Evidence for alternative trapping strategies in two forms of the pitcher plant, Nepenthes rafflesiana

Nepenthes pitchers are specialized leaves that function as insect traps. Several pitcher components may contribute to trapping, including the pitcher fluid, slippery wax crystals and downward-pointing epidermal cells on the inner pitcher wall, and the wetness-dependent pitcher rim (peristome), but the relative importance of these traits is unclear. Mechanisms of prey capture and retention in the field were investigated by quantifying the effect of 'knock-out' manipulations of individual pitcher structures, and by testing the ability of pitcher fluids and water to retain insects. Two forms of Nepenthes rafflesiana Jack ('elongate' and 'typical') with contrasting combinations of pitcher traits were compared. Wax crystals on the inner pitcher wall were found to be the most important trapping structure in the elongate form, whereas the typical form relied primarily on the peristome. The pitcher fluids of both forms, differing markedly in the degree of viscoelasticity, retained significantly more ants than water. The present results show that pitcher plants utilize several mechanisms for prey capture and retention, varying in efficiency and relative importance between forms. It is proposed that these differences represent alternative prey capture strategies that may provide a mechanism to reduce competition and facilitate species co-existence in nutrient-limited habitats.     

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     

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.     

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     

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.     

A novel resource-service mutualism between bats and pitcher plants

Mutualistic relationships between vertebrates and plants apart from the pollen and seed-dispersal syndromes are rare. At first view, carnivorous pitcher plants of the genus Nepenthes seem to be highly unlikely candidates for mutualistic interactions with animals, as they form dimorphic terrestrial and aerial pitchers that trap arthropods and small vertebrates. Surprisingly, however, the aerial pitchers of Nepenthes rafflesiana variety elongata are poor insect traps, with low amounts of insect-attractive volatile compounds and low amounts of digestive fluid. Here, we show that N. rafflesiana elongata gains an estimated 33.8 per cent of the total foliar nitrogen from the faeces of Hardwicke's woolly bats (Kerivoula hardwickii hardwickii) that exclusively roost in its aerial pitchers. This is the first case in which the faeces-trapping syndrome has been documented in a pitcher plant that attracts bats and only the second case of a mutualistic association between a carnivorous plant and a mammal to date.     

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.     

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     

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.     

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.     

AN S.E.M. SURVEY OF THE FIVE CARNIVOROUS PITCHER PLANT GENERA

Scanning electron microscopy was used to observe features of representative species of the five carnivorous pitcher plant genera which allow them to lure, capture, and digest insects. Nepenthes rafflesiana, Sarracenia purpurea, Darlingtonia californica, Heliamphora heterodoxa, and Cephalotus follicularis were studied. The many morphological similarities observed in the phylogenetically unrelated plants show evidence supporting the concept of their convergent evolution. Several previously undescribed features of some plants were resolved which help elucidate their insect trapping mechanisms.     

A viscoelastic deadly fluid in carnivorous pitcher plants

Background

The carnivorous plants of the genus Nepenthes, widely distributed in the Asian tropics, rely mostly on nutrients derived from arthropods trapped in their pitcher-shaped leaves and digested by their enzymatic fluid. The genus exhibits a great diversity of prey and pitcher forms and its mechanism of trapping has long intrigued scientists. The slippery inner surfaces of the pitchers, which can be waxy or highly wettable, have so far been considered as the key trapping devices. However, the occurrence of species lacking such epidermal specializations but still effective at trapping insects suggests the possible implication of other mechanisms.

Methodology/Principal Findings

Using a combination of insect bioassays, high-speed video and rheological measurements, we show that the digestive fluid of Nepenthes rafflesiana is highly viscoelastic and that this physical property is crucial for the retention of insects in its traps. Trapping efficiency is shown to remain strong even when the fluid is highly diluted by water, as long as the elastic relaxation time of the fluid is higher than the typical time scale of insect movements.

Conclusions/Significance

This finding challenges the common classification of Nepenthes pitchers as simple passive traps and is of great adaptive significance for these tropical plants, which are often submitted to high rainfalls and variations in fluid concentration. The viscoelastic trap constitutes a cryptic but potentially widespread adaptation of Nepenthes species and could be a homologous trait shared through common ancestry with the sundew (Drosera) flypaper plants. Such large production of a highly viscoelastic biopolymer fluid in permanent pools is nevertheless unique in the plant kingdom and suggests novel applications for pest control.     

Carnivorous pitcher plants: insights in an old topic

Plant insect interactions are usually recognized as a scenario where herbivorous insects feed on a host plant. However, also the opposite situation is known, where plants feed on insects. Carnivorous pitcher plants of the genus Nepenthes as well as other pitcher plants obtain many nutrients from caught insect prey. Special features of the pitcher traps’ surface are responsible for attraction and trapping insects. Once caught, the prey is digested in the fluid of the pitchers to release nutrients and make them available for the plant. Nutrients are taken up by special glands localized on the inner surface of the pitchers. These glands also secrete the hydrolyzing enzymes into the digestion fluid. Although this is known for more than 100 years, our knowledge of the pitcher fluid composition is still limited. Only in recent years some enzymes have been purified from the pitcher fluid and their corresponding genes could be identified. Among them, many pathogenesis-related proteins have been identified, most of which exhibiting hydrolytic activities. The role of these proteins as well as the role of secondary metabolites, which have been identified in the pitcher fluid, is discussed in general and in the context of further studies on carnivorous plants that might give answers to basic questions in plant biology.     

Local and Regional-Scale Food Web Structure in Nepenthes alata Pitchers1

Tropical Nepenthes pitcher plants provide small, isolated aquatic habitats. We examined inter-pitcher variation in the community structure of the inhabitants of Nepenthes alata Blanco in West Sumatra, focusing on the conditions of the pitchers, bacterial density in the pitcher fluid, density and biomass of metazoan inhabitants, and the frequencies of interspecific encounters. Older pitchers contained more insect carcasses. The bacterial density increased with the age of the pitchers, but decreased in withered pitchers that contained finely decomposed detritus. In live pitchers, the bacterial density, the density, mass and species richness of metazoa, and the number of trophic levels per pitcher were positively correlated with detrital mass, which was correlated with volume of pitcher fluid. The metazoan fauna from N. alata consisted of 4 predators and 12 saprophages, among the richest known for Nepenthes species. However, each individual pitcher harbored a limited numbers of species, owing to (1) the low incidence of many species, and (2) the aggregated distribution and different temporal colonization pattern of major species. Six dipteran taxa (one predator and five saprophages) accounted for the bulk of metazoan inhabitant biomass. Of 48 combinations of predator-prey encountered, only four occurred frequently (in > 30% of pitchers), which included two predators and three saprophages. Thus, individual pitchers harbored relatively simple communities despite the regional species richness, and only limited kinds of predator-prey encounters seemed to occur frequently in the regional food web. The local-scale properties of the subdivided communities presented here provide the basic information for understanding the maintenance of regional species richness and food web complexity.     

Fenestration: a window of opportunity for carnivorous plants

A long-standing but controversial hypothesis assumes that carnivorous plants employ aggressive mimicry to increase their prey capture success. A possible mechanism is that pitcher plants use aggressive mimicry to deceive prey about the location of the pitcher''s exit. Specifically, species from unrelated families sport fenestration, i.e. transparent windows on the upper surfaces of pitchers which might function to mimic the exit of the pitcher. This hypothesis has not been evaluated against alternative hypotheses predicting that fenestration functions to attract insects from afar. By manipulating fenestration, we show that it does not increase the number of Drosophila flies or of two ant species entering pitchers in Sarracenia minor nor their retention time or a pitcher''s capture success. However, fenestration increased the number of Drosophila flies alighting on the pitcher compared with pitchers of the same plant without fenestration. We thus suggest that fenestration in S. minor is not an example of aggressive mimicry but rather functions in long-range attraction of prey. We highlight the need to evaluate aggressive mimicry relative to alternative concepts of plant–animal communication.     

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     

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.