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.     

Fluid physico-chemical properties influence capture and diet in Nepenthes pitcher plants

Background and AimsNepenthes pitcher plants have evolved modified leaves with slippery surfaces and enzymatic fluids that trap and digest prey, faeces and/or plant detritus. Although the fluid’s contribution to insect capture is recognized, the physico-chemical properties involved remain underexplored and may vary among species, influencing their diet type. This study investigates the contributions of acidity and viscoelasticity in the fluid’s capture efficiency of two ant and two fly species in four Nepenthes species with different nutrition strategies.Methods Four Nepenthes species were studied, namely N. rafflesiana, N. gracilis, N. hemsleyana and N. ampullaria. Fluid was collected from pitchers of varying ages from plants growing in the field and immediately transferred to glass vials, and individual ants (tribe Campotini, Fomicinae) and flies (Calliphora vomitoria and Drosophila melanogaster) were dropped in and observed for 5 min. Water-filled vials were used as controls. Survival and lifetime data were analysed using models applied to right-censored observations. Additional laboratory experiments were carried out in which C. vomitoria flies were immersed in pH-controlled aqueous solutions and observed for 5 min.Key Results Pitcher fluid differed among Nepenthes species as regards insect retention capacity and time-to-kill, with differences observed between prey types. Only the fluids of the reputedly insectivorous species were very acidic and/or viscoelastic and retained significantly more insects than the water controls. Viscoelastic fluids were fatal to flies and were able to trap the broadest diversity of insects. Younger viscoelastic fluids showed a better retention ability than older fluids, although with less rapid killing ability, suggesting that a chemical action follows a mechanical one. Insect retention increased exponentially with fluid viscoelasticity, and this happened more abruptly and at a lower threshold for flies compared with ants. Flies were more often retained if they fell into the traps on their backs, thus wetting their wings. Insect retention and death rate increased with fluid acidity, with a lower threshold for ants than for flies, and the time-to-kill decreased with increasing acidity. The laboratory experiments showed that fewer flies escaped from acidic solutions compared with water.Conclusions In addition to viscoelasticity, the pitcher’s fluid acidity and wetting ability influence the fate of insects and hence the diet of Nepenthes. The plants might select the prey that they retain by manipulating the secretion of H⁺ ions and polysaccharides in their pitcher fluid. This in turn might participate in possible adaptive radiation of this genus with regard to nutrient sequestration strategy. These plants might even structurally influence insect fall-orientation and capture-probability, inspiring biomimetic designs for pest control.     

Adaptive radiation with regard to nutrient sequestration strategies in the carnivorous plants of the genus Nepenthes

Carnivorous pitcher plants of the genus Nepenthes have evolved a great diversity of pitcher morphologies. Selective pressures for maximizing nutrient uptake have driven speciation and diversification of the genus in a process known as adaptive radiation. This leads to the evolution of pitchers adapted to specific and often bizarre source of nutrients, which are not strictly animal-derived. One example is Nepenthes ampullaria with unusual growth pattern and pitcher morphology what enables the plant to capture a leaf litter from the canopy above. We showed that the plant benefits from nitrogen uptake by increased rate of photosynthesis and growth what may provide competitive advantage over others co-habiting plants. A possible impact of such specialization toward hybridization, an important mechanism in speciation, is discussed.     

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.     

Structure and development of the pitchers from the carnivorous plantNepenthes alata (Nepenthaceae)

The pitchers of the tropical carnivorous plant Nepenthes alata are highly specialized organs for the attraction and capture of insects and absorption of nutrients from them. This study examined the structure and development of these pitchers, with particular focus on the nectaries and digestive glands. Immature pitchers developed at the tips of tendrils and were tightly sealed by a lid structure that opened during the end of pitcher elongation. Opened pitchers exposed a ridged peristome containing large nectaries. Like other members of the genus, a thick coating of epicuticular waxy scales covered the upper one-third of the pitcher. Scattered within this zone were cells resembling a stomatal complex with a protruding ridge. Cross sections showed that this ridge was formed by asymmetric divisions of the epidermal cells and lacked an underlying pore. The basal region of the trap had large multicellular glands that developed from single epidermal cells. These glands were closely associated with underlying vascular traces and provided a mechanism for supplying fluid to closed immature pitchers.     

Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth

Background and Aims

Carnivorous plants of the genus Nepenthes possess modified leaves that form pitfall traps in order to capture prey, mainly arthropods, to make additional nutrients available for the plant. These pitchers contain a digestive fluid due to the presence of hydrolytic enzymes. In this study, the composition of the digestive fluid was further analysed with regard to mineral nutrients and low molecular-weight compounds. A potential contribution of microbes to the composition of pitcher fluid was investigated.

Methods

Fluids from closed pitchers were harvested and analysed for mineral nutrients using analytical techniques based on ion-chromatography and inductively coupled plasma–optical emission spectroscopy. Secondary metabolites were identified by a combination of LC-MS and NMR. The presence of bacteria in the pitcher fluid was investigated by PCR of 16S-rRNA genes. Growth analyses of bacteria and yeast were performed in vitro with harvested pitcher fluid and in vivo within pitchers with injected microbes.

Key Results

The pitcher fluid from closed pitchers was found to be primarily an approx. 25-mm KCl solution, which is free of bacteria and unsuitable for microbial growth probably due to the lack of essential mineral nutrients such as phosphate and inorganic nitrogen. The fluid also contained antimicrobial naphthoquinones, plumbagin and 7-methyl-juglone, and defensive proteins such as the thaumatin-like protein. Challenging with bacteria or yeast caused bactericide as well as fungistatic properties in the fluid. Our results reveal that Nepenthes pitcher fluids represent a dynamic system that is able to react to the presence of microbes.

Conclusions

The secreted liquid of closed and freshly opened Nepenthes pitchers is exclusively plant-derived. It is unsuitable to serve as an environment for microbial growth. Thus, Nepenthes plants can avoid and control, at least to some extent, the microbial colonization of their pitfall traps and, thereby, reduce the need to vie with microbes for the prey-derived nutrients.     

Nitrogen uptake from prey and substrate as affected by prey capture level and plant reproductive status in four carnivorous plant species

Uptake of nitrogen from prey and substrate and partitioning of prey-derived nitrogen were studied in the carnivorous plant species Pinguicula alpina, P. villosa, P. vulgaris and Drosera rotundifolia in a subarctic environment. Efficiency in nitrogen uptake from prey was evaluated by tracing ¹⁵N from ¹⁵N-enriched Drosophila flies fed to the plants. The in situ uptake efficiency differed somewhat between species and ranged from 29 to 41% of prey N. This efficiency was not affected by different feeding levels or plant reproductive status (flowering or non-flowering). A test of the amount of N absorbed from prey caught on flower stalks of Pinguicula villosa and P. vulgaris showed that both species took up little of what was available in prey (2.5% or less). The uptake efficiency found in greenhouse grown plants was higher than in plants in situ (40–50% vs. 30–40% respectively). This could probably best be explained by the absence of rain and a higher temperature in the greenhouse. The prey-derived ¹⁵N was traced to reproductive organs and winter buds. Non-flowering individuals allocated 58–97% of the N derived from prey to their winter buds. Flowering individuals allocated 17–43% of the N income from prey to reproduction, while 34–71% were allocated to buds. Root uptake of nitrogen was stimulated by increased prey capture. This increase in uptake of nitrogen from the substrate was larger than the potential direct uptake of nitrogen from captured prey.     

The influence of prey behaviour on prey selection of the carnivorous plant Utricularia vulgaris

The mechanisms underlying differential prey selection of two microcrustaceans by the common bladderwort (Utricularia vulgaris) were studied in the laboratory. Functional response experiments with single prey showed that Utricularia had a higher attack rate coefficient and a longer handling time coefficient with the cladoceran Polyphemus pediculus than with the cyclopoid copepod Eucyclops serrulatus. Observation of predation rate, defined as number of prey eaten per unit time, from direct behavioural observation on single prey species, showed a higher predation rate with Polyphemus than on Eucyclops, at low prey densities. The opposite pattern was found at high prey density. When the two prey were presented simultaneously to the predator, Eucyclops was preferred over Polyphemus. Results from the situation with two prey and some of the results from the direct behavioural observations support field data on the diet of Utricularia, which shows that cyclopoid copepods are selected more frequently than Polyphemus.     

Traps of carnivorous pitcher plants as a habitat: composition of the fluid, biodiversity and mutualistic activities

BACKGROUND: Carnivorous pitcher plants (CPPs) use cone-shaped leaves to trap animals for nutrient supply but are not able to kill all intruders of their traps. Numerous species, ranging from bacteria to vertrebrates, survive and propagate in the otherwise deadly traps. This paper reviews the literature on phytotelmata of CPPs. PITCHER: Fluid as a Habitat The volumes of pitchers range from 0·2 mL to 1·5 L. In Nepenthes and Cephalotus, the fluid is secreted by the trap; the other genera collect rain water. The fluid is usually acidic, rich in O(2) and contains digestive enzymes. In some taxa, toxins or detergents are found, or the fluid is extremely viscous. In Heliamphora or Sarracenia, the fluid differs little from pure water. INQUILINE: Diversity Pitcher inquilines comprise bacteria, protozoa, algae, fungi, rotifers, crustaceans, arachnids, insects and amphibia. The dominant groups are protists and Dipteran larvae. The various species of CPPs host different sets of inquilines. Sarracenia purpurea hosts up to 165 species of inquilines, followed by Nepenthes ampullaria with 59 species, compared with only three species from Brocchinia reducta. Reasons for these differences include size, the life span of the pitcher as well as its fluid. MUTUALISTIC: Activities Inquilines closely interact with their host. Some live as parasites, but the vast majority are mutualists. Beneficial activities include secretion of enzymes, feeding on the plant's prey and successive excretion of inorganic nutrients, mechanical break up of the prey, removal of excessive prey and assimilation of atmospheric N(2). CONCLUSIONS: There is strong evidence that CPPs influence their phytotelm. Two strategies can be distinguished: (1) Nepenthes and Cephalotus produce acidic, toxic or digestive fluids and host a limited diversity of inquilines. (2) Genera without efficient enzymes such as Sarracenia or Heliamphora host diverse organisms and depend to a large extent on their symbionts for prey utilization.     

Photosynthetic CO2 affinity of the aquatic carnivorous plant Utricularia australis (Lentibulariaceae) and its investment in carnivory

Aquatic carnivorous plants usually grow in shallow dystrophic waters poor in inorganic N and P. Utricularia australis was chosen as a model plant for its prolific distribution and great ecological plasticity. The photosynthetic CO₂ compensation point and factors associated with investment in carnivory and capture of prey were measured in 17 U. australis micropopulations in Třeboň basin, Czech Republic, together with water chemistry factors at these sites differing greatly in their trophic level, water hardness, and prey availability. Apical shoot growth rate was estimated at some oligotrophic sites. The micropopulations differed greatly in the proportion of traps with animal prey (2.7–70%, mean 26%), trap proportion to total biomass (1.4–42%, mean 26%), mean trap biomass (0.7–63 μg trap⁻¹, mean 19 μg), and maximum trap size (1–3 mm, mean 2.0 mm). CO₂ compensation points ranged from 0.7 to 6.1 μM (mean 2.6 μM). A weak HCO₃ ⁻ use (compensation point 0.51 mM) was found in plants growing in alkaline water. Trap biomass proportion did not correlate significantly with prey capture and CO₂ compensation points with ambient [CO₂]. A very rapid apical growth (2.5–4.2 new nodes day⁻¹) occurred in sand pits. Thus, HCO₃ ⁻ use in U. australis can be induced by growing at very high pH. CO₂ compensation points resembled those known in other aquatic non-carnivorous plants. They did not reflect carnivory. In spite of very rapid apical shoot growth, the relative growth rate of U. australis can be zero in oligotrophic habitats without prey.     

Red trap colour of the carnivorous plant Drosera rotundifolia does not serve a prey attraction or camouflage function

The traps of many carnivorous plants are red in colour. This has been widely hypothesized to serve a prey attraction function; colour has also been hypothesized to function as camouflage, preventing prey avoidance. We tested these two hypotheses in situ for the carnivorous plant Drosera rotundifolia. We conducted three separate studies: (i) prey attraction to artificial traps to isolate the influence of colour; (ii) prey attraction to artificial traps on artificial backgrounds to control the degree of contrast and (iii) observation of prey capture by D. rotundifolia to determine the effects of colour on prey capture. Prey were not attracted to green traps and were deterred from red traps. There was no evidence that camouflaged traps caught more prey. For D. rotundifolia, there was a relationship between trap colour and prey capture. However, trap colour may be confounded with other leaf traits. Thus, we conclude that for D. rotundifolia, red trap colour does not serve a prey attraction or camouflage function.     

High diversity of diazotrophic bacteria associated with the carnivorous plant Drosera villosa var. villosa growing in oligotrophic habitats in Brazil

Drosera villosa var. villosa A. St.-Hil is a carnivorous plant that grows in Brazilian flooded soils very poor on nutrients, including low levels of N. Under these conditions, the plant shows vigorous growth, low root number, low number of captured prey (less than 50%) and a great assemblage of bacteria associated with the roots and leaves that grow in N-free medium. These preliminary results have led us to investigate the number of colony forming units (log CFU) in the roots (rhizosphere and endorhizosphere) and leaves (phyllosphere and endophyllosphere) of D. villosa var. villosa by the tenfold serial dilution technique in two N-free culture media. The results showed that the phyllosphere had 6.65 log CFU g dry leaf⁻¹ and the rhizosphere 6.47 log CFU g dry soil⁻¹, with the lowest value detected in the endophyllosphere (4.39 log CFU g dry leaf⁻¹). Sixty-three different bacteria morphotypes were isolated from the surface and interior of the roots and leaves and the amplification of the DNA with specific primers detected the nifH gene in 34 of those strains. The DNAs of the 34 strains were compared by the BOX-PCR technique and a great diversity was observed, with the bacteria clustering at a final level of similarity of only 12%. The strains were also submitted to the partial sequencing of the 16S rRNA gene and several genera of N₂-fixing bacteria were detected, including Bacillus, Burkholderia, Methylobacterium, Paenibacillus, Pseudomonas and Sphingomonas.     

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.     

The effect of plant chemistry on the acceptability of caterpillar prey to the argentine antIridomyrmex humilils (Hymenoptera: Formicidae)

Experiments were performed to test the acceptability of two palatable, cryptic caterpillars, the tobacco hornworm,Manduca sexta, and the cabbage looper,Trichoplusia ni, reared on different diets, to the Argentine ant,Iridomyrmex humilis. Ants preferred larvae reared on artificial diet, groundcherry, or cowpea to tobacco-reared larvae. Ants also preferred larvae reared on artificial diet without nicotine to larvae reared on diet containing nicotine (5% dry wt). Experiments were also performed to test the response of ants to larval extracts and chemicals applied to the surface of palatable prey. Ants did not respond differently to larvae of the potato tuber moth,Phthorimaea operculella, treated with larval extracts or regurgitate from tobacco-reared larvae compared to artificialdiet-reared larvae, but ants were deterred byP. operculella larvae treated with nicotine compared to untreated larvae. The results of this study indicate that caterpillars can derive at least some degree of chemical protection from their food plant without sequestering and storing plant compounds and without the development of elaborate aposematic characteristics.     

Responses of a carnivorous plant to prey and inorganic nutrients in a Mediterranean environment

We have analysed the effect of prey and fertilization by inorganic nutrients on the survival, growth, reproduction (sexual and vegetative) and mucilage secretion of Pinguicula vallisneriifolia (Lentibulariaceae), a carnivorous plant inhabiting rocky substrates of southern Spain. We tested the hypothesis that carnivorous plants are more prey dependent when root access to nutrients is strongly limited by (1) analysing the importance of the carnivorous habit to the fitness of P. vallisneriifolia in its natural rocky habitat, and (2) determining whether the effect of trapped prey varies with soil nutrient levels. Our 2-year experimental results indicated prey to be limiting to P. vallisneriifolia growth on its natural rocky substrate. Animal food supply substantially increased the chance of survival, growth, vegetative propagation, sexual reproductive success and mucilage secretion. The differences between prey levels were more evident at the end of the experiment when all the surviving Prey-exclusion plants had lost weight, and the probability of sexual reproduction and of vegetative propagation by axillary buds had accordingly diminished. Furthermore, there were clear benefits from carnivory at the population level, since both the expected individual life span and the lifetime vegetative and sexual output correlated positively with the quantity of prey trapped. Application of insects to non-fertilized plants stimulated growth, but similar application to fertilized plants grown on a complete nutrient solution failed to enhance growth. There was no obvious benefit from the provision of a balanced mineral nutrient solution (alone or with prey). The greatest absolute growth and sexual and vegetative output resulted from providing a surplus of insects to plants on their natural rocky substrate. The strong dependence of P. vallisneriifolia on prey can therefore be considered a useful preadaptation enabling colonization of rocky substrates. Received: 11 November 1996 / Accepted: 31 March 1997     

The feeding ecology of a carnivorous plant (Pinguicula nevadense): prey analysis and capture constraints

Summary The taxonomic composition and size of arthropods captured by Pinguicula nevadense, an endemic carnivorous plant of the high-mountain zone of the Sierra Nevada (southern Spain), are analysed. The actual prey of P. nevadense and the available arthropods trapped by mimic-traps are compared, in order to identify the capture constraints of the plant. The results show that P. nevadense captures various arthropod taxa. Winged insects, especially Nematocera, make up the main component of the diet. The range of prey sizes in all P. nevadense populations studied is similar. The taxonomic composition of arthropods trapped by the mimic-traps is similar to that of the actual prey of P. nevadense. However, the plant captures prey only below a specific size threshold. These size constraints appear to be the principal factor determining the actual prey of this carnivorous plant.     

Orientation behaviour of the predatory hemipteran Perillus bioculatus to plant and prey odours

The pentatomid predator P. bioculatus responded by positive odour-conditioned anemotaxis when exposed to airborne volatiles emitted by potato plants damaged by Colorado potato beetle larvae, whereas intact potato plants and non-feeding larvae as odour sources failed to elicit anemotaxis. Walking tracks of adult predators had higher values of straightness and upwind fixation when odours emanating from mechanically damaged plants were encountered than tracks registered in response to air carrying volatiles from intact plants, but these parameters returned to control values within 1–2 h after damage was caused. In contrast, air led over plants damaged by beetle larvae elicited orientation responses at least 3 hours after feeding damage ceased. The combination of chemical data on headspace composition, olfactory sensitivity established in electro-antennogram studies and behavioural data presented here imply a role of sesquiterpenoid plant volatiles as odorous cues eliciting attraction of this predator to damaged potato plants.     

Callus induction and plant regeneration from shoot portions of mature embryos of high tannin sorghums

Callus and plant regenertion were induced from shoot portions of mature embryos (dry seeds) of five high tannin Sorghum bicolor (L.) Moench cultivars. The explants were cultured on Murashige and Skoog medium with altered concentrations of 5 salts, supplemented with 150 mg/L L-asparagine, 5mg/L 2,4-Dichlorophenoxyacetic acid and 0.05mg/L kinetin. Calli which were yellow and globular were formed with 70–90% frequencies. The subculture medium which gave best results was MS with 2mg/L 2,4-Dichlorophenoxyacetic acid and 0.5mg/L kinetin. Plants were regenerated on MS medium supplemented with 150mg/L L-asparagine and 0.2mg/L kinetin with regeneration frequencies of 11–48%.Abbreviations 2,4-D dichlorophenoxyacetic acid     

Interactive effects of mycorrhizae and a root hemiparasite on plant community productivity and diversity

Plant communities can be affected both by arbuscular mycorrhizal fungi (AMF) and hemiparasitic plants. However, little is known about the interactive effects of these two biotic factors on the productivity and diversity of plant communities. To address this question, we set up a greenhouse study in which different AMF inocula and a hemiparasitic plant (Rhinanthus minor) were added to experimental grassland communities in a fully factorial design. In addition, single plants of each species in the grassland community were grown with the same treatments to distinguish direct AMF effects from indirect effects via plant competition. We found that AMF changed plant community structure by influencing the plant species differently. At the community level, AMF decreased the productivity by 15–24%, depending on the particular AMF treatment, mainly because two dominant species, Holcus lanatus and Plantago lanceolata, showed a negative mycorrhizal dependency. Concomitantly, plant diversity increased due to AMF inoculation and was highest in the treatment with a combination of two commercial AM strains. AMF had a positive effect on growth of the hemiparasite, and thereby induced a negative impact of the hemiparasite on host plant biomass which was not found in non-inoculated communities. However, the hemiparasite did not increase plant diversity. Our results highlight the importance of interactions with soil microbes for plant community structure and that these indirect effects can vary among AMF treatments. We conclude that mutualistic interactions with AMF, but not antagonistic interactions with a root hemiparasite, promote plant diversity in this grassland community. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.