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.     

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.     

A unique resource mutualism between the giant Bornean pitcher plant, Nepenthes rajah, and members of a small mammal community

The carnivorous pitcher plant genus Nepenthes grows in nutrient-deficient substrates and produce jug-shaped leaf organs (pitchers) that trap arthropods as a source of N and P. A number of Bornean Nepenthes demonstrate novel nutrient acquisition strategies. Notably, three giant montane species are engaged in a mutualistic association with the mountain treeshrew, Tupaia montana, in which the treeshrew defecates into the pitchers while visiting them to feed on nectar secretions on the pitchers' lids.Although the basis of this resource mutualism has been elucidated, many aspects are yet to be investigated. We sought to provide insights into the value of the mutualism to each participant. During initial observations we discovered that the summit rat, R. baluensis, also feeds on sugary exudates of N. rajah pitchers and defecates into them, and that this behavior appears to be habitual. The scope of the study was therefore expanded to assess to what degree N. rajah interacts with the small mammal community.We found that both T. montana and R. baluensis are engaged in a mutualistic interaction with N. rajah. T .montana visit pitchers more frequently than R. baluensis, but daily scat deposition rates within pitchers do not differ, suggesting that the mutualistic relationships are of a similar strength. This study is the first to demonstrate that a mutualism exists between a carnivorous plant species and multiple members of a small mammal community. Further, the newly discovered mutualism between R. baluensis and N. rajah represents only the second ever example of a multidirectional resource-based mutualism between a mammal and a carnivorous plant.     

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.     

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.     

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.     

Carnivorous syndrome in Asian pitcher plants of the genus Nepenthes

BACKGROUND AND AIMS: Pitcher plants Nepenthes alata and N. mirabilis are carnivorous species with leaves composed of a photosynthetic part (lamina) and a pitcher trap. This characteristic permitted direct physiological and anatomical comparison between these two distinct parts of the leaves to determine those features involved in the 'carnivorous syndrome', which include low net photosynthetic assimilation rate (A(N)) and low photosynthetic nitrogen use efficiency (PNUE). METHODS: Photosynthetic rate (A(N)) and respiration rate (R(d)) were measured gasometrically, chlorophyll concentration was determined spectrophotometrically and nitrogen concentration was determined using a CHN elemental analyser in lamina and trap separately. Anatomy of N. alata was observed using light, fluorescence and transmission electron microscopy. A(N), foliar nitrogen and chlorophyll concentration were also compared with values for other carnivorous plant species (genera Sarracenia, Drosera) that combine both autotrophic and carnivorous functions into the same physical organ. KEY RESULTS: It was found that the A(N) in Nepenthes lamina was low and PNUE was only slightly higher or similar in comparison with other carnivorous plants. It was not observed that the pitcher had a higher R(d) than the lamina, but A(N) in the pitcher was significantly lower than in the lamina. Nepenthes possesses a cluster of characters that could result in reduced photosynthesis in the pitcher and be responsible for carnivorous function of the leaf: replacement of chlorophyll-containing cells with digestive glands, low chlorophyll and nitrogen concentration, compact mesophyll with a small portion of intercellular spaces, absence of palisade parenchyma and low stomatal density. CONCLUSION: Low photosynthetic capacity, nitrogen efficiency, chlorophyll and nitrogen concentration of Nepenthes pitchers was found, together with a set of features that characterized the carnivorous syndrome. Dual use of leaves for photosynthesis and nutrient gain can decrease photosynthetic efficiency in carnivorous plants in general.     

Nepenthes insignis uses a C2-portion of the carbon skeleton of L-alanine acquired via its carnivorous organs, to build up the allelochemical plumbagin

Tropical pitcher plants (Nepenthes) catch animals in their specialized cup-shaped leaves, digest the prey by secreting enzymes, and actively take up the resulting compounds. The benefit of this behaviour is the ability to grow and compete in nutrient-poor habitats. Our present in vitro study shows that not only the nitrogen of alanine fed to the carnivorous organs is used by the plant but that in addition intact C2-units derived from C-2 and C-3 of stable isotope labelled L-alanine serve as building blocks, here exemplarily for the synthesis of the secondary metabolite plumbagin, a potent allelochemical. This result adds a new facet to the benefit of carnivory for plants. The availability of plumbagin by a de novo synthesis probably enhances the plants' fitness in their defence against phytophagous and pathogenic organisms. A missing specific uptake or CoA activation mechanism might be the reason that acetate fed to the pitchers was not incorporated into the naphthoquinone plumbagin. The dihydronaphthoquinone glucosides rossoliside and plumbaside A, here isolated for the first time from Nepenthes, by contrast, showed no incorporation after feeding of any of the two precursors, suggesting these compounds to be storage forms with probably very low turnover rates.     

Nectar,not colour,may lure insects to their death

We experimentally demonstrate in the field that prey of the carnivorous plant Sarracenia purpurea are attracted to sugar, not to colour. Prey capture (either all taxa summed or individual common taxa considered separately) was not associated with total red area or patterning on pitchers of living pitcher plants. We separated effects of nectar availability and coloration using painted ‘pseudopitchers’, half of which were coated with sugar solution. Unsugared pseudopitchers captured virtually no prey, whereas pseudopitchers with sugar solution captured the same amount of prey as living pitchers. In contrast to a recent study that associated red coloration with prey capture but that lacked controls for nectar availability, we infer that nectar, not colour, is the primary means by which pitcher plants attract prey.     

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.     

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 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     

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.     

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.     

猪笼草消化液成分及其应用价值研究进展

猪笼草是一类食虫植物,通过捕虫囊内消化液分解猎物,为自身生长提供营养。猪笼草消化液中含天冬氨酸蛋白酶、几丁质酶等水解酶类,还有萘醌、自由基及一些无机离子。猪笼草消化液具有抗真菌,治疗创伤、头痛等药用功能,并有抗肿瘤、降血压、抗疟疾等潜在药用开发价值。对猪笼草消化液的成分及活性进行归纳,为其药用开发提供思路。     

Degradation of a peptide in pitcher fluid of the carnivorous plant Nepenthes alata Blanco

Carnivorous plants acquire substantial amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes produces trapping organs called pitchers at the tips of tendrils elongated from leaf ends. Acidic fluid is secreted at the bottoms of the pitchers. The pitcher fluid includes several hydrolytic enzymes, and some, such as aspartic proteinase, are thought to be involved in nitrogen acquisition from insect proteins. To understand the nitrogen-acquisition process, it is essential to identify the protein-degradation products in the pitcher fluid. To gain insight into protein degradation in pitcher fluid, we used the oxidized B-chain of bovine insulin as a model substrate, and its degradation by the pitcher fluid of N. alata was investigated using liquid chromatography-mass spectrometry (LC-MS). LC-MS analysis of the degradation products revealed that the oxidized B-chain of bovine insulin was initially cleaved at aromatic amino acids such as phenylalanine and tyrosine. These cleavage sites are similar to those of aspartic proteinases from other plants and animals. The presence of a series of peptide fragments as degradation products suggests that exopeptidase(s) is also present in the pitcher fluid. Amino acid analysis and peptide fragment analysis of the degradation products demonstrated that three amino acids plus small peptides were released from the oxidized B-chain of bovine insulin, suggesting that insect proteins are readily degraded to small peptides and amino acids in the pitcher fluid of N. alata.     

Supply determines demand: influence of partner quality and quantity on the interactions between bats and pitcher plants

Interspecific relationships such as mutualism and parasitism are major drivers of biodiversity. Because such interactions often comprise more than two species, ecological studies increasingly focus on complex multispecies systems. However, the spatial heterogeneity of multi-species interactions is often poorly understood. Here, we investigate the unusual interaction of a bat (Kerivoula hardwickii hardwickii) and two pitcher plant species (Nepenthes hemsleyana and N. bicalcarata) whose pitchers serve as roost for bats. Nepenthes hemsleyana offers roosts of higher quality, indicated by a more stable microclimate compared to N. bicalcarata but occurs at lower abundance and is less common than the latter. Whereas N. hemsleyana benefits from the roosting bats by gaining nitrogen from their feces, the bats’ interaction with N. bicalcarata seems to be commensal or even parasitic. Bats stayed longer in roosts of higher quality provided by N. hemsleyana and preferred them to pitchers of N. bicalcarata in a disturbance experiment. Moreover, bats roosting only in pitchers of N. hemsleyana had a higher body condition and were less infested with parasites compared to bats roosting in pitchers of N. bicalcarata. Our study shows how the local supply of roosts with different qualities affects the behavior and status of their inhabitants and—as a consequence—how the demand of the inhabitants can influence evolutionary adaptations of the roost providing species.     

Fatal attraction: carnivorous plants roll out the red carpet to lure insects

We provide the first experimental test of the hypothesis that the coloration of carnivorous plants can act as a signal to lure insects and thus enhance capture rates. An experimental approach was needed to separate effects of the visual appearance of plants from those of traits that may correlate with appearance and also affect capture rates. We compared insect capture rates of pitcher plants with artificially coloured red and green pitchers in a paired design, and found that plants with red pitchers captured significantly more flying insects. Thus, we present the first experimental evidence of visual signalling in carnivorous plants. Further, it has previously been suggested that carnivorous plants use contrasting stripes or UV marks on their pitchers to lure insects; our results emphasize that insect traps do not need to sport contrasting colours to be attractive; it might be sufficient to be different from the background.