Volatile organic compounds influence prey composition in Sarracenia carnivorous plants.

Sarracenia pitcher plants display interspecific differences in prey, so far only explained by pitcher morphology. We hypothesized that pitcher odours play a role in prey composition. We first compared odour and prey compositions among Sarracenia taxa grown together, forming a kinship gradient from S. purpurea known to capture primarily ants towards S. leucophylla known to capture many flying insects: S. purpurea, S. X mitchelliana, and S. X Juthatip soper & S. X leucophylla horticultural hybrids. We then measured several pitcher traits to disentangle the contributions of morphology and odour to prey variation. The pitcher odours were as diverse as those of generalist-pollinated flowers but with notable differences among taxa, reflecting their relatedness. VOC similarity analyses revealed taxon specificities, that mirrored those revealed by prey similarity analyses. S. X leucophylla stood out by being more specialised in flying insects like bees and moths and by releasing more monoterpenes known to attract flower visitors. S. X Juthatip soper trapped as many bees but fewer moths, sesquiterpenes contributing less to its scent. Ants and Diptera were the main prey of the other two with fatty-acid-derivative-dominated scents. Quantities of the different prey groups can be inferred 98% from quantities of the odour classes and pitcher dimensions. Two syndromes were revealed: ants associated with fatty-acid-derivatives and short pitchers; flying insects associated with monoterpenes, benzenoids and tall pitchers. In S. X leucophylla, emission rate of fatty-acid-derivatives and pitcher length explained most variation in ant captures; monoterpenes and pitcher length explained most variation in bee and moth captures; monoterpenes alone explained most variation in Diptera and wasp captures. Our results suggest that odours are key factors of the diet composition of pitcher plants. They support the hypothesis of perceptual exploitation of insect biases in carnivorous plants and provide new insights into the olfactory preferences of insect groups.

Comment on "Meta-analysis reveals declines in terrestrial but increases in freshwater insect abundances".

Van Klink et al (Reports, 24 April 2020, p. 417) argue for a more nuanced view of insect decline, and of human responsibility for this decline, than previously suggested. However, shortcomings in data selection and methodology raise questions about their conclusions on trends and drivers. We call for more rigorous methodology to be applied in meta-analyses of ecological data.

Proteome analysis of digestive fluids in Nepenthes pitchers.

BACKGROUND AND AIMS: Carnivorous plants have developed strategies to enable growth in nutrient-poor soils. For the genus Nepenthes, this strategy represents producing pitcher-modified leaves that can trap and digest various prey. These pitchers produce a digestive fluid composed of proteins, including hydrolytic enzymes. The focus of this study was on the identification of these proteins. METHODS: In order to better characterize and have an overview of these proteins, digestive fluid was sampled from pitchers at different stages of maturity from five species of Nepenthes (N. mirabilis, N. alata, N. sanguinea, N. bicalcarata and N. albomarginata) that vary in their ecological niches and grew under different conditions. Three complementary approaches based on transcriptomic resources, mass spectrometry and in silico analysis were used. KEY RESULTS: This study permitted the identification of 29 proteins excreted in the pitchers. Twenty of these proteins were never reported in Nepenthes previously and included serine carboxypeptidases, α- and ß-galactosidases, lipid transfer proteins and esterases/lipases. These 20 proteins display sequence signals allowing their secretion into the pitcher fluid. CONCLUSIONS: Nepenthes pitcher plants have evolved an arsenal of enzymes to digest prey caught in their traps. The panel of new proteins identified in this study provides new insights into the digestive process of these carnivorous plants.

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.

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

BACKGROUND AND AIMS: Nepenthes 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.

A simple SDS-PAGE protein pattern from pitcher secretions as a new tool to distinguish Nepenthes species (Nepenthaceae).

PREMISE OF THE STUDY: Carnivorous plants have always fascinated scientists because these plants are able to attract, capture, and digest animal prey using their remarkable traps that contain digestive secretions. Nepenthes is one of the largest genera of carnivorous plants, with 120 species described thus far. Despite an outstanding diversity of trap designs, many species are often confused with each other and remain difficult to classify because they resemble pitchers or of the occurrence of interspecific hybrids. METHODS: Here, we propose a new method to easily distinguish Nepenthes species based on a SDS PAGE protein pattern analysis of their pitcher secretions. Intraspecific comparisons were performed among specimens growing in different environmental conditions to ascertain the robustness of this method. KEY RESULTS: Our results show that, at the juvenile stage and in the absence of prey in the pitcher, an examined species is characterized by a specific and stable profile, whatever the environmental conditions. CONCLUSIONS: The method we describe here can be used as a reliable tool to easily distinguish between Nepenthes species and to help with potential identification based on the species-specific protein pattern of their pitcher secretions, which is complementary to the monograph information.

A carnivorous plant fed by its ant symbiont: a unique multi-faceted nutritional mutualism.

Scarcity of essential nutrients has led plants to evolve alternative nutritional strategies, such as myrmecotrophy (ant-waste-derived nutrition) and carnivory (invertebrate predation). The carnivorous plant Nepenthes bicalcarata grows in the Bornean peatswamp forests and is believed to have a mutualistic relationship with its symbiotic ant Camponotus schmitzi. However, the benefits provided by the ant have not been quantified. We tested the hypothesis of a nutritional mutualism, using foliar isotopic and reflectance analyses and by comparing fitness-related traits between ant-inhabited and uninhabited plants. Plants inhabited by C. schmitzi produced more leaves of greater area and nitrogen content than unoccupied plants. The ants were estimated to provide a 200% increase in foliar nitrogen to adult plants. Inhabited plants also produced more and larger pitchers containing higher prey biomass. C. schmitzi-occupied pitchers differed qualitatively in containing C. schmitzi wastes and captured large ants and flying insects. Pitcher abortion rates were lower in inhabited plants partly because of herbivore deterrence as herbivory-aborted buds decreased with ant occupation rate. Lower abortion was also attributed to ant nutritional service. The ants had higher δ(15)N values than any tested prey, and foliar δ(15)N increased with ant occupation rate, confirming their predatory behaviour and demonstrating their direct contribution to the plant-recycled N. We estimated that N. bicalcarata derives on average 42% of its foliar N from C. schmitzi wastes, (76% in highly-occupied plants). According to the Structure Independent Pigment Index, plants without C. schmitzi were nutrient stressed compared to both occupied plants, and pitcher-lacking plants. This attests to the physiological cost of pitcher production and poor nutrient assimilation in the absence of the symbiont. Hence C. schmitzi contributes crucially to the nutrition of N. bicalcarata, via protection of assimilatory organs, enhancement of prey capture, and myrmecotrophy. This combination of carnivory and myrmecotrophy represents an outstanding strategy of nutrient sequestration.

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.

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.

Range expansion drives dispersal evolution in an equatorial three-species symbiosis.

BACKGROUND: Recurrent climatic oscillations have produced dramatic changes in species distributions. This process has been proposed to be a major evolutionary force, shaping many life history traits of species, and to govern global patterns of biodiversity at different scales. During range expansions selection may favor the evolution of higher dispersal, and symbiotic interactions may be affected. It has been argued that a weakness of climate fluctuation-driven range dynamics at equatorial latitudes has facilitated the persistence there of more specialized species and interactions. However, how much the biology and ecology of species is changed by range dynamics has seldom been investigated, particularly in equatorial regions. METHODOLOGY/PRINCIPAL FINDINGS: We studied a three-species symbiosis endemic to coastal equatorial rainforests in Cameroon, where the impact of range dynamics is supposed to be limited, comprised of two species-specific obligate mutualists--an ant-plant and its protective ant--and a species-specific ant parasite of this mutualism. We combined analyses of within-species genetic diversity and of phenotypic variation in a transect at the southern range limit of this ant-plant system. All three species present congruent genetic signatures of recent gradual southward expansion, a result compatible with available regional paleoclimatic data. As predicted, this expansion has been accompanied by the evolution of more dispersive traits in the two ant species. In contrast, we detected no evidence of change in lifetime reproductive strategy in the tree, nor in its investment in food resources provided to its symbiotic ants. CONCLUSIONS/SIGNIFICANCE: Despite the decreasing investment in protective workers and the increasing investment in dispersing females by both the mutualistic and the parasitic ant species, there was no evidence of destabilization of the symbiosis at the colonization front. To our knowledge, we provide here the first evidence at equatorial latitudes that biological traits associated with dispersal are affected by the range expansion dynamics of a set of interacting species.

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.

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.

The fitness consequences of bearing domatia and having the right ant partner: experiments with protective and non-protective ants in a semi-myrmecophyte.

The fitness advantage provided by caulinary domatia to myrmecophytes has never been directly demonstrated because most myrmecophytic species do not present any individual variation in the presence of domatia and the removal of domatia from entire plants is a destructive process. The semi-myrmecophytic tree, Humboldtia brunonis (Fabaceae: Caesalpinioideae), is an ideal species to investigate the selective advantage conferred by domatia because within the same population, some plants are devoid of domatia while others bear them. Several ant species patrol the plant for extra-floral nectar. Fruit production was found to be enhanced in domatia-bearing trees compared to trees devoid of domatia independent of the ant associate. However, this domatium effect was most conspicuous for trees associated with the populous and nomadic ant, Technomyrmex albipes. This species is a frequent associate of H. brunonis, inhabiting its domatia or building carton nests on it. Ant exclusion experiments revealed that T. albipes was the only ant to provide efficient anti-herbivore protection to the leaves of its host tree. Measures of ant activity as well as experiments using caterpillars revealed that the higher efficiency of T. albipes was due to its greater patrolling density and consequent shorter lag time in attacking the larvae. T. albipes also provided efficient anti-herbivore protection to flowers since fruit initiation was greater on ant-patrolled inflorescences than on those from which ants were excluded. We therefore demonstrated that caulinary domatia provide a selective advantage to their host-plant and that biotic defence is potentially the main fitness benefit mediated by domatia. However, it is not the sole advantage. The general positive effect of domatia on fruit set in this ant-plant could reflect other benefits conferred by domatia-inhabitants, which are not restricted to ants in this myrmecophyte, but comprise a large diversity of other invertebrates. Our results indicate that mutualisms enhance the evolution of myrmecophytism.

Structure and properties of the glandular surface in the digestive zone of the pitcher in the carnivorous plant Nepenthes ventrata and its role in insect trapping and retention.

Carnivorous plants of the genus Nepenthes grow in nutrient-poor habitats and have evolved specialised trapping organs, known as pitchers. These are composed of different surface zones serving the functions of attraction, capture and digestion of insects, which represent a main source of nitrogen. To investigate the role of the glandular digestive zone in the trapping mechanism of the pitcher, structural, mechanical and physico-chemical studies were applied to N. ventrata and combined with insect behavioural experiments. It was found that the glandular surface is microscopically rough since it is regularly structured with multicellular glands situated in epidermal depressions. The presence of downward-directed 'hoods' over the upper part of glands and sloped depressions in the proximal direction of the pitcher causes a marked anisotropy of the surface. The glandular zone surface is composed of relatively stiff material (Young's modulus, 637.19+/-213.44 kPa). It is not homogeneous, in terms of adhesive properties, and contains numerous areas without adhesion as well as adhesive areas differing greatly in tenacity values (range, 1.39-28.24 kPa). The surface is readily wettable with water (contact angle, 31.9-36.0 degrees C) and has a high surface free energy (56.84-61.93 mN m(-1)) with a relatively high polar component (33.09-52.70 mN m(-1)). To examine the effect of the glandular secretion on attachment systems of insects having hairy and smooth adhesive pads, forces generated on different surfaces by Calliphora vicina flies and Pyrrhocoris apterus bugs, respectively, were measured. Flies attached equally well to both fresh and air-dried glandular surfaces whereas bugs generated a significantly lower force on the fresh glandular surface compared with the air-dried one. It is assumed that the contribution of the glandular surface to insect retention, due to its effect on insect attachment, differs depending on insect weight and the type of insect attachment system. Surface anisotropy does not facilitate effective claw interlocking so that insects possessing only claws are probably not able to cling to the glandular surface. However, stiffness of the pitcher wall material in the digestive zone can provide claw clinging via punching of the pitcher wall by claws. Small insects lacking pads may use adhesive areas on the plant surface to attach themselves, but such solitary points with very strong adhesion possibly impede their overall locomotion and chance of escape. Pad-bearing insects are presumably able to attach to smooth parts of the glandular surface located between glands. High free surface energy of the plant substrate may promote adhesion. Gland secretion may decrease attachment ability in insects with smooth adhesive pads but not influence attachment of insects with hairy attachment systems.

Function of epidermal surfaces in the trapping efficiency of Nepenthes alata pitchers.

• Several epidermal microstructures characterize surfaces of pitcher plants and are presumably involved in their trapping function. Here we report the effects of Nepenthes alata surfaces on insect locomotion and trapping efficiency. • The architectural designs of pitcher surfaces were characterized using scanning electron microscopy. Two insect species - fruitfly (Drosophila melanogaster) and ant (Iridomyrmex humilis) - were tested for their ability to remain and walk on them. The relative contributions of various epidermal structures to trapping ability were quantified. • Pitchers were very effective traps for both insect species. They were slightly more efficient in capturing the ants, but slightly more effective in retaining captured flies. Trapping efficiency was attributed to the combined effects of several surfaces displaying different functions. The waxy zone played a key role in the slippery syndrome: in addition to the wax itself, the subjacent layer of convex lunate cells interfered considerably with insect locomotion. The unsubmersed glandular zone displayed an important retentive effect and secretions of the digestive glands are suspected to be adhesive. • Pad performances of the hairy and smooth system of attachment are discussed to explain the differences between the two insect species. This study aims to encourage biomechanical studies of plant-insect surface mechanisms.

Benefits conferred by "timid" ants: active anti-herbivore protection of the rainforest tree Leonardoxa africana by the minute ant Petalomyrmex phylax.

In this study, we demonstrate that an important benefit provided by the small host-specific ant Petalomyrmex phylax to its host plant Leonardoxa africana is efficient protection against herbivores. We estimate that in the absence of ants, insect herbivory would reduce the leaf area by about one-third. This contributes considerably to the fitness of the plant. Our estimates take into account not only direct damage, such as removal of leaf surface by chewing insects, but also the effects of sucking insects on leaf growth and expansion. Sucking insects are numerically predominant in this system, and the hitherto cryptic effects of ant protection against the growth-reducing effects of sucking insects accounted for half of the total estimated benefit of ant protection. We propose that the small size of workers confers a distinct advantage in this system. Assuming that resource limitation implies a trade off between size and number of ants, and given the small size of phytophagous insects that attack Leonardoxa, we conclude that fine-grained patrolling by a large number of small workers maximises protection of young leaves of this plant. Since herbivores are small and must complete their development on the young leaves of Leonardoxa, and since a high patrolling density is required for a fine-grained search for these enemies, numerous small ants should provide the most effective protection of young leaves of Leonardoxa. We also discuss other factors that may have influenced worker size in this ant.