Seasonal heterophylly and leaf gland features in Triphyophyllum (Dioncophyllaceae), a new carnivorous plant genus

Triphyophyllum peltatum (Dioncophyllaceae), a tropical-West African liane, is heterophyllous, bearing leaves of three types, two on juvenile, non-climbing, short shoots, and the third on the mature axis. The short shoots produce mostly eglandular, lanceolate leaves, but just before the height of the rainy season small clusters of relatively ephemeral, glandular, filiform leaves are formed. These glandular leaves bear both stalked and sessile glands, and they act as efficient trapping organs for the prey, mostly flying insects and other small animals. The stalked glands, of which there are two size classes, are the most anatomically elaborate known in the plant kingdom. Structurally there are similarities with the stalked glands of Drosophyllum and Drosera. At maturity they carry secretion droplets, and stimulation by the prey leads to further secretion. The secretions contain a range of hydrolytic enzymes.
During sonic periods of its development Triphyophyllum is probably carnivorous. An important source of nutrients is thus tapped which could be significant in making possible an earlier transition from the juvenile to the rapidly-climbing adult form.
The Dioncophyllaceae, contains two other monotypic genera, neither with the carnivorous habit. Triphyophyllum is considered as an ecological African analogue of Nepenthes.     

Catapulting Tentacles in a Sticky Carnivorous Plant

Among trapping mechanisms in carnivorous plants, those termed ‘active’ have especially fascinated scientists since Charles Darwin’s early works because trap movements are involved. Fast snap-trapping and suction of prey are two of the most spectacular examples for how these plants actively catch animals, mainly arthropods, for a substantial nutrient supply. We show that Drosera glanduligera, a sundew from southern Australia, features a sophisticated catapult mechanism: Prey animals walking near the edge of the sundew trigger a touch-sensitive snap-tentacle, which swiftly catapults them onto adjacent sticky glue-tentacles; the insects are then slowly drawn within the concave trap leaf by sticky tentacles. This is the first detailed documentation and analysis of such catapult-flypaper traps in action and highlights a unique and surprisingly complex mechanical adaptation to carnivory.     

Do carnivorous plants use volatiles for attracting prey insects?

1.  Scientists have been fascinated by carnivorous plants for centuries and they have thoroughly investigated how these plants can benefit from insect capture for example through increased growth, earlier flowering, and increased seed production. How prey is actually lured into the traps, however, is less well understood. Trapping prey may be achieved in a random way, for example by camouflaging the traps (hiding them in the surrounding vegetation), so that prey is trapped by accidental landing on the trap leaves or wind drift, or in the other extreme, trapping may involve mimicry of other attractive resources such as fruits or flowers by using specific visual or olfactory signals to attract a specific prey assemblage.
2.  We investigated for the first time volatiles of the trapping leaves of carnivorous plant species by dynamic headspace methods. We present data on the venus flytrap Dionaea muscipula , the sundew Drosera binata , and the North American pitcher plants Sarracenia flava , Sarracenia leucophylla , Sarracenia minor , and Sarracenia purpurea . A large number of compounds and relatively high emission rates were found in three of the North American pitcher plants ( S. flava , S. leucophylla , and S. minor ) with compounds typically found in flowers or fruits. This suggests together with other features (e.g. colour, nectar production) that these traps are possibly flower or fruit mimics. The leaves of S. purpurea , Dionaea muscipula , and Drosera binata emitted much weaker scents with lower numbers of components, consisting mainly of volatiles typically emitted from green leaves.
3.  We discuss whether or not the use of volatiles for attracting prey animals is linked with specific trapping mechanisms and whether carnivorous plants can be grouped into specialized 'olfactory syndromes'.     

Group foraging in carnivorous plants: Carnivorous plant Drosera makinoi (Droseraceae) is more effective at trapping larger prey in large groups

Some predatory animals, represented by large carnivores, forage in groups and benefit from this behavior. We tested the hypothesis that carnivorous plants also benefit from group foraging to improve the efficiency of trapping large prey using Drosera makinoi (Droseraceae). As a result of our field observations, it was found that larger neighboring D. makinoi density yields a greater number of large preys (≥3 mm) and total prey biomass per plant. Results showed that a total of 43.4% of the events to trap large prey was achieved by two trap leaves belonging to two neighboring D. makinoi plants. Our results proved that group foraging in D. makinoi enables them to trap large prey.     

Minor pollinator–prey conflict in the carnivorous plant, Drosera anglica

We studied the physical and temporal isolation of two arthropod guilds interacting with Drosera anglica Huds., a terrestrial carnivorous plant. Flowers are separated from basal trap leaves by a leafless stalk. Since arthropods are potentially employed both as prey and pollinators, we asked whether separation of traps from flowers reduces the frequency with which flower visitors are captured by the leaves. Plants captured prey throughout the season, with peak trapping activity occurring before flowering began. The diverse prey spectrum included at least 109 species in 94 genera in 26 of 37 identified families representing 11 arthropod orders. The most common prey were adult flies of Nematocera, particularly Ceratopogonidae (50%) and Chironomidae (42%). The following taxa were periodically abundant: Acarina, Diptera–Cecidomyiidae, Chloropidae, Sciaridae, Hemiptera nymphs and Thysanoptera–Thripidae. Flies (Diptera) were chief flower visitors (95%), dominated by Syrphidae (66%), Bombyliidae and Muscidae (10% each), Calliphoridae (7%), Tachinidae and Dolichopodidae (3% each). Additionally, visitors were a bee (Hymenoptera–Halictidae) and thrips (Thysanoptera–Thripidae). Four families were common to both guilds: Diptera–Dolichopodidae, Muscidae, Tachinidae; and Thysanoptera–Thripidae. However, direct comparisons of identified taxa within these families showed that overlap between flower visitors and prey occurred for Thrips sp. larvae alone, which comprised only 3% of all flower visitors and 0.5% of prey. Drosera anglica exploits distinct guilds of insects for pollinators and prey.     

A Rapid and Efficient Method for Isolating High Quality DNA from Leaves of Carnivorous Plants from the Drosera Genus

Drosera rotundifolia, Drosera capensis, and Drosera regia are carnivorous plants of the sundew family, characterized by the presence of stalked and sticky glands on the upper leaf surface, to attract, trap, and digest insects. These plants contain exceptionally high amounts of polysaccharides, polyphenols, and other secondary metabolites that interfere with DNA isolation and subsequent enzymatic reactions such as PCR amplification. We present here a protocol for quick isolation of Drosera DNA with high yield and a high level of purity, by combining a borate extraction buffer with a commercial DNA extraction kit, and a proteinase K treatment during extraction. The yield of genomic DNA is from 13.36 μg/g of fresh weight to 35.29 μg/g depending of the species of Drosera, with a A???/A??? ratio of 1.43-1.92. Moreover, the procedure is quick and can be completed in 2.5 h.     

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.     

Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants

Abstract: A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as “proto‐carnivores”, lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional “carnivorous organ”, which can trap a prey, digest it, and finally absorb available nutrients.     

Tentacles of in vitro-grown round-leaf sundew (<Emphasis Type="Italic">Drosera rotundifolia</Emphasis>L.) show induction of chitinase activity upon mimicking the presence of prey

Induction of plant-derived chitinases in the leaves of a carnivorous plant was demonstrated using aseptically grown round-leaf sundew (Drosera rotundifolia L.). The presence of insect prey was mimicked by placing the chemical inducers gelatine, salicylic acid and crustacean chitin on leaves. In addition, mechanical stirring of tentacles was performed. Chitinase activity was markedly increased in leaf exudates upon application of notably chitin. Application of gelatine increased the proteolytic activity of leaf exudates, indicating that the reaction of sundew leaves depends on the molecular nature of the inducer applied. In situ hybridization of sundew leaves with a Drosera chitinase probe showed chitinase gene expression in different cell types of non-treated leaves, but not in the secretory cells of the glandular heads. Upon induction, chitinase mRNA was also present in the secretory cells of the sundew leaf. The combined results indicate that chitinase is likely to be involved in the decomposition of insect prey by carnivorous plants. This adds a novel role to the already broad function of chitinases in the plant kingdom and may contribute to our understanding of the molecular mechanisms behind the ecological success of carnivorous plants in nutritionally poor environments.     

Pollinator‐prey conflict in carnivorous plants

Most carnivorous plants utilize insects in two ways: the flowers attract insects as pollen vectors for sexual reproduction, and the leaves trap insects for nutrients. Feeding on insects has been explained as an adaptation to nutrient‐poor soil, and carnivorous plants have been shown to benefit from insect capture through increased growth, earlier flowering and increased seed production. Most carnivorous plant species seem to benefit from insect pollination, although many species autonomously self‐pollinate and some propagate vegetatively. However, assuming that outcross pollen is advantageous and is a more important determinant of reproductive success than the nutrients gained from prey, there should be a selective pressure on carnivorous plants not to feed on their potential pollen vectors. Therefore, it has been suggested that carnivorous plants are subject to a conflict, often called the pollinator‐prey conflict (PPC). The conflict results from a trade‐off of the benefits from feeding on potentially pollinating insects versus the need to use them as pollen vectors for sexual reproduction. In this review we analyze the conditions under which a PPC may occur, review the evidence for the existence of PPCs in carnivorous plants, and explore the mechanisms that may be in place to prevent or alleviate a PPC. With respect to the latter, we discuss how plant signals such as olfactory and visual cues may play a role in separating the functions of pollinator attraction and prey capture.     

Isolation of High Quality DNA and RNA from Leaves of the Carnivorous Plant Drosera rotundifolia

Drosera rotundifolia belongs to the family of the sundews, a large group of carnivorous plants that carry stalked glands on the upper leaf surface to attract, trap and digest insects for food. Therefore, such plants can live in relatively poor ecosystems. They are frequently used as medicinal herbs and have various other interesting characteristics associated with them. In attempts to evaluate the gene pool of these plants, we experienced that many published protocols for nucleic acid isolation failed to yield DNA and RNA of sufficient quality for analysis. Therefore, we have developed CTAB (hexadecyltrimethylammoniumbromide)-based extraction protocols for the routine isolation of high-quality DNA and RNA from small amounts of in vitro-grown Drosera rotundifolia leaves. The methods developed are simple, fast and effective. The obtained DNA could be analyzed by PCR, restriction endonucleases and DNA gel blotting, and the obtained RNA was of sufficient quality for RT-PCR and RNA gel blotting.     

Evolving Darwin's 'most wonderful' plant: ecological steps to a snap-trap

Among carnivorous plants, Darwin was particularly fascinated by the speed and sensitivity of snap-traps in Dionaea and Aldrovanda . Recent molecular work confirms Darwin's conjecture that these monotypic taxa are sister to Drosera , meaning that snap-traps evolved from a 'flypaper' trap. Transitions include tentacles being modified into trigger hairs and marginal 'teeth', the loss of sticky tentacles, depressed digestive glands, and rapid leaf movement. Pre-adaptations are known for all these traits in Drosera yet snap-traps only evolved once. We hypothesize that selection to catch and retain large insects favored the evolution of elongate leaves and snap-tentacles in Drosera and snap-traps. Although sticky traps efficiently capture small prey, they allow larger prey to escape and may lose nutrients. Dionaea 's snap-trap efficiently captures and processes larger prey providing higher, but variable, rewards. We develop a size-selective model and parametrize it with field data to demonstrate how selection to capture larger prey strongly favors snap-traps. As prey become larger, they also become rarer and gain the power to rip leaves, causing returns to larger snap-traps to plateau. We propose testing these hypotheses with specific field data and Darwin-like experiments. The complexity of snap-traps, competition with pitfall traps, and their association with ephemeral habitats all help to explain why this curious adaptation only evolved once.     

S-like ribonuclease gene expression in carnivorous plants

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

Quantification of insect nitrogen utilization by the venus fly trap Dionaea muscipula catching prey with highly variable isotope signatures.

Dionaea is a highly specialized carnivorous plant species with a unique mechanism for insect capture. The leaf is converted into an osmotically driven trap that closes when an insect triggers sensory trichomes. This study investigates the significance of insect capture for growth of Dionaea at different successional stages after a fire, under conditions where the prey is highly variable in its isotope signature. The contribution of insect-derived nitrogen (N) was estimated using the natural abundance of 15N. In contrast to previous 15N studies on carnivorous plants, the problem emerges that delta15N values of prey insects ranged between -4.47 per thousand (grasshoppers) and +7.21 per thousand (ants), a range that exceeds the delta15N values of non carnivorous reference plants (-4.2 per thousand) and soils (+3 per thousand). Thus, the isotope-mixing model used by Shearer and Kohl to estimate the amount of insect-derived N is not applicable. In a novel approach, the relationships of delta15N values of different organs with delta15N of trapping leaves were used to estimate N partitioning within the plant. It is estimated that soon after fire approximately 75% of the nitrogen is obtained from insects, regardless of plant size or developmental stage. The estimates are verified by calculating the average isotope signatures of insects from an isotope mass balance and comparing this with the average measured delta15N values of insects. It appears that for Dionaea to survive and reach the flowering stage, seedlings must first reach the 6th-leaf rosette stage, in which trap surface area nearly doubles and facilitates the capture of large insects. Large amounts of nitrogen thus made available to plants may facilitate an enhanced growth rate and the progressive production of additional large traps. Dionaea reaches a maximum abundance after fire when growth of the competing vegetation is suppressed. About 10 years after fire, when grasses and shrubs recover, Dionaea becomes overtopped by other species. This would not only reduce carbon assimilation but also the probability of catching larger prey. The amount of insect-derived nitrogen decreases to 46%, and Dionaea becomes increasingly dependent on N-supply from the soil. Competition for both light and N may cause the near disappearance of Dionaea in older stages of the fire succession.     

Food or sex; pollinator–prey conflict in carnivorous plants

Carnivorous plants potentially trap their own pollinators and it has been argued that considerable spatial separation of flowers and traps has evolved to protect pollinators. We investigated flower-trap separation of Drosera and Utricularia . Short Drosera had a greater element of floral–trap separation than tall Drosera . Such a relationship is unexpected for plants whose peduncles were evolved to protect their pollinators. Utricularia can not trap pollinators but this genus still produces exceptionally long peduncles. We propose that flower-trap separation evolved because carnivorous plants are often short and need to project their flowers well above ground level to make them more attractive to pollinators.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

Abstract

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

Endocytotic uptake of nutrients in carnivorous plants

Carnivorous plants trap, digest and absorb animals in order to supplement their mineral nutrition. Nutrients absorbed by the plant include different nitrogen species, phosphate, potassium, trace elements and small organic compounds. Uptake is usually thought to be performed via specific channels, but this study provides evidence that endocytosis is involved as well. Traps of the carnivorous plants Nepenthes coccinea, Nepenthes ventrata, Cephalotus follicularis, Drosophyllum lusitanicum, Drosera capensis, Dionaea muscipula, Aldrovanda vesiculosa, Genlisea violacea × lobata, Sarracenia psittacina and Sarracenia purpurea were stained with methylene blue in order to identify possible sites of uptake. The permeable parts of the traps were incubated with fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) and other fluorescent endocytosis markers, combined with the soluble protein BSA or respiratory inhibitors. Uptake was studied by confocal microscopy. In Nepenthes, small fluorescent vesicles became visible 1 h after incubation with FITC-BSA. These vesicles fused to larger compartments within 30 h. A similar behaviour was found in the related genera Drosera, Dionaea, Aldrovanda and Drosophyllum but also in Cephalotus with glands of different evolutionary origin. In Genlisea and Sarracenia, no evidence for endocytosis was found. We propose that in many carnivorous plants, nutrient uptake by carriers is supplemented by endocytosis, which enables absorption and intracellular digestion of whole proteins. The advantage for the plant of reducing secretion of enzymes for extracellular digestion is evident.     

Reliance on prey-derived nitrogen by the carnivorous plant Drosera rotundifolia decreases with increasing nitrogen deposition

? Carnivory in plants is presumed to be an adaptation to a low-nutrient environment. Nitrogen (N) from carnivory is expected to become a less important component of the N budget as root N availability increases. ? Here, we investigated the uptake of N via roots versus prey of the carnivorous plant Drosera rotundifolia growing in ombrotrophic bogs along a latitudinal N deposition gradient through Sweden, using a natural abundance stable isotope mass balance technique. ? Drosera rotundifolia plants receiving the lowest level of N deposition obtained a greater proportion of N from prey (57%) than did plants on bogs with higher N deposition (22% at intermediate and 33% at the highest deposition). When adjusted for differences in plant mass, this pattern was also present when considering total prey N uptake (66, 26 and 26 μg prey N per plant at the low, intermediate and high N deposition sites, respectively). The pattern of mass-adjusted root N uptake was opposite to this (47, 75 and 86 μg N per plant). ? Drosera rotundifolia plants in this study switched from reliance on prey N to reliance on root-derived N as a result of increasing N availability from atmospheric N deposition.     

Hoverflies can sense the risk of being trapped by carnivorous plants: An empirical study using Sphaerophoria menthastri and Drosera toyoakensis

Carnivorous plants are major predators of small insects in some habitats. Because traps of carnivorous plants are serious threats for small insects, it is probable to evolve a mechanism to sense a cue of carnivorous plants and avoid being trapped. However, such a sensing behavior of small insects has never been described. Here we report that a hoverfly species Sphaerophoria menthastri, a major pollinator species of carnivorous sundew Drosera toyoakensis, exhibits a behavior to sense a cue of trap leaves and avoids landing there. In a quadrat (5?m?×?5?m) where D. toyoakensis and other non-carnivorous plant species co-occur, we observed behaviors of hoverflies approaching D. toyoakensis and other plants. The numbers of approaches to trap leaves, flowers of D. toyoakensis, flowers of non-carnivorous Lysimachia fortunei and leaves of Poaceae and Cyperaceae were 9, 60, 52 and 54, respectively, and the numbers of landings to those four organs were 2, 55, 49 and 49, respectively. When S. menthastri approached trap leaves, almost all individuals successfully avoided landing there by 1 or 2 hesitation behaviors. These findings suggest that S. menthastri can sense the cue of trap leaves during an approach.