With a flick of the lid: a novel trapping mechanism in Nepenthes gracilis pitcher plants

Carnivorous pitcher plants capture prey with modified leaves (pitchers), using diverse mechanisms such as 'insect aquaplaning' on the wet pitcher rim, slippery wax crystals on the inner pitcher wall, and viscoelastic retentive fluids. Here we describe a new trapping mechanism for Nepenthes gracilis which has evolved a unique, semi-slippery wax crystal surface on the underside of the pitcher lid and utilises the impact of rain drops to 'flick' insects into the trap. Depending on the experimental conditions (simulated 'rain', wet after 'rain', or dry), insects were captured mainly by the lid, the peristome, or the inner pitcher wall, respectively. The application of an anti-slip coating to the lower lid surface reduced prey capture in the field. Compared to sympatric N. rafflesiana, N. gracilis pitchers secreted more nectar under the lid and less on the peristome, thereby directing prey mainly towards the lid. The direct contribution to prey capture represents a novel function of the pitcher lid.     

Form follows function: morphological diversification and alternative trapping strategies in carnivorous Nepenthes pitcher plants

Carnivorous plants of the genus Nepenthes have evolved a striking diversity of pitcher traps that rely on specialized slippery surfaces for prey capture. With a comparative study of trap morphology, we show that Nepenthes pitcher plants have evolved specific adaptations for the use of either one of two distinct trapping mechanisms: slippery wax crystals on the inner pitcher wall and 'insect aquaplaning' on the wet upper rim (peristome). Species without wax crystals had wider peristomes with a longer inward slope. Ancestral state reconstructions identified wax crystal layers and narrow, symmetrical peristomes as ancestral, indicating that wax crystals have been reduced or lost multiple times independently. Our results complement recent reports of nutrient source specializations in Nepenthes and suggest that these specializations may have driven speciation and rapid diversification in this genus.     

Contribution of pitcher fragrance and fluid viscosity to high prey diversity in a Nepenthes carnivorous plant from borneo

Mechanisms that improve prey richness in carnivorous plants may involve three crucial phases of trapping:attraction, capture and retention.Nepenthes rafflesiana var. typica is an insectivorous pitcher plant that is widespread in northern Borneo.It exhibits ontogenetic pitcher dimorphism with the upper pitchers trapping more flying prey than the lower pitchers.While this difference in prey composition has been ascribed to differences in attraction,the contribution of capture and retention has been overlooked.This study focused on distinguishing between the prey trapping mechanisms, and assessing their relative contribution to prey diversity.Arthropod richness and diversity of both visitors and prey in the two types of pitchers were analysed to quantify the relative contribution of attraction to prey trapping.Rate of insect visits to the different pitcher parts and the presence or absence of a sweet fragrance was recorded to clarify the origin and mechanism of attraction.The mechanism of retention was studied by insect bioassays and measurements of fluid viscosity. Nepenthes rafflesiana was found to trap a broader prey spectrum than that previously described for any Nepenthes species,with the upper pitchers attracting and trapping a greater quantity and diversity of prey items than the lower pitchers.Capture efficiency was low compared with attraction or retention efficiency.Fragrance of the peristome,or nectar rim,accounted mainly for the observed non-specific, better prey attraction by the upper pitchers, while the retentive properties of the viscous fluid in these upper pitchers arguably explains the species richness of their flying prey.The pitchers of N. rafflesiana are therefore more than simple pitfall traps and the digestive fluid plays an important yet unsuspected role in the ecological success of the species.     

Harmless nectar source or deadly trap: Nepenthes pitchers are activated by rain, condensation and nectar

The leaves of Nepenthes pitcher plants are specialized pitfall traps which capture and digest arthropod prey. In many species, insects become trapped by 'aquaplaning' on the wet pitcher rim (peristome). Here we investigate the ecological implications of this capture mechanism in Nepenthes rafflesiana var. typica. We combine meteorological data and continuous field measurements of peristome wetness using electrical conductance with experimental assessments of the pitchers' capture efficiency. Our results demonstrate that pitchers can be highly effective traps with capture rates as high as 80% but completely ineffective at other times. These dramatic changes are due to the wetting condition of the peristome. Variation of peristome wetness and capture efficiency was perfectly synchronous, and caused by rain, condensation and nectar secreted from peristome nectaries. The presence of nectar on the peristome increased surface wetness mainly indirectly by its hygroscopic properties. Experiments confirmed that pitchers with removed peristome nectaries remained generally drier and captured prey less efficiently than untreated controls. This role of nectar in prey capture represents a novel function of plant nectar. We propose that the intermittent and unpredictable activation of Nepenthes pitcher traps facilitates ant recruitment and constitutes a strategy to maximize prey capture.     

The insect-trapping rim of Nepenthes pitchers: Surface structure and function

Carnivorous pitcher plants of the genus Nepenthes capture prey with a pitfall trap that relies on a micro-structured, slippery surface. The upper pitcher rim (peristome) is fully wettable and causes insects to slip by aquaplaning on a thin water film. The high wettability of the peristome is probably achieved by a combination of hydrophilic surface chemistry, surface roughness and the presence of hygroscopic nectar. Insect foot attachment could be prevented by the delayed drainage of the thin water film between the adhesive pad and the surface. Drainage should be faster for insects with a hairy adhesive system; however, they slip equally on the wet peristome. Therefore the stability of the water film against dewetting appears to be the key factor for aquaplaning. New experimental techniques may help to clarify the detailed function of the pitcher plant peristome and to explore its potential for biomimetic applications.Key words: carnivorous plants, insect aquaplaning, superhydrophilic leaves, Nepenthes, peristome     

Effect of pitcher age on trapping efficiency and natural prey capture in carnivorous Nepenthes rafflesiana plants

Background and Aims

Nepenthes pitchers are sophisticated traps that employ a variety of mechanisms to attract, capture and retain prey. The underlying morphological structures and physiological processes are subject to change over the lifetime of a pitcher. Here an investigation was carried out on how pitcher properties and capture efficiency change over the first 2 weeks after pitcher opening.

Methods

Prey capture, trapping efficiency, extrafloral nectar secretion, pitcher odour, as well as pH and viscoelasticity of the digestive fluid in N. rafflesiana pitchers were monitored in the natural habitat from pitcher opening up to an age of 2 weeks.

Key Results

Pitchers not only increased their attractiveness over this period by becoming more fragrant and secreting more nectar, but also gained mechanical trapping efficiency via an enhanced wettability of the upper pitcher rim (peristome). Consistently, natural prey capture was initially low and increased 3–6 d after opening. It was, however, highly variable within and among pitchers. At the same time, the pH and viscoelasticity of the digestive fluid decreased, suggesting that the latter is not essential for effective prey capture.

Conclusions

Prey capture and attraction by Nepenthes are dynamic processes strongly influenced by the changing properties of the pitcher. The results confirm insect aquaplaning on the peristome as the main capture mechanism in N. rafflesiana.Key words: Carnivorous plants, pitcher development, prey attraction, prey capture, insect aquaplaning, extrafloral nectar, Nepenthes rafflesiana     

Slippery or sticky? Functional diversity in the trapping strategy of Nepenthes carnivorous plants

The pitcher-shaped leaves of Nepenthes carnivorous plants have been considered as pitfall traps that essentially rely on slippery surfaces to capture insects. But a recent study of Nepenthes rafflesiana has shown that the viscoelasticity of the digestive fluid inside the pitchers plays a key role. Here, we investigated whether Nepenthes species exhibit diverse trapping strategies. We measured the amount of slippery wax on the pitcher walls of 23 taxa and the viscoelasticity of their digestive liquid and compared their retention efficiency on ants and flies. The amount of wax was shown to vary greatly between species. Most mountain species exhibited viscoelastic digestive fluids while water-like fluids were predominant in lowland species. Both characteristics contributed to insect trapping but wax was more efficient at trapping ants while viscoelasticity was key in trapping insects and was even more efficient than wax on flies. Trap waxiness and fluid viscoelasticity were inversely related, suggesting the possibility of an investment trade-off for the plants. Therefore Nepenthes pitcher plants do not solely employ slippery devices to trap insects but often employ a viscoelastic strategy. The entomofauna specific to the plant's habitat may exert selective pressures, favouring one trapping strategy at the expense of the other.     

The carnivorous syndrome in Nepenthes pitcher plants: Current state of knowledge and potential future directions

Nepenthes is the largest genus of pitcher plants, with its center of diversity in SE Asia. The plants grow in substrates that are deficient in N and offset this deficiency by trapping animal prey, primarily arthropods. Recent research has provided new insights into the function of the pitchers, particularly with regard to prey tapping and retention. Species examined to date use combinations of wettable peristomes, wax layers and viscoelastic fluid to trap and retain prey. In many respects, this has redefined our understanding of the functioning of Nepenthes pitchers. In addition, recent research has shown that several Nepenthes species target specific groups of prey animals, or are even evolving away from a strictly carnivorous mode of operation. Future research into nutrient sequestration strategies and mechanisms of prey attraction would no doubt further enhance our knowledge of the ecology of this remarkable genus.Key words: carnivory, mutualism, Nepenthes, pitcher plants     

Distribution of naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone in chitin-induced and uninduced Nepenthes khasiana: molecular events in prey capture

Prey capture and digestion in Nepenthes spp. through their leaf-evolved biological traps involve a sequence of exciting events. Sugar-rich nectar, aroma chemicals, narcotic alkaloid secretions, slippery wax crystals, and other biochemicals take part in attracting, capturing, and digesting preys in Nepenthes pitchers. Here we report the distribution of three potent naphthoquinones in Nepenthes khasiana and their roles in prey capture. Plumbagin was first detected in N. khasiana, and its content (root: 1.33 ± 0.02%, dry wt.) was the highest found in any natural source. Chitin induction enhanced plumbagin levels in N. khasiana (root: 2.17 ± 0.02%, dry wt.). Potted N. khasiana plants with limited growth of roots and aerial parts, showed higher levels of plumbagin accumulation (root: 1.92 ± 0.02%; root, chitin induction: 3.30 ± 0.21%, dry wt.) compared with field plants. Plumbagin, a known toxin, insect ecdysis inhibitor, and antimicrobial, was also found embedded in the waxy layers at the top prey capture region of N. khasiana pitchers. Chitin induction, mimicking prey capture, produced droserone and 5-O-methyl droserone in N. khasiana pitcher fluid. Both these naphthoquinone derivatives provide antimicrobial protection to the pitcher fluid from visiting preys. A two-way barrier was found between plumbagin and its two derivatives. Plumbagin was never detected in the pitcher fluid whereas both its derivatives were only found in the pitcher fluid on chitin induction or prey capture. The three naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone, act as molecular triggers in prey capture and digestion in the carnivorous plant, N. khasiana.     

Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata

The Nepenthes species are carnivorous plants that have evolved a specialized leaf organ, the 'pitcher', to attract, capture, and digest insects. The digested insects provide nutrients for growth, allowing these plants to grow even in poor soil. Several proteins have been identified in the pitcher fluid, including aspartic proteases (nepenthesin I and II) and pathogenesis-related (PR) proteins (β-1,3-glucanase, class IV chitinase, and thaumatin-like protein). In this study, we collected and concentrated pitcher fluid to identify minor proteins. In addition, we tried to identify the protein secreted in response to trapping the insect. To make a similar situation in which the insect falls into the pitcher, chitin which was a major component of the insect exoskeleton was added to the fluid in the pitcher. Three PR proteins, class III peroxidase (Prx), β-1,3-glucanase, and class III chitinase, were newly identified. Prx was induced after the addition of chitin to the pitcher fluid. Proteins in the pitcher fluid of the carnivorous plant Nepenthes alata probably have two roles in nutrient supply: digestion of prey and the antibacterial effect. These results suggest that the system for digesting prey has evolved from the defense system against pathogens in the carnivorous plant Nepenthes.     

Physicochemical properties of functional surfaces in pitchers of the carnivorous plant Nepenthes alata Blanco (Nepenthaceae)

Pitchers of the carnivorous plant Nepenthes alata are highly specialized organs adapted to attract, capture, and digest animals, mostly insects. They consist of several well distinguishable zones, differing in macro-morphology, surface microstructure, and functions. Since physicochemical properties of these surfaces may influence insect adhesion, we measured contact angles of non-polar (diiodomethane) and polar liquids (water and ethylene glycol) and estimated the free surface energy of 1) the lid, 2) the peristome, 3) the waxy surface of the slippery zone, and 4) the glandular surface of the digestive zone in N. alata pitchers. As a control, the external surface of the pitcher, as well as abaxial and adaxial surfaces of the leaf blade, was measured. Both leaf surfaces, both lid surfaces, and the external pitcher surface showed similar contact angles and had rather high values of surface free energy with relatively high dispersion component. These surfaces are considered to support strong adhesion forces based on the capillary interaction, and by this, to promote successful attachment of insects. The waxy surface is almost unwettable, has extremely low surface energy, and therefore, must essentially decrease insect adhesion. Both the peristome and glandular surfaces are wetted readily with both non-polar and polar liquids and have very high surface energy with a predominating polar component. These properties result in the preclusion of insect adhesion due to the hydrophilic lubricating film covering the surfaces. The obtained results support field observations and laboratory experiments of previous authors that demonstrated the possible role of different pitcher surfaces in insect trapping and retention.     

Pitcher geometry facilitates extrinsically powered ‘springboard trapping' in carnivorous Nepenthes gracilis pitcher plants

Carnivorous pitcher plants capture insects in cup-shaped leaves that function as motionless pitfall traps. Nepenthes gracilis evolved a unique ‘springboard'' trapping mechanism that exploits the impact energy of falling raindrops to actuate a fast pivoting motion of the canopy-like pitcher lid. We superimposed multiple computed micro-tomography images of the same pitcher to reveal distinct deformation patterns in lid-trapping N. gracilis and closely related pitfall-trapping N. rafflesiana. We found prominent differences between downward and upward lid displacement in N. gracilis only. Downward displacement was characterized by bending in two distinct deformation zones whist upward displacement was accomplished by evenly distributed straightening of the entire upper rear section of the pitcher. This suggests an anisotropic impact response, which may help to maximize initial jerk forces for prey capture, as well as the subsequent damping of the oscillation. Our results point to a key role of pitcher geometry for effective ‘springboard'' trapping in N. gracilis.     

The use of light in prey capture by the tropical pitcher plant Nepenthes aristolochioides

Nepenthes pitcher plants deploy tube-shaped pitchers to catch invertebrate prey; those of Nepenthes aristolochioides possess an unusual translucent dome. The hypothesis was tested that N. aristolochioides pitchers operate as light traps, by quantifying prey capture under three shade treatments. Flies are red-blind, with visual sensitivity maxima in the UV, blue, and green wavebands. Red celluloid filters were used to reduce the transmission of these wavebands into the interior of the pitchers. Those that were shaded at the rear showed a 3-fold reduction in Drosophila caught, relative to either unshaded control pitchers, or pitchers that were shaded at the front. Thus, light transmitted through the translucent dome is a fundamental component of N. aristolochioides' trapping mechanism.     

Adaptive significance and ontogenetic variability of the waxy zone in Nepenthes rafflesiana

Background and Aims

The slippery waxy zone in the upper part of pitchers has long been considered the key trapping structure of the Nepenthes carnivorous plants; however, the presence of wax is reported to be variable within and between species of this species-rich genus. This study raises the question of the adaptive significance of the waxy zone and investigates the basis for an ontogenetic cause of its variability and correlation with pitcher shape.

Methods

In Brunei (Borneo) the expression of the waxy zone throughout plant ontogeny was studied in two taxa of the Nepenthes rafflesiana complex, typica and elongata, which differ in pitcher shape and size. We also tested the adaptive significance of this zone by comparing the trapping efficiency and the number of prey captured of wax-bearing and wax-lacking plants.

Key Results

In elongata, the waxy zone is always well expanded and the elongated pitchers change little in form during plant development. Wax efficiently traps experimental ants but the number of captured prey in pitchers is low. In contrast, in typica, the waxy zone is reduced in successively produced pitchers until it is lost at the end of the plant''s juvenile stage. The form of pitchers thus changes continuously throughout plant ontogeny, from elongated to ovoid. In typica, the number of captured prey is greater, but the role of wax in trapping is minor compared with that of the digestive liquid, and waxy plants do not show a higher insect retention and prey abundance as compared with non-waxy plants.

Conclusions

The waxy zone is not always a key trapping structure in Nepenthes and can be lost when supplanted by more efficient features. This study points out how pitcher structure is submitted to selection, and that evolutionary changes in developmental mechanisms could play a role in the morphological diversity of Nepenthes.Key words: Carnivorous plant, developmental evolution, digestive liquid, epicuticular wax, insect trapping, heteroblasty, heterochrony, leaf form, morphological diversity, Nepenthes rafflesiana, ontogenetic change, pitcher plant     

Different pitcher shapes and trapping syndromes explain resource partitioning in Nepenthes species

Nepenthes pitcher plants display interspecific diversity in pitcher form and diets. This species‐rich genus might be a conspicuous candidate for an adaptive radiation. However, the pitcher traits of different species have never been quantified in a comparative study, nor have their possible adaptations to the resources they exploit been tested. In this study, we compare the pitcher features and prey composition of the seven Nepenthes taxa that grow in the heath forest of Brunei (Borneo) and investigate whether these species display different trapping syndromes that target different prey. The Nepenthes species are shown to display species‐specific combinations of pitcher shapes, volumes, rewards, attraction and capture traits, and different degrees of ontogenetic pitcher dimorphism. The prey spectra also differ among plant species and between ontogenetic morphotypes in their combinations of ants, flying insects, termites, and noninsect guilds. According to a discriminant analysis, the Nepenthes species collected at the same site differ significantly in prey abundance and composition at the level of order, showing niche segregation but with varying degrees of niche overlap according to pairwise species comparisons. Weakly carnivorous species are first characterized by an absence of attractive traits. Generalist carnivorous species have a sweet odor, a wide pitcher aperture, and an acidic pitcher fluid. Guild specializations are explained by different combinations of morpho‐functional traits. Ant captures increase with extrafloral nectar, fluid acidity, and slippery waxy walls. Termite captures increase with narrowness of pitchers, presence of a rim of edible trichomes, and symbiotic association with ants. The abundance of flying insects is primarily correlated with pitcher conicity, pitcher aperture diameter, and odor presence. Such species‐specific syndromes favoring resource partitioning may result from local character displacement by competition and/or previous adaptations to geographically distinct environments.     

Plasma-membrane H+-ATPases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco

Nepenthes is a unique genus of carnivorous plants that can capture insects in trapping organs called pitchers and digest them in pitcher fluid. The pitcher fluid includes digestive enzymes and is strongly acidic. We found that the fluid pH decreased when prey accumulates in the pitcher fluid of Nepenthes alata. The pH decrease may be important for prey digestion and the absorption of prey-derived nutrients. To identify the proton pump involved in the acidification of pitcher fluid, plant proton-pump homologs were cloned and their expressions were examined. In the lower part of pitchers with natural prey, expression of one putative plasma-membrane (PM) H⁺-ATPase gene, NaPHA3, was considerably higher than that of the putative vacuolar H⁺-ATPase (subunit A) gene, NaVHA1, or the putative vacuolar H⁺-pyrophosphatase gene, NaVHP1. Expression of one PM H⁺-ATPase gene, NaPHA1, was detected in the head cells of digestive glands in the lower part of pitchers, where proton extrusion may occur. Involvement of the PM H⁺-ATPase in the acidification of pitcher fluid was also supported by experiments with proton-pump modulators; vanadate inhibited proton extrusion from the inner surface of pitchers, whereas bafilomycin A₁ did not, and fusicoccin induced proton extrusion. These results strongly suggest that the PM H⁺-ATPase is responsible for acidification of the pitcher fluid of Nepenthes. Received: 8 June 2000 / Accepted: 8 August 2000     

Tree shrew lavatories: a novel nitrogen sequestration strategy in a tropical pitcher plant

Nepenthes pitcher plants are typically carnivorous, producing pitchers with varying combinations of epicuticular wax crystals, viscoelastic fluids and slippery peristomes to trap arthropod prey, especially ants. However, ant densities are low in tropical montane habitats, thereby limiting the potential benefits of the carnivorous syndrome. Nepenthes lowii, a montane species from Borneo, produces two types of pitchers that differ greatly in form and function. Pitchers produced by immature plants conform to the ‘typical’ Nepenthes pattern, catching arthropod prey. However, pitchers produced by mature N. lowii plants lack the features associated with carnivory and are instead visited by tree shrews, which defaecate into them after feeding on exudates that accumulate on the pitcher lid. We tested the hypothesis that tree shrew faeces represent a significant nitrogen (N) source for N. lowii, finding that it accounts for between 57 and 100 per cent of foliar N in mature N. lowii plants. Thus, N. lowii employs a diversified N sequestration strategy, gaining access to a N source that is not available to sympatric congeners. The interaction between N. lowii and tree shrews appears to be a mutualism based on the exchange of food sources that are scarce in their montane habitat.     

In situ enzyme activity in the dissolved and particulate fraction of the fluid from four pitcher plant species of the genus Nepenthes

The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×10(6) to 2.2×10(8) cells ml(-1). We measured the activity of three common enzymes in the fluid: acid phosphatases, β-D-glucosidases, and β-D-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant.