layer and examined in a Philips FEI XL30-FEG SEM (accelerating voltage: 5.0 kV). Trichome dimensions and densities were measured using the software Scion Image (Scion Corporation, Frederick, MD, USA).

(b) Running experiments

Observations of ants on three different H. nutans plants showed that the hairy inner pitcher wall becomes very slippery when wet. To quantify this effect, we performed running experiments with free-running Camponotus rufifemur worker ants (body length: 5–8 mm, body weight: 5–10 mg, kept at 24–29°C and 60–80% relative humidity) on a freshly harvested pitcher. Immediately before the experiments, 200–300 ants were collected, transported to the greenhouse in a plastic container with slippery Fluon (Whitford, Dietz) coating on the walls and allowed to acclimatize for 1 h. Pitchers, plant surfaces and greenhouse benches were dry when we started the experiment (i.e. during the day, not directly after plants had been watered).

A H. nutans pitcher was cut at the base, thereby draining the fluid without wetting the trichomes, and placed upright inside the container with ants. The ants were given 10 min to explore the pitcher before they were videotaped for 100 s while running on the pubescent surface. The surface was wetted using an atomizer with distilled water, and filming resumed for another 100 s. The trapping efficiency was calculated as the number of fallen ants per number of ‘visitors’ (defined as ants entering the inner wall with all six legs from the pitcher outside).

(c) Friction force measurements

We tested the effect of wetness and pulling direction on the friction forces of a total of 21 Oecophylla smaragdina workers (body length: 6–8 mm, body weight: 4–15 mg, kept as C. rufifemur) on the pubescent inner pitcher wall surface of H. nutans. Oecophylla smaragdina are ideal for friction force measurements because of their very high attachment forces [22,23]. Both ant species have been shown to ‘aquaplane’ on the Nepenthes peristome, and were chosen to allow comparisons with previous studies [19,20].

Each ant was anaesthetized with CO2, and a human hair with a loop at one end was glued to its thorax. To remove variation caused by different numbers of legs holding on to the surface, we ablated the pretarsi of the front and middle legs so that the ant could use only the hind legs [19]. In addition, we manipulated the pretarsi of the hind legs of seven ants each by removing the arolia or clipping the claw tips, respectively. The remaining seven ants were kept with the hind legs intact. Following the anaesthesia, each ant was allowed to recover for a minimum of 1 h in a Petri dish with a moist tissue paper.

A 55 × 25 mm piece (height × width) of freshly cut inner pitcher wall was glued flat onto a microscope slide and stored between trials in an airtight container with moist tissue paper to avoid desiccation. The inner wall sample was replaced at the first sign of drying from the edges. An ant was tethered via the hair to a one-dimensional force transducer (2 × 350 Ω foil strain gauges in half-bridge configuration; resolution 0.15 mN) with a hook on the end. The ant was placed on the test surface which was horizontally pulled away over a distance of 20 mm at a velocity of 2.5 mm s−1 using a software-controlled motor stage. Each ant was tested under four different conditions, varying both surface wetness (wet versus dry) and pulling direction (inward versus outward). A minimum of three pulls (mean = 3.75) per ant and condition was performed. We recorded peak forces for each pull and calculated the mean for each ant/condition combination.

3. Results

(a) Micromorphology and trichome density

The inward-pointing hairs covered the upper third to one-half of the H. nutans pitchers and increased in length towards the lower end of the pubescent zone (table 1). Long hairs were interspersed with shorter trichomes of variable length (figure 2a–c). Each hair had a surface microstructure of longitudinal ridges with a spacing of 11.5±0.4 µm (n = 36). Roughly constant ridge spacing on the tapering hair was achieved by different lengths of the individual ridges (figure 2b,d). We counted 218 trichomes on four pieces of inner wall surface with a total area of 7.02 mm2, corresponding to an average trichome density of 31.05 mm−2 (table 1).

View this table:
Table 1.

Size and spacing of the trichomes at the upper and lower end of the pubescent zone on the inner wall of a H. nutans pitcher (mean±s.e.).

Figure 2.

SEM images of the trichomes on the pubescent inner wall surface of H. nutans. All images are oriented to resemble an upright pitcher. (a,b) Upper pubescent zone close to the pitcher rim (visible in (a)), showing short trichomes of different length and the underlying epidermis interspersed with nectaries (arrows). (b) Detail of the microstructure formed by longitudinal cuticular folds on the trichome surface. (c,d) Long trichomes at the lower margin of the pubescent zone just above the smooth zone (visible below the trichomes in (c)). (d) Surface microstructure of a long trichome.

(b) Effect of wetness and surface anisotropy on walking ability and friction forces of ants

Under dry conditions, 28.8 per cent of all visiting ants fell from the pubescent inner wall surface. The ants experienced little difficulty to walk on the short trichomes close to the pitcher rim but were increasingly likely to fall when they approached the longer hairs at the bottom end of the pubescent zone (see the electronic supplementary material, video S2). Wetting of the inner wall did not affect the visiting rate (105 versus 104 ants per 100 s) but dramatically increased the capture rate to 87.6 per cent (chi-square test with Yates correction, χ2 = 19.22, p < 0.001).

Force measurements were performed to disentangle the effects of wetness and surface anisotropy on the ants' attachment organs (arolia, claws). When pulled inwards (along the trichomes), ants with intact hind feet generated significantly higher friction forces under dry than under wet conditions. By contrast, forces in the outward pulling direction (against the trichome orientation) were uniformly high, regardless of wetness (figure 3a; statistics in table 2; raw data shown in the electronic supplementary material, table S3). Ablation of the adhesive pads neutralized the effect of wetness, leading to similarly low inward friction forces on both dry and wet trichomes while the direction dependence (higher outward forces) remained unaffected (figure 3b). Ants with clipped claw tips produced similar results to untreated individuals (figure 3c).

View this table:
Table 2.

Effect of surface wetness and pulling direction on the friction forces of O. smaragdina ants with three different tarsus manipulations (n = 3 × 7) on the pubescent inner pitcher surface of H. nutans (Friedman test with pairwise Conover comparisons, p-values corrected for multiple testing).

Figure 3.

Effect of surface wetness and pulling direction on the friction forces of O. smaragdina ants with (a) intact tarsi, (b) ablated arolia and (c) clipped claws (n = 7 each) on the dry (left side, white bars) and wet (right side, grey bars) pubescent inner wall surface of a H. nutans pitcher. Bars denote medians, boxes the inner and whiskers the outer quartiles of the data.

4. Discussion

The slipperiness of the H. nutans inner pitcher wall is strongly enhanced by wetness. A strikingly similar ‘aquaplaning’ mechanism has been described for Nepenthes [19,21]. Similar to the pubescent H. nutans surface, the Nepenthes peristome is fully wettable and, under humid conditions, covered by a thin, stable fluid film. The evolution of specialized superhydrophilic trapping surfaces in two different plant orders provides a striking example of trait convergence.

In both taxa, experimental ablation of arolia neutralized the effect of wetness on the friction forces of ant tarsi, confirming that wetting is effective against the insects' adhesive pads [19]. In addition, the microstructure of both surfaces shows a strong inward–outward anisotropy that allows interlocking only in one direction. In contrast to the findings for Nepenthes, the direction dependence in our experiment remained present even when the claw tips were clipped. This can be explained by the much larger scale of the inward-pointing hairs of H. nutans compared with the step-like structure on the Nepenthes peristome, allowing the whole tarsus and not just the claws to interlock.

Interestingly, and in contrast to the Nepenthes peristome, the investigated H. nutans pitchers captured ants even when dry (albeit in much smaller numbers and only on the lower part of the pubescent zone; see electronic supplementary material, video S2). Lloyd [13] suggested that the flexibility of the longer trichomes causes them to ‘give way’ and destabilize the grip of the insects; however, the observations may also be explained by the reduced contact area for the ants' adhesive pads. The width of the ants' arolia (mean±s.e. = 227.2±7.7 µm for O. smaragdina and 121.5±7.2 µm for C. rufifemur, n = 6 each) is significantly larger than the diameter of the H. nutans trichomes (table 1) while the claw span (mean±s.e. = 415.7±29.9 µm for O. smaragdina, n = 5, and 331.5±9.8 µm for C. rufifemur, n = 6) exceeds the spacing of the hairs (table 1), preventing the tarsi from making contact with the underlying epidermis. Both O. smaragdina and C. rufifemur are relatively large-bodied ant species. Further experiments need to clarify how ants of different body sizes are affected by the pubescent surface of H. nutans.

Wetting caused a dramatic increase in the capture rate from 29 to 88 per cent. Jaffe et al. [15] concluded from the sinking of insects that the pitcher fluid has a lower surface tension than water, which may in turn facilitate the wetting of the trichomes. In line with this, Jaffe et al. [15] reported that excess fluid inside the pitcher is wicked up by the trichomes. In contrast to most Nepenthes species, Heliamphora pitchers do not have a lid to protect them against flooding by rain. Instead, many species possess a drainage hole (or slit) that allows excess water to run out (figure 1b) so that, despite copious rainfalls (2000–4000 mm annually), the pitchers are never flooded. The position of the hole slightly above the lower margin of the pubescent zone ensures that water is wicked up if the fluid level is sufficiently high (own observations).

Interestingly, Jaffe et al. [15] observed that pitchers stopped capturing prey at the end of the dry season when pitcher fluid levels were low, but filling them up to the usual level restored the trapping ability. This strongly supports the hypothesis that the ‘aquaplaning’ mechanism for prey capture is important for H. nutans, and that the drainage function of the trichomes is directly linked to trapping. Aided by the hydrophilic trichomes and the low surface tension of the pitcher fluid, the inner pitcher wall may be covered by water films and slippery at most times. By contrast, the peristome of Nepenthes pitchers is only intermittently wet and slippery, and its trapping efficiency is linked to environmental factors such as rain and air humidity [20]. While the intermittent activity of Nepenthes traps might represent an adaptive strategy to capture ants [20], uncoupling this link might be better for H. nutans that grows in a more seasonal climate than most Nepenthes. We propose that by using the fluid depot inside the pitcher for surface wetting, the plant is able to extend ‘active’ trapping periods beyond the times of actual rainfalls and stay continuously slippery during the wet (growth) season, thereby maximizing nutrient acquisition when it is most needed. On the other hand, the observed slipperiness of the longer trichomes even under dry conditions could help to maintain a base level of nutrient intake during dry spells.

The discovery of a superhydrophilic hairy surface is interesting as many hairy plant surfaces are highly water-repellent [48], in particular when the hair density exceeds 25 mm−2 as is the case in H. nutans [4]. Water-absorbing trichomes occur in several plant families but they are morphologically specialized, e.g. short, glandular-stalked trichomes with a low density (1–14 mm−2) in some Orchidaceae [10], sunken into the epidermal surface in Peperomia (Piperaceae) [24] or peltate in Bromeliaceae [9]. Hydrophilic hairs (in the typical sense) have been reported to cover the leaves of the Lady's Mantle (Alchemilla vulgaris); however, the underlying cuticle is hydrophobic, and in combination with the elastic properties of the hairs, the leaf is ultimately rendered water-repellent [6]. By contrast, the hydrophobic trichomes of the Jerusalem Sage (Phlomis fruticosa) are able to entrap and retain water droplets [25] but do not support a stable water film. These findings suggest that both the cuticular surface and the emergent trichomes need to be hydrophilic to allow the formation of stable water films as observed in H. nutans. The cuticular folds on the trichomes (figure 2b,d) are likely to enhance their wettability, similar to the small-scale radial ridges that provide capillarity on the Nepenthes peristome [21]. This interpretation is supported by the roughly similar dimensions for both structures: 11.5±0.4 µm in H. nutans compared with 36.75±1.4 µm across 61 Nepenthes species [26]. Further experiments are needed to quantify the wettability of the trichomes and the underlying cuticle, and to elucidate the role of the nectar for wetting [20].

Wetness-dependent slipperiness might also play a role in other Heliamphora species although the extent of the pubescent zone and the length, diameter and spacing of the trichomes vary considerably. Some species (e.g. H. tatei and H. ceracea) even have slippery wax crystals instead of trichomes on the inner pitcher wall [14,15], similar to many Nepenthes. Trichomes with similar micromorphology are also found on the inside of the ‘hood’ of North American Sarracenia (Sarraceniaceae) pitcher plants [12]. Newell & Nastase [27] observed that ants could walk on the hood trichomes of S. purpurea without difficulty except in two cases when they repeatedly lost footing and fell into the pitchers. Preliminary tests indicate that the hood trichomes are indeed wettable, and variable surface wetness may explain the observations of Newell & Nastase. In contrast to H. nutans, the pubescent surface on the S. purpurea hood does not extend down to the fluid level. The fluid is, however, reached by the shorter, imbricate trichomes in the subjacent zone which possess a similar ridged microstructure [12], rendering a wicking mechanism for wetting possible. Finally, the peristome of Cephalotus follicularis with its striking resemblance of the Nepenthes peristome might represent yet another case of convergently evolved adaptations for a wetness-based trapping mechanism. Further research is needed to investigate whether ‘insect aquaplaning’ on superhydrophilic, micropatterned surfaces is a widespread phenomenon among carnivorous plants.

Acknowledgements

We thank the Royal Botanical Gardens at Kew for providing plant material, Ch'ien C. Lee and Robert Severitt for contributing photographs, and two anonymous referees for helpful comments including the observation of wettable trichomes under the S. purpurea hood. This study was supported by a Henslow Research Fellowship of the Cambridge Philosophical Society to U.B. and a grant from the Leverhulme Trust (F/09 364/G) to W.F.

  • Received October 30, 2012.
  • Accepted November 23, 2012.

References

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